95369245>IL-2 gene expression and <n>NF-kappa B</n> activation through <n>CD28</n> requires reactive oxygen production by <n>5-lipoxygenase</n>. Activation of the <n>CD28</n> surface receptor provides a major costimulatory signal for T cell activation resulting in enhanced production of <n>interleukin-2</n> (<n>IL-2</n>) and cell proliferation. In primary T lymphocytes we show that <n>CD28</n> ligation leads to the rapid intracellular formation of reactive oxygen intermediates (ROIs) which are required for <n>CD28</n>- mediated activation of the <n>NF-kappa B</n>/<n>CD28</n>-responsive complex and <n>IL-2</n> expression. Delineation of the <n>CD28</n> signaling cascade was found to involve protein tyrosine kinase activity, followed by the activation of <n>phospholipase A2</n> and <n>5-lipoxygenase</n>. Our data suggest that <n>lipoxygenase</n> metabolites activate ROI formation which then induce <n>IL-2</n> expression via <n>NF-kappa B</n> activation. These findings should be useful for therapeutic strategies and the development of immunosuppressants targeting the <n>CD28</n> costimulatory pathway.
95333264>The peri-kappa B site mediates human immunodeficiency virus type 2 enhancer activation in monocytes but not in T cells. Human immunodeficiency virus type 2 (HIV-2), like HIV-1, causes AIDS and is associated with AIDS cases primarily in West Africa. HIV-1 and HIV-2 display significant differences in nucleic acid sequence and in the natural history of clinical disease. Consistent with these differences, we have previously demonstrated that the enhancer/promoter region of HIV-2 functions quite differently from that of HIV-1. Whereas activation of the HIV-1 enhancer following T-cell stimulation is mediated largely through binding of the transcription factor <n>NF-kappa B</n> to two adjacent kappa B sites in the HIV-1 long terminal repeat, activation of the HIV-2 enhancer in monocytes and T cells is dependent on four cis-acting elements: a single kappa B site, two purine-rich binding sites, PuB1 and PuB2, and a pets site. We have now identified a novel cis-acting element within the HIV-2 enhancer, immediately upstream of the kappa B site, designated peri-kappa B. This site is conserved among isolates of HIV-2 and the closely related simian immunodeficiency virus, and transfection assays show this site to mediate HIV-2 enhancer activation following stimulation of monocytic but not T-cell lines. This is the first description of an HIV-2 enhancer element which displays such monocyte specificity, and no comparable enhancer element has been clearly defined for HIV-1. While a nuclear factor(s) from both peripheral blood monocytes and T cells binds the peri-kappa B site, electrophoretic mobility shift assays suggest that either a different protein binds to this site in monocytes versus T cells or that the protein recognizing this enhancer element undergoes differential modification in monocytes and T cells, thus supporting the transfection data. Further, while specific constitutive binding to the peri-kappa B site is seen in monocytes, stimulation with phorbol esters induces additional, specific binding. Understanding the monocyte-specific function of the <n>peri-kappa B factor</n> may ultimately provide insight into the different role monocytes and T cells play in HIV pathogenesis.
95343554>E1A gene expression induces susceptibility to killing by NK cells following immortalization but not adenovirus infection of human cells. Adenovirus (Ad) infection and <n>E1A</n> transfection were used to model changes in susceptibility to NK cell killing caused by transient vs stable <n>E1A</n> expression in human cells. Only stably transfected target cells exhibited cytolytic susceptibility, despite expression of equivalent levels of E1A proteins in Ad-infected targets. The inability of E1A gene products to induce cytolytic susceptibility during infection was not explained by an inhibitory effect of viral infection on otherwise susceptible target cells or by viral gene effects on class I MHC antigen expression on target cells. This differential effect of <n>E1A</n> expression on the cytolytic phenotypes of infected and stably transfected human cells suggests that human NK cells provide an effective immunologic barrier against the in vivo survival and neoplastic progression of E1A-immortalized cells that may emerge from the reservoir of persistently infected cells in the human host.
95347379>Distinct signaling properties identify functionally different CD4 epitopes. The <n>CD4 coreceptor</n> interacts with non-polymorphic regions of major histocompatibility complex class II molecules on antigen-presenting cells and contributes to T cell activation. We have investigated the effect of <n>CD4</n> triggering on T cell activating signals in a lymphoma model using monoclonal antibodies (mAb) which recognize different CD4 epitopes. We demonstrate that <n>CD4</n> triggering delivers signals capable of activating the <n>NF-AT transcription factor</n> which is required for <n>interleukin-2</n> gene expression. Whereas different anti-CD4 mAb or <n>HIV-1 gp120</n> could all trigger activation of the protein tyrosine kinases <n>p56lck</n> and <n>p59fyn</n> and phosphorylation of the <n>Shc adaptor protein</n>, which mediates signals to Ras, they differed significantly in their ability to activate <n>NF-AT</n>. Lack of full activation of <n>NF-AT</n> could be correlated to a dramatically reduced capacity to induce calcium flux and could be complemented with a calcium ionophore. The results identify functionally distinct epitopes on the <n>CD4 coreceptor</n> involved in activation of the <n>Ras</n>/<n>protein kinase C</n> and calcium pathways.
95280913>Ligand-dependent repression of the erythroid transcription factor <n>GATA- 1</n> by the estrogen receptor. High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the <n>erythroid cell-specific histone</n> <n>H5</n>. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on <n>GATA-1</n>, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of <n>GATA-1</n> is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of <n>GATA-1</n> activity occurs on an artificial promoter containing a single GATA- binding site, as well as in the context of an intact promoter which is normally regulated by <n>GATA-1</n>. <n>GATA-1</n> and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, <n>GATA-1</n> and ER associate in a ligand-dependent manner. Mapping experiments indicate that <n>GATA-1</n> and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of <n>GATA-1</n>. We speculate that estrogens exert effects on erythropoiesis by modulating <n>GATA-1</n> activity through protein-protein interaction with the ER. (ABSTRACT TRUNCATED AT 250 WORDS)
95256242>Mouse <n>interleukin-2</n> receptor alpha gene expression. <n>Interleukin-1</n> and <n>interleukin-2</n> control transcription via distinct cis-acting elements. We have shown that <n>interleukin-1</n> (<n>IL-1</n>) and <n>IL-2</n> control <n>IL-2 receptor alpha</n> (<n>IL-2R alpha</n>) gene transcription in CD4-CD8- murine T lymphocyte precursors. Here we map the cis-acting elements that mediate interleukin responsiveness of the <n>mouse IL-2R alpha</n> gene using a thymic lymphoma-derived hybridoma (PC60). The transcriptional response of the IL-2R alpha gene to stimulation by <n>IL-1</n> + <n>IL-2</n> is biphasic. <n>IL-1</n> induces a rapid, protein synthesis-independent appearance of <n>IL-2R alpha</n> mRNA that is blocked by inhibitors of <n>NF-kappa B</n> activation. It also primes cells to become <n>IL-2</n> responsive and thereby prepares the second phase, in which <n>IL-2</n> induces a 100-fold further increase in <n>IL- 2R alpha</n> transcripts. Transient transfection experiments show that several elements in the promoter-proximal region of the <n>IL-2R alpha</n> gene contribute to <n>IL-1</n> responsiveness, most importantly an <n>NF-kappa B</n> site conserved in the human and mouse gene. <n>IL-2</n> responsiveness, on the other hand, depends on a 78-nucleotide segment 1.3 kilobases upstream of the major transcription start site. This segment functions as an IL- 2-inducible enhancer and lies within a region that becomes <n>DNase I</n> hypersensitive in normal T cells in which <n>IL-2R alpha</n> expression has been induced. <n>IL-2</n> responsiveness requires three distinct elements within the enhancer. Two of these are potential binding sites for STAT proteins.
95338146>Hematopoietic lineage commitment: role of transcription factors. This review focuses on the roles of transcription factors in hematopoietic lineage commitment. A brief introduction to lineage commitment and asymmetric cell division is followed by a discussion of several methods used to identify transcription factors important in specifying hematopoietic cell types. Next is presented a discussion of the use of embryonic stem cells in the analysis of hematopoietic gene expression and the use of targeted gene disruption to analyze the role of transcription factors in hematopoiesis. Finally, the status of our current knowledge concerning the roles of transcription factors in the commitment to erythroid, myeloid and lymphoid cell types is summarized.
95266275>Epstein-Barr virus replicative gene transcription during de novo infection of human thymocytes: simultaneous early expression of <n>BZLF-1</n> and its repressor <n>RAZ</n>. Epstein-Barr virus (EBV) is known to infect B cells and epithelial cells. We and others have shown that EBV can also infect a subset of thymocytes. Infection of thymocytes was accompanied by the appearance of linear EBV genome within 8 hr of infection. Circularization of the EBV genome was not detected. This is in contrast to the infection in B cells where the genome can circularize within 24 hr of infection. The appearance of the <n>BamHI ZLF-1 gene product</n>, <n>ZEBRA</n>, by RT-PCR, was observed within 8 hr of infection. The appearance of a novel fusion transcript (<n>RAZ</n>), which comprised regions of the <n>BZLF-1</n> locus and the adjacent BRLF-1 locus, was detected by RT-PCR. <n>ZEBRA</n> protein was also identified in infected thymocytes by immunoprecipitation. In addition, we demonstrated that the <n>EBNA-1</n> gene in infected thymocytes was transcribed from the Fp promoter, rather than from the Cp/Wp promoter which is used in latently infected B cells. Transcripts encoding <n>gp350/220</n>, the major coat protein of EBV, were identified, but we did not find any evidence of transcription from the LMP-2A or EBER-1 loci in infected thymocytes. These observations suggest that de novo EBV infection of thymocytes differs from infection of B cells. The main difference is that with thymocytes, no evidence could be found that the virus ever circularizes. Rather, EBV remains in a linear configuration from which replicative genes are transcribed.
95236292>Identification and purification of human Stat proteins activated in response to <n>interleukin-2</n>. A key cytokine induced during the immune response is <n>IL-2</n>. Following T cell activation, the genes encoding <n>IL-2</n> and the various chains of its receptor are transcriptionally induced. In turn, secreted <n>IL-2</n> serves to stimulate the proliferation and differentiation of T lymphocytes. Several recent studies have implicated Jak kinases in the signaling pathway induced by <n>IL-2</n>. Following this lead, we set out to identify transcription factors induced in response to <n>IL-2</n>. Human peripheral blood lymphocytes were observed to contain several <n>IL-2</n>-inducible DNA binding activities. Similar activities were also observed in a transformed human lymphocyte line, termed YT. We have purified these activities and found that the principal <n>IL-2</n>-inducible component bears significant relatedness to a <n>prolactin-induced transcription factor</n> first identified in sheep mammary gland tissue. We hypothesize that activation of this protein, designated <n>hStat5</n>, helps govern the biological effects of <n>IL-2</n> during the immune response.
95197524><n>E2F-1</n> and a cyclin-like DNA repair enzyme, <n>uracil-DNA glycosylase</n>, provide evidence for an autoregulatory mechanism for transcription. The cell cycle-dependent transcription factor, <n>E2F-1</n>, regulates the cyclin-like species of the DNA repair enzyme <n>uracil-DNA glycosylase</n> (<n>UDG</n>) gene in human osteosarcoma (Saos-2) cells. We demonstrate, through the deletion of the human <n>UDG</n> promoter sequences, that expression of <n>E2F-1</n> activates the <n>UDG</n> promoter through several E2F sites. The major putative downstream site for E2F, located in the first exon, serves as a target for E2F-1/DP1 complex binding in vitro. We also provide evidence for the functional relationship between the cyclin-like <n>UDG</n> gene product and E2F. High levels of <n>UDG</n> expression in a transient transfection assay result in the down-regulation of transcriptional activity through elements specific for E2F-mediated transcription. Overexpression of <n>UDG</n> in Saos 2 cells was observed to delay growth late in G1 phase and transiently arrest these cells from progressing into the S phase. This hypothetical model integrates one mechanism of DNA repair with the cell cycle control of gene transcription, likely through E2F. This implicates E2F as a multifunctional target for proteins and enzymes, possibly, responsive to DNA damage through the negative effect of <n>UDG</n> on E2F-mediated transcriptional activity
95370702>Cellular and molecular mechanisms of <n>IFN-gamma</n> production induced by <n>IL- 2</n> and <n>IL-12</n> in a human NK cell line. <n>Interferon-gamma</n> (<n>IFN-gamma</n>) is an important <n>immunoregulatory protein</n> produced predominantly by T cells and large granular lymphocytes (LGL) in response to different extracellular signals. In particular, two interleukins (ILs), <n>IL-2</n> and <n>IL-12</n>, have been shown to be potent inducers of <n>IFN-gamma</n> gene expression in both T cells and LGL. Although it has been reported that there are some T cell lines that produce <n>IFN- gamma</n> in response to IL-2 and IL-12 stimulation, there has as yet been no report of a natural killer (NK) cell line that responds in a similar manner. In this report we present evidence that the cell line NK3.3 derived from human NK cells, responds to both <n>IL-2</n> and <n>IL-12</n>, as measured by increases in <n>IFN-gamma</n> and <n>granulocyte-macrophage colony- stimulating factor</n> (<n>GM-CSF</n>) cytoplasmic mRNA and protein expression. In addition, when used together <n>IL-2</n> and <n>IL-12</n> synergized in the induction of <n>IFN-gamma</n> and <n>GM-CSF</n> and this synergy was attributed to an increased accumulation and stability of the <n>IFN-gamma</n> and <n>GM-CSF</n> mRNAs. To investigate the signaling pathways involved in the gene induction, five inhibitors, cyclosporin A (CsA), <n>transforming growth factor-beta</n>, cycloheximide, genistein, and staurosporine A, were used in analyzing the effects of <n>IL-2</n> and <n>IL-12</n> on NK3.3 cells. The results suggest that activation of <n>protein kinase C</n>, but not new protein synthesis, is required for <n>IL-2</n> induction of IFN-gamma and GM-CSF cytoplasmic mRNA. In contrast, <n>IL-12</n> induction of <n>IFN-gamma</n> cytoplasmic mRNA appears to only partially depend on activation of <n>protein kinase C</n>. Furthermore, both <n>transforming growth factor-beta</n> and genistein, a tyrosine kinase inhibitor, could suppress <n>IL-2</n> and <n>IL-12</n> signaling but CsA was generally inactive. It also was observed that suppression of cytokine gene expression by these agents was independent of the inhibition of proliferation. In addition, <n>IL-2</n> but not <n>IL-12</n> induced nuclear factors <n>NF-kappa B</n> and <n>AP1</n>, and regulation of the nuclear levels of these two DNA binding protein complexes is correlated with IFN-gamma and GM-CSF gene expression. These data indicate that <n>IL-2</n> and <n>IL-12</n> may have distinct signaling pathways leading to the induction of <n>IFN-gamma</n> and <n>GM-CSF</n> gene expression, and that the NK3.3 cell line may serve as a novel model for dissecting the biochemical and molecular events involved in these pathways.
95349600>A functional T-cell receptor signaling pathway is required for <n>p95vav</n> activity. Stimulation of the <n>T-cell antigen receptor</n> (<n>TCR</n>) induces activation of multiple <n>tyrosine kinases</n>, resulting in phosphorylation of numerous intracellular substrates. One substrate is <n>p95vav</n>, which is expressed exclusively in hematopoietic and trophoblast cells. It contains a number of structural motifs, including Src homology 2, Src homology 3, and pleckstrin homology domains and a putative guanine nucleotide exchange domain. The role of <n>p95vav</n> in <n>TCR</n>-mediated signaling processes is unclear. Here, we show that overexpression of <n>p95vav</n> alone in Jurkat T cells leads to activation of the nuclear factors, including NFAT, involved in <n>interleukin-2</n> expression. Furthermore, <n>p95vav</n> synergizes with <n>TCR</n> stimulation in inducing NFAT- and interleukin-2-dependent transcription. In contrast, NFAT activation by a G-protein-coupled receptor is not modulated by <n>p95vav</n> overexpression, suggesting that the effect is specific to the <n>TCR</n> signaling pathways. Although removal of the first 67 amino acids of <n>p95vav</n> activates its transforming potential in NIH 3T3 cells, this region appears to be required for its function in T cells. We further demonstrate that the <n>p95vav</n>-induced NFAT activation is not mimicked by Ras activation, though its function is dependent upon Ras and Raf. Furthermore, the activating function of <n>p95vav</n> is blocked by FK506, suggesting that its activity also depends on <n>calcineurin</n>. To further dissect <n>p95vav</n> involvement in <n>TCR</n> signaling, we analyzed various Jurkat mutants deficient in <n>TCR</n> signaling function or <n>TCR</n> expression and showed that an intact <n>TCR</n> signaling pathway is required for <n>p95vav</n> to function. However, overexpression of <n>p95vav</n> does not appear to influence <n>TCR</n>-induced protein tyrosine phosphorylation or increases in cytoplasmic free calcium. Taken together, our data suggest that <n>p95vav</n> plays an important role at an yet unidentified proximal position in the <n>TCR</n> signaling cascade.
95329717>Positive and negative regulation of <n>granulocyte-macrophage colony- stimulating factor</n> promoter activity by <n>AML1-related transcription factor</n>, <n>PEBP2</n>. The <n>granulocyte-macrophage colony-stimulating factor</n> (<n>GM-CSF</n>) gene promoter contains a consensus sequence for the <n>polyomavirus enhancer binding-protein 2</n> (<n>PEBP2</n>) transcription factor, which consists of alpha and beta subunits. There are at least two genes, alpha A and alpha B, encoding the alpha subunit. alpha B is the mouse homologue of human AML1 gene detected at the breakpoints of t(8;21) and t(3;21) myeloid leukemias. We examined alpha A1 (an alpha A-gene product) and alpha B1 and alpha B2 (two alpha B-encoded isomers) for their effects on the GM- CSF promoter. <n>PEBP2</n> alpha A1, alpha B1, and alpha B2 proteins bound the <n>PEBP2</n> site within the mouse <n>GM-CSF</n> promoter. <n>PEBP2</n> alpha A1 and alpha B1 enhanced the expression of the <n>GM-CSF</n> promoter-driven reporter plasmid in unstimulated and 12-O-tetradecanoylphorbol 13- acetate/phytohemagglutinin-stimulated human Jurkat T cells. In contrast, the promoter activity was suppressed by alpha B2. Coexpression of alpha B1 and alpha B2 showed that the promoter activity could be determined by the alpha B1/alpha B2 ratio. Jurkat cell extract contained <n>PEBP2</n> site-binding protein(s) that cross-reacted with antimouse alpha A1 antibodies. Northern blot analysis indicated the expression of human <n>PEBP2</n> alpha A, alpha B (AML1), and beta genes in Jurkat cells. Although further studies are required to determine the precise role of <n>PEBP2</n> in the <n>GM-CSF</n> promoter activity, the present findings suggested the importance of the relative ratio of different <n>PEBP2</n> isoforms in regulating the levels of the promoter activity.
96075792><n>IFN-gamma</n> priming of monocytes enhances LPS-induced TNF production by augmenting both transcription and MRNA stability. The induction of cytokine expression in monocytes/macrophages by bacterial endotoxin or lipopolysaccharide is a critical, highly regulated host defence response. The augmentation of LPS responses by <n>interferon gamma</n> (<n>IFN-gamma</n>), referred to as priming, is well established. However, the mechanism(s) by which priming occurs is poorly defined. Using tumour necrosis factor (TNF) induction as a model, experiments were designed to analyse in detail the priming effect on the LPS response in human monocytes. Priming by <n>IFN-gamma</n> was primarily manifested at the level of TNF mRNA accumulation. <n>IFN-gamma</n> pre-treatment affected the magnitude rather than the sensitivity of the LPS response. Priming occurred after several hours of treatment, and the primed state was induced by either <n>IFN-gamma</n> or <n>GM-CSF</n>, but not <n>M- CSF</n>. Primed monocytes transcribed TNF mRNA at a higher rate than freshly isolated monocytes upon activation with LPS. The increased transcriptional rate correlated with a marked increase in <n>nuclear factor-kappa B</n> activity in these cells as determined by electrophoretic mobility shift assay using a consensus <n>NF-kappa B</n> oligonucleotide. An additional significant finding was than TNF mRNA induced in primed cells was much more stable than in unprimed cells (T1/2 increased 6-8- fold). Consistent with the increased mRNA stability, the duration of mRNA accumulation was longer following LPS stimulation in primed monocytes, in addition to being of greater magnitude. Finally, primed and unprimed cells possessed a differential sensitivity to the kinase inhibitor H-89. H-89 substantially suppressed LPS-induced TNF mRNA accumulation in unprimed cells, but had no effect on primed monocytes following LPS stimulation. (ABSTRACT TRUNCATED AT 250 WORDS)
95280917>A Myc-associated zinc finger protein binding site is one of four important functional regions in the <n>CD4</n> promoter. The <n>CD4</n> promoter plays an important role in the developmental control of <n>CD4</n> transcription. In this report, we show that the minimal <n>CD4</n> promoter has four factor binding sites, each of which is required for full function. Using biochemical and mutagenesis analyses, we determined that multiple nuclear factors bind to these independent sites. We determined that an initiator-like sequence present at the cap site and an Ets consensus sequence are required for full promoter function. We also demonstrate that the <n>Myc-associated zinc finger protein</n> (<n>MAZ</n>) appears to be the predominant factor binding to one of these sites. This last site closely resembles the ME1a1 G3AG4AG3 motif previously shown to be a critical element in the P2 promoter of the c- myc gene. We therefore believe that the <n>MAZ</n> transcription factor is also likely to play an important role in the control of developmental expression of the <n>CD4</n> gene.
95263486><n>Erythropoietin</n> stimulates transcription of the TAL1/SCL gene and phosphorylation of its protein products. Activation of the TAL1 (or SCL) gene, originally identified through its involvement by a recurrent chromosomal translocation, is the most frequent molecular lesion recognized in T-cell acute lymphoblastic leukemia. The protein products of this gene contain the basic-helix- loop-helix motif characteristic of a large family of transcription factors that bind to the canonical DNA sequence CANNTG as protein heterodimers. <n>TAL1</n> expression by erythroid cells in vivo and in chemical-induced erythroleukemia cell lines in vivo suggested the gene might regulate aspects of erythroid differentiation. Since the terminal events of erythropoiesis are controlled by the <n>glycoprotein hormone erythropoietin</n> (<n>Epo</n>), we investigated whether the expression or activity of the <n>TAL1</n> gene and its protein products were affected by <n>Epo</n> in splenic erythroblasts from mice infected with an anemia-inducing strain of Friend virus (FVA cells). <n>Epo</n> elicited a rapid, dose-related increase in <n>TAL1</n> mRNA by increasing transcription of the gene and stabilizing one of its mRNAs. An <n>Epo</n>-inducible <n>TAL1</n> DNA binding activity was identified in FVA cell nuclear extracts that subsequently decayed despite accumulating mRNA and protein. Induction of DNA binding activity was associated temporally with <n>Epo</n>-induced phosphorylation of nuclear <n>TAL1</n> protein. These results indicate that <n>Epo</n> acts at both transcriptional and posttranscriptional levels on the <n>TAL1</n> locus in Friend virus-induced erythroblasts and establish a link between <n>Epo</n> signaling mechanisms and a member of a family of transcription factors involved in the differentiation of diverse cell lineages.
95397944>Nonradioactive quantification of glucocorticoid receptor expression during differentiation of human monocytic cells (U937). We describe a method for relative quantification of specific mRNA using a nonradioactive assay based on DNA strand competition between identical sequences of biotin- and fluorescein-labeled amplicon (probe) and unlabeled amplicon (target) during hybridization. As the target quantity increased, that of the double-labeled probe decreased in accordance with the mass action law. This technique was successfully applied to evaluate differences in glucocorticoid receptor expression in U937 cells before and after the addition of potent differentiation inducers: 12-O-tetradecanoylphorbol 13-acetate (TPA) and a combination of all-trans retinoic acid (RA) and 1,25-dihydroxyvitamin D2 (VD). We observed that TPA treatment was associated with an increase in specific binding of [3H]dexamethasone and up-regulation of GR mRNA while no enhanced GR expression was perceived with RA/VD treatment.
95266282>Induction of <n>Sp1</n> phosphorylation and <n>NF-kappa B</n>-independent HIV promoter domain activity in T lymphocytes stimulated by okadaic acid. In contrast to the purely enhancer-dependent effect of cytokines such as TNF on the activity of the HIV regulatory region (LTR), we observed that okadaic acid (OKA) activates HIV transcription through both the enhancer, responding to the factor <n>NF-kappa B</n>, and the promoter domain of the LTR. The inducibility of HIV LTR-driven <n>luciferase</n> expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional <n>Sp1</n> binding elements and the ability of the TATA box to bind the protein <n>TBP</n>. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed <n>Sp1</n> protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or <n>PHA</n> and <n>interleukin 2</n>. Responsiveness of LTR constructs deleted of kappa B elements to <n>HIV Tat</n> expression was increased upon OKA but not TNF stimulation. Our results suggest that <n>SP1</n> phosphorylation induced by OKA, a selective inhibitor of the serine- threonine phosphatase <n>PP2A</n>, facilitates the formation of a transcription complex involving general transcription factors, <n>HIV Tat</n>, and <n>Sp1</n> proteins. The formation of this complex would increase, independently of an in synergy with <n>NF-kappa B</n>, the low basal activity of the HIV LTR observed in normal T lymphocytes.
95236293>The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by <n>IL-2</n>, <n>IL-4</n>, <n>IL-7</n>, <n>IL-13</n>, and <n>IL-15</n>. To understand the molecular bases for cytokine redundancy and pleiotropy, we have compared the Stat proteins activated in peripheral blood lymphocytes (PBLs) by cytokines with shared and distinct actions. <n>Interleukin-2</n> (<n>IL-2</n>) rapidly activated <n>Stat5</n> in fresh PBL, and <n>Stat3</n> and <n>Stat5</n> in preactivated PBL. <n>IL-7</n> and <n>IL-15</n> induced the same complexes as <n>IL-2</n>, a feature explained by the existence of similar tyrosine-phosphorylated motifs in the cytoplasmic domains of <n>IL-2R beta</n> and <n>IL-7R</n> that can serve as docking sites for Stat proteins. <n>IL-13</n> Induced the same complexes as <n>IL-4</n>, a finding explained by our studies implicating <n>IL-4R</n> as a shared component of the receptors. These studies demonstrate that a single cytokine can activate different combinations of Stat proteins under different physiological conditions, and also indicate two mechanisms by which distinct cytokines can activate the same Stat protein.
95184007>Control of <n>I kappa B-alpha</n> proteolysis by site-specific, signal-induced phosphorylation. <n>I kappa B-alpha</n> inhibits transcription factor <n>NF-kappa B</n> by retaining it in the cytoplasm. Various stimuli, typically those associated with stress or pathogens, rapidly inactivate <n>I kappa B-alpha</n>. This liberates <n>NF-kappa B</n> to translocate to the nucleus and initiate transcription of genes important for the defense of the organism. Activation of <n>NF-kappa B</n> correlates with phosphorylation of <n>I kappa B-alpha</n> and requires the proteolysis of this inhibitor. When either serine-32 or serine-36 of <n>I kappa B-alpha</n> was mutated, the protein did not undergo signal-induced phosphorylation or degradation, and <n>NF-kappa B</n> could not be activated. These results suggest that phosphorylation at one or both of these residues is critical for activation of <n>NF-kappa B</n>
95388524>Regulation of transcription of the human erythropoietin receptor gene by proteins binding to GATA-1 and Sp1 motifs. <n>Erythropoietin</n> (<n>Epo</n>), the primary regulator of the production of erythroid cells, acts by binding to a cell surface receptor (<n>EpoR</n>) on erythroid progenitors. We used deletion analysis and transfection assays with reporter gene constructs to examine the transcription control elements in the 5' flanking region of the human <n>EpoR</n> gene. In erythroid cells most of the transcription activity was contained in a 150 bp promoter fragment with binding sites for transcription factors <n>AP2</n>, <n>Sp1</n> and the <n>erythroid-specific GATA-1</n>. The 150 bp hEpoR promoter exhibited high and low activity in erythroid OCIM1 and K562 cells, respectively, reflecting the high and low levels of constitutive h<n>EpoR</n> expression. The GATA-1 and Sp1 binding sites in this promoter lacking a TATA sequence were necessary for a high level of transcription activation. Protein-DNA binding studies suggested that <n>Sp1</n> and two other CCGCCC binding proteins from erythroid and non-erythroid cells could bind to the <n>Sp1</n> binding motif. By increasing <n>GATA-1</n> levels via co- transfection, we were able to transactivate the hEpoR promoter in K562 cells and non-erythroid cells, but not in the highly active OCIM1 cells, although <n>GATA-1</n> mRNA levels were comparable in OCIM1 and K562. Interestingly, when we mutated the <n>Sp1</n> site, resulting in a marked decrease in hEpoR promoter activity, we could restore transactivation by increasing <n>GATA-1</n> levels in OCIM1 cells. These data suggest that while <n>GATA-1</n> can transactivate the <n>EpoR</n> promoter, the level of hEpoR gene expression does not depend on <n>GATA-1</n> alone. Rather, <n>hEpoR</n> transcription activity depends on coordination between <n>Sp1</n> and <n>GATA-1</n> with other cell-specific factors, including possibly other <n>Sp1</n>-like binding proteins, to provide high level, tissue-specific expression.
95365382>Overexpression of <n>DR-nm23</n>, a protein encoded by a member of the nm23 gene family, inhibits granulocyte differentiation and induces apoptosis in 32Dc13 myeloid cells. Chronic myelogenous leukemia evolves in two clinically distinct stages: a chronic and a blast crisis phase. The molecular changes associated with chronic phase to blast crisis transition are largely unknown. We have identified a cDNA clone, <n>DR-nm23</n>, differentially expressed in a blast-crisis cDNA library, which has approximately 70% sequence similarity to the putative metastatic suppressor genes, nm23-H1 and nm23-H2. The deduced amino acid sequence similarity to the proteins encoded by these two latter genes is approximately 65% and includes domains and amino acid residues (the leucine zipper-like and the RGD domain, a serine and a histidine residue in the NH2- and in the COOH- terminal portion of the protein, respectively) postulated to be important for <n>nm23</n> function. <n>DR-nm23</n> mRNA is preferentially expressed at early stages of myeloid differentiation of highly purified CD34+ cells. Its constitutive expression in the myeloid precursor 32Dc13 cell line, which is growth-factor dependent for both proliferation and differentiation, results in inhibition of granulocytic differentiation induced by <n>granulocyte colony-stimulating factor</n> and causes apoptotic cell death. These results are consistent with a role for <n>DR-nm23</n> in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia.
96032864>An interferon-gamma activation sequence mediates the transcriptional regulation of the IgG Fc receptor type IC gene by <n>interferon-gamma</n>. Expression of the <n>IgG Fc receptor type I</n> (<n>Fc gamma RI</n>) on myeloid cells is dramatically increased by treatment with <n>interferon-gamma</n> (<n>IFN- gamma</n>). We observed that <n>Fc gamma RI</n> transcript levels in monoblast- like U937 cells were elevated within 3 hr and peaked 12 hr after exposure to <n>IFN-gamma</n>. Treatment of U937 with <n>IFN-gamma</n> for 9 hr in the presence of cycloheximide led to super-induction of <n>Fc gamma RI</n> expression. Nuclear run-on analysis revealed that the rate of <n>Fc gamma RI</n> transcription was increased by <n>IFN-gamma</n>. Genomic sequence upstream of the Fc gamma RIC gene was cloned and subjected to primer extension analysis, which demonstrated a single transcription initiation site without a TATA box. Transient transfections of CAT reporter gene constructs containing various Fc gamma RIC promoter sequences into U937 cells revealed that a 20-bp region surrounding the transcription start site (-7 to +13) was capable of mediating transcription initiation and that an IFN-gamma responsive element (GIRE) was present within 74 bp upstream of the transcription initiation site. A 17-bp sequence between positions -51 and -35 conferred <n>IFN-gamma</n> responsiveness on a heterologous promoter. Double-stranded GIRE sequence, but not a scrambled sequence, was specifically bound by nuclear proteins from <n>IFN- gamma</n> treated U937 cells. Gel shift experiments further showed that the <n>STAT1 alpha protein</n> bound to the Fc gamma RIC GIRE in response to <n>IFN- gamma</n> treatment of U937 cells. The Fc gamma RIC GIRE is homologous to the <n>IFN-gamma</n> activation sequence (GAS) of the <n>guanylate binding protein</n> and to X box elements of class II MHC genes. Our results demonstrate that transcriptional regulation of the Fc gamma RIC gene by <n>IFN-gamma</n> involves the binding of <n>STAT1 alpha</n> to a 17-bp GAS homology in the proximal promoter.
95327953>Constitutively activated Jak-STAT pathway in T cells transformed with HTLV-I. Human T cell lymphotropic virus I (HTLV-I) is the etiological agent for adult T cell leukemia and tropical spastic paraparesis (also termed HTLV-I-associated myelopathy). HTLV-I-infected peripheral blood T cells exhibit an initial phase of <n>interleukin-2</n> (<n>IL-2</n>)-dependent growth; over time, by an unknown mechanism, the cells become <n>IL-2</n>-independent. Whereas the Jak kinases <n>Jak1</n> and <n>Jak3</n> and the signal transducer and activator of transcription proteins <n>Stat3</n> and <n>Stat5</n> are activated in normal T cells in response to <n>IL-2</n>, this signaling pathway was constitutively activated in HTLV-I-transformed cells. In HTLV-I- infected cord blood lymphocytes, the transition from <n>IL-2</n>-dependent to <n>IL-2</n>-independent growth correlated with the acquisition of a constitutively activated Jak-STAT pathway, which suggests that this pathway participates in HTLV-I-mediated T cell transformation.
95340873>Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. To test the hypothesis that nitric oxide (NO) limits endothelial activation, we treated cytokine-stimulated human saphenous vein endothelial cells with several NO donors and assessed their effects on the inducible expression of <n>vascular cell adhesion molecule-1</n> (<n>VCAM-1</n>). In a concentration-dependent manner, NO inhibited <n>interleukin (IL)-1</n> alpha-stimulated <n>VCAM-1</n> expression by 35-55% as determined by cell surface enzyme immunoassays and flow cytometry. This inhibition was paralleled by reduced monocyte adhesion to endothelial monolayers in nonstatic assays, was unaffected by cGMP analogues, and was quantitatively similar after stimulation by either IL-1 alpha, <n>IL-1 beta</n>, <n>IL-4</n>, tumor necrosis factor (<n>TNF alpha</n>), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (<n>E-selectin</n> and to a lesser extent, <n>intercellular adhesion molecule-1</n>) and secretable cytokines (<n>IL-6</n> and <n>IL-8</n>). Inhibition of endogenous NO production by L-N-monomethyl- arginine also induced the expression of <n>VCAM-1</n>, but did not augment cytokine-induced <n>VCAM-1</n> expression. Nuclear run-on assays, transfection studies using various <n>VCAM-1</n> promoter reporter gene constructs, and electrophoretic mobility shift assays indicated that NO represses <n>VCAM- 1</n> gene transcription, in part, by inhibiting <n>NF-kappa B</n>. We propose that NO's ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall.
95280909><n>MIP1 alpha nuclear protein</n> (<n>MNP</n>), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the human MIP-1 alpha gene. <n>Murine macrophage inflammatory protein 1 alpha</n> (<n>MIP-1 alpha</n>) and its human equivalent (<n>GOS19</n>, <n>LD78</n>, or <n>AT464</n>) are members of the -C-C family of low-molecular-weight chemokines. Secreted from activated T cells and macrophages, bone marrow-derived <n>MIP-1 alpha</n>/<n>GOS19</n> inhibits primitive hematopoietic stem cells and appears to be involved in the homeostatic control of stem cell proliferation. It also induces chemotaxis and inflammatory responses in mature cell types. Therefore, it is important to understand the mechanisms which control the expression of <n>MIP-1 alpha</n>/<n>GOS19</n>. Previous work has shown that in Jurkat T cells, a set of widely expressed transcription factors (the ICK-1 family) affect the <n>GOS19</n> promoter. One member, <n>ICK-1A</n>, behaves as a strong negative regulator. In this communication, we provide evidence that the pathway of induction in the macrophage cell line U937 is different from that in Jurkat cells. Furthermore, we show that the ICK-1 binding site does not confer negative regulation in U937 cells. We provide evidence for an additional binding site, the <n>MIP-1 alpha</n> nuclear protein (MNP) site, which overlaps the ICK-1 site. Interaction of nuclear extracts from various cell lines and tissue with the MNP site leads to the formation of fast-migrating protein-DNA complexes with similar but distinct electrophoretic mobilities. A mutation of the MNP site which does not abrogate ICK-1 binding inactivates the GOS19.1 promoter in U937 cells and reduces its activity by fourfold in Jurkat cells. We propose that the MNP protein(s) binding at the MNP site constitutes a novel transcription factor(s) expressed in hematopoietic cells.
96055286>The effect of Toremifene on the expression of some genes in human mononuclear cells. Toremifene exerts multiple and varied effects on the gene expression of human peripheral mononuclear cells. After short-term, in vitro exposure to therapeutical levels, distinct changes in P-glycoprotein, steroid receptors, p53 and Bcl-2 expression take place. In view of the increasing use of antiestrogens in cancer therapy and prevention, there is obvious merit in long-term in vivo studies to be conducted.
95337737>The interleukin-5/receptor interaction activates Lyn and Jak2 tyrosine kinases and propagates signals via the <n>Ras-Raf-1-MAP kinase</n> and the Jak- STAT pathways in eosinophils. We have shown that the interaction of <n>interleukin (IL)-5</n> with the receptor activates <n>Lyn tyrosine kinase</n> within 1 min and <n>Jak2 tyrosine kinase</n> within 1-3 min. <n>IL-5</n> also stimulates GTP binding to <n>p21ras</n>. The signal is subsequently propagated through the activation of <n>Raf-1</n>, MEK, and MAP kinases as shown by their increased autophosphorylation in vitro and phosphorylation in situ. <n>Jak2 kinase</n> has been shown to phosphorylate STAT nuclear proteins. The activation of STAT nuclear factors was studied by electrophoretic mobility shift assay using a gamma activation site (GAS) probe. We found that <n>IL-5</n> induces two GAS- binding proteins in eosinophils, one of which is <n>STAT1</n>. We conclude that <n>IL-5</n> induced signals are propagated through two distinct pathways: (1) Lyn--&gt;Ras--&gt;<n>Raf-1</n>--&gt;MEK--&gt;MAP kinase and (2) <n>Jak2</n>--&gt;<n>STAT1</n>.
95266250>The <n>retinoblastoma gene product</n> negatively regulates transcriptional activation mediated by the <n>human cytomegalovirus IE2 protein</n>. The <n>IE2 gene product</n> of human cytomegalovirus (HCMV) is one of a few viral regulatory proteins expressed immediately upon infection of the host cell. It is a potent transcriptional activator of many viral and cellular promoters. We found that the <n>retinoblastoma susceptibility gene product</n> (<n>Rb</n>) dramatically suppressed this <n>IE2</n> transactivation of various promoters. However, unlike another tumor suppressor protein, <n>p53</n>, <n>Rb</n> did not have any significant effect on basal levels of transcription, suggesting that <n>Rb</n> specifically interacts with <n>IE2</n> rather than other cellular factors involved in the general transcription machinery. We found by protein-affinity chromatography that <n>Rb</n> in nuclear extracts or produced by in vitro translation directly bound to <n>IE2</n>. Our results suggest that <n>Rb</n> may regulate the life cycle of HCMV, which is endemic in the human population. Furthermore, these data may provide new insights into the slow rate of HCMV DNA replication in cells and the possible involvement of HCMV in tumorigenesis.
96082382>Expression of the <n>nucleoside diphosphate kinase</n> in human skin cancers: an immunohistochemical study. Expression of <n>nucleoside diphosphate(NDP) kinase</n>, which is homologous to the <n>nm23 gene product</n> in a variety of species, has been found to be inversely associated with metastatic potential. However, the relationship remains controversial according to the tumor cell types and experimental system, with conflicting results from different research groups. In order to determine whether <n>NDP kinase</n> expression serves as a marker for metastatic potential in human skin cancer, we assessed the levels of <n>NDP kinase</n> expression in 9 keratoacanthomas (KAs), 26 squamous cell carcinomas (SCCs), and 25 basal cell carcinomas (BCCs) using immunohistochemistry. The expression of <n>NDP kinase</n> was intense in KA and SCC compared with BCC. However, the difference of <n>NDP kinase</n> expression between KA and SCC was not statistically significant. And there was no statistically significant difference in <n>NDP kinase</n> expression between SCC with metastasis and SCC without metastasis. Our results contradict the hypothesis concerning the possible role of nm23 gene as a metastatic suppressor gene in human skin cancer. The mechanism of overexpression in various tumor cell types and its biological significance in cutaneous carcinogenesis
95362365><n>RB</n> and a novel E2F-1 binding protein in MHC class II deficient B-cell lines and normal <n>IFN-gamma</n> induction of the class IL transactivator CIITA in class II non-inducible <n>RB</n>-defective tumor lines. The major histocompatibility (MHC) class II genes encode cell surface proteins that bind antigenic peptide for presentation to T-cells. The class II proteins are expressed constitutively on B-cells and EBV- transformed B-cells, and are inducible by <n>IFN-gamma</n> on a wide variety of cell types. Retinoblastoma protein (<n>RB</n>) is a tumor suppressor and functions as a transcriptional repressor by binding and inactivating the transactivator E2F-I. <n>RB</n>-defective tumor lines are non-inducible for MHC class II by <n>IFN-gamma</n>, or very weakly inducible, but transfection of 2 different lines with <n>RB</n> expression vectors re- establishes or substantially enhances class II inducibility. Therefore, we examined the <n>RB</n> status of a series of B-cell mutants that are defective in class II expression, generated either in vitro or derived from Bare Lymphocyte Syndrome (BLS) patients. Nuclear matrix-bound <n>RB</n> was detectable in all cases, indicating that loss of <n>RB</n> is not responsible for decreased class II expression in these lines. A second E2F-I binding protein, most likely DP-I, was also apparently normal in both class II-positive and -negative B-cell lines. We also examined the <n>IFN-gamma</n> induction of CIITA in <n>RB</n>-defective lines. CIITA is a class II gene transactivator known to be defective in one form of BLS and to be required for the induction of MHC class II by <n>IFN-gamma</n>. CIITA mRNA is normally inducible by <n>IFN-gamma</n> in class II non-inducible, <n>RB</n>-defective lines, and in one line, re-expression of <n>RB</n> has no effect on CIITA mRNA induction levels. Thus, the block in MHC class II inducibility in <n>RB</n>- defective cells is not due to a block in CIITA inducibility.
95365357><n>Interleukin 12</n> induces tyrosine phosphorylation and activation of <n>STAT4</n> in human lymphocytes. <n>Interleukin 12</n> (<n>IL-12</n>) is an important immunoregulatory cytokine whose receptor is a member of the hematopoietin receptor superfamily. We have recently demonstrated that stimulation of human T and natural killer cells with <n>IL-12</n> induces tyrosine phosphorylation of the Janus family tyrosine kinase <n>JAK2</n> and <n>Tyk2</n>, implicating these kinases in the immediate biochemical response to <n>IL-12</n>. Recently, transcription factors known as STATs (signal transducers and activators of transcription) have been shown to be tyrosine phosphorylated and activated in response to a number of cytokines that bind hematopoietin receptors and activate JAK kinases. In this report we demonstrate that <n>IL-12</n> induces tyrosine phosphorylation of a recently identified STAT family member, <n>STAT4</n>, and show that <n>STAT4</n> expression is regulated by T- cell activation. Furthermore, we show that <n>IL-12</n> stimulates formation of a DNA-binding complex that recognizes a DNA sequence previously shown to bind STAT proteins and that this complex contains <n>STAT4</n>. These data, and the recent demonstration of JAK phosphorylation by <n>IL-12</n>, identify a rapid signal-transduction pathway likely to mediate <n>IL-12</n>- induced gene expression.
96007705>Temperature-induced down-regulation of the glucocorticoid receptor in peripheral blood mononuclear leucocyte in patients with sepsis or septic shock. OBJECTIVE: Activation of the hypothalamic-pituitary-adrenal axis is of vital importance during critical illness. We have studied the adaptive mechanisms which occur at the level of the glucocorticoid receptor in glucocorticoid target tissues in patients with sepsis or septic shock. DESIGN: The effects of hypercortisolaemia, hyperthermia and cellular composition on number of glucocorticoid receptors per cell and their affinity were evaluated, both in vitro and in vivo, in peripheral blood mononuclear leucocytes of control subjects and in patients with sepsis or septic shock. SUBJECTS: Fifteen patients (age 25-79) with sepsis or septic shock who were admitted to an intensive care unit were studied. The control group consisted of 24 healthy laboratory employees. MEASUREMENTS: The binding capacity and affinity of the glucocorticoid receptors were measured and compared to clinical data and the plasma cortisol concentrations. RESULTS: Hypercortisolaemia, in vitro, resulted in a decreased affinity and a decreased binding capacity of the glucocorticoid receptor. In vitro, hyperthermia as well as variations in the cellular composition did not influence the glucocorticoid receptor. In vivo, there was no change in the number of receptors per cell in patients with sepsis or septic shock as compared to healthy controls. However, a decreased affinity of the glucocorticoid receptor was observed. There was a weak but significant negative correlation between body temperature and the number of glucocorticoid receptors in the patient group. There was no relation between circulating cortisol concentrations and glucocorticoid receptor affinity and number. CONCLUSIONS: There is no obvious regulation of the number of glucocorticoid receptors by plasma cortisol concentrations in vivo. The decreased affinity of the glucocorticoid receptor together with the negative correlation between hyperthermia and the number of glucocorticoid receptors in patients with sepsis or septic shock suggest that hypothalamic-pituitary-adrenal axis activation during critical illness is accompanied by peripheral adaptation in glucocorticoid receptor number and affinity.
95332324>Integrin-mediated tyrosine phosphorylation and cytokine message induction in monocytic cells. A possible signaling role for the Syk tyrosine kinase. Activation of cytoplasmic tyrosine kinases is an important aspect of signal transduction mediated by integrins. In the human monocytic cell line THP-1, either integrin-dependent cell adhesion to fibronectin or ligation of beta 1 integrins with antibodies causes a rapid and intense tyrosine phosphorylation of two sets of proteins of about 65-75 and 120- 125 kDa. In addition, integrin ligation leads to nuclear translocation of the p50 and p65 subunits of the NF-kappa B transcription factor, to activation of a reporter gene driven by a promoter containing NF-kappa B sites, and to increased levels of mRNAs for immediate-early genes, including the <n>cytokine interleukin (IL)-1 beta</n>. The tyrosine kinase inhibitors genistein and herbimycin A block both integrin-mediated tyrosine phosphorylation and increases in <n>IL-1 beta</n> message levels, indicating a causal relationship between the two events. The components tyrosine phosphorylated subsequent to cell adhesion include <n>paxillin</n>, <n>pp125FAK</n>, and the <n>SH2 domain containing tyrosine kinase Syk</n>. In contrast, integrin ligation with antibodies induces tyrosine phosphorylation of <n>Syk</n> but not of <n>FAK</n> or <n>paxillin</n>. In adhering cells, pre-treatment with cytochalasin D suppresses tyrosine phosphorylation of <n>FAK</n> and <n>paxillin</n> but not of <n>Syk</n>, while <n>IL-1 beta</n> message induction is unaffected. These observations indicate that the <n>Syk</n> tyrosine kinase may be an important component of an integrin signaling pathway in monocytic cells, leading to activation of <n>NF-kappa B</n> and to increased levels of cytokine messages.
96065933>Inhibition of dexamethasone binding to human glucocorticoid receptor by New World primate cell extracts. To determine if New World primates express an inhibitor that influences glucocorticoid receptor (GR) binding characteristics, we examined [3H]dexamethasone binding in cytosol prepared from B95-8 lymphoid cells, derived from the cotton top tamarin (Saguinus oedipus), in combination with cytosol prepared from human or rat tissues. B95-8 cytosol inhibited specific binding of [3H]dexamethasone (P &lt; 0.01) when mixed with cytosol prepared from either a human lymphoid cell line (HL) or rat thymus. The inhibitory activity was heat labile and <n>trypsin</n> sensitive. Peak inhibitory activity was found in the 150-200 kd fractions after Sephacryl G-200 ultrafiltration. Scatchard analysis of [3H]dexamethasone binding using mixed cytosol showed a diminished GR apparent binding affinity when compared to HL cytosol. Kinetic studies using mixed cytosol indicated that B95-8 cytosol did not affect the apparent dissociation rate of [3H]dexamethasone. These data demonstrate that B95-8 cells contain a competitive inhibitor that prevents binding of dexamethasone to its cognate receptor.
95392577>Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy-negative individuals. The mRNA for the <n>Duffy blood group antigen</n>, the erythrocyte receptor for the Plasmodium vivax malaria parasite, has recently been cloned and shown to encode a widely expressed chemokine receptor. Here, we show that the <n>Duffy antigen/chemokine receptor</n> gene (DARC) is composed of a single exon and that most Duffy-negative blacks carry a silent FY*B allele with a single T to C substitution at nucleotide -46. This mutation impairs the promoter activity in erythroid cells by disrupting a binding site for the GATA1 erythroid transcription factor. With the recent characterization of the FY*A and FY*B alleles, these findings provide the molecular basis of the Duffy blood group system and an explanation for the erythroid-specific repression of the DARC gene in Duffy-negative individuals.
95287633>Activation of <n>pp90rsk</n> and early growth response-1 gene expression by <n>pokeweed mitogen</n> in human B cells. The present studies have examined the effects of <n>pokeweed mitogen</n> (<n>PWM</n>) on the induction of early growth response-1 gene (EGR-1) in normal human B cells. <n>PWM</n> regulates EGR-1 gene expression by both transcriptional and post-transcriptional mechanisms. Transient transfection assays with EGR-1 promoter fragments linked to the <n>chloramphenicol acetyltransferase</n> (<n>CAT</n>) gene demonstrated that <n>PWM</n> induced EGR-1 transcription is conferred by the CArG motif (C C[AT]6GG) in the EGR-1 promoter. The results further demonstrated the activation of <n>S6 kinase</n> (<n>pp90rsk</n>), evidenced by phosphorylation of <n>S6</n> and serum response factor (SRF) peptides, in <n>PWM</n> treated B cells. Taken together, these findings suggest that <n>PWM</n> is able to initiate an intracytoplasmic signalling cascade and EGR-1 induction in normal human B cells.
95337734>A conserved motif in the promoters of several cytokines expressed by human Th2-type lymphocytes. We have recently found a novel conserved motif in the promoters of several T-cell-expressed cytokines [human interleukin-2, -4, -5 and -13 and human and mouse granulocyte/macrophage-colony stimulating factor (<n>GM-CSF</n>)]. It contains a core sequence CTTGG ... CCAAG which is present as part of larger palindromic sequences in each gene. This suggest that they may interact with a new family of trans-acting factors. In transfection assays, the human <n>GM-CSF</n> element has a strong positive effect on the expression of a reporter gene by the human T cell line Jurkat J6 upon stimulation. In DNA mobility shift assays, this sequence can give either six different specific bands which are competed out by different parts of the sequence or one specific band which is competed out by each of the inverted repeats, depending on the reconstitution conditions. In different genes, the core sequences are separated by integer numbers of helical turns. Considering the strong positive regulatory effect of this element and its presence in several T-cell- expressed cytokine genes, it may be crucial to the coordinated expression of these cytokines in T helper cells.
95221892>cDNA cloning of a NGFI-B/nur77-related transcription factor from an apoptotic human T cell line. A human T lymphoid cell line, PEER, dies by apoptosis in the presence of PMA and calcium ionophore. A new gene, TINUR, was cloned from apoptotic PEER cells. The expression of the TINUR gene is induced within 1 h after the cross-linking of the T cell Ag receptor complex. TINUR belongs to the NGFI-B/nur77 family of the steroid receptor superfamily and is an orphan receptor. TINUR binds to the same DNA sequence as <n>NGFI-B/nur77</n>. We also propose that the NGFI-B/nur77 family can be classified into two subtypes.
95202809>Aspirin inhibits <n>nuclear factor-kappa B</n> mobilization and monocyte adhesion in stimulated human endothelial cells. BACKGROUND: The induction of <n>vascular cell adhesion molecule-1</n> (<n>VCAM-1</n>) and <n>E-selectin</n> by <n>tumor necrosis factor-alpha</n> (<n>TNF</n>) is mediated by mobilization of the transcription factor <n>nuclear factor-kappa B</n> (<n>NF- kappa B</n>). Since salicylates have been reported to inhibit <n>NF-kappa B</n> activation by preventing the degradation of its inhibitor <n>I kappa B</n>, we studied a potential inhibition of this pathway by acetylsalicylate (aspirin) in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Gel-shift analyses demonstrated dose-dependent inhibition of <n>TNF</n>-induced <n>NF-kappa B</n> mobilization by aspirin at concentrations ranging from 1 to 10 mmol/L. Induction of <n>VCAM-1</n> and <n>E-selectin</n> surface expression by <n>TNF</n> was dose-dependently reduced by aspirin over the same range, while induction of <n>intercellular adhesion molecule-1</n> (<n>ICAM-1</n>) was hardly affected. Aspirin appeared to prevent <n>VCAM-1</n> transcription, since it dose-dependently inhibited induction of <n>VCAM-1</n> mRNA by <n>TNF</n>. As a functional consequence, adhesion of U937 monocytes to <n>TNF</n>-stimulated HUVECs was markedly reduced by aspirin due to suppression of <n>VCAM-1</n> and <n>E-selectin</n> upregulation. These effects of aspirin were not related to the inhibition of <n>cyclooxygenase</n> activity, since indomethacin was ineffective. CONCLUSIONS: Our data suggest that aspirin inhibits <n>NF- kappa B</n> mobilization, induction of <n>VCAM-1</n> and <n>E-selectin</n>, and subsequent monocyte adhesion in endothelial cells stimulated by <n>TNF</n>, thereby providing an additional mechanism for therapeutic effects of aspirin
95355456>Activation of <n>NF-kappa B</n> by phosphatase inhibitors involves the phosphorylation of <n>I kappa B alpha</n> at phosphatase 2A-sensitive sites. Activation of <n>NF-kappa B</n> by various cellular stimuli involves the phosphorylation and subsequent degradation of its inhibitor, <n>I kappa B alpha</n>, although the underlying mechanism remains unclear. In the present study, the role of <n>serine/threonine phosphatases</n> in the regulation of <n>I kappa B alpha</n> phosphorylation was investigated. Our studies demonstrate that incubation of human T cells with low concentrations (approximately 1-5 nM) of calyculin A or okadaic acid, potent inhibitors of protein phosphatase type 1 (PP-1) and type 2A (PP- 2A), induces the phosphorylation of <n>I kappa B alpha</n> even in the absence of any cellular stimulus. This action of the phosphatase inhibitors, which is associated with the activation of the RelA.p50 <n>NF-kappa B</n> heterodimer, is not affected by agents that block the induction of <n>I kappa B alpha</n> phosphorylation by <n>tumor necrosis factor alpha</n> (<n>TNF- alpha</n>). Furthermore, the phosphorylated <n>I kappa B alpha</n> from calyculin A-treated cells, but not that from <n>TNF-alpha</n>-stimulated cells, is sensitive to PP-2A in vitro, suggesting the existence of fundamental differences in the phosphorylation of <n>I kappa B alpha</n> induced by the two different <n>NF-kappa B</n> inducers. However, induction of <n>I kappa B alpha</n> phosphorylation by both <n>TNF-alpha</n> and the phosphatase inhibitors is associated with the subsequent degradation of <n>I kappa B alpha</n>. We further demonstrate that TNF-alpha- and calyculin A-induced I kappa B alpha degradation exhibits similar but not identical sensitivities to a proteasome inhibitor. Together, these results suggest that phosphorylation of <n>I kappa B alpha</n>, mediated through both the <n>TNF-alpha</n>- inducible and the PP-2A-opposing kinases, may serve to target <n>I kappa B alpha</n> for proteasome-mediated degradation.
95365343><n>Sp1</n> functions in a chromatin-dependent manner to augment human alpha- globin promoter activity. The 5' flanking region of the human alpha-globin gene is highly G + C rich and contains multiple copies of the consensus sequence for the <n>Sp1</n> binding site. We investigated the role of this G + C-rich region in augmenting alpha-globin promoter activity in the presence of the far- upstream alpha-globin enhancer, HS-40. We show that in transiently transfected erythroid cells, deletion of the alpha-globin G + C-rich 5' flanking region has no effect on alpha-globin promoter activity. However, upon stable integration into chromatin, deletion of this region causes a nearly 90% decrease in promoter activity compared with expression from an alpha-globin promoter retaining this region. These results suggest that the alpha-globin G + C-rich 5' flanking region augments alpha-globin promoter activity in a chromatin-dependent manner. We further show that this G + C-rich region is required for the activation of alpha-globin gene expression during erythroid differentiation. Finally, we show by both footprint analysis and functional assays that the ability of the G + C-rich region to increase alpha-globin promoter activity from a stably integrated alpha-globin gene is mediated by its multiple binding sites for the transcription factor <n>Sp1</n>.
95363078>Costimulation of human CD4+ T cells with <n>LFA-3</n> and <n>B7</n> induce distinct effects on AP-1 and <n>NF-kappa B</n> transcription factors. We have earlier shown that stimulation of human CD4+ T cells with SEA presented on Chinese hamster ovary (CHO)-DR transfectants coexpressing either <n>B7</n> or <n>LFA-3</n> resulted in distinct cytokine profiles. We now demonstrate that <n>B7</n>, but not <n>LFA-3</n>, strongly costimulated <n>IL-2</n> transcription and mRNA expression in CD4+ T cells. Maximal increase in <n>IL-2</n> transcription was recorded with CHO-DR/<n>B7</n>/<n>LFA-3</n>, suggesting a cooperative effect of <n>B7</n> and <n>LFA-3</n> at the transcriptional level. Gel- shift analysis demonstrated that stimulation of CD4+ T cells with <n>CHO- DR</n> and staphylococcal enterotoxin A was sufficient to induce significant amounts of <n>NF-kappa B</n> binding proteins, whereas induction of AP-1 binding proteins required costimulation. <n>LFA-3</n> induced moderate levels of <n>AP-1</n>, but did not influence the levels of <n>NF-kappa B</n>, while <n>B7</n> costimulation strongly induced both <n>AP-1</n> and substantially enhanced <n>NF-kappa B</n> binding proteins. The CHO-DR/<n>B7</n>/<n>LFA-3</n> triple transfectant induced a further increase in <n>AP-1</n> and <n>NF-kappa B</n> binding proteins compared with the double transfectants. The level of Oct-1 binding proteins remained similar in all samples. Super-shift analysis revealed that the <n>NF-kappa B</n> complex of costimulated CD4+ T cells contained large amounts of p50, substantial amounts of p65, and marginal levels of c-Rel proteins. The <n>AP-1</n> binding proteins contained <n>c-Jun</n>, <n>Jun-D</n>, and <n>Fra-1</n>, but marginal amounts of <n>Jun-B</n> and <n>c-Fos</n>. Our results indicate distinct effects of <n>B7</n> and <n>LFA-3</n> costimulation on the activity of <n>AP-1</n> and <n>NF-kappa B</n>. These may partly account for the differential effects of <n>B7</n> and <n>LFA-3</n> costimulation on <n>IL-2</n> expression.
95336453>The <n>Ah receptor</n> recognizes DNA binding sites for the B cell transcription factor, <n>BSAP</n>: a possible mechanism for dioxin-mediated alteration of CD19 gene expression in human B lymphocytes. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits murine and human B lymphocyte immunoglobulin production through an unknown mechanism. This study investigated the effect of TCDD on expression of the CD19 gene in a human B lymphocyte cell line. Northern blot analysis showed that TCDD treatment decreased steady state levels of CD19 mRNA by 67% in the IM-9 cell line. Using a gel mobility shift assay, we identified a DNA- binding complex in IM-9 nuclear extracts that by several criteria appears to be the <n>Ah receptor</n>. In addition, the <n>Ah receptor</n> complex recognized a DNA binding site for B cell lineage-specific activator protein (<n>BSAP</n>) in the promoter region of the human CD19 gene which is similar to the consensus <n>Ah receptor</n> DNA binding site. These results suggest that the <n>AhR</n> could interfere with <n>BSAP</n>-stimulated CD19 gene transcription by competition for a common DNA binding site.
95337142>Inhibitory action of nm23 proteins on induction of erythroid differentiation of human leukemia cells. We recently identified a differentiation inhibitory factor (I-factor) in mouse myeloid leukemia M1 cells as a murine homolog of the <n>human nm23-H2 gene product</n>. nm23 genes encode proteins that participate in tumor metastasis regulation and in various fundamental cellular processes, although their mechanisms of action are still unknown. Although all nm23 proteins contain nucleoside diphosphate (NDP) kinase activity, it has not been established that the enzyme activity mediated the various functions of nm23 proteins. In the present experiment, we examined the effect of nm23 proteins on various differentiation induction systems of human leukemic cells including HL-60, U937, HEL/S, KU812F, K562, and HEL cells. Native human erythrocyte NDP kinase protein inhibited the induction of erythroid differentiation of HEL, KU812 and K562 cells, but not the induction of monocytic or granulocytic differentiation of HL-60, U937 and HEL/S cells. The erythroid differentiation of HEL cells was inhibited by recombinant human nm23-H1, -H2, mouse nm23-M1, and -M2 proteins. Moreover, both the <n>mutant nm23-H2His protein</n> and <n>truncated nm23-H2 protein</n> containing N- terminal (1-60) peptide, which do not have <n>NDP kinase</n> activity, also inhibited erythroid differentiation of HEL cells. These results suggest that (1) the differentiation inhibitory activity of <n>I-factor/nm23 protein</n> is not restricted to monocytic differentiation of M1 cells, (2) the inhibitory activity is exhibited without species specificity, and (3) the differentiation inhibitory activity of the <n>nm23/NDP kinase protein</n> is independent of its enzyme activity and requires the presence of N-terminal peptides.
95355137>Lipopolysaccharide-induced <n>E-selectin</n> expression requires continuous presence of LPS and is inhibited by bactericidal/permeability- increasing protein. Endothelial cells stimulated by LPS express <n>E-selectin</n>, which plays an important role in mediating neutrophil adhesion during inflammation. <n>E- selectin</n> is induced within 1-2 h, peaks at 4-6 h, and gradually returns to basal level by 24 h. <n>rBPI21</n>, a recombinant N-terminal fragment of <n>human bactericidal/permeability-increasing protein</n> (<n>BPI</n>), inhibited LPS- induced <n>E-selectin</n> expression when added at the same time as, and up to 6 h after, LPS. Delayed administration of <n>rBPI21</n> also affected LPS- mediated activation of the nuclear factor, <n>NF-kappa B</n>. Two to 4 h following LPS addition to endothelial cells, when <n>NF-kappa B</n> was already activated, addition of <n>rBPI21</n> resulted in marked reduction of <n>NF-kappa B</n> detectable at 4 or 6 h. These results indicate that endothelial activation requires continuous presence of LPS, and <n>rBPI21</n> acts to reverse LPS-mediated endothelial activation by interrupting the on-going LPS signal.
95279882><n>GM-CSF</n> and <n>IL-2</n> share common control mechanisms in response to costimulatory signals in T cells. Antigen complexed with major histocompatibility complex class I or II molecules on the surface of antigen presenting cells interacts with the T cell receptor (TCR) on the surface of T cells and initiates an activation cascade. So called costimulatory signals, mediated by other cell surface interactions or soluble cytokines produced by antigen presenting cells, are also required for complete T cell activation. High levels of cytokine gene expression in T cells also required both TCR and costimulatory signals. The <n>granulocyte-macrophage colony- stimulating factor</n> requires sequences in the promoter as well as a powerful enhancer located 3kb upstream to respond to TCR-like signals. These promoter and enhancer regions are mainly activated by the transcription factor nuclear factor of activated T cells (NFAT). The activation of NFAT by TCR signals has been well described for interleukin-2 (<n>IL-2</n>) and IL-4 gene transcription in T cells. Costimulatory signals, such as activation of the <n>CD28 cell surface molecule</n> on T cells, lead to activation through a distinct region of the <n>granulocyte-macrophage colony-stimulating factor</n> (<n>GM-CSF</n>) promoter. This region is termed the CK-1 or CD28RE and appears to bind specific members of the <n>NF-kappa B</n> family of transcription factors. Human T leukemia virus type 1 (HTLV-1) infects T cells and can lead to increase <n>GM-CSF</n> expression. We have found that the HTLV-1 transactivator protein, <n>tax</n>, acts as a costimulatory signal for GM-CSF and IL-2 gene transcription, in that it can cooperate with TCR signals to mediate high level gene expression. Tax activates the <n>GM-CSF</n> promoter through the CK-1/CD28RE region and also activates <n>nuclear factor-kappa B</n> binding to this region. However, other transcription factors or coactivators of <n>NF-kappa B</n> are required for <n>tax</n> activation but these remain to be identified. The CK-1/CD28RE of <n>GM-CSF</n> shows a high degree of similarity to the <n>IL-2</n> CD28RE and the IL-3 gene also contains a related region. This observation, together with the fact that both <n>GM- CSF</n> and <n>IL-2</n> respond to TCR signals via NFAT, implies a high degree of conservation in the regulation of cytokine gene expression in T cells.
95309632>Danazol decreases transcription of estrogen receptor gene in human monocytes. 1. Administration of danazol for over one month reduced the levels of estrogen receptor (ER) and its mRNA to approximately 50 and 20%, respectively in monocytes. 2. Danazol did not alter the degradation rate of ER mRNA in monocytes. 3. Danazol decreased the transcription rate of ER gene to approximately 50% in monocytes in a run-on assay. 4. Danazol may release estrogen predominance via the reduction of transcription for ER gene, which leads to the reduction of ER mRNA and ER expressions in monocytes.
95221870>Functional characterization of novel <n>IL-2</n> transcriptional inhibitors. <n>IL-2</n>-mediated T cell proliferation is a critical early event in the inflammatory process. Formation of the NFAT-1 transcriptional complex on the <n>IL-2</n> promoter is essential for <n>IL-2</n> transcription. Using a cell line that is stably transfected with a trimer of the NFAT-1 regulatory element linked to a lac-Z reporter gene, we screened for inhibitors of NFAT-1-mediated <n>beta-galactosidase</n> activity. WIN 61058 and WIN 53071 were identified as microM inhibitors. These compounds also inhibited <n>beta-galactosidase</n> mRNA levels. Similar inhibition of NFAT-1-mediated gene expression was observed in a second cell line, which is stably transfected with NFAT-1 regulatory elements linked to the reporter gene for sCD8. At 10 microM, both compounds inhibited IL-2 mRNA and protein levels in the NFAT-1-linked lac-Z transfectants, and in human lymphocytes. Both compounds inhibited the mixed lymphocyte reaction, and this inhibition was reversed by exogenous <n>IL-2</n>. WIN 53071 inhibited <n>IL-2</n> production induced in the calcium-dependent PMA and ionomycin pathway. Conversely, <n>calcium-independent anti-CD28 Ab</n> and PMA-induced <n>IL-2</n> production was resistant. Both compounds altered the NFAT-1 transcriptional complex, causing its retarded mobility on gels. By these functional criteria, we believe we have identified two structurally distinct, novel inhibitors of NFAT-1-mediated transcription.
95221386>Transcriptional regulation of the vacuolar H(+)-ATPase B2 subunit gene in differentiating THP-1 cells. Monocyte-macrophage differentiation was used as a model system for studying gene regulation of the <n>human vacuolar H(+)-ATPase</n> (<n>V-ATPase</n>). We examined mRNA levels of various <n>V-ATPase</n> subunits during differentiation of both native monocytes and the cell line THP-1, and found that transcriptional and post-transcriptional mechanisms could account for increases in cell <n>V-ATPase</n> content. From nuclear runoff experiments, we found that one subunit in particular, the B2 isoform (Mr = 56,000), was amplified primarily by transcriptional means. We have begun to examine the structure of the B2 subunit promoter region. Isolation and sequencing of the first exon and 5'-flanking region of this gene reveal a TATA-less promoter with a high G + C content. Primer extension and <n>ribonuclease</n> protection analyses indicate a single major transcriptional start site. We transfected promoter-luciferase reporter plasmids into THP-1 cells to define sequences that mediate transcriptional control during monocyte differentiation. We found that sequences downstream from the transcriptional start site were sufficient to confer increased expression during THP-1 differentiation. <n>DNase I</n> footprinting and sequence analysis revealed the existence of multiple AP2 and Sp1 binding sites in the 5'-untranslated and proximal coding regions
96089572>Synergy between signal transduction pathways is obligatory for expression of c-fos in B and T cell lines: implication for c-fos control via surface immunoglobulin and T cell antigen receptors. Expression of the protooncogene c-fos is controlled by three main regulatory pathways involving <n>kinase C</n>, cAMP, and calcium. Kinase C mediates its effects via phosphorylation of <n>serum response factor</n> (<n>SRF</n>) which interacts with the serum response element (SRE); cAMP and calcium mediate their effects via phosphorylation of <n>CREB</n> (<n>cAMP regulatory element binding protein</n>) presumably by activation of a <n>protein kinase A</n> or calmodulin-regulated kinase. We have examined the function of these elements in Burkitt's lymphoma cells (Ramos and Daudi) as well as a T lymphocytic cell line (Jurkat). We have found that stimulation of any one of these pathways alone has little or no effect on c-fos induction. However, <n>kinase C</n> activation (PMA stimulation) combined with either cAMP (forskolin plus MIX) or calcium stimulation (ionophore) leads to greatly enhanced c-fos induction. By contrast, cAMP in the presence of calcium shows no synergy in c-fos induction. Okadaic acid augments PMA- as well as calcium-mediated activation of c-fos, and has little or no effect when combined with cAMP. The main difference between Ramos (B cells) and Jurkat (T cells) in the regulation of c-fos is that cAMP plus calcium is strongly synergistic in Jurkat and is without effect in Ramos. Analysis of <n>AP-1</n> activity using gel mobility shift assays confirms that the requirements for synergy in c-fos mRNA induction are paralleled by requirements for synergy in induction of <n>AP-1</n> activity. Signaling in B cells due to anti-Ig stimulation involves both <n>kinase C</n> activation and release of intracellular calcium, and results in c-fos mRNA induction. Our results indicate that synergy between the <n>kinase C</n> activation and calcium is needed for efficient c-fos induction since neither of these two alone induces c-fos well. That synergy of signaling pathways is relevant for the anti-Ig induction of c-fos is supported by the fact that cAMP-inducing agents and okadaic acid further enhance anti-Ig induction of c-fos. These results suggest that cell-specific patterns of synergy are an essential feature for c-fos induction and may be relevant for c-fos control through B and T cell antigen receptors.
95363089>Multiple signals are required for function of the <n>human granulocyte- macrophage colony-stimulating factor</n> gene promoter in T cells. The <n>human granulocyte-macrophage CSF</n> (<n>GM-CSF</n>) gene is expressed in T cells in response to TCR activation that can be mimicked by treatment of the cells with PMA and Ca2+ ionophore. The gene contains a proximal functional promoter region (-620 to +34), as well as a powerful enhancer located 3 kb upstream, both of which are involved in the response of the gene to TCR activation. The proximal promoter contains a region termed CLEO (-54 to -31) that consists of a purine-rich element abutting an <n>activator protein-1</n> (<n>AP-1</n>)-like site, as well as an upstream <n>nuclear factor-kappa B</n> (<n>NF-kappa B</n>) site (-85 to -76) and a CK- 1 element (-101 to -92). We show in this work that mutations in either the purine-rich region of the CLEO element or the <n>NF-kappa B</n> site result in reduced PMA/Ca2+ activation of a 620-bp human <n>GM-CSF</n> promoter- <n>luciferase</n> reporter construct in Jurkat T cells by 65% and 50%, respectively. The major inducible protein complex that binds to the human CLEO (hCLEO) element is an <n>AP-1</n>-like complex that is inducible by PMA alone, but shows increased binding in response to PMA together with Ca2+ ionophore. Although the binding of this complex is not cyclosporin- sensitive, promoter induction is inhibited by cyclosporin treatment. A second weak inducible complex resembling nuclear factor of activated T cells (NF-AT) was also observed binding to the hCLEO region. By using recombinant proteins, we confirmed that <n>AP-1</n>, <n>NF-ATp</n>, and a higher order <n>NF-ATp</n>/<n>AP-1</n> complex could all form with the hCLEO element, and we have also defined the sequence requirements for binding of each of these complexes. We found that expression of a constitutively active form of <n>calcineurin</n> could substitute for Ca2+ ionophore and synergize with PMA to activate the <n>GM-CSF</n> promoter, and conversely that <n>mutant- activated Ras</n> could substitute for PMA and cooperate with Ca2+ ionophore. Co-expression of <n>Ras</n> and <n>calcineurin</n>, however, did not activate the <n>GM-CSF</n> promoter, but required the additional expression of <n>NF-kappa B</n> p65. These results imply that at least three signals are required to activate the <n>GM-CSF</n> proximal promoter, and that the signals impinge on distinct transcription factors that bind to the hCLEO and <n>NF- kappa B</n> regions of the promoter.
95363073>IL-10 induces the tyrosine phosphorylation of <n>tyk2</n> and <n>Jak1</n> and the differential assembly of <n>STAT1 alpha</n> and STAT3 complexes in human T cells and monocytes. <n>IL-10</n> affects monocytes and T cells by driving the progression of immune responsiveness such that Th2 lymphocyte-mediated effects predominate. In this report, we show that in monocytes and T cells <n>IL- 10</n> stimulates tyrosine phosphorylation of the signal transducers and activators of transcription, <n>STAT1 alpha</n> and <n>STAT3</n>, in a differential manner such that the relative formation of homo- and heterodimers varies between the two cell types. Moreover, monocytes express a novel <n>IL-10</n>-stimulated STAT protein with an M(r) of <n>70 kDa</n> that is recognized by the anti-<n>STAT3</n> Ab but is not observed in T cells. <n>IL-10</n> treatment of both T cells and monocytes results in the ligand-induced tyrosine phosphorylation of <n>tyk2</n> and <n>Jak1</n>, but not <n>Jak2</n> or <n>Jak3</n>. Selective modulation of immune responsiveness by <n>IL-10</n> in cells such as monocytes and T cells may result in part from the differential activation of STAT protein pairs.
97253017>Use of new biologic markers in the ovulation induction. Biological markers of ovulation, after a great in the past, have been fallen into disuse for the large diffusion of biochemical and biophysical ones. However, the real effect of hormones involved in ovulation is expressed by biological modifications on target tissues. To explore the modifications of not reproductive target tissues as ovulation markers we studied the behaviour of Albuminemia, <n>Platelet Factor IV</n> (as indicator of Platelet Aggregation), Type II estrogenic receptors in 42 ovulation induced women, undergoing our observation. 33 of them had ovulation and 9 developed a LUF syndrome, constituting two biological models of an opposite situation for the three markers observed. All the markers considered were sufficiently sensitive, but among them, <n>Platelet Factor IV</n> was the most reliable to the hormonal ovulatory situation.
95279790>Abnormal regulation of the <n>IL-2</n> promoter in lpr CD4-CD8- T lymphocytes results in constitutive expression of a novel nuclear factor of activated T cells-binding factor. The inert quality of MRL-Ipr/Ipr (Ipr) peripheral CD4-CD8- (CD4-8-) T cells manifests primarily as an inability to proliferate or produce <n>IL- 2</n> in response to TCR or mitogenic stimulation. Yet these same cells do initiate early TCR-mediated signaling events, such as generation of inositol phosphates and increased intracellular calcium. They also display constitutively high levels of <n>p59fyn</n> and <n>CD3 zeta</n> tyrosine phosphorylation. The generation of second messengers in T cells normally leads to downstream signaling that results in transcriptional activation of the <n>IL-2</n> gene. We, therefore, compared the activation state of the <n>IL-2</n> gene promoter region in freshly isolated and stimulated Ipr CD4-8- T cells with that of normal T lymphocytes. Levels of the octamer, <n>NF-kappa B</n> (<n>p50-p65 heterodimer</n>), and <n>AP-1</n> transcriptional factors are constitutively elevated in freshly isolated Ipr CD4-8- T cells, consistent with the activated phenotype of these cells. Upon stimulation with mitogens, formation of the transactivating complex, nuclear factor of activated T cells (NF-AT), occurs with normal kinetics in Ipr CD4-8- T cells. Yet, the levels of the activating NF-AT complex never reach those observed in similarly stimulated normal T cells. Furthermore, nuclear extracts from Ipr CD4-8- T cells display high levels of a novel specific binding activity at the NF-AT site, which is present at much lower levels in freshly isolated normal T lymphocytes. Upon mitogenic stimulation, the binding activity of the novel NF-AT-binding factor is rapidly down-regulated in normal T cells, but persists at high levels in Ipr CD4-8- T cells. These two abnormalities at the NF-AT site provide a potential mechanism to account for the defect in <n>IL-2</n> production from Ipr CD4-8- T cells.
95316714>Cross-linking of <n>CD30</n> induces HIV expression in chronically infected T cells. <n>CD30</n>, a member of the tumor necrosis factor (TNF) receptor family, is expressed constitutively on the surface of the human T cell line ACH-2, which is chronically infected with human immunodeficiency virus type-1 (HIV)-1. We demonstrate that cross-linking <n>CD30</n> with an anti-<n>CD30</n>- specific monoclonal antibody, which mimics the described biological activities of the <n>CD30 ligand</n> (<n>CD30L</n>), results in HIV expression. <n>CD30</n> cross-linking does not alter proliferation of ACH-2 cells and the induction of HIV expression is not mediated by endogenous <n>TNF alpha/beta</n>. Furthermore, cross-linking of <n>CD30</n> leads to <n>NF-kappa B</n> activation and enhanced HIV transcription. Thus, <n>CD30</n>-<n>CD30L</n> interactions mediate the induction of HIV expression by a kappa B- dependent pathway that is independent of TNF. This mechanism may be important in the activation of HIV expression from latently infected CD4+ T cells, especially in lymphoid organs where cell to cell contact is conducive to receptor-ligand interactions.
95269137>Human MHC class II gene transcription directed by the carboxyl terminus of CIITA, one of the defective genes in type II MHC combined immune deficiency. Type II major histocompatibility complex combined immune deficiency (type II MHC CID or bare lymphocyte syndrome) is a congenital immunodeficiency disease characterized by absent MHC class II expression. Four distinct complementation groups have been identified. Recently, the defective gene in group II type II MHC CID has been isolated and termed CIITA. Here, we demonstrate that CIITA is an MHC class II gene-specific transcription activator. The transcription activation function is provided by the N-terminal acidic domain (amino acids 26-137), which is experimentally exchangeable with a heterologous viral transcription-activating domain. The specificity of <n>CIITA</n> for three major MHC class II genes, DR, DQ and DP, is mediated by its remaining C-terminal residues (amino acids 317-1130). The transactivation of multiple cis elements, especially S and X2, of the DR alpha proximal promoter in group II CID cells is <n>CIITA</n> dependent. Since <n>CIITA</n> overexpression in normal cells did not increase class II expression, we propose that initiation of <n>CIITA</n> expression serves as the on-off switch, while availability of downstream interactor(s) limits transcription.
95256455>Monocyte tethering by <n>P-selectin</n> regulates <n>monocyte chemotactic protein- 1</n> and <n>tumor necrosis factor-alpha</n> secretion. Signal integration and <n>NF- kappa B</n> translocation [see comments] Adhesion molecules that tether circulating leukocytes to endothelial cells may also transduce or modulate outside-in signals for cellular activation, providing an initial regulatory point in the inflammatory response. Adhesion of human monocytes to <n>P-selectin</n>, the most rapidly expressed <n>endothelial tethering factor</n>, increased the secretion of <n>monocyte chemotactic protein-1</n> (<n>MCP-1</n>) and <n>tumor necrosis factor-alpha</n> (<n>TNF-alpha</n>) by the leukocytes when they were stimulated with <n>platelet- activating factor</n>. Increased cytokine secretion was specifically inhibited by <n>G1</n>, an anti-<n>P-selectin</n> mAb that prevents <n>P-selectin</n> from binding to its ligand (<n>P-selectin</n> glycoprotein ligand-1) on myeloid cells. Moreover, tethering by <n>P-selectin</n> specifically enhanced nuclear translocation of <n>nuclear factor-kappa B</n> (<n>NF-kappa B</n>), a transcription factor required for expression of <n>MCP-1</n>, <n>TNF-alpha</n>, and other immediate- early genes. These results demonstrate that <n>P-selectin</n>, through its ligands on monocytes, may locally regulate cytokine secretion in inflamed tissues.
95238333>Functional roles of in vivo footprinted DNA motifs within an alpha- globin enhancer. Erythroid lineage and developmental stage specificities. Transcriptional regulation of the human alpha-like globin genes, embryonic zeta 2 and adult alpha, during erythroid development is mediated by a distal enhancer, HS-40. Previous protein-DNA binding studies have shown that HS-40 consists of multiple nuclear factor binding motifs that are occupied in vivo in an erythroid lineage- and developmental stage-specific manner. We have systematically analyzed the functional roles of these factor binding motifs of HS-40 by site- directed mutagenesis and transient expression assay in erythroid cell cultures. Three of these HS-40 enhancer motifs, 5'NF-E2/AP1, GT II, and GATA-1(c), positively regulate the zeta 2-globin promoter activity in embryonic/fetal erythroid K562 cells and the adult alpha-globin promoter activity in adult erythroid MEL cells. On the other hand, the 3'NF-E2/AP1 motif is able to exert both positive and negative regulatory effects on the zeta 2-globin promoter activity in K562 cells, and this dual function appears to be modulated through differential binding of the ubiquitous AP1 factors and the <n>erythroid- enriched NF-E2 factor</n>. Mutation in the GATA-1(d) motif, which exhibits an adult erythroid-specific genomic footprint, decreases the HS-40 enhancer function in dimethyl sulfoxide-induced MEL cells but not in K562 cells. These studies have defined the regulatory roles of the different HS-40 motifs. The remarkable correlation between genomic footprinting data and the mutagenesis results also suggests that the erythroid lineage- and developmental stage-specific regulation of human alpha-like globin promoters is indeed modulated by stable binding of specific nuclear factors in vivo.
95221342>Nitric oxide-stimulated guanine nucleotide exchange on <n>p21ras</n>. The protooncogene p21ras, a monomeric G protein family member, plays a critical role in converting extracellular signals into intracellular biochemical events. Here, we report that nitric oxide (NO) activates p21ras in human T cells as evidenced by an increase in <n>GTP-bound p21ras</n>. In vitro studies using <n>pure recombinant p21ras</n> demonstrate that the activation is direct and reversible. Circular dichroism analysis reveals that NO induces a profound conformational change in p21ras in association with GDP/GTP exchange. The mechanism of activation is due to S-nitrosylation of a critical cysteine residue which stimulates guanine nucleotide exchange. Furthermore, we demonstrate that <n>p21ras</n> is essential for NO-induced downstream signaling, such as <n>NF-kappa B</n> activation, and that endogenous NO can activate <n>p21ras</n> in the same cell. These studies identify <n>p21ras</n> as a target of the same cell. These studies identify <n>p21ras</n> as a target of NO in T cells and suggest that NO activates <n>p21ras</n> by an action which mimics that of guanine nucleotide exchange factors
95394020>Interleukin-2 promoter activity in Epstein-Barr virus-transformed B lymphocytes is controlled by <n>nuclear factor-chi B</n>. The regulation of interleukin (IL)-2 gene expression has been investigated mainly in T lymphocytes, the predominant producers of <n>IL- 2</n>. However, B cells can also synthesize <n>IL-2</n>. In the present study we analyzed the control of <n>IL-2</n> promoter activity in Epstein-Barr virus (EBV)-transformed B cell clones which are capable of secreting <n>IL-2</n> at a low level after stimulation with phorbol 12-myristate 13-acetate and the Ca2+ ionophore ionomycin. Transient transfections using reporter constructs with multiples of transcription factor binding sites from the <n>IL-2</n> promoter [<n>distal nuclear factor (NF)-AT</n>, <n>proximal NF-AT</n>, <n>AP- 1/Octamer</n> (<n>UPS</n>) or <n>NF-chi B</n> (<n>TCEd</n>) sites] were performed. In EBV- transformed B clones, the chi B site exerted the strongest inducible activity; the NF-AT binding sites showed either no or only weak activity compared to Jurkat T cells. An <n>IL-2</n> promoter bearing a defective <n>NF-chi B</n> site was completely inactive in EBV-transformed B cells, while it still had activity in Jurkat T cells. In seven EBV-B cell clones or lines differing in their capacity to secrete <n>IL-2</n>, the activity of the <n>IL-2</n> promoter correlated well with the status of <n>IL-2</n> secretion. Similarly, a human immunodeficiency virus promoter, whose activity is controlled through chi B factors, was found to be active in the <n>IL-2</n> producing EBV-B cells, but inactive in the non-<n>IL-2</n>-producing cells. Electrophoretic mobility shift assays using protein extracts from EBV-B cells and the <n>IL-2</n> <n>NF-chi B</n> probe revealed the constitutive generation of chi B complexes in <n>IL-2</n>-secreting cells consisting mainly of heterodimeric p50/p65 complexes. A weaker chi B complex formation and faster-migrating complexes were detected in non-<n>IL-2</n>-secreting cells. These results demonstrate that the <n>IL-2</n> <n>NF-chi B</n> site is indispensable for the activity of the <n>IL-2</n> promoter in EBV-transformed B cells, whereas other transcription factors appear to be less important for <n>IL-2</n> expression in these cells.
95363069><n>Latent membrane protein-1</n> induces <n>cyclin D2</n> expression, <n>pRb</n> hyperphosphorylation, and loss of <n>TGF-beta 1</n>-mediated growth inhibition in EBV-positive B cells. The normal cell cycle is regulated by several molecules, such as the tumor-suppressor protein <n>pRb</n>, the G1 cyclins, the cyclin-dependent kinases, and their inhibitors. These regulators are targeted by negative growth regulatory signals, such as that provided by <n>TGF-beta</n>. Here, we show that the presence of either wild-type EBV or its <n>transforming latent membrane protein-1</n> (<n>LMP-1</n>) results in the loss of <n>TGF-beta</n> 1-mediated growth inhibition in human B cells. Chemical cross- linking with 125I-labeled <n>TGF-beta</n> 1 showed an essentially normal <n>TGF- beta receptor</n> profile in EBV-positive and EBV-negative Burkitt's lymphoma cell lines, and these receptors were shown to be functional in transducing signals, as evidenced by the <n>TGF-beta 1</n>-mediated modulation of junB gene expression. However, <n>TGF-beta 1</n> did not induce dephosphorylation of <n>pRb</n> in EBV (or <n>LMP-1</n>)-positive cells as opposed to EBV-negative cells, suggesting a dichotomy in the <n>TGF-beta 1</n> signaling pathway leading to separable gene regulatory and growth inhibitory responses. Furthermore, <n>LMP-1</n> was found to induce the expression of <n>cyclin D2</n>; normal B cells or EBV-negative Burkitt's lymphoma cells do not express D-type cyclins. Taken together, these data point to a potential mechanism underlying EBV-mediated B cell transformation whereby constitutive induction of key cell cycle regulators by <n>LMP-1</n> can lead to <n>pRb</n> hyperphosphorylation and uncontrolled cell proliferation.
95355533>Does thyroidectomy, radioactive iodine therapy, or antithyroid drug treatment alter reactivity of patients' T cells to epitopes of thyrotropin receptor in autoimmune thyroid diseases? The effect of treatment on thyroid antibody production and T cell reactivity to thyroid antigens was studied in 15 patients with Graves' disease (GD) before and after thyroidectomy, 19 patients with GD before and after radioactive iodine (RAI) therapy, and 9 patients maintained euthyroid on antithyroid drugs (ATD). Twenty subjects matched for age and sex without known thyroid disease served as controls. In GD patients, the responses of peripheral blood mononuclear cells (PBMC) and TSH receptor (TSHR)-specific T cell lines to recombinant human TSHR extracellular domain, <n>thyroglobulin</n>, and TSHR peptides were examined on the day of surgery or RAI therapy (day 0) and also 6-8 weeks and 3-6 months thereafter. Reactivity to TSHR peptides before surgery was heterogeneous and spanned the entire extracellular domain. Six to 8 weeks after subtotal thyroidectomy, the number of patients' PBMC responding to any peptide and the average number of recognized peptides decreased. A further decrease in the T cell reactivity to TSHR peptides was observed 3-6 months after surgery. The responses of PBMC from Graves' patients before RAI therapy were less than those in the presurgical group. Six to 8 weeks after RAI therapy, the number of patients responding to any peptide and the average number of recognized peptides increased. Three to 6 months after RAI, T cell responses to TSHR peptides were less than those 6-8 weeks after RAI therapy, but still higher than the values on day 0. Responses of PBMC from patients with GD, maintained euthyroid on ATD, were lower than those before surgery or RAI therapy. The reactivity of T cell lines in different groups reflected a pattern similar to PBMC after treatment. TSHR antibody and microsomal antibody levels decreased after surgery, but increased after RAI therapy. The difference in the number of recognized peptides by patients' PBMC before RAI and surgery may reflect the effect of long term therapy with ATD in the patients before RAI vs. the shorter period in patients before surgery. The decreased T cell reactivity to thyroid antigens after thyroidectomy could be the result of removal of a major part of the thyroid gland or redistribution of suppressor-inducer T cells. The increased T cell response after RAI therapy is probably epitope specific, rather than a response to the whole TSHR molecule. Synchronous recognition of peptides 158-176 and 248-263 is important for the development of GD, and the loss of recognition of one of these epitopes may be an early sign of immune remission and a predictor of euthyroidism.
96030053>Circumvention of tolerance for the nuclear T cell protein <n>TCF-1</n> by immunization of <n>TCF-1</n> knock-out mice. Molecular events that underlie the well-defined phenotypic changes of the differentiating thymocyte are poorly understood. A candidate gene to control thymocyte differentiation, <n>T cell factor-1</n> (<n>TCF-1</n>)* encodes a DNA-binding protein. Its mRNA expression pattern is complex during embryogenesis, yet restricted to lymphocytes postnatally. Expression studies on <n>TCF-1</n> protein have been hampered by the difficulty to raise antibodies due to extreme evolutionary conservation. <n>TCF-1</n> knock-out mice, generated recently in our laboratory, have strongly decreased numbers of thymocytes, but are otherwise normal. We have used these mice to generate anti-<n>TCF-1</n> antibodies. By immunization with a recombinant fusion protein, we show that <n>TCF-1</n> knock-out mice readily yield antiserum titers against human and mouse TCF-1 protein. Wild-type littermates remain unresponsive to <n>TCF-1</n> while they mount a high-titer antibody response to the fusion protein, <n>Maltose Binding Protein</n> (<n>MBP</n>). Subsequently, <n>TCF-1</n>-specific hybridomas could be prepared from the spleens of immunized knock-out mice. This study illustrates the almost complete tolerance of mice for human <n>TCF-1</n> and demonstrates that this tolerance is readily broken by gene knock-out. Furthermore, the usefulness of knock-out mice for the generation of monoclonal antibodies against the gene product of interest is underscored.
95286632>The transcription factor, <n>Nm23H2</n>, binds to and activates the translocated c-myc allele in Burkitt's lymphoma. We have identified an in vivo footprint over the PuF site on the translocated c-myc allele in Burkitt's lymphoma cells. The PuF site on the silent normal c-myc allele was unoccupied. We demonstrated by electrophoretic mobility shift assay, electrophoretic mobility shift assay with antibody, UV cross-linking followed by SDS-gel electrophoresis, and Western analysis that <n>Nm23H2</n> in B cell nuclear extracts bound to the c-myc PuF site. Transfection experiments with c- myc promoter constructs in both DHL-9 and Raji cells revealed that the PuF site functioned as a positive regulatory element in B cells with a drop in activity with mutation of this site. Access to this site is blocked in the normal silent c-myc allele; these data suggest that the <n>Nm23H2</n> protein is involved in deregulation of the translocated c-myc allele in Burkitt's lymphoma cells.
95279435>Identification of two novel regulatory elements within the 5'- untranslated region of the human A gamma-globin gene. Interaction between the stage selector element (SSE) in the proximal gamma-globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the beta-globin promoter in the fetal developmental stage. We have now demonstrated that a second SSE-like element in the 5'-untranslated region of the gamma-gene also contributes to this competitive silencing of the beta- gene. Utilizing transient transfection assays in the fetal erythroid cell line, K562, we have shown that the core enhancer of hypersensitivity site 2 can preferentially interact with the proximal gamma-promoter in the absence of the SSE, completely silencing a linked beta-promoter. Mutation of a 20-base pair sequence of the gamma-gene 5'- untranslated region (UTR) led to derepression of beta-promoter activity. A marked activation of gamma-promoter activity was also observed with this mutation, suggesting the presence of a repressor. Fine mutagenesis dissected these activities to different regions of the 5'-UTR. The stage selector activity was localized to a region centered on nucleotides +13 to +15. Electromobility shift assays utilizing this sequence demonstrated binding of a fetal and erythroid-specific protein. The repressor activity of the 5'-UTR was localized to tandem GATA-like sites, which appear to bind a complex of two proteins, one of which is the erythroid transcription factor <n>GATA-1</n>. These results indicate that the 5'-UTR of the gamma-gene contains sequences that may be important for its transcriptional and developmental regulation.
95257963>Coupling of a signal response domain in <n>I kappa B alpha</n> to multiple pathways for <n>NF-kappa B</n> activation. The eukaryotic transcription factor <n>NF-kappa B</n> plays a central role in the induced expression of human immunodeficiency virus type 1 and in many aspects of the genetic program mediating normal T-cell activation and growth. The nuclear activity of <n>NF-kappa B</n> is tightly regulated from the cytoplasmic compartment by an inhibitory subunit called I kappa B alpha. This cytoplasmic inhibitor is rapidly phosphorylated and degraded in response to a diverse set of <n>NF-kappa B</n>-inducing agents, including T-cell mitogens, proinflammatory cytokines, and viral transactivators such as the <n>Tax protein</n> of human T-cell leukemia virus type 1. To explore these I kappa B alpha-dependent mechanisms for <n>NF- kappa B</n> induction, we identified novel mutants of <n>I kappa B alpha</n> that uncouple its inhibitory and signal-transducing functions in human T lymphocytes. Specifically, removal of the N-terminal 36 amino acids of <n>I kappa B alpha</n> failed to disrupt its ability to form latent complexes with <n>NF-kappa B</n> in the cytoplasm. However, this deletion mutation prevented the induced phosphorylation, degradative loss, and functional release of <n>I kappa B alpha</n> from <n>NF-kappa B</n> in <n>Tax</n>-expressing cells. Alanine substitutions introduced at two serine residues positioned within this N-terminal regulatory region of <n>I kappa B alpha</n> also yielded constitutive repressors that escaped from <n>Tax</n>-induced turnover and that potently inhibited immune activation pathways for <n>NF-kappa B</n> induction, including those initiated from antigen and cytokine receptors. In contrast, introduction of a phosphoserine mimetic at these sites rectified this functional defect, a finding consistent with a causal linkage between the phosphorylation status and proteolytic stability of this cytoplasmic inhibitor. Together, these in vivo studies define a critical signal response domain in <n>I kappa B alpha</n> that coordinately controls the biologic activities of <n>I kappa B alpha</n> and <n>NF-kappa B</n> in response to viral and immune stimuli.
95255485>Growth regulation and cellular changes during differentiation of human prostatic cancer LNCaP cells as induced by T lymphocyte-conditioned medium. Human prostatic epithelial cells from an androgen-dependent LNCaP cell line were examined in response to conditioned medium (CM) derived from <n>phytohemagglutinin</n> (<n>PHA</n>)-stimulated lymphocytes. Addition of CM caused a greater than 70% reduction of cell proliferation by cell counting and cell cycle. These cells showed G1 phase arrest and the clonogenicity was reduced. The growth-modulating effect was dose-dependent and not due to cell lysis or apoptosis. The binding of androgen to androgen receptor on these cells showed approximately 50% reduction, underlining a proliferation reduction mechanism. The <n>prostate-specific antigen</n> (<n>PSA</n>) was downregulated to approximately 75% during the process. Cell morphology showed dendritic processes extending from cytoplasm and other neuroendocrine cell characteristics. The expression of several cytoskeleton and intracellular proteins increased as determined by immunostaining on slides and by ELISA procedures. These included <n>vimentin</n>, correlating to cell shape changes, cytokeratins 8 and 18, associated with differentiated cell types of prostate epithelia, and <n>neuron-specific enolase</n> and serotonin, associated with neuroendocrine cells. From these cellular changes, we can infer that the cell growth was modulated along with induction of terminal differentiation. Activated T cells were demonstrated to be important in providing the modulating activity. This growth modulator was semipurified and had an estimated molecular weight 13,000 to 24,000 Da. The activity was determined to be distinct from TGF, TNF, and some commonly known lymphokines. The interaction between lymphoid and prostatic cells in growth and development is described.
95229576>Platelet-activating factor stimulates transcription of the <n>heparin- binding epidermal growth factor-like growth factor</n> in monocytes. Correlation with an increased kappa B binding activity. Human peripheral blood monocytes responded to stimulation of <n>platelet- activating factor</n> (<n>PAF</n>) with up-regulation of the transcript for <n>heparin-binding epidermal growth factor-like growth factor</n> (<n>HB-EGF</n>), a potent mitogen for vascular smooth muscle cells. This function of <n>PAF</n> was observed at nanomolar concentrations of the ligand, starting at 30 min after stimulation. The <n>PAF</n>-induced up-regulation of <n>HB-EGF</n> mRNA was accompanied by an increase in kappa B binding activity. These functions of <n>PAF</n> appeared to be mediated through the cell surface <n>PAF</n> receptors, as two <n>PAF</n> receptor antagonists, WEB 2086 and L-659,989, blocked both the up-regulation of <n>HB-EGF</n> mRNA and kappa B binding activity induced by <n>PAF</n>. The antagonists, however, had no effect on phorbol ester- induced up-regulation of <n>HB-EGF</n> mRNA and kappa B binding activity. Pretreatment of monocytes with pertussis toxin inhibited these functions of <n>PAF</n>, whereas cholera toxin had no inhibitory effect. Pyrrolidine dithiocarbamate, an inhibitor for <n>NF-kappa B</n> activation, markedly reduced <n>PAF</n>-stimulated kappa B binding activity as well as up- regulation of <n>HB-EGF</n> mRNA. These results suggest a potential role of <n>PAF</n> in <n>HB-EGF</n> expression and provide evidence that this stimulation may occur through increased kappa B binding activity.
95197681>Mapping of the interaction site of the defective transcription factor in the class II major histocompatibility complex mutant cell line clone- 13 to the divergent X2-box. We have previously described a mutant B lymphoblastoid cell line, Clone- 13, that expresses <n>HLA-DQ</n> in the absence of HLA-DR and -DP. Several criteria indicated that the defect in this cell line influences the activity of an isotype-specific transcription factor. Indeed, transient transfection of HLA-DRA and DQB reporter constructs indicated that the affected factor operates via cis-elements located between -141 base pairs and the transcription initiation site. A series of hybrid DRA/DQB reporter constructs was generated to further map the relevant cis- elements in this system. Insertion of oligonucleotides spanning the DQB X-box (but not the DQB-W region or the DQB Y-box) upstream of -141 in a DRA reporter plasmid rescued expression to nearly wild-type levels. Substitution promoters were then generated where the entire X-box, or only the X1- or X2-boxes of HLA-DRA were replaced with the analogous regions of HLA-DQB. The DQB X2-box was able to restore expression to the silent DRA reporter construct. Moreover, replacement of the DQB X2- box with the DRA X2-box markedly diminished the activity of the DQB promoter in the mutant cell. None of the hybrid reporter constructs were defective when transfected into the wild-type, HLA-DR/-DQ positive parental cell line, Jijoye. These studies suggest that the divergent X2- box of the class II major histocompatibility complex promoters plays an important role in influencing differential expression of the human class II isotypes
95373165>Restoration of the <n>Epstein-Barr virus ZEBRA protein</n>'s capacity to disrupt latency by the addition of heterologous activation regions. The <n>ZEBRA protein</n> has a unique biological function among herpesviral proteins. It is responsible for the disruption of Epstein-Barr virus (EBV) latency and the induction of the lytic cycle. <n>ZEBRA</n> is a bZIP transcriptional activator which binds as a dimer to 7-bp response elements within EBV promoters and is directly involved in the stimulation of virus replication at the EBV lytic origin. We have employed the <n>ZEBRA</n>/EBV biological system to test whether a heterologous activation domain can substitute for another activation domain (the <n>ZEBRA</n> domain). The <n>ZEBRA</n> activation region was replaced with the potent acid activation region from the <n>herpes simplex virus VP16 protein</n> or with the activation region of the <n>EBV R protein</n>. Both chimeras were found to transactivate model and native promoters at equivalent or better levels than <n>ZEBRA</n> itself. Activation was not target- or cell- type dependent, nor was it dependent on the presence of virus. These activation domains restored <n>ZEBRA</n>'s ability to induce early antigen and to stimulate origin replication to levels that were equal to or greater than those of wild type. These studies suggest that the specificities of some of the known biological functions of <n>ZEBRA</n> are not dependent upon the nature of the activation domain present within <n>ZEBRA</n>.
95362854><n>Interleukin 4</n> activates a signal transducer and activator of transcription (Stat) protein which interacts with an interferon-gamma activation site-like sequence upstream of the I epsilon exon in a human B cell line. Evidence for the involvement of <n>Janus kinase 3</n> and <n>interleukin-4 Stat</n>. Germ line C transcripts can be induced by <n>IL-4</n> in the human B cell line, BL-2. Utilizing a IFN-gamma activation site-like DNA sequence element located upstream of the I epsilon exon, we demonstrated by gel mobility shift assays that <n>IL-4</n> induced a binding activity in the cytosol and nucleus of BL-2 cells. This factor was designated <n>IL-4</n> NAF (<n>IL-4</n>-induced nuclear-activating factors) and was identified as a tyrosine phosphoprotein, which translocates from the cytosol to the nucleus upon <n>IL-4</n> treatment. Because these are the characteristics of a signal transducer and activator of transcription (Stat) protein, we determined whether antibodies to Stat proteins will interfere with gel mobility shift and found that antibodies to <n>IL-4</n> Stat, also known as <n>Stat6</n>, but not antibodies to other Stat proteins, interfere with the formation of the <n>IL-4</n> NAF complex. Congruous with the involvement of a Stat protein, <n>IL-4</n> induced robust <n>Janus kinase 3</n> (<n>JAK3</n>) activity in BL- 2 cells. Cotransfection of <n>JAK3</n> with <n>IL-4</n> Stat into COS-7 cells produced an intracellular activity which bound the same IFN-gamma activation site-like sequence and comigrated with <n>IL-4</n> NAF in electrophoretic mobility shift assay. These results show that <n>IL-4</n> NAF is <n>IL-4</n> Stat, which is activated by <n>JAK3</n> in response to <n>IL-4</n> receptor engagement.
95329724>Does activation of the TAL1 gene occur in a majority of patients with T- cell acute lymphoblastic leukemia? A pediatric oncology group study. Almost 25% of patients with T-cell acute lymphoblastic leukemia (T-ALL) have tumor-specific rearrangements of the TAL1 gene. Although <n>TAL1</n> expression has not been observed in normal lymphocytes, TAL1 gene products are readily detected in leukemic cells that harbor a rearranged TAL1 allele. Hence, it has been proposed that ectopic expression of <n>TAL1</n> promotes the development of T-ALL. In this report, we show that <n>TAL1</n> is expressed in the leukemic cells of most patients with T-ALL, including many that do not display an apparent <n>TAL1</n> gene alteration. A polymorphic dinucleotide repeat in the transcribed sequences of <n>TAL1</n> was used to determine the allele specificity of <n>TAL1</n> transcription in primary T-ALL cells. Monoallelic expression of <n>TAL1</n> was observed in the leukemic cells of all patients (8 of 8) bearing a <n>TAL1</n> gene rearrangement. In the leukemic cells of patients without detectable <n>TAL1</n> rearrangements, <n>TAL1</n> transcription occurred in either a monoallelic (3 of 7 patients) or a biallelic (4 of 7 patients) fashion. Thus, <n>TAL1</n> activation in these patients may result from subtle alterations in cis-acting regulatory sequences (affecting expression of a single <n>TAL1</n> allele) or changes in trans-acting factors that control <n>TAL1</n> transcription (affecting expression of both <n>TAL1</n> alleles).
95374911>Expression of erythroid-specific genes in acute megakaryoblastic leukaemia and transient myeloproliferative disorder in Down's syndrome. Acute megakaryoblastic leukaemia (M7) and transient myeloproliferative disorder in Down's syndrome (TMD) are characterized by rapid growth of abnormal blast cells which express megakaryocytic markers. To clarify properties of the blast cells in M7 and TMD cases, we examined erythroid markers expression in blasts from six cases with M7 and seven cases with TMD in this study. Erythroid-specific mRNAs encoding gamma- globin and <n>erythroid delta-aminolevulinate synthase</n> were found to be expressed in blasts from most of these cases, indicating that majorities of the blasts in M7 and TMD cases have erythroid and megakaryocytic phenotypes. We also found that mRNAs encoding <n>GATA-1</n> and <n>GATA-2</n> are expressed in all these cases. These results suggest that M7 blasts and TMD blasts correspond to the erythroid/megakaryocytic bipotential progenitor cells.
95308691>Expression of <n>Ah receptor</n> (<n>TCDD receptor</n>) during human monocytic differentiation. We have previously found a high expression of human <n>Ah receptor</n> (<n>TCDD receptor</n>) mRNA in peripheral blood cells of individuals. In this paper, the expression of this gene in blood cells was first investigated in fractions of nucleated cells, revealing predominant expression of the <n>Ah receptor</n> gene in the monocyte fraction. Then the expression levels of AhR mRNA in various hematopoietic cell lines were examined together with those of <n>Arnt</n> and <n>P450IA1</n>. <n>AhR</n> was expressed at high levels in monocytoid U937, THP1, and HEL/S cells, and at moderate levels in promyelocytic HL60 cells and erythroblastic HEL cells. However, it was not detected in lymphoid cells MOLT4 (T cell) and BALL1 (B cell), nor in K562 erythroblasts. Furthermore, a specific induction of <n>AhR</n> during monocytic differentiation was investigated in HL60 and HEL cells. HL60 cells were induced to differentiate toward monocytes-macrophages by incubation with phorbol ester, showing a 5- to 2-fold increase of <n>AhR</n> mRNA. The incubation with <n>transforming growth factor beta 1</n> and 1 alpha,25-dihydroxyvitamin D3 resulted in a 5- to 7-fold increase of <n>AhR</n> mRNA. The HEL cells also exhibited a similar elevation of <n>AhR</n> mRNA level, when they had differentiated toward monocyte-macrophage cells by these combined inducers, but little change in the mRNA level was observed when the cells were induced to differentiate into other cell types. Treatment of the differentiated HL60 cells with 3- methylcholanthrene, a ligand of <n>AhR</n>, induced the expression of the <n>P450IA1</n> gene. These results indicated that expression of <n>AhR</n> mRNA was significantly induced during monocytic differentiation and that the differentiated cells were responsive to xenobiotics. Our results suggest that <n>AhR</n> may play an important role in the function of monocytes and also in the eventual activation of environmental carcinogens.
95261059>Neutrophils and monocytes express high levels of <n>PU.1</n> (<n>Spi-1</n>) but not <n>Spi-B</n>. <n>PU.1</n> (the Spi-1 oncogene) and <n>Spi-B</n> are closely related members of the ets transcription factor family, sharing similar DNA binding specificities mediated by similar DNA binding domains. <n>PU.1</n> and <n>Spi-B</n> have been previously described as being predominantly expressed coordinately in macrophages and B cells, but their expression in early hematopoietic stages and during the course of myeloid differentiation to monocytes and macrophages or to neutrophils has not been extensively investigated. Here, we report that <n>PU.1</n> mRNA is upregulated during myeloid differentiation of human purified CD34+ cells and murine multipotential FDCP-mix A4 cells, suggesting that <n>PU.1</n> is upregulated as an early event during differentiation of multipotential progenitor cells. <n>PU.1</n> expression is maintained at stable levels during differentiation of myeloid cell lines U937 and HL-60 to monocytic and neutrophilic cells. <n>PU.1</n> is expressed at highest levels in mature human monocytes and human peripheral blood neutrophils. In contrast to <n>PU.1</n>, significant levels of <n>Spi-B</n> mRNA and protein are found only in some B- cell lines and spleen but are not found in myeloid cell lines, neutrophils, or macrophages. In vitro translated <n>Spi-B</n> protein can bind to <n>PU.1</n> binding sites in myeloid promoters and transactivate these promoters in nonmyeloid cells. Therefore, although <n>PU.1</n> and <n>Spi-B</n> may bind to similar DNA control elements and have redundancy of transactivation function in vitro, the lack of significant levels of <n>Spi-B</n> in myeloid cells makes it unlikely that <n>Spi-B</n> plays a significant role in myeloid lineage development and gene expression. In contrast, <n>PU.1</n> is expressed at high levels not only in monocytes and macrophages but also in neutrophils, indicating that <n>PU.1</n> can activate gene expression in both major myeloid lineages.
95244883>Identification of a major positive regulatory element located 5' to the human zeta-globin gene. The function of the zeta-globin promoter was studied using a series of zeta-globin promoter deletion constructs to drive <n>luciferase</n> expression in transiently transfected human erythroleukemia cells. The promoters were used without enhancers, or with enhancers derived from the beta- globin locus control region and the alpha-globin HS-40 enhancer. When transfected into K562 cells, which express zeta-globin, comparable amounts of activity were obtained from the -557 and -417 zeta- <n>luciferase</n> constructs and the alpha-<n>luciferase</n> constructs when no enhancers or the alpha-globin locus enhancers were used. When the constructs were transfected into OCIM1 cells, which do not express zeta- globin, the zeta-globin promoters were at best 20% as active as the alpha-globin promoters. When sequences from -417 to -207 5' to the zeta- globin mRNA cap site were deleted, up to 95% of the zeta-globin promoter activity was lost in K562 cells. Reinsertion of these sequences into zeta-<n>luciferase</n> constructs missing the -417 to -207 region showed that the sequences lack classical enhancer activity. Point mutation of a <n>GATA-1</n> site at -230 reduced promoter activity by 37%. Point mutation of a CCACC site at -240 had no effect. Electrophoretic mobility shift assays indicated that the -230 <n>GATA-1</n> site has a relatively low affinity for <n>GATA-1</n>. These experiments show the presence of a strong positive-acting element, located between -417 and -207 bp 5' to the zeta-globin mRNA cap site, is necessary for high- level promoter activity in K562 cells. This element requires <n>GATA-1</n> and additional unknown factors for maximal activity.
95247773>Analysis of the role of protein kinase C-alpha, -epsilon, and -zeta in T cell activation. T cells express multiple isotypes of <n>protein kinase C</n> (<n>PKC</n>) and although it is well accepted that <n>PKCs</n> have an important role in T cell activation, little is known about the function of individual PKC isotypes. To address this issue, mutationally active PKC-alpha, - epsilon, or -zeta have been transfected into T cells and the consequences for T cell activation determined. <n>p21ras</n> plays an essential role in T cell activation. Accordingly, the effects of the constitutively active PKCs were compared to the effects of mutationally activated <n>p21ras</n>. The data indicate that <n>PKC-epsilon</n> and, to a lesser extent <n>PKC-alpha</n> but not <n>-zeta</n>, can regulate the transcription factors <n>AP-1</n> and nuclear factor of activated T cells (<n>NF-AT-1</n>). The ability of <n>PKC-epsilon</n> to induce transactivation of <n>NF-AT-1</n> and <n>AP-1</n> was similar to the stimulatory effect of a constitutively activated <n>p21ras</n>. <n>PKC- epsilon</n>, but not <n>PKC-alpha</n> nor activated <n>p21ras</n>, was able to induce <n>NF- KB</n> activity. Phorbol esters induce expression of <n>CD69</n> whereas none of the activated PKC isotypes tested were able to have this effect. Activated Src and <n>p21ras</n> were able to induce <n>CD69</n> expression. These results indicate selective functions for different PKC isotypes in T cells. Moreover, the data comparing the effects of activated Ras and PKC mutants suggest that <n>PKC-alpha</n>, <n>p21ras</n>, and <n>PKC-epsilon</n> are not positioned linearly on a single signal transduction pathway.
95204946>Differences in binding of glucocorticoid receptor to DNA in steroid- resistant asthma. Although glucocorticosteroids are a very effective treatment for asthma and other chronic inflammatory diseases, a small proportion of patients are resistant to their therapeutic effects. The molecular mechanism for this steroid resistance is unclear. Steroid resistance cannot be explained by pharmacokinetic mechanisms, by a defect in the binding of steroids to glucocorticoid receptors, nor by defective nuclear translocation of this receptor, thereby suggesting that the molecular abnormality lies distal to nuclear translocation. We examined the ability of nuclear translocated glucocorticoid receptors to bind to their DNA binding sites (GRE) using electrophoretic mobility shift assays in PBMC from patients with steroid-sensitive and steroid- resistant asthma. The binding of the glucocorticoid receptor to DNA in these patients was also studied using Scatchard analysis. Dexamethasone induced a significant rapid and sustained twofold increase in GRE binding in PBMCs from steroid-sensitive asthmatic patients and nonasthmatic individuals, but this was markedly reduced in steroid- resistant asthmatic patients. Scatchard analysis of glucocorticoid receptor-GRE binding showed no change in binding affinity but did show a reduced number of receptors available for DNA binding in the steroid- resistant patients. These results suggest that the ability of the glucocorticoid receptor to bind to GRE is impaired in steroid-resistant patients because of a reduced number of receptors available for binding to DNA.
95195208><n>Interleukin-5</n> signaling in human eosinophils involves <n>JAK2</n> tyrosine kinase and <n>Stat1 alpha</n>. Signaling by a wide variety of cytokines, including interferons, interleukins, and growth factors, involves activation of JAK kinases and Stat (Signal transducers and activators of transcription) proteins. At present, not much is known about the molecular mechanisms by which <n>interleukin-5</n> (<n>IL-5</n>) exerts its diverse biologic effects. Human eosinophils are one of the most important target cells for <n>IL-5</n> and were used here to study <n>IL-5</n> signaling in a primary human cell. <n>IL-5</n> induced rapid and transient tyrosine phosphorylation of <n>JAK2</n>. Moreover, <n>IL-5</n> induced at least two DNA-binding complexes, using nuclear extracts from normal human eosinophils and the IL-6/interferon-gamma response element of the ICAM-1 promoter (ICAM-1 pIRE) in an electromobility shift assay. From supershift experiments it was concluded that one DNA- binding complex contained <n>Stat1 alpha</n>, probably as a homodimer. Both DNA-binding complexes were inhibited by a phosphotyrosine antibody (<n>4G10</n>), suggesting that tyrosine phosphorylation is required for complex formation. IL-3 and <n>granulocyte-macrophage colony-stimulating factor</n> induced, similar to <n>IL-5</n>, two DNA-binding complexes in human eosinophils, including <n>Stat1 alpha</n>. These data show for the first time that molecular mechanisms of <n>IL-5</n> signaling in human eosinophils involve members of the JAK kinase family as well as members of the Stat family
95373132>Infection and replication of Tat- human immunodeficiency viruses: genetic analyses of LTR and tat mutations in primary and long-term human lymphoid cells. <n>Tat</n> is an essential regulatory protein for the replication of human immunodeficiency virus (HIV). Mutations in the tat gene have been shown to block HIV replication in human T cells. Several studies have established that <n>Tat</n> releases an elongation block to the transcription of HIV long terminal repeat (LTR); however, it is not known whether this mechanism alone is sufficient to explain the block to HIV replication in human T cells when <n>Tat</n> is absent. It is possible that <n>Tat</n> is also needed for other functions during HIV replication. To test these hypotheses, we studied several tat mutants, including two stop codon mutants and one deletion mutant using replication-competent HIV-1 constructs carrying wild-type or mutant LTRs with modifications in the NF-kappa B and/or Sp1 binding sites. In this study, we show that Tat- HIV-1 with wild-type LTRs can replicate in HeLa cells, and the virus produced from HeLa cells can infect primary peripheral blood lymphocytes and macrophages. It was found that the propagation of the <n>Tat</n> mutants containing wild-type LTRs was less efficient than that of the LTR-modified <n>Tat</n> mutants. Large amounts of viral RNA and particles were synthesized in infections established using the tat mutants that contain modified LTRs. However, this efficient propagation of the LTR- modified tat mutants was restricted to some lymphoid cell lines that have been transformed with other viruses. Thus, despite its essential role for releasing an elongation block, <n>Tat</n> is not otherwise absolutely required for synthesis of full-length HIV transcripts and assembly of virus particles. Direct sequencing of the viral genomes and reinfection kinetics showed no evidence of wild-type reversion even after prolonged infection with the <n>Tat</n>- virus. The implications for in vivo HIV-1 replication and potential application of this system to the study of alternative <n>Tat</n> function are discussed.
95355848>Functional roles of the transcription factor <n>Oct-2A</n> and the high mobility group protein <n>I/Y</n> in HLA-DRA gene expression. The class II major histocompatibility complex gene HLA-DRA is expressed in B cells, activated T lymphocytes, and in antigen-presenting cells. In addition, HLA-DRA gene expression is inducible in a variety of cell types by <n>interferon-gamma</n> (<n>IFN-gamma</n>). Here we show that the lymphoid- specific transcription factor <n>Oct-2A</n> plays a critical role in HLA-DRA gene expression in class II-positive B cell lines, and that the high mobility group protein (HMG) <n>I/Y</n> binds to multiple sites within the DRA promoter, including the <n>Oct-2A</n> binding site. Coexpression of HMG <n>I/Y</n> and Oct-2 in cell lines lacking Oct-2 results in high levels of HLA-DRA gene expression, and in vitro DNA-binding studies reveal that HMG <n>I/Y</n> stimulates <n>Oct-2A</n> binding to the HLA-DRA promoter. Thus, <n>Oct-2A</n> and HMG <n>I/Y</n> may synergize to activate HLA-DRA expression in B cells. By contrast, <n>Oct-2A</n> is not involved in the <n>IFN-gamma</n> induction of the HLA- DRA gene in HeLa cells, but antisense HMG <n>I/Y</n> dramatically decreases the level of induction. We conclude that distinct sets of transcription factors are involved in the two modes of HLA-DRA expression, and that <n>HMG I/Y</n> may be important for B cell-specific expression, and is essential for <n>IFN-gamma</n> induction.
95329723>Heterogeneous expression of Epstein-Barr virus latent proteins in endemic Burkitt's lymphoma. Epstein-Barr virus (EBV)-infected cells may sustain three distinct forms of virus latency. In lymphoblastoid cell lines, six <n>EBV-encoded nuclear antigens</n> (EBNA1, 2, 3A, 3B, 3C, -LP), three latent membrane proteins (<n>LMP1</n>, <n>2A</n>, <n>2B</n>), and two nuclear RNAs (EBERs) are expressed. This form of latency, termed latency III, is also encountered in some posttransplant lymphoproliferative disorders. In EBV-positive cases of Hodgkin's disease, the EBERs, <n>EBNA1</n>, and the LMPs are expressed (latency II), whereas in Burkitt's lymphoma (BL) only the EBERs and <n>EBNA1</n> have been detected (latency I). We have studied the expression of EBV proteins in 17 cases of EBV-positive endemic BL by immunohistology. Expression of <n>LMP1</n> was seen in variable proportions of tumor cells in two cases and <n>EBNA2</n> was detected in some tumor cells in three other cases. Also, the <n>BZLF1 trans-activator protein</n> was expressed in a few tumor cells in 6 cases, indicating entry into the lytic cycle. A phenotypic drift from latency I to latency III has been observed previously in some BL cell lines. Our results suggest that a similar phenomenon may occur in BL in vivo and indicate that the operational definition of EBV latencies is not easily applied to human tumors.
95325614>The activation of the Jak-<n>STAT 1</n> signaling pathway by <n>IL-5</n> in eosinophils. The intracellular signal transduction of <n>IL-5</n> in eosinophils is unknown. The objective of this study was to investigate the involvement of the newly discovered Jak-STAT pathway in the <n>IL-5</n> signal transduction mechanism. Eosinophils were purified from peripheral blood by discontinuous Percoll gradients and stimulated with <n>IL-5</n>. The involvement of Jak 2 was investigated by immunoprecipitation followed by immunoblotting for tyrosine phosphorylation. The activation of Jak 2 was studied by autophosphorylation of the immunoprecipitated kinase. Jak 2 was tyrosine phosphorylated within 1 to 3 min after stimulation of eosinophils with <n>IL-5</n>. Further, the <n>immunoprecipitated Jak 2</n> obtained from <n>IL-5</n>-stimulated cells underwent autophosphorylation. Jak 2 coprecipitated with the beta-subunit of the <n>IL-5</n> receptor, suggesting a physical association of the kinase with the receptor. The <n>nuclear factor STAT-1</n> (<n>p91</n>) was investigated by immunoprecipitation followed by immunoblotting for tyrosine phosphorylation. <n>STAT-1</n> was tyrosine phosphorylated within 15 min of <n>IL-5</n> stimulation. The presence of <n>STAT- 1</n> in the nuclear extract was studied by electrophoretic mobility shift assay. <n>IL-5</n> induced two proteins that bound to the gamma-activating sequence. In the presence of an anti-<n>STAT-1</n> Ab, the band was supershifted. Thus, we demonstrated that <n>IL-5</n> activated the Jak 2-<n>STAT 1</n> signaling pathway in eosinophils. We speculate that the Jak 2-<n>STAT 1</n> pathway may be involved in the activation of <n>IL-5</n>-inducible genes in eosinophils.
95298682>Reduced mitogenic stimulation of peripheral blood mononuclear cells as a prognostic parameter for the course of breast cancer: a prospective longitudinal study. Immunosuppression has been often associated with the course of malignant diseases. In the present study, the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogenic stimulation with <n>phytohaemagglutinin</n> (<n>PHA</n>) was assessed prospectively in 90 patients with stage I-III breast cancer. Whereas <n>PHA</n>-induced proliferation of PBMCs derived from patients with breast cancer preoperatively was significantly decreased when compared with data obtained in healthy control individuals (P &lt; 0.001), the degree of the defect in <n>PHA</n>-induced proliferation of PBMCs depended upon the tumour burden as manifested by tumour size and axillary lymph node involvement (P &lt; 0.003 in each case). <n>PHA</n>-induced proliferation of PBMCs dropped significantly in patients who received adjuvant chemotherapy consisting of cyclophosphamide, methotrexate and fluorouracil (CMF) after an observation period of 6 months (P &lt; 0.01), but not in patients under adjuvant treatment with tamoxifen only. After an additional 6 months (i.e. 12 months after surgery), <n>PHA</n>-induced proliferation of PBMCs was similar in patients after adjuvant chemotherapy with CMF and in those receiving continued adjuvant tamoxifen treatment (P &gt; 0.1), but in all patients still significantly decreased as compared with healthy controls (P &lt; 0.001). When data obtained preoperatively and after 12 months were compared, it was found that out of 23 patients whose PBMCs had experienced a drop in their <n>PHA</n>-induced proliferation, 14 (61%) had developed metastatic disease within the subsequent 24 months (i.e. 36 months after surgery). In contrast, out of 59 patients whose PBMCs showed an increase in their <n>PHA</n>-induced proliferation within the first 12 months after surgery, only one (2%) presented with disease progression. We thus conclude that <n>PHA</n>-induced proliferation of PBMCs derived from patients with breast cancer depends upon the tumour load and is a good clinical predictor for the further course of the disease.
95248083>Activation of transcription by binding of <n>NF-E1</n> (<n>YY1</n>) to a newly identified element in the first exon of the human DR alpha gene. A previously unrecognized element, located downstream of the start site of transcription in the first exon of the DR alpha gene, has been defined that enhances promoter activity up to eightfold in a position- dependent manner. Mutations in this DNA-binding site abolished binding of a nuclear factor in human B cell nuclear extract and decreased the activity of the DR alpha promoter to a basal level. Significant sequence homology of this element was found in the DNA of the DR beta, DP alpha and -beta, and DQ alpha genes, always located downstream of the transcriptional start site. The nuclear factor binds to the DR alpha and DP alpha element but not to the element in the DQ alpha gene. It was identified as <n>NF-E1</n> (<n>YY1</n>). This protein, previously identified by its binding to the Ig kappa 3' enhancer and the Ig heavy chain mu E1 site, thus also appears to be quite important in the regulation of MHC class II gene expression.
95222739>Transcriptional activity of core binding factor-alpha (AML1) and beta subunits on murine leukemia virus enhancer cores. Core binding factor (<n>CBF</n>), also known as polyomavirus enhancer-binding protein 2 and <n>SL3 enhancer factor 1</n>, is a mammalian transcription factor that binds to an element termed the core within the enhancers of the murine leukemia virus family of retroviruses. The core elements of the SL3 virus are important genetic determinants of the ability of this virus to induce T-cell lymphomas and the transcriptional activity of the viral long terminal repeat in T lymphocytes. <n>CBF</n> consists of two subunits, a DNA binding subunit, CBF alpha, and a second subunit, CBF beta, that stimulates the DNA binding activity of CBF alpha. One of the genes that encodes a CBF alpha subunit is AML1, also called <n>Cbf alpha 2</n>. This locus is rearranged by chromosomal translocations in human myeloproliferative disorders and leukemias. An exogenously expressed <n>Cbf alpha 2</n>-encoded subunit (CBF alpha 2-451) stimulated transcription from the SL3 enhancer in <n>P19</n> and HeLa cells. Activity was mediated through the core elements. Three different isoforms of CBF beta were also tested for transcriptional activity on the SL3 enhancer. The longest form, CBF beta-187, increased the transcriptional stimulation by CBF alpha 2-451 twofold in HeLa cells, although it had no effect in <n>P19</n> cells. Transcriptional activation by CBF beta required binding to the CBF alpha subunit, as a form of CBF beta that lacked binding ability, CBF beta-148, failed to increase activity. These results indicated that at least in certain cell types, the maximum activity of <n>CBF</n> required both subunits. They also provided support for the hypothesis that <n>CBF</n> is a factor in T lymphocytes that is responsible for recognition of the SL3 cores. We also examined whether <n>CBF</n> could distinguish a 1-bp difference between the enhancer core of SL3 and the core of the nonleukemogenic virus, Akv. This difference strongly affects transcription in T cells and leukemogenicity of SL3. However, no combination of CBF alpha and CBF beta subunits that we tested was able to distinguish the 1-bp difference in transcription assays. Thus, a complete understanding of how T cells recognize the SL3 core remains to be elucidated.
95238477><n>Interleukin (IL)-10</n> inhibits <n>nuclear factor kappa B</n> (<n>NF kappa B</n>) activation in human monocytes. <n>IL-10</n> and <n>IL-4</n> suppress cytokine synthesis by different mechanisms. Our previous studies in human monocytes have demonstrated that <n>interleukin (IL)-10</n> inhibits lipopolysaccharide (LPS)-stimulated production of inflammatory cytokines, <n>IL-1 beta</n>, <n>IL-6</n>, <n>IL-8</n>, and <n>tumor necrosis factor (TNF)-alpha</n> by blocking gene transcription. Using electrophoretic mobility shift assays (EMSA), we now show that, in monocytes stimulated with LPS or <n>TNF alpha</n>, <n>IL-10</n> inhibits nuclear stimulation of <n>nuclear factor kappa B</n> (<n>NF kappa B</n>), a transcription factor involved in the expression of inflammatory cytokine genes. Several other transcription factors including NF-<n>IL-6</n>, <n>AP-1</n>, <n>AP-2</n>, GR, CREB, <n>Oct-1</n>, and <n>Sp-1</n> are not affected by <n>IL-10</n>. This selective inhibition by <n>IL-10</n> of <n>NF kappa B</n> activation occurs rapidly and in a dose-dependent manner and correlates well with <n>IL-10</n>'s cytokine synthesis inhibitory activity in terms of both kinetics and dose responsiveness. Furthermore, compounds such as tosylphenylalanyl chloromethyl ketone and pyrrolidinedithiocarbamate that are known to selectively inhibit <n>NF kappa B</n> activation block cytokine gene transcription in LPS-stimulated monocytes. Taken together, these results suggest that inhibition of <n>NF kappa B</n> activation may be an important mechanism for <n>IL-10</n> suppression of cytokine gene transcription in human monocytes. <n>IL-4</n>, another cytokine that inhibits cytokine mRNA accumulation in monocytes, shows little inhibitory effect on LPS-induced <n>NF kappa B</n> activation. Further examination reveals that, unlike <n>IL-10</n>, <n>IL-4</n> enhances mRNA degradation and does not suppress cytokine gene transcription. These data indicate that <n>IL-10</n> and <n>IL-4</n> inhibit cytokine production by different mechanisms.
95190988><n>LMP-1</n> activates <n>NF-kappa B</n> by targeting the inhibitory molecule <n>I kappa B alpha</n>. <n>LMP-1</n>, an Epstein-Barr virus membrane protein expressed during latent infection, has oncogenic properties, as judged from its ability to transform B lymphocytes and rodent fibroblasts. <n>LMP-1</n> induces the expression of bcl2, an oncogene which protects cells from apoptosis, as well as of genes encoding other proteins involved in cell regulation and growth control. The mechanisms by which <n>LMP-1</n> upregulates these proteins is unknown, but it is plausible that <n>LMP-1</n> modifies signal transduction pathways that result in the activation of one or more transcription factors that ultimately regulate transcription of oncogenic genes. <n>NF-kappa B</n>, a transcription factor controlling the expression of genes involved in cell activation and growth control, has been shown to be activated by <n>LMP-1</n>. The mechanism(s) regulating this activation remains unknown. Our data indicate that increased <n>NF-kappa B</n> DNA binding and functional activity are present in B-lymphoid cells stably or transiently expressing <n>LMP-1</n>. <n>I kappa B alpha</n> is selectively modified in <n>LMP-1</n>-expressing B cells. A phosphorylated form of <n>I kappa B alpha</n> and increased protein turnover-degradation correlate with increased <n>NF-kappa B</n> nuclear translocation. This results in increased transcription of <n>NF-kappa B</n>-dependent-genes, including those encoding <n>p105</n> and <n>I kappa B alpha</n> (MAD3). These results indicate that <n>LMP-1</n> activates <n>NF-kappa B</n> in B-cell lines by targeting <n>I kappa B alpha</n>. Identification of the pathways activated by <n>LMP-1</n> to result in posttranslational modifications of <n>I kappa B alpha</n> will aid in determining the role of this virus-host cell protein interaction in Epstein-Barr virus-mediated oncogenesis.
95197545>Identification of <n>human TR2 orphan receptor</n> response element in the transcriptional initiation site of the simian virus 40 major late promoter [published erratum appears in J Biol Chem 1995 Nov 3;270(44):26721] A DNA response element (TR2RE-SV40) for the <n>TR2 orphan receptor</n>, a member of the steroid-thyroid hormone receptor superfamily, has been identified in the simian virus 40 (SV40) +55 region (nucleotide numbers 368-389, 5'-GTTAAGGTTCGTAGGTCATGGA-3'). Electrophoretic mobility shift assay, using in vitro translated <n>TR2 orphan receptor</n> with a molecular mass of <n>67 kilodaltons</n>, showed a specific binding with high affinity (dissociation constant = 9 nM) for this DNA sequence. DNA-swap experiments using <n>chloramphenicol acetyl-transferase</n> assay demonstrated that androgen can suppress the transcriptional activities of SV40 early promoter via the interaction between this TR2RE-SV40 and the <n>chimeric receptor AR/TR2/AR</n> with the DNA-binding domain of the <n>TR2 orphan receptor</n> flanked by the N-terminal and androgen-binding domains of the <n>androgen receptor</n>. In addition, this TR2RE-SV40 can function as a repressor to suppress the transcriptional activities of both SV40 early and late promoters. Together, these data suggest the TR2RE-SV40 may represent the first identified natural DNA response element for the <n>TR2 orphan receptor</n> that may function as a repressor for the SV40 gene expression
95161757>Detection of the chromosome 16 CBF beta-MYH11 fusion transcript in myelomonocytic leukemias. Karyotypic detection of chromosomal 16 abnormalities classically associated with AML M4Eo can be difficult. Characterization of the two genes involved in the inv(16)(p13q22), CBF beta and MYH11, has allowed the detection of fusion transcripts by reverse-transcriptase polymerase chain reaction (RT-PCR). We have analyzed CBF beta-MYH11 fusion transcripts by RT-PCR in myelomonocytic leukemias, with or without eosinophilia, to determine whether their presence correlates with morphology. Fifty-three cases (11 AML M4Eo; 1 AML M4 with atypical abnormal eosinophils (AML M4 "Eo"); 29 AML M4; 8 AML M5; 3 CMML; and 1 AML M2 with eosinophilia) were analyzed. All 11 typical AML M4Eo were CBF beta-MYH11 positive. The single case of AML M4 with distinctive eosinophil abnormalities was negative by karyotype, RT-PCR and fluorescent in situ hybridization (FISH). Three of 29 (10%) AML M4 without abnormal eosinophils were CBF beta-MYH11 positive, 1 of which did not show any apparent chromosome 16 abnormalities by classical metaphase analysis (2 not tested). Both cases tested also showed MYH11 genomic rearrangement. None of the other leukemias were RT-PCR positive. Follow-up of three patient showed residual positivity in apparent complete remission. These data show that CBF beta-MYH11 fusion transcripts occur not only in the vast majority of typical AML M4Eo, but also in approximately 10% of AML M4 without eosinophilic abnormalities, a much higher incidence than the sporadic reports of chromosome 16 abnormalities in AML M4 would suggest. Taken together with the detection of CBF beta-MYH11 transcripts in the absence of apparent chromosome 16 abnormalities by classical banding techniques, these data show that additional screening by either RT-PCR or FISH should be performed in all AML M4, regardless of morphologic features, to allow accurate evaluation of the prognostic importance of this fusion transcript.
95163587><n>HIV-1 Tat</n> potentiates TNF-induced <n>NF-kappa B</n> activation and cytotoxicity by altering the cellular redox state. This study demonstrates that <n>human immunodeficiency virus type 1 (HIV- 1) Tat protein</n> amplifies the activity of tumor necrosis factor (<n>TNF</n>), a cytokine that stimulates HIV-1 replication through activation of <n>NF- kappa B</n>. In HeLa cells stably transfected with the HIV-1 tat gene (HeLa- tat cells), expression of the <n>Tat protein</n> enhanced both <n>TNF</n>-induced activation of <n>NF-kappa B</n> and <n>TNF</n>-mediated cytotoxicity. A similar potentiation of <n>TNF</n> effects was observed in Jurkat T cells and HeLa cells treated with soluble <n>Tat protein</n>. <n>TNF</n>-mediated activation of <n>NF- kappa B</n> and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, <n>Tat</n>-mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells <n>HIV- 1 Tat</n> suppressed the expression of <n>Mn-dependent superoxide dismutase</n> (<n>Mn-SOD</n>), a <n>mitochondrial enzyme</n> that is part of the cellular defense system against oxidative stress. Thus, <n>Mn-SOD</n> RNA protein levels and activity were markedly reduced in the presence of <n>Tat</n>. Decreased <n>Mn-SOD</n> expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated <n>Tat</n> protein (<n>Tat1-72</n>), known to transactivate the HIV-1 long terminal repeat (LTR), no longer affected <n>Mn-SOD</n> expression, the cellular redox state or <n>TNF</n>- mediated cytotoxicity. Thus, our experiments demonstrate that the C- terminal region of HIV-1 <n>Tat</n> is required to suppress <n>Mn-SOD</n> expression and to induce pro-oxidative conditions reflected by a drop in reduced glutathione (<n>GSH</n>) and the GSH:oxidized GSH (GSSG) ratio. (ABSTRACT TRUNCATED AT 250 WORDS)
95261816>The use of glucocorticoids in acute lymphoblastic leukemia of childhood. Molecular, cellular, and clinical considerations. Glucocorticoids have been included in almost all treatment regimens for childhood acute lymphoblastic leukemia for decades. However, optimal agents, doses, and/or schedules have yet to be defined despite extensive clinical application. New data on the pharmacokinetics, pharmacodynamics, and molecular mechanisms of action of glucocorticoids have suggested alternative approaches in ALL. These suggest that prolonged, i.e. 28 day, glucocorticoid therapy may be unnecessary as exposure to glucocorticoid induces down-regulation of glucocorticoid receptors. Dexamethasone may be superior to prednisone in conventional equi-effective doses. Blast sensitivity to glucocorticoids correlates closely with sensitivity to other, putatively non-cross-resisting agents and with outcome after multi-agent therapy, suggesting overlapping mechanisms of action, and focusing attention on the determinants of the threshold for apoptosis. Increasing success in the treatment of childhood acute lymphoblastic leukemia has led to increasing awareness of avascular necrosis of bone as a potentially disabling sequela of glucocorticoid therapy, especially in adolescent and young adult patients.
95074873><n>Epstein-Barr virus nuclear protein 2</n> transactivation of the latent membrane protein 1 promoter is mediated by <n>J kappa</n> and <n>PU.1</n>. Expression of the Epstein-Barr virus (EBV) latent membrane protein 1 (<n>LMP-1</n>) oncogene is regulated by the EBV nuclear protein 2 (<n>EBNA-2</n>) transactivator. <n>EBNA-2</n> is known to interact with the cellular DNA- binding protein <n>J kappa</n> and is recruited to promoters containing the GTGGGAA <n>J kappa</n> recognition sequence. The minimal <n>EBNA-2</n>-responsive LMP- 1 promoter includes one <n>J kappa</n>-binding site, and we now show that mutation of that site, such that <n>J kappa</n> cannot bind, reduces <n>EBNA-2</n> responsiveness by 60%. To identify other factors which interact with the LMP-1 <n>EBNA-2</n> response element (E2RE), a -236/-145 minimal E2RE was used as a probe in an electrophoretic mobility shift assay. The previously characterized factors <n>J kappa</n>, <n>PU.1</n>, and <n>AML1</n> bind to the LMP-1 E2RE, along with six other unidentified factors (<n>LBF2</n> to <n>LBF7</n>). Binding sites were mapped for each factor. <n>LBF4</n> is B- and T-cell specific and recognizes the <n>PU.1</n> GGAA core sequence as shown by methylation interference. <n>LBF4</n> has a molecular mass of 105 kDa and is probably unrelated to <n>PU.1</n>. <n>LBF2</n> was found only in epithelial cell lines, whereas <n>LBF3</n>, <n>LBF5</n>, <n>LBF6</n>, and <n>LBF7</n> were not cell type specific. Mutations of the AML1- or LBF4-binding sites had no effect on <n>EBNA-2</n> transactivation, whereas mutation of the <n>PU.1</n>-binding site completely eliminated <n>EBNA-2</n> responses. A gst-<n>EBNA-2</n> fusion protein specifically depleted <n>PU.1</n> from nuclear extracts and bound in vitro translated <n>PU.1</n>, providing biochemical evidence for a direct <n>EBNA-2</n>-<n>PU.1</n> interaction. Thus, <n>EBNA-2</n> transactivation of the LMP-1 promoter is dependent on interaction with at least two distinct sequence-specific DNA-binding proteins, <n>J kappa</n> and <n>PU.1</n>. <n>LBF3</n>, <n>LBF5</n>, <n>LBF6</n>, or <n>LBF7</n> may also be involved, since their binding sites also contribute to <n>EBNA-2</n> responsiveness.
96094166>Lymphocyte glucocorticoid receptor: predictor of sertraline response in adolescent major depressive disorder (MDD). Major depressive disorder (MDD) in adolescents demonstrates resistance to tricyclic antidepressants and absence of hypercortisolemia. The efficacy of serotonin reuptake inhibitors (SRIs) is uncertain, and response predictors are unavailable. Abnormal fast feedback and negative feedback of the hypothalamic-pituitary-adrenal axis implicates a dampened <n>limbic-hippocampal glucocorticoid type II receptor</n> (<n>GCII</n>). We hypothesized that lymphocyte <n>GCII</n> is altered in adolescent MDD and could serve as a marker for response to SRIs. In an open-label study, adolescents (n = 20) meeting DSM-III-R criteria for MDD showed baseline lymphocyte <n>GCII</n> sites per cell (sites/cell) values of 793 +/- 106 versus 2,563 +/- 499 (+/- SEM) for matched controls (n = 18) (t = 3.5; df = 36; p &lt; .001). <n>GCII</n> was bimodally distributed, with SRI responders differing from nonresponders (t = 3.9; df = 14; p &lt; .001). <n>GCII</n> accurately classified 90 percent of sertraline responders and 80 percent of nonresponders. Only SRI responders showed <n>GCII</n> sites/cell upregulated after 6 weeks of treatment (t = 2.1, df = 10; p &lt; .05).
95074049>The role of <n>NFATp</n> in <n>cyclosporin A-sensitive tumor necrosis factor</n>- alpha gene transcription. The <n>tumor necrosis factor-alpha</n> (<n>TNF</n> alpha) gene is an immediate early gene in activated T cells, in that it is rapidly induced without a requirement for protein synthesis. Maximal induction of <n>TNF</n> alpha mRNA can be induced by treatment of T cells with calcium ionophores alone, via a <n>calcineurin</n>-dependent process that is blocked by cyclosporin A. We have previously identified a promoter element, kappa 3, that is required for calcium-stimulated, cyclosporin A-sensitive induction of the <n>TNF</n> alpha gene in activated T cells. Here, we demonstrate that the <n>kappa 3 binding factor</n> contains <n>NFATp</n>, a cyclosporin-sensitive DNA- binding protein required for interleukin-2 gene transcription. <n>NFATp</n> binds to two sites within the kappa 3 element, and occupancy of both sites is required for <n>TNF</n> alpha gene induction. Thus, although the kappa 3 element has little sequence similarity to other <n>NFATp</n>-binding sites, it appears to function as a cyclosporin-sensitive promoter element in T cells by virtue of its ability to bind <n>NFATp</n>. The involvement of <n>NFATp</n> in transcriptional activation of both the <n>interleukin-2</n> and TNF alpha genes suggests that this factor plays an important role in the coordinate induction of multiple cytokine genes, starting at the earliest stages of T cell activation.
95197720>Regulation and specificity of <n>MNDA</n> expression in monocytes, macrophages, and leukemia/B lymphoma cell lines. The expression of the <n>human myeloid cell nuclear differentiation antigen</n> (<n>MNDA</n>) was observed specifically in cells of the granulocyte- macrophage lineage in our earlier reports. The specificity of <n>MNDA</n> expression for cells in the granulocyte-macrophage lineage was reexamined in cell lines established from patients with Philadelphia chromosome-positive chronic myeloid leukemia. Cell lines that expressed <n>MNDA</n> exhibited myeloid cell features and granulocyte or monocyte differentiation could be induced in vitro, while cell lines exhibiting properties of very early stage cells or multipotential cells did not express <n>MNDA</n>. Cells originating from cases of Burkitt's lymphoma were negative. By contrast, three lymphoblastoid cell lines (immortalized in vitro with Epstein-Barr virus) were weakly positive and <n>MNDA</n> was up- regulated by <n>interferon-alpha</n> (<n>IFN-alpha</n>) treatment. As we reported previously, <n>MNDA</n> mRNA level in adherent monocytes is elevated by <n>IFN- alpha</n>; in this study, we further assessed <n>MNDA</n> expression in in vitro monocyte-derived macrophages. Three additional agents (endotoxin, <n>phytohemagglutinin</n>, and phorbol ester) and other conditions that affect function, cytokine production, differentiation, and/or growth of monocytes were examined for their ability to alter <n>MNDA</n> expression. The results varied with the agent, cell type, and stage of differentiation. Changes in <n>MNDA</n> expression occurred slowly (hours to days), suggesting that <n>MNDA</n> could mediate changes realized over a long period. The results also reveal a discordance in certain <n>MNDA</n> positive cells between steady-state levels or changes in levels of protein and mRNA indicating that the regulation of <n>MNDA</n> expression occurs at more than one point. Changes in <n>MNDA</n> expression are consistent with a role in opposing macrophage differentiation and activation of monocytes/macrophages.
95053891>DNA-binding studies of the <n>Epstein-Barr virus nuclear antigen 2</n> (<n>EBNA- 2</n>): evidence for complex formation by latent membrane protein gene promoter-binding proteins in <n>EBNA-2</n>-positive cell lines. The <n>Epstein-Barr virus (EBV) nuclear antigen 2</n> (<n>EBNA-2</n>) protein is essential for the immortalization of human primary B cells by EBV. <n>EBNA- 2</n> trans-activates cellular and viral genes like CD23, c-fgr, <n>latent membrane protein 1</n> (<n>LMP1</n>) and <n>terminal protein 1</n> (<n>TP1</n>). Trans- activation of the TP1 promoter and of the BamHI C promoter has already been investigated in detail and appears to be mediated via protein- protein interactions and not by direct binding of <n>EBNA-2</n> type A (of EBV type 1) to the DNA. <n>EBNA-2</n> is able to trans-activate the expression of the LMP gene in several cell lines. Various reports have delineated the cis-acting elements of the LMP promoter through which <n>EBNA-2</n> mediates trans-activation. To determine whether <n>EBNA-2</n> also trans-activates the LMP promoter by protein-protein interactions, we performed a series of gel retardation assays and competition experiments with LMP promoter fragments of different sizes. We determined that the protein-binding region on the LMP promoter was within a 42 bp fragment encompassing nucleotides -135 to -176 relative to the LMP transcriptional start site. None of the DNA fragments investigated indicated interaction of <n>EBNA-2</n> with the DNA via protein-protein interactions. No significant differences between <n>EBNA-2</n>-positive and <n>EBNA-2</n>-negative nuclear extracts could be seen in the gel retardation assay under conditions that clearly showed binding of <n>EBNA-2A</n> to the TP1 promoter. However, analysis of sucrose gradient fractions in the gel retardation assay provided evidence that the LMP promoter-binding proteins form a complex of higher M(r) in <n>EBNA-2</n>-positive cell extracts. These complexes were destroyed by detergent. We deduce from these results that <n>EBNA-2</n>- positive cells might indeed contain specific complexes bound to the LMP promoter which are, however, too labile to be detected in a standard gel retardation assay.
95015835>Simultaneous activation of Ig and <n>Oct-2</n> synthesis and reduction of surface MHC class II expression by <n>IL-6</n>. Terminal differentiation of B cells to plasma cells in vivo is characterized by secretion of Ig and extinction of MHC class II expression on the cell surface. We show that <n>IL-6</n> signaling leads to marked increases in the synthesis and secretion of Ig in clonal human B cell lines and newly isolated polyclonal B lymphocytes in vitro. The IL- 6-induced cells resemble plasma cells in ultrastructure and in reduced expression of surface MHC class II. Enhanced Ig synthesis is a result of coordinated transcriptional activation of Ig genes without promoter or isotype specificity, and differential accumulation of the mRNA encoding the secreted form of Ig heavy chain. It is saturable and subject to negative control when <n>IL-6</n> stimulation is prolonged. Coordinate with temporal changes in Ig synthesis, the DNA-binding activity and the synthesis of the B cell-enriched transcription factor <n>Oct-2</n> are regulated. Thus, differentiation of B cells with <n>IL-6</n> in vitro recapitulates the hallmarks of terminal B differentiation in vivo; <n>Oct-2</n> may have a role in this process.
95127210>T-cell functional regions of the human IL-3 proximal promoter. The human interleukin-3 (<n>IL-3</n>) gene is expressed almost exclusively in activated T cells. Its expression is regulated at both the transcriptional and post-transcriptional level. We have previously shown that treatment of Jurkat T cells with <n>phytohemaglutinin</n> (<n>PHA</n>) and the phorbol ester, PMA, activated transcription initiation from the IL- 3 gene. To define the regions of the gene required for transcription activation, we generated a series of reporter constructs containing different regions of the IL-3 gene 5' and 3' flanking sequences. Both positive and negative regulatory elements were identified in the proximal 5' flanking region of the IL-3 gene. The promoter region between -173 and -60 contained the strongest activating elements. The transcription factor <n>AP-1</n> could bind to this positive activator region of the promoter. We also examined the function of the IL-3 CK-1/CK-2 elements that are present in many cytokine genes and found that they acted as a repressor of basal level expression when cloned upstream of a heterologous promoter but were also inducible by PMA/<n>PHA</n>
95166251>Expression of the Runt domain-encoding PEBP2 alpha genes in T cells during thymic development. The PEBP2 alpha A and PEBP2 alpha B genes encode the DNA-binding subunit of a murine transcription factor, <n>PEBP2</n>, which is implicated as a T-cell-specific transcriptional regulator. These two related genes share the evolutionarily conserved region encoding the Runt domain. <n>PEBP2</n> alpha B is the murine counterpart of <n>human AML1</n>, which is located at the breakpoints of the 8;21 and 3;21 chromosome translocations associated with acute myeloid leukemia. Northern (RNA) blots of various adult mouse tissues revealed that the levels of expression of both genes were most prominent in the thymus. Furthermore, transcripts of <n>PEBP2</n> alpha A and mouse AML1/<n>PEBP2</n> alpha B were detected in T lymphocytes in the thymuses from day 16 embryos and newborns, as well as 4-week-old adult mice, by in situ hybridization. The expression of the genes persisted in peripheral lymph nodes of adult mice. The transcripts were detected in all the CD4- CD8-, CD4+ CD8+, CD4+ CD8-, and CD4- CD8+ cell populations. The results indicated that both genes are expressed in T cells throughout their development, supporting the notion that <n>PEBP2</n> is a T-cell-specific transcription factor. Transcripts of mouse AML1/<n>PEBP2</n> alpha B were also detected in day 12 fetal hematopoietic liver and in the bone marrow cells of newborn mice. The implication of mouse AML1/<n>PEBP2</n> alpha B expression in hematopoietic cells other than those of T-cell lineage is discussed in relation to myeloid leukemogenesis.
95168272>Effects of glucocorticoids on transcription factor activation in human peripheral blood mononuclear cells. Glucocorticoids have an inhibitory effect on inflammatory and immune responses, and this may be through the modulation of transcription factor binding to DNA. The interaction of the transcription factors, <n>activator protein-1</n> (<n>AP-1</n>), <n>nuclear factor kappa B</n> (<n>NF kappa B</n>), and <n>cAMP-responsive element binding protein</n> (<n>CREB</n>) with DNA and glucocorticoid receptors (GR) was analyzed in human peripheral blood mononuclear cells by gel mobility shift assays. TNF-alpha, IL-1 beta and phorbol myristate acetate (PMA) treatment increased <n>AP-1</n> and NF kappa B DNA binding by up to 200% but decreased <n>CREB</n> binding (38%) over a 60-min time course. Dexamethasone produced a rapid and sustained increase in glucocorticoid response element binding and a concomitant 40-50% decrease in AP-1, NF kappa B, and CREB DNA binding that was blocked by combined dexamethasone and cytokine or PMA treatment. These latter effects were due to increases in the nuclear localization of GR, not to reduced amounts of the other transcription factors. This suggests that in these cells GR within the nucleus interacts with cytokine-stimulated transcription factors by the process of cross coupling. This may be an important molecular site of steroid action.
95263289>Differential induction of the NF-AT complex during restimulation and the induction of T-cell anergy. Stimulation of human CD4+ T-cell clones through the T-cell receptor (TcR) by high doses of specific peptide results in the induction of a long-lived state of nonresponsiveness that has been called anergy. During the induction of anergy, T cells are phenotypically similar to cells responding to an immunogenic stimulus. The amount of TcR at the cell surface is downmodulated, whereas the CD2 and CD25 receptors are increased. When restimulated, however, anergic T cells fail to up- regulate transcription of the IL-2 gene and in consequence do not produce <n>IL-2</n>. In this study, we have compared the ability of various transcription factors to bind to their appropriate site on DNA. Factors were isolated from the nuclei of T cells that were in the induction phase of anergy or were undergoing activation. The pattern of binding activity in restimulated T cells is consistent with the pattern that has previously been shown to regulate T-cell-specific expression of the <n>IL-2</n> and the beta chain of the TcR genes. The measured binding to a TCF- 1 site is the same in the nuclei of resting, activated, and anergized cells. The inducible factors <n>NK-kappa B</n>, beta E2, <n>CD28RC</n>, and <n>AP-1</n> are not expressed in resting cells and are twofold lower in anergized as compared with activated cells. In contrast, anergic T cells express approximately eightfold lower amounts of NF-AT, a member of the class of inducible factors that regulates <n>IL-2</n> gene transcription. (ABSTRACT TRUNCATED AT 250 WORDS)
96126094>The number of glucocorticoid receptors in peripheral human lymphocytes is elevated by a zinc containing trace element preparation. A trace element preparation (Beres Drops Plus, BDP) elevates the number of glucocorticoid receptors (gcR) in peripheral lymphocytes isolated both from healthy blood donors and rheumatoid arthritis patients. This enhancement by BDP was found either for constitutive expression of gcRs or in experiments when the lymphocytes were stimulated by <n>interleukin (IL)-6</n>. There was no significant effect of BDP on IL-1 and <n>tumour necrosis factor alpha</n> (<n>TNF alpha</n>)-induced changes of gcRs. The effect of BDP was greatly dependent on the presence of Zn++ ions in the preparation, since the augmenting effect was abolished if BDP did not contain zinc.
96063336>The DNA and steroid binding domains of the glucocorticoid receptor are not altered in mononuclear cells of treated CLL patients. The aim of this study was to investigate whether mutations in the glucocorticoid receptor could account for the increasing unresponsiveness of patients with chronic lymphatic leukemia (CLL) to combination chemotherapy. The receptor was tested immunocytochemically, in steroid binding assays, and by a mutation screening (denaturing gradient gel electrophoresis) of the receptor-cDNA. The receptor concentration, as measured by staining and steroid binding test, varied considerably but showed no clear correlation to clinical response. Using a highly sensitive mutation screening assay of the DNA- and the steroid-binding region, none of the treated patients revealed any mutation, suggesting that the glucocorticoid receptor in the CLL patients tested is not altered in these domains. In one individual who had not been treated before analysis a silent mutation was found in one receptor allele. The results suggest that mechanisms other than altered ligand or DNA binding of the receptor may be responsible for the lack of response to chemotherapy. This conclusion is discussed in relation to the mechanism of corticoid resistance in mouse and human lymphoma cells in culture.
95116336>An AP1 binding site upstream of the kappa immunoglobulin intron enhancer binds inducible factors and contributes to expression. Expression of the kappa immunoglobulin light chain gene requires developmental- and tissue-specific regulation by trans-acting factors which interact with two distinct enhancer elements. A new protein-DNA interaction has been identified upstream of the intron enhancer, within the matrix-associated region of the J-C intron. The binding activity is greatly inducible in pre-B cells by bacterial lipopolysaccharide and <n>interleukin-1</n> but specific complexes are found at all stages of B cell development tested. The footprinted binding site is homologous to the consensus AP1 motif. The protein components of this complex are specifically competed by an AP1 consensus motif and were shown by supershift to include <n>c-Jun</n> and <n>c-Fos</n>, suggesting that this binding site is an AP1 motif and that the Jun and Fos families of transcription factors play a role in the regulation of the kappa light chain gene. Mutation of the AP1 motif in the context of the intron enhancer was shown to decrease enhancer-mediated activation of the promoter in both pre-B cells induced with LPS and constitutive expression in mature B cells.
95036290>Distinct DNase-I hypersensitive sites are associated with <n>TAL-1</n> transcription in erythroid and T-cell lines. The tal-1 gene, frequently activated in human T-cell acute lymphoblastic leukemia (T-ALL), is expressed in the erythroid, megakaryocytic, and mast cell lineages during normal hematopoiesis. To gain further insight into the molecular mechanisms that control tal-1 expression, we investigated tal-1 chromatin structure in erythroid/megakaryocytic cell lines and in T-cell lines either with or without tal-1 rearrangements. Tal-1 transcription was shown to be monoallelic in Jurkat, a T-cell line that expresses tal-1 in the absence of apparent genomic alteration of the locus. Methylation studies indicated that the tal-15' GC-rich region behaves like a CpG island, hypomethylated in normal cells, and methylated de novo on transcriptionally inactive alleles in established cell lines. Five major DNase-I hypersensitive sites (HS) were mapped in the tal-1 locus. HS I, IV, and V were exclusively observed in the erythroid/megakaryocytic cell lines that express tal-1 from the promoters 1a and 1b. HS II was weak in hematopoietic cell lines, absent in Hela, and greatly enhanced in Jurkat, suggesting that this region might be implicated in the cis-activation of tal-1 promoter 1b in this cell line. HS III was weak in HEL and Jurkat, and greatly enhanced in DU528, a T-cell line that bears a t (1;14) and initiates tal-1 transcription within exon 4. These results suggest that distinct regulatory elements are associated with the use of the different tal-1 promoters.
95050523><n>Erythropoietin</n>-dependent induction of hemoglobin synthesis in a cytokine-dependent cell line M-TAT. M-TAT is a cytokine-dependent cell line with the potential to differentiate along the erythroid and megakaryocytic lineages. We cultured M-TAT cells long term (&gt; 1 year) in the continuous presence of <n>erythropoietin</n> (<n>EPO</n>), <n>granulocyte-macrophage colony-stimulating factor</n> (<n>GM-CSF</n>), or <n>stem cell factor</n> (<n>SCF</n>). These long term cultures are referred to as M-TAT/EPO, M-TAT/GM-CSF, and M-TAT/SCF cells, respectively. <n>Hemoglobin</n> concentration and gamma-globin and <n>erythroid delta-aminolevulinate synthase</n> mRNA levels were significantly higher in M-TAT/EPO cells than in M-TAT/GM-CSF cells. When the supplemented cytokine was switched from <n>GM-CSF</n> to <n>EPO</n>, hemoglobin synthesis in M- TAT/GM-CSF cells increased rapidly (within 5 h), and the level of GATA- 1 mRNA increased. In contrast, the addition of <n>GM-CSF</n> to the M-TAT/EPO cell culture decreased the amount of hemoglobin, even in the presence of <n>EPO</n>, indicating that the <n>EPO</n> signal for erythroid differentiation is suppressed by <n>GM-CSF</n>. Thus, erythroid development of M-TAT cells is promoted by <n>EPO</n> and suppressed by <n>GM-CSF</n>. These results support the hypothesis that <n>EPO</n> actively influences the programming of gene expression required for erythroid progenitor cell differentiation.
95014458>The role of <n>cellular transcription factor E2F</n> in the regulation of cdc2 mRNA expression and cell cycle control of human hematopoietic cells. cdc2 mRNA transcripts were detected in immature bone marrow cells and became undetectable along with differentiation. Peripheral blood resting cells did not express cdc2 mRNA, but it was induced in T- lymphocytes when the cells reentered the cell cycle in response to specific mitogens. In contrast, cdc2 mRNA could not be induced in granulocytes and monocytes even after the culture with the appropriate stimulants. In order to investigate the mechanism of the regulation of cdc2 mRNA expression in hematopoietic cells, we isolated the 5'- flanking sequence of the cdc2 gene and found the putative E2F binding site at the position of nucleotides -124 to -117. The binding of <n>E2F</n> at this region was detected by a gel-retardation assay and <n>DNaseI</n> footprinting in <n>phytohemagglutinin</n>-stimulated T-lymphocytes, which was coincident with the expression of cdc2 mRNA. <n>E2F</n> binding was not observed in both granulocytes and monocytes. Transient <n>chloramphenicol acetyltransferase</n> assay revealed that the region containing <n>E2F</n> binding site had a strong promoter activity, and introduction of the mutation at the <n>E2F</n> binding site resulted in a significant loss of the activity. E2F-1 and DP-1 mRNAs were not detectable in granulocytes, monocytes and resting T-lymphocytes but were induced after the mitogenic stimulation of T-lymphocytes. The induction of <n>E2F</n> activity preceded the appearance of cdc2 mRNA, which is consistent with the role of <n>E2F</n> in the regulation of cdc2 mRNA expression. These results suggest that cdc2 mRNA expression is related to the cell cycling of normal human hematopoietic cells and that <n>E2F</n> plays some roles in the regulation of its expression.
95151358>Association of alterations in NF-kappa B moieties with HIV type 1 proviral latency in certain monocytic cells. Human immunodeficiency virus type 1 (HIV-1) replication is controlled by a complex array of virally encoded and cellular proteins. A wide spectrum of levels of HIV-1 expression have been demonstrated in various cells, both in cell culture and in vivo. Molecular mechanisms leading to restricted HIV-1 replication may differ between certain cell types. It is now demonstrated that HIV-1 proviral latency in the monocytic cell line U1, in which only extremely low levels of HIV-1 expression are detected in the baseline unstimulated state, is associated with alterations in nuclear factor-kappa B (<n>NF-kappa B</n>) moieties demonstrated in these cells by electrophoretic mobility shift assays (EMSAs) and in situ UV cross-linking studies. A predominance of p50 NF-kappa B moieties and possibly p50 homodimers or closely related species, rather than the p50-p56 heterodimer of <n>NF-kappa B</n> that is the predominant <n>NF-kappa B</n> species in most T lymphocytic and monocytic cells, is demonstrated in the nuclei of U1 cells. This pattern of NF- kappa B-related moieties differs from the latently infected T lymphocytic cell line ACH-2, and from the U937 monocytic line, the parental cell line of the U1 cellular clone. As such, these data suggest that different proximal mechanisms may lead to restricted HIV-1 replication
95199654>[Regulation of transcription of the <n>interleukin-2</n> gene in B-lymphocytes] Since most B cell clones immortalized with EBV virus can be induced to produce <n>interleukin-2</n>, a typical <n>T cell cytokine</n>, we studied the role of different elements of the IL-2 promoter in such clones by transfection. It was found, in particular, that the element TCEd, which binds the transcription factor <n>NF-kB</n>, is very active in all three B clones tested. This element has no activity in T cells of the Jurkat line. The NFATd element, which binds the transcription factor <n>NFAT-1</n> and is very active in T cells, is only weakly active in one B clone and not at all in another. Different elements thus contribute to IL-2 promoter activity in different cells.
95156575>Regulation of <n>I kappa B alpha</n> and <n>p105</n> in monocytes and macrophages persistently infected with human immunodeficiency virus. The mechanisms regulating human immunodeficiency virus (HIV) persistence in human monocytes/macrophages are partially understood. Persistent HIV infection of U937 monocytic cells results in <n>NF-kappa B</n> activation. Whether virus-induced <n>NF-kappa B</n> activation is a mechanism that favors continuous viral replication in macrophages remains unknown. To further delineate the molecular mechanisms involved in the activation of <n>NF-kappa B</n> in HIV-infected monocytes and macrophages, we have focused on the regulation of the I kappa B molecules. First, we show that persistent HIV infection results in the activation of NF- kappa B not only in monocytic cells but also in macrophages. In HIV- infected cells, <n>I kappa B alpha</n> protein levels are decreased secondary to enhanced protein degradation. This parallels the increased <n>I kappa B alpha</n> synthesis secondary to increased <n>I kappa B alpha</n> gene transcription, i.e., increased RNA and transcriptional activity of its promoter-enhancer. Another protein with I kappa B function, <n>p105</n>, is also modified in HIV-infected cells: <n>p105</n> and <n>p50</n> steady-state protein levels are increased as a result of increased synthesis and proteolytic processing of <n>p105</n>. Transcriptional activity of <n>p105</n> is also increased in infected cells and is also mediated by <n>NF-kappa B</n> through a specific kappa B motif. These results demonstrate the existence of a triple autoregulatory loop in monocytes and macrophages involving HIV, <n>p105</n> and <n>p50</n>, and <n>MAD3</n>, with the end result of persistent NF-kappa B activation and viral persistence. Furthermore, persistent HIV infection of monocytes and macrophages provides a useful model with which to study concomitant modifications of different I kappa B molecules.
95163578>Glucocorticoid-induced apoptosis of human leukemic cells is caused by the repressive function of the glucocorticoid receptor. Induction of apoptosis in lymphocytes, which may account for the therapeutic effects of glucocorticoids in various diseases including leukemia, depends on the glucocorticoid receptor. However, the events leading from the activated receptor to cell lysis are not understood. A prevailing hypothesis postulates induction of so-called 'lysis genes' by the activated receptor. In this study, we show that an <n>activation- deficient glucocorticoid receptor mutant</n> is as effective as the wild- type receptor in repression of <n>AP-1</n> activity, inhibition of <n>interleukin- 2</n> production, inhibition of c-myc expression and induction of apoptosis. Furthermore, we show that retinoic acid can also induce apoptosis in these cells through the retinoic acid receptor, whose repressive functions but not target site specificity, are similar to those of the glucocorticoid receptor. Therefore, the primary effect of the receptor in glucocorticoid-mediated apoptosis correlates with transcriptional repression rather than activation and could be mediated by interference with other transcription factors required for cell survival.
95260531>HIV type 1 protease activation of <n>NF-kappa B</n> within T lymphoid cells. <n>NF-kappa B</n> is a nuclear protein of the rel oncogene family capable of enhancing transcription of several cellular genes, including <n>IL-2</n> and the <n>IL-2</n> receptor, and viral genes transcribed from the HIV-1 LTR. It has been reported that <n>HIV-1 protease</n> may cleave the <n>NF-kappa B</n> precursor to its active form in vitro. In this study the effects of HIV protease on <n>NF-kappa B</n> precursor activation were examined in Jurkat T cells by introducing a protease expression vector into the cells. Increased <n>NF-kappa B</n> activity was observed and this increased activity was blocked by a specific inhibitor of the <n>viral protease</n>. Viral transcription, as measured using LTR-<n>CAT</n> assays, was only slightly enhanced in the HIV-protease expressing cells, while secretion of <n>IL-2</n> and expression of the <n>IL-2</n> receptor were not affected. The limited activation of <n>NF-kappa B</n> by <n>HIV protease</n> appears unlikely to have a significant effect on virus expression or T cell function.
95074854>Inhibition of human immunodeficiency virus type 1 replication by a Tat- activated, transduced interferon gene: targeted expression to human immunodeficiency virus type 1-infected cells. We have examined the feasibility of using interferon (IFN) gene transfer as a novel approach to anti-human immunodeficiency virus type 1 (HIV-1) therapy in this study. To limit expression of a transduced HIV-1 long terminal repeat (LTR)-IFNA2 (the new approved nomenclature for IFN genes is used throughout this article) hybrid gene to the HIV-1- infected cells, HIV-1 LTR was modified. Deletion of the <n>NF-kappa B</n> elements of the HIV-1 LTR significantly inhibited <n>Tat</n>-mediated transactivation in T-cell lines, as well as in a monocyte line, U937. Replacement of the <n>NF-kappa B</n> elements in the HIV-1 LTR by a DNA fragment derived from the 5'-flanking region of IFN-stimulated gene 15 (ISG15), containing the IFN-stimulated response element, partially restored <n>Tat</n>-mediated activation of LTR in T cells as well as in monocytes. Insertion of this chimeric promoter (ISG15 LTR) upstream of the human IFNA2 gene directed high levels of IFN synthesis in Tat- expressing cells, while this promoter was not responsive to <n>tumor necrosis factor alpha</n>-mediated activation. ISG15-LTR-IFN hybrid gene inserted into the retrovirus vector was transduced into Jurkat and U937 cells. Selected transfected clones produced low levels of <n>IFN A</n> (<n>IFNA</n>) constitutively, and their abilities to express <n>interleukin-2</n> and <n>interleukin-2</n> receptor upon stimulation with <n>phytohemagglutinin</n> and phorbol myristate acetate were retained. Enhancement of <n>IFNA</n> synthesis observed upon HIV-1 infection resulted in significant inhibition of HIV- 1 replication for a period of at least 30 days. Virus isolated from <n>IFNA</n>-producing cells was able to replicate in the U937 cells but did not replicate efficiently in U937 cells transduced with the <n>IFNA</n> gene. These results suggest that targeting IFN synthesis to HIV-1-infected cells is an attainable goal and that autocrine IFN synthesis results in a long-lasting and permanent suppression of HIV-1 replication.
96000378>Chromosomal localization of two KOX zinc finger genes on chromosome bands 7q21-q22. Human cDNAs encoding Kruppel-type zinc finger domains, designated KOX 1- 32, have been cloned from human T lymphocyte cell line libraries. We report here the regional localizations by in situ hybridization of KOX 18 and KOX 25 on chromosome bands 7q21q22. Pulse-field gel electrophoresis (PFGE) analysis showed that these genes are physically located within a DNA fragment of 250 kb. The genes KOX 4 and KOX 9, mapped on chromosome 8q24, were found to be located within a DNA fragment of 450 kb. From the present and previous data, eighteen different KOX genes have been located at least two by two within nine DNA fragments of 200 to 580 kb.
95088506>Influence of age on the production of <n>Fos</n> and <n>Jun</n> by influenza virus- exposed T cells. This study investigated age-related T cell responses after in vitro exposure to influenza A virus. Mononuclear leukocytes from young or elderly persons were sham-exposed or exposed to influenza virus for 1, 24, and 72 h. Immunofluorescent staining and flow cytometric analysis were then used to detect T cells producing the transcriptional regulating proteins <n>Fos</n> and <n>Jun</n>. Fewer virus-exposed cells from elderly donors stained for <n>Fos</n> and <n>Jun</n> at each data point compared with cells from young donors. Flow cytometric analysis also showed that at 72 h of virus exposure fewer T cells from the elderly produced <n>interferon-gamma</n> (<n>IFN-gamma</n>), suggesting a link between the magnitude of the <n>Fos</n> and <n>Jun</n> and <n>IFN-gamma</n> responses. Thus, failure of virus-exposed T cells to produce <n>Fos</n> and <n>Jun</n> could contribute to the increase in illness due to influenza virus in the elderly.
95194613>The regulation of HIV by retinoic acid correlates with cellular expression of the retinoic acid receptors. OBJECTIVES: To analyze the effect of retinoic acids (RA) on HIV-1 expression and correlate this effect with expression levels of RA receptors (RARs) in T-lymphoid and monocytoid cell lines. DESIGN AND METHODS: The effect of all-trans and 9-cis RA on HIV-1 production in T- lymphoid (H9, CEM) and monocytoid (U937,THP-1) cell lines was measured during acute and chronic infection. The expression levels of <n>human RAR alpha</n> (<n>hRAR alpha, receptor</n> for all-trans RA) and the <n>human retinoid-X receptor alpha</n> (<n>hRXR alpha receptor</n> for 9-cis RA) were determined by Northern blot analysis. RESULTS: Both all-trans and 9-cis RA inhibited virus replication in HIV-1 IIIB-infected monocytoid cells, in the presence and absence of the co-stimulatory agent phorbol myristate acetate (PMA). The retinoids had weak or no stimulatory effects on HIV production by T-cell lines. HIV production by PMA-stimulated T-cell lines was inhibited by these retinoids. The 9-cis RA was generally more effective than all-trans RA in inhibiting HIV production and in combination generally more effective than the single agents alone. <n>Human RAR alpha</n> was expressed in H9, U937 and THP-1 cells, but almost undetectable in CEM cells. <n>Human RXR alpha</n> was significantly expressed in U937 and THP-1 cells, weakly expressed in H9 cells and not detectable in CEM cells. After stimulation by PMA, <n>RXR alpha</n> expression increased in H9 and U937 cells but not in CEM cells. Human RAR alpha expression was unchanged in H9 and CEM cells, and elevated in U937 cells, after PMA stimulation. CONCLUSION: The effect of RA on HIV-1 expression was cell-type-dependent and partially correlated with cellular expression of RARs. Endogenous or exogenously administered RA may have a significant role in HIV regulation.
95047083>Functions of glutathione and glutathione disulfide in immunology and immunopathology. Even a moderate increase in the cellular cysteine supply elevates the intracellular glutathione (GSH) and glutathione disulfide (GSSG) levels and potentiates immunological functions of lymphocytes in vitro. At low GSSG levels, T cells cannot optimally activate the immunologically important transcription factor <n>NF kappa B</n>, whereas high GSSG levels inhibit the DNA binding activity of <n>NF kappa B</n>. The effects of GSSG are antagonized by reduced <n>thioredoxin</n> (<n>TRX</n>). As the protein tyrosine kinase activities <n>p56lck</n> and <n>p59fyn</n> are activated in intact cells by hydrogen peroxide, they are likely targets for GSSG action. These redox- regulated enzymes trigger signal cascades for <n>NF kappa B</n> activation and transduce signals from the T cell antigen receptor, from <n>CD4</n> and <n>CD8 molecules</n>, and from the <n>IL-2</n> receptor beta-chain. The effector phase of cytotoxic T cell responses and <n>IL-2</n>-dependent functions are inhibited even by a partial depletion of the intracellular GSH pool. As signal transduction is facilitated by prooxidant conditions, we propose that the well-known immunological consequences of GSH depletion ultimately may be results of the accompanying GSSG deficiency. As HIV-infected patients and SIV-infected rhesus macaques have, on the average, significantly decreased plasma cyst(e)ine and intracellular GSH levels, we also hypothesize that AIDS may be the consequence of a GSSG deficiency as well.
95007565>T cells from renal cell carcinoma patients exhibit an abnormal pattern of kappa B-specific DNA-binding activity: a preliminary report. Recent data suggest that the poor induction of a T-cell response to human renal cell carcinoma (RCC) may be related to alterations in signal transduction pathways. We report that T cells from RCC patients have two alterations in kappa B motif-specific DNA-binding activity. The first alteration involves the constitutive expression of substantial kappa B-binding activity in nuclear extracts, which was observed in the electrophoretic mobility shift assay. The magnitude of kappa B activity in unstimulated patient T cells was similar to that observed in T cells from normal individuals that had been activated in vitro. On the basis of Western blotting experiments using antibodies to kappa B/Rel family proteins, the kappa B-binding activity constitutively expressed in T cells from RCC patients is composed mostly of the NF-kappa B1 (<n>p50</n>) subunit. The second abnormality in kappa B-binding activity in T cells from these patients is that <n>RelA</n>, a member of the Rel homology family which is part of the normal NF-kappa B complex, was not induced in the nucleus following activation. Western blotting analysis did not detect any <n>RelA</n> in nuclear extracts either before or after stimulation of T cells. The altered kappa B-binding activity in T cells from RCC patients
96091353><n>Hemoglobin</n> switching in humans is accompanied by changes in the ratio of the transcription factors, <n>GATA-1</n> and <n>SP1</n>. BACKGROUND: Understanding the mechanism of developmental regulation of <n>hemoglobin</n> switching has scientific as well as clinical relevance because of the influence of fetal <n>hemoglobin</n> (<n>HbF</n>) production in adulthood on the clinical manifestation of thalassemia and sickle cell anemia. We have previously found that the normal developmental patterns of globin gene expression are recapitulated in an experimental system of primary cultures that support differentiation of erythroid progenitors. We further found that high activities of the transcriptional activators, <n>GATA-1</n> and <n>SP1</n>, are associated with normal adult erythroid differentiation. MATERIALS AND METHODS: In the present work, we have studied, the activities of <n>GATA-1</n> and <n>SP1</n> during differentiation of cultured erythroid progenitors derived from cord blood and from fetal livers, as well as from beta zero-thalassemia patients. RESULTS: The results showed high <n>GATA-1</n> binding activity and very low <n>SP1</n> activity in the fetal liver cultures. This pattern was in contrast to cultures derived from normal adult peripheral blood, in which both <n>GATA-1</n> and <n>SP1</n> activities were high. Cord blood cultures showed an additive combination of "adult" and "fetal" patterns. The progenitors derived from a beta zero-thalassemia patient with high <n>HbF</n> production showed "fetal" pattern. On the other hand, in cultures of 2 beta zero-thalassemia patients without high <n>HbF</n>, "adult" pattern was observed. CONCLUSIONS: In the present work, we show that human fetal and adult erythroid progenitors are distinct in their transcription factors, and that the commitment to fetal or adult program occurs at a very early differentiation stage. Our studies also demonstrate that under anemic stress, recruitment of fetal progenitors may occur in adulthood.
95166259>Transcription-independent turnover of <n>I kappa B alpha</n> during monocyte adherence: implications for a translational component regulating <n>I kappa B alpha</n>/<n>MAD-3</n> mRNA levels. We identified <n>I kappa B alpha</n>/MAD-3 as an immediate-early gene in human monocytes that is expressed in response to a variety of signals, including adhesion, lipopolysaccharide, and phorbol myristate acetate. Within 5 min of monocyte adhesion, the level of the <n>I kappa B alpha</n> protein is markedly diminished but is rapidly replaced in a cycloheximide-sensitive manner within 20 min. Accompanying the rapid turnover of the <n>I kappa B alpha</n> protein is simultaneous translocation of NF-kappa B-related transcription factors to nuclei of adhered monocytes. The demonstration that <n>NF-kappa B</n> can regulate <n>I kappa B alpha</n>/MAD-3 gene transcription in other cell types suggested that the rapid increase in steady-state <n>I kappa B alpha</n>/MAD-3 mRNA levels we observed within 30 min of monocyte adherence would result from <n>NF-kappa B</n>-dependent transcriptional stimulation of the <n>I kappa B alpha</n>/MAD-3 gene. Nuclear run-on analyses indicated that, instead, while several immediate-early cytokine genes, such as the <n>interleukin 1 beta</n> (<n>IL-1 beta</n>) gene, were transcriptionally activated during monocyte adhesion, the rate of <n>I kappa B alpha</n>/MAD-3 gene transcription remained constant. The adherence-dependent increase in <n>I kappa B alpha</n>/MAD-3 mRNA levels was also not a consequence of mRNA stabilization events. Interestingly, while increases in both IL-1 beta and I kappa B alpha/MAD-3 mRNA levels were detected in nuclei of adherent monocytes, cytoplasmic levels of <n>IL- 1 beta</n> mRNA increased during adherence whereas those of <n>I kappa B alpha</n>/MAD-3 mRNA did not. Taken together, our data suggest that two interactive mechanisms regulate monocytic <n>I kappa B alpha</n>/MAD-3 mRNA levels. We propose that adherent monocytes regulate nuclear processing (or decay) of <n>I kappa B alpha</n>/MAD-3 mRNA, thereby increasing mRNA levels without stimulating <n>I kappa B alpha</n>/MAD-3 gene transcription. Moreover, since inhibition of protein synthesis leads to accumulation of <n>I kappa B alpha</n>/MAD-3 mRNA without stimulating <n>I kappa B alpha</n>/MAD-3 gene transcription, we suggest that low cytoplasmic levels of <n>I kappa B alpha</n>/MAD-3 mRNA are maintained by a translation-dependent degradation mechanism.
95124333>Distinct roles of the molecular <n>chaperone hsp90</n> in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains. The <n>intracellular dioxin receptor</n> mediates signal transduction by dioxin and functions as a ligand-activated transcription factor. It contains a basic helix-loop-helix (bHLH) motif contiguous with a Per- Arnt-Sim (PAS) homology region. In extracts from nonstimulated cells the receptor is recovered in an inducible cytoplasmic form associated with the <n>90-kDa heat shock protein</n> (<n>hsp90</n>), a molecular chaperone. We have reconstituted ligand-dependent activation of the receptor to a DNA- binding form by using the dioxin receptor and its <n>bHLH-PAS partner factor</n> <n>Arnt</n> expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with <n>Arnt</n>. In contrast, this receptor mutant showed low levels of xenobiotic response element-binding activity, indicating that the PAS domain may be important for DNA-binding affinity and/or specificity of the receptor. It was not possible to reconstitute dioxin receptor function with proteins expressed in wheat germ lysate. In line with these observations, reticulocyte lysate but not wheat germ lysate promoted the association of de novo synthesized dioxin receptor with <n>hsp90</n>. At least two distinct domains of the receptor mediated interaction with <n>hsp90</n>: the ligand-binding domain located within the PAS region and, surprisingly, the bHLH domain. Whereas ligand-binding activity correlated with association with <n>hsp90</n>, bHLH-<n>hsp90</n> interaction appeared to be important for DNA-binding activity but not for dimerization of the receptor. Several distinct roles for <n>hsp90</n> in modulating dioxin receptor function are therefore likely: correct folding of the ligand-binding domain, interference with <n>Arnt</n> heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system provides a complex and interesting model of the regulation of transcription factors by <n>hsp90</n>.
95236467>Induction of transcription factors in human T lymphocytes by aspirin- like drugs. Aspirin-like drugs (ALD) induce calcium mobilization, an essential component of T cell activation, but do not induce the biosynthesis of <n>IL-2</n>. To understand the extent to which ALD may mimic mitogenic stimulation, we studied cytoplasmic and nuclear signaling steps in ALD- treated T cells. We found that ALD induce a transient activation of protein kinase (<n>PKC</n>) but have no effect (in comparison to anti-CD3 antibodies) on protein tyrosine phosphorylation nor on PCL gamma 1 tyrosine phosphorylation. ALD-induced calcium mobilization and <n>PKC</n> activation are independent of tyrosine protein kinase activity as shown by the lack of effect of herbimycin, a tyrosine-protein kinase-specific inhibitor. Although we detected no <n>IL-2</n> mRNA in ALD-treated cells, the nuclei of these cells contain proteins capable of binding to three regulatory sequences in the <n>IL-2</n> promoter region: <n>NFAT</n>, <n>NF kappa B</n>, and <n>AP-1</n>. These binding activities are expressed only in activated T cells. The expression of <n>AP-1</n> depended on calcium mobilization and <n>PKC</n> activation. These data suggest that ALD cause transient but significant changes in T cell transmembrane signaling, although some events induced by stimulation with anti-CD3 antibodies are not induced by ALD. The signal is transmitted to the nucleus and induces DNA-binding activity by several transcription factors. However, the ALD stimulus is not capable of causing complete T cell activation.
95145550><n>Protein kinase C</n> is not a downstream effector of p21ras in activated T cells. The aim of this present study was to investigate the role of <n>protein kinase C</n> (<n>PKC</n>), downstream of p21ras, in activating interleukin-2 (IL- 2) gene expression. It has been reported that <n>PKC</n> is an effector of p21ras in T cells. Data is presented, using the potent and selective <n>PKC</n> inhibitor Ro 31-8425 and transient expression of a constitutively active ras mutant, which clearly shows that <n>PKC</n> is not downstream of p21ras in the induction of <n>NF-AT</n> and <n>AP-1</n> transcriptional activity and in the expression of <n>IL-2</n> in human Jurkat T cells. Reporter gene experiments demonstrated that <n>NF-kappa B</n> transcriptional activity is not affected by expression of activated p21ras. The signaling pathways involving <n>PKC</n> activation, calcium mobilization and <n>ras</n> activation combine to provide the necessary components for production of <n>IL-2</n> during T cell activation.
95110353>The C-terminus of the B cell activator <n>Oct-2</n> functions as an activation domain in yeast. <n>Oct-1</n> and <n>Oct-2</n> are human transcriptional activators that bind to the same DNA element but activate distinct sets of genes. We expressed these factors in S. cerevisiae and observed greater than 5-fold stimulation of a lacZ reporter gene only with <n>Oct-2</n>. Transfer of the <n>Oct-2</n> C-terminal domain onto either <n>Oct-1</n> (<n>Oct1.2</n>) or a nonactivating DNA-binding domain from <n>GAL4</n> created activators capable of greater than 15 and 10-fold stimulation of activity, respectively. Thus, the C- terminus of <n>Oct-2</n> is sufficient to confer activation potential to nonactive DNA-binding fragments in yeast.
95072469>Glucocorticoid-induced apoptosis of lymphoid cells. The induction of cell death in lymphoid cells by glucocorticoids is one of the earliest and most thoroughly studied models of apoptosis. Although the exact mechanism by which apoptosis occurs in lymphocytes is unknown many biochemical and molecular changes have been shown to occur in these cells in response to glucocorticoids. The role of chromatin degradation and endonucleases in the apoptotic process has been closely studied, as well as the involvement of several oncogenes in glucocorticoid-induced cell lysis. In addition, the clinical importance of glucocorticoid-induced apoptosis in the treatment of lymphoid neoplasms has recently received increased attention.
95104296><n>Interleukin-2</n> induces tyrosine phosphorylation and nuclear translocation of <n>stat3</n> in human T lymphocytes. An early biochemical event associated with T cell activation through the interleukin-2 receptor (IL-2R) is tyrosine phosphorylation of several intracellular substrates. The exact mechanism by which <n>IL-2</n> regulates transcription of different genes is presently unknown. Here, we report that stimulation through the IL-2R induced tyrosine phosphorylation and subsequent nuclear translocation of <n>stat3</n>, a newly identified member of the signal transducers and activators of transcription (STAT) family of proteins. In contrast, stat1 proteins were not tyrosine phosphorylated after <n>IL-2</n> ligation, whereas tyrosine- phosphorylated stat1 proteins (91 and 84 kDa proteins) were translocated to the nucleus following <n>interferon-gamma</n> treatment of HeLa cells. Apart from <n>stat3</n>, another cytoplasmic protein was tyrosine phosphorylated and subsequently translocated to the nucleus in response to <n>IL-2</n>. This protein had an apparent molecular mass of 84 kDa and was not recognized by <n>stat3</n> or stat1 mAb or antisera. Since <n>IL-2</n> induced nuclear translocation of the <n>84 kDa protein</n> and <n>stat3</n> followed identical kinetics, <n>p84</n> is a candidate for a new, yet undefined, member of the STAT family. Taken together, we report that <n>IL-2</n> induces tyrosine phosphorylation and subsequent nuclear translocation of <n>stat3</n> and an as yet undefined <n>84-kDa protein</n> in antigen-specific human T cell lines.
95021266>Human T-cell leukemia virus type I <n>Tax</n> activation of <n>NF-kappa B</n>/Rel involves phosphorylation and degradation of <n>I kappa B alpha</n> and <n>RelA</n> (p65)-mediated induction of the c-rel gene. The tax gene product of human T-cell leukemia virus type I (HTLV-I) is a potent transcriptional activator that both stimulates viral gene expression and activates an array of cellular genes involved in T-cell growth. Tax acts indirectly by inducing or modifying the action of various host transcription factors, including members of the <n>NF-kappa B</n>/Rel family of enhancer-binding proteins. In resting T cells, many of these <n>NF-kappa B</n>/Rel factors are sequestered in the cytoplasm by various ankyrin-rich inhibitory proteins, including <n>I kappa B alpha</n>. HTLV-I <n>Tax</n> expression leads to the constitutive nuclear expression of biologically active <n>NF-kappa B</n> and c-Rel complexes; however, the biochemical mechanism(s) underlying this response remains poorly understood. In this study, we demonstrate that <n>Tax</n>-stimulated nuclear expression of <n>NF-kappa B</n> in both HTLV-I-infected and <n>Tax</n>-transfected human T cells is associated with the phosphorylation and rapid proteolytic degradation of <n>I kappa B alpha</n>. In contrast to prior in vitro studies, at least a fraction of the phosphorylated form of <n>I kappa B alpha</n> remains physically associated with the <n>NF-kappa B</n> complex in vivo but is subject to rapid degradation, thereby promoting the nuclear translocation of the active <n>NF-kappa B</n> complex. We further demonstrate that <n>Tax</n> induction of nuclear <n>c-Rel</n> expression is activated by the RelA (p65) subunit of <n>NF-kappa B</n>, which activates transcription of the c-rel gene through an intrinsic kappa B enhancer element. In normal cells, the subsequent accumulation of nuclear <n>c-Rel</n> acts to inhibit its own continued production, indicating the presence of an autoregulatory loop. (ABSTRACT TRUNCATED AT 250 WORDS)
95009960>Activation of <n>NF-kappa B</n> in vivo is regulated by multiple phosphorylations. The activation of <n>nuclear factor kappa B</n> (<n>NF-kappa B</n>) in intact cells is mechanistically not well understood. Therefore we investigated the modifications imposed on <n>NF-kappa B</n>/I kappa B components following stimulation and show that the final step of <n>NF-kappa B</n> induction in vivo involves phosphorylation of several members of the <n>NF-kappa B</n>/I kappa B protein families. In HeLa cells as well as in B cells, TNF- alpha rapidly induced nuclear translocation primarily of p50-p65, but not of <n>c-rel</n>. Both <n>NF-kappa B</n> precursors and <n>I kappa B alpha</n> became strongly phosphorylated with the same kinetics. In addition to the inducible phosphorylation after stimulation, B lymphocytes containing constitutive nuclear <n>NF-kappa B</n> revealed constitutively phosphorylated p65 and <n>I kappa B alpha</n>. Phosphorylation was accompanied by induced processing of the precursors <n>p100</n> and <n>p105</n> and by degradation of <n>I kappa B alpha</n>. As an in vitro model we show that phosphorylation of <n>p105</n> impedes its ability to interact with <n>NF-kappa B</n>, as has been shown before for <n>I kappa B alpha</n>. Surprisingly, even p65, but not <n>c-rel</n>, was phosphorylated after induction in vivo, suggesting that <n>TNF-alpha</n> selectively activates only specific <n>NF-kappa B</n> heteromers and that modifications regulate not only <n>I kappa B</n> molecules but also <n>NF-kappa B</n> molecules. In fact, cellular <n>NF-kappa B</n> activity was phosphorylation- dependent and the DNA binding activity of p65-containing <n>NF-kappa B</n> was enhanced by phosphorylation in vitro. Furthermore, we found that the induction by hydrogen peroxide of <n>NF-kappa B</n> translocation to the nucleus, which is assumed to be triggered by reactive oxygen intermediates, also coincided with incorporation of phosphate into the same subunits that were modified after stimulation by <n>TNF-alpha</n>. Thus, phosphorylation appears to be a general mechanism for activation of <n>NF- kappa B</n>
95191179>Treatment of HL60 cells with various combinations of retinoids and 1 alpha,25 dihydroxyvitamin D3 results in differentiation towards neutrophils or monocytes or a failure to differentiate and apoptosis. It is well documented that treatment of serum-grown HL60 cells with 10(- 7) M all-trans retinoic acid (all-trans RA) induces neutrophil differentiation, whereas treatment with 10(-7) M 1 alpha,25 dihydroxyvitamin D3(D3) induces differentiation towards monocytes. In recent investigations, using serum-free grown HL60 cells, we observed that all-trans RA, at 10(-7) M, did not induce neutrophil differentiation and that all-trans RA, at 10(-8) M, reduced the D3 concentration required for monocyte differentiation to 5 x 10(-9) M. In this study, co-operative interactions between all-trans and 9-cis RA and D3 which promote neutrophil and monocyte differentiation of HL60 cells have been analysed in detail. Treatment of serum-free grown HL60 cells with 5 x 10(-7) M all-trans RA or 9-cis RA resulted in sub- optimal neutrophil differentiation (up to 25% mature cells). As shown for all-trans RA, 9-cis RA cooperated with D3 to promote monocyte differentiation. Culture of HL60 cells in 5 x 10(-7) M 9-cis RA together with a wide range of concentrations of D3 resulted in promotion of neutrophil differentiation at 10(-15)-10(-12) D3, a failure to differentiate and apoptosis at 10(-11)-10(-10) M D3, followed by co-operativity between 9-cis RA and 5 x 10(-9) M D3 in inducing monocyte differentiation in the absence of neutrophil differentiation. Similar results were obtained when HL60 cells were treated with 5 x 10(-7) all-trans RA together with a wide range of concentrations of D3. Cross titration analyses of the effects of 9-cis RA and D3 on HL60 cell differentiation were undertaken to determine the boundaries of the concentrations of each agent, alone and in combination, that give rise to optimal neutrophil and monocyte differentiation of HL60 cells. The observed cooperativities between either 9-cis RA or all-trans RA and D3 have important implications for the use of combinations of these agents in differentiation therapy.
95278601>Isolation of differentially expressed sequence tags from steroid- responsive cells using mRNA differential display. Transcriptional control of steroid-regulated gene networks by nuclear receptor proteins results in the coordinate expression of a limited number of target genes. Although much is known about the structure and function of steroid receptors, relatively few cell-specific steroid- regulated genes have been isolated and characterized. In this paper we describe results using mRNA differential display <n>reverse transcriptase</n> PCR (DDPCR) to identify and isolate short cDNA sequence tags from thymocyte and prostate cells under various hormone conditions. Using this technique we have isolated several differentially expressed sequence tags (DESTs) from the mouse thymocyte cell line WEHI 7.2. Two of these DESTs, GIG10 and GIG18, are rapidly induced by dexamethasone within 2 h of treatment. GIG10 is a novel sequence and GIG18 is the mouse homologue of a human expressed sequence tag isolated from activated B lymphocytes. We also used DDPCR to isolate DESTs from androgen-modulated rat ventral prostate tissue, one of which we characterized and found to correspond to the 3' end of <n>prostatic spermine binding protein</n> mRNA, a known androgen-regulated gene. Modifications of the original DDPCR protocol, which we found can potentially decrease the frequency of isolating false-positive DESTs, are described and the merits of DDPCR, relative to other differential cDNA cloning strategies, are discussed.
95123097>Activation of human thymocytes after infection by EBV. The discovery of EBV in certain T cell malignancies and the expression of the <n>EBV receptor</n>, <n>CR2/CD21</n>, on a population of immature thymocytes, T lymphoblastoid cell lines, and childhood acute T lymphoblastic leukemia cells suggested that EBV-receptor interactions on T cells may be of importance. We have shown that, within the thymus, a population of large, immature cells expresses <n>CD21</n>. EBV altered the activation responses of immature thymocytes in vitro. Triggering through <n>CD2</n> is mitogenic for mature, but not immature, T cells. However, during infection by EBV, ligation of <n>CD2</n> caused thymocytes to proliferate in the absence of exogenous cytokines. This function was a result of the interaction of EBV with its receptor, <n>CD21</n>, but was caused by infection rather than surface signaling, because neither specific mAb nor the P3HR-1 strain of virus mimicked the effect of B95-8. Immature thymocytes were infected by EBV, as determined by the internalization of the viral genome and its transcriptional activity. Consistent with the activity of <n>B95-8</n>, EBNA-2 transcripts were identified within infected thymocyte populations. In addition, components of the viral replicative pathway were expressed during infection of thymocytes. These components included transcription of BZLF-1, an early gene that characterizes EBV-infected B cells after disruption of latency. A second transcript was identified as encoding the recently characterized <n>RAZ</n>, which also is associated with replicative infection. The consequences of EBV infection of T cells at an early stage of differentiation may lead to failure of normal T cell repertoire development, autoimmunity, or malignancy.
95222176>Effects of intranasal glucocorticoids on endogenous glucocorticoid peripheral and central function. Glucocorticoids are among the most potent antiinflammatory agents that can be used in the treatment of rhinitis. Their mechanisms of action are multiple and complex and a number of reports describe significant systemic effects of locally administered glucocorticoids. In order to evaluate the short-term systemic effects of intranasally administered glucocorticoids, 14 normal healthy subjects were treated with two doses of either budesonide (BUD) or fluticasone propionate (FP) for 2 weeks. Before treatment, at regular intervals during the treatment, 1 week and finally 6 weeks after termination of treatment, the effects on glucocorticoid receptor (GR) and methallothionein (MTIIa) mRNA expression levels were examined in peripheral lymphocytes using a solution hybridization assay. Serum cortisol, osteocalcin and urinary cortisol levels were also determined. An insulin tolerance test (ITT) was performed at the end of the second week of treatment and at the end of the 6-week washout period with no statistically significant change in cortisol response. In peripheral lymphocytes, GR mRNA levels were significantly down-regulated. <n>MTIIa</n> mRNA levels increased significantly. Serum <n>osteocalcin</n> decreased significantly during treatment with both BUD and FP. Serum cortisol decreased after 1 week of treatment whereas urinary cortisol was not affected until the second week of treatment. In conclusion, intranasal glucocorticoids at clinically recommended doses have not only significant systemic effects on adrenal function, but also have an effect on specific gene expression in peripheral lymphocytes. These effects are receptor- dependent, reversible, and according to serum and urinary cortisol levels and ITT, leave the hypothalamic-pituitary-adrenal function intact. Finally, these short-term systemic effects were not associated with any of the noticeable side-effects usually observed during long- term treatment with glucocorticoids.
95145523>Biphasic control of <n>nuclear factor-kappa B</n> activation by the T cell receptor complex: role of <n>tumor necrosis factor alpha</n>. The regulation of <n>nuclear factor (NF)-kappa B</n> activation by the T cell receptor (TcR)/CD3 complex in primary human T cells has been studied at various times after activation. Only <n>p50 NF-kappa B protein</n> bound the kappa B element of <n>interleukin-2 receptor</n> (<n>IL-2R</n>) alpha chain promoter on resting T cells. However, immediately after TcR/CD3 cross-linking (after approximately 1 h; immediate) binding of p50.p65 heterodimers was observed. p50.c-rel heterodimers were also detected bound to this sequence at early time points (7-16 h; early), and both remained active at later time points (40 h; late) after activation. This regulation takes place mainly at the level of nuclear translocation of p65 and c- rel, at immediate and early time points. Activation also induced c-rel and p105/p50 mRNA synthesis, but not p65 mRNA whose expression was constitutive. Interestingly, all those early and late events, but not the immediate ones, were inhibited by a neutralizing anti-<n>tumor necrosis factor alpha</n> (TNF-alpha) monoclonal antibody. Similarly, cycloheximide prevented the p65 and c-rel translocation and consequent formation of active binding heterodimers, at early and late times. Cyclosporin A impaired not only early and late, but also immediate events; however, addition of <n>TNF-alpha</n> prevented all inhibition. These results indicate that the regulation of <n>NF-kappa B</n> activation during T cell activation by TcR/CD3 signals is biphasic: TcR/CD3 triggers its immediate translocation, which is transient if no <n>TNF-alpha</n> is present. <n>TNF-alpha</n>, therefore, emerges as the main factor responsible for a second phase of <n>NF-kappa B</n> regulation, controlling both translocation of p65 and c-rel, and new mRNA synthesis for c-rel and p105/p50.
95110287>The transcription factor <n>Sp1</n> is required for induction of the murine GM- CSF promoter in T cells. The cis-acting region, GM-kappa B/GC-box (positions -95 and -73), within the murine GM-CSF gene promoter is required for maximal induction by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in T cells. <n>GM-kappa B</n> defines a binding site for <n>NF-kappa B</n>, and GC-box defines a binding site for three (<n>A1</n>, <n>A2</n>, <n>B</n>) constitutive proteins. We report here that three copies of the GC-box can functionally compensate for the GM-kappa B/GC-box region, suggesting that the GC-motif can function independently of the GM-kappa B motif. The major GC-box binding activity <n>A1</n> was purified and identified as the transcription factor <n>Sp1</n>. We show that depletion of <n>Sp1</n> (<n>A1</n>) from nuclear extracts specifically decreases in vitro transcription activity on <n>GM-CSF</n> templates. Since the human GM-CSF promoter has a base difference within the GC-box, we speculate that this may explain why the human promoter is weak and that an upstream enhancer is required for the induction of the human GM-CSF gene.
95059073>A factor that regulates the class II major histocompatibility complex gene DPA is a member of a subfamily of zinc finger proteins that includes a Drosophila developmental control protein. A novel DNA sequence element termed the J element involved in the regulated expression of class II major histocompatibility complex genes was recently described. To study this element and its role in class II gene regulation further, a cDNA library was screened with oligonucleotide probes containing both the S element and the nearby J element of the human DPA gene. Several DNA clones were obtained by this procedure, one of which, clone 18, is reported and characterized here. It encodes a protein predicted to contain 688 amino acid residues, including 11 zinc finger motifs of the C2H2 type in the C-terminal region, that are Kruppel-like in the conservation of the H/C link sequence connecting them. The 160 N-terminal amino acids in the nonfinger region of <n>clone 18</n> are highly homologous with similar regions of several other human, mouse, and Drosophila sequences, defining a subfamily of Kruppel-like zinc finger proteins termed TAB (tramtrack [ttk]-associated box) here. One of the Drosophila sequences, ttk, is a developmental control gene, while a second does not contain a zinc finger region but encodes a structure important in oocyte development. An acidic activation domain is located between the N-terminal conserved region of <n>clone 18</n> and its zinc fingers. This protein appears to require both the S and J elements, which are separated by 10 bp for optimal binding. Antisense cDNA to <n>clone 18</n> inhibited the expression of a reporter construct containing the DPA promoter, indicating its functional importance in the expression of this class II gene.
95062301>Separation of oxidant-initiated and redox-regulated steps in the <n>NF- kappa B</n> signal transduction pathway. Studies presented here show that overall <n>NF-kappa B</n> signal transduction begins with a parallel series of stimuli-specific pathways through which cytokines (<n>tumor necrosis factor alpha</n>), oxidants (hydrogen peroxide and mitomycin C), and phorbol ester (phorbol 12-myristate 13- acetate) individually initiate signaling. These initial pathways culminate in a common pathway through which all of the stimulating agents ultimately signal <n>NF-kappa B</n> activation. We distinguish the stimuli-specific pathways by showing that the oxidative stimuli trigger <n>NF-kappa B</n> activation in only one of two human T-cell lines (Wurzburg but not Jurkat), whereas <n>tumor necrosis factor alpha</n> and phorbol 12- myristate 13-acetate readily stimulate in both lines. We propose the common pathway as the simplest way of accounting for the common requirements and properties of the signaling pathway. We include a redox-regulatory mechanism(s) in this common pathway to account for the previously demonstrated redox regulation of <n>NF-kappa B</n> activation in Jurkat cells (in which oxidants don't activate <n>NF-kappa B</n>); we put tyrosine phosphorylation in the common pathway by showing that kinase activity (inhibitable by herbimycin A and tyrphostin 47) is required for <n>NF-kappa B</n> activation by all stimuli tested in both cell lines. Since internal sites of oxidant production have been shown to play a key role in the cytokine-stimulated activation of <n>NF-kappa B</n>, and since tyrosine kinase and phosphatase activities are known to be altered by oxidants, these findings suggest that intracellular redox status controls <n>NF-kappa B</n> activation by regulating tyrosine phosphorylation event(s) within the common step of the <n>NF-kappa B</n> signal transduction pathway.
95178839>Deleted chromosome 20 from a patient with Alagille syndrome isolated in a cell hybrid through leucine transport selection: study of three candidate genes. Alagille syndrome (AGS) is a well-defined genetic entity assigned to the short arm of Chromosome (Chr) 20 by a series of observations of AGS patients associated with microdeletions in this region. By fusing lymphoblastoid cells of an AGS patient that exhibited a microdeletion in the short arm of Chr 20 encompassing bands p11.23 to p12.3 with rodent thermosensitive mutant cells (CHOtsH1-1) deficient <n>in-leucyl- tRNA synthetase</n>, we isolated a somatic cell hybrid segregating the deleted human Chr 20. This hybrid clone, designated NR2, was characterized by several methods, including PCR, with eight pairs of oligonucleotides mapped to Chr 20: D20S5, D20S41, D20S42, D20S56, D20S57, D20S58, <n>adenosine deaminase</n> (<n>ADA</n>), and <n>Prion protein</n> (<n>PRIP</n>); Restriction Fragment Length Polymorphism (RFLP) analyses with four genomic anonymous probes (D20S5, cD3H12, D20S17, D20S18); and fluorescent in situ hybridization (FISH) with total human DNA and D20Z1, a sequence specific to the human Chr 20 centromere, as probes. The NR2 hybrid allowed us to exclude three candidate genes for AGS: hepatic nuclear factor 3 beta (HNF3 beta), paired box 1 (PAX1), and cystatin C (CST3) as shown by their localization outside of the deletion. The NR2 hybrid is a powerful tool for the mapping of new probes of this region, as well as for obtaining new informative probes specific for the deletion by subtractive cloning of the region. Such markers will be useful for linkage analysis and screening of cDNA libraries.
95016439>Positive and negative regulation of the composite octamer motif of the interleukin 2 enhancer by <n>AP-1</n>, <n>Oct-2</n>, and <n>retinoic acid receptor</n>. The differentiating agent retinoic acid (RA) has been previously reported to interfere with 12-O-tetradecanoyl-phorbol-13-acetate (TPA)/Ca(2+)-induced signals for the regulation of the -96 to -66-bp octamer motif found in the enhancer for the interleukin (IL)-2 gene, which encodes a major T lymphocyte growth factor. The <n>IL-2</n> octamer motif is a composite cis-element which binds <n>Oct-1</n> and <n>Oct-2</n> as well as a TPA/Ca(2+)-inducible nuclear factor, previously termed octamer- associated protein (<n>OAP40</n>). We show here that <n>Oct-2</n>, despite the presence of an active transcriptional activation domain, requires TPA/Ca(2+)-induced signals to strongly transactivate the <n>IL-2</n> octamer motif in Jurkat T cells. This <n>Oct-2</n>-dependent transactivation is inhibited by RA. The presence of an intact COOH-terminal domain of <n>Oct- 2</n> contributes to both TPA/Ca(2+)-induced transactivation and the RA- mediated repression. We also show that both Fos and Jun components of the <n>AP-1</n> factors participate in the OAP40 complex. Furthermore, transfected c-jun, jun-B, jun-D, c-fos, or Fos-B expression vectors partially substitute for TPA and Ca2+ and cooperate with <n>Oct-2</n> for the transactivation of the combined OAP/octamer cis-element. Mutations of the genuine octamer-binding site abrogate both the binding of <n>Oct-1</n> and <n>Oct-2</n> and the TPA/Ca(2+)-induced transactivation of the OAP/octamer motif. OAP confers to <n>Oct-2</n> responsivity to both TPA/Ca2+ and RA, since specific mutations of the <n>AP-1</n>/OAP-binding site significantly reduce the transactivation by <n>Oct-2</n> in response to TPA and Ca2+ and abolish the inhibition by RA. Furthermore, <n>retinoic acid receptor</n> (<n>RAR</n>) alpha is able to inhibit in vitro the formation of the complex between the nuclear <n>AP-1</n>/OAP and its specific binding site, resulting in the interference with <n>Oct-2</n>-dependent cis-regulatory function of this <n>AP-1</n> element. Therefore, we propose that the TPA/calcium-activated <n>AP-1</n>/OAP element is the main target of positive or negative regulatory signals influencing the <n>IL-2</n> octamer motif, through synergism with <n>Oct-2</n> and antagonism by RAR
95190013>Missense mutation in exon 7 of the common gamma chain gene causes a moderate form of X-linked combined immunodeficiency. Clinical and immunologic features of a recently recognized X-linked combined immunodeficiency disease (XCID) suggested that XCID and X- linked severe combined immunodeficiency (XSCID) might arise from different genetic defects. The recent discovery of mutations in the common gamma chain (gamma c) gene, a constituent of several cytokine receptors, in XSCID provided an opportunity to test directly whether a previously unrecognized mutation in this same gene was responsible for XCID. The status of X chromosome inactivation in blood leukocytes from obligate carriers of XCID was determined from the polymorphic, short tandem repeats (CAG), in the androgen receptor gene, which also contains a methylation-sensitive HpaII site. As in XSCID, X-chromosome inactivation in obligate carriers of XCID was nonrandom in T and B lymphocytes. In addition, X chromosome inactivation in PMNs was variable. Findings from this analysis prompted sequencing of the gamma c gene in this pedigree. A missense mutation in the region coding for the cytoplasmic portion of the gamma c gene was found in three affected males but not in a normal brother. Therefore, this point mutation in the gamma c gene leads to a less severe degree of deficiency in cellular and humoral immunity than that seen in XSCID.
95152043>Posttranscriptional regulation of macrophage tissue factor expression by antioxidants. Tissue factor (TF) expression by cells of monocyte/macrophage lineage represents an important mechanism underlying the initiation of <n>fibrin</n> deposition at sites of extravascular inflammation. Recent evidence suggests a role for oxidant stress in the signalling pathway of various cell types by virtue of its ability to induce DNA binding of various transcription factors, including <n>nuclear factor kappa B</n> and <n>AP-1</n>. The effect of antioxidant treatment on lipopolysaccharide (LPS)-induced TF expression was examined in murine peritoneal macrophages and human monocytes. Both pyrrolidine dithiocarbamate, an oxidant scavenger, and N-acetyl-cysteine, a precursor of the endogenous antioxidant glutathione, inhibited stimulation of macrophage procoagulant activity by LPS. Northern blot analysis showed that neither of these agents reduced LPS-stimulated TF mRNA accumulation, thereby suggesting a posttranscriptional mechanism for the effect. Immunofluorescence studies of human monocytes using polyclonal anti-TF antibody showed that N-acetyl-cysteine treatment prevented the characteristic plasmalemmal localization of TF antigen that occurs in response to LPS. Western blot analysis showed that N-acetyl-cysteine reduced the accumulation of the <n>47-kD mature glycoprotein</n> in LPS-treated cells, a finding consistent with the results of the immunofluorescence studies. Furthermore, these conditions did not result in an accumulation of the less mature forms of TF. When considered together, these data suggest that antioxidants exert their effects by impairing translation and/or by causing degradation of newly translated protein. The effect of antioxidants on tumor necrosis factor appeared to be species specific, with no effect on LPS-induced tumor necrosis factor in murine cells, but with inhibition in human monocytes. The posttranscriptional effect of antioxidants on TF expression data suggests a novel mechanism whereby these agents might modulate monocyte/macrophage activation.
95115141>Characterization of the CD48 gene demonstrates a positive element that is specific to Epstein-Barr virus-immortalized B-cell lines and contains an essential <n>NF-kappa B</n> site. Epstein-Barr virus (EBV) infection of mature, resting B cells drives them to become lymphoblasts expressing high levels of cell surface molecules, such as <n>CD48</n>, characteristically expressed on normal activated B cells. Here, we report on the identification of an enhancer element in the <n>CD48</n> gene which reproducibly confers strong transcriptional activity only in EBV-positive B-lymphoblastoid cell lines. The element is not activated upon infection of established EBV- negative B-cell lines, indicating that EBV fails to drive these cells to a fully lymphoblastoid phenotype. An <n>NF-kappa B</n> binding site is an essential component of the element but alone is not sufficient to account for the activity or the specificity of the element. We have detected a specific nuclear protein complex that binds to the element and show that NF-kappa B1 (p50) is a part of this complex. The <n>EBV- encoded latent membrane protein 1</n> is capable of transactivating the isolated <n>CD48</n> <n>NF-kappa B</n> site but not the intact element, suggesting that the <n>latent membrane protein 1</n>-driven activation of <n>NF-kappa B/Rel</n> must interact with other regulatory pathways to control expression of cellular genes as EBV drives resting B cells into the cell cycle.
95347767>Thapsigargin induces IL-2 receptor alpha-chain in human peripheral and Jurkat T cells via a <n>protein kinase C</n>-independent mechanism. Thapsigargin (TG), an inhibitor of Ca(2+)-ATPase, depletes intracellular Ca2+ stores and induces a sustained Ca2+ influx without altering phosphatidyl inositol levels. TG plus phorbol myristate acetate (PMA) but not TG alone induced <n>IL-2</n> in Jurkat T cells, suggesting that TG had no effect on <n>protein kinase C</n> (<n>PKC</n>). However, TG induced increases in <n>IL-2R alpha protein</n> as well as IL-2R alpha mRNA in Jurkat T cells in a dose-dependent manner. A similar increase in <n>IL-2R alpha</n> by TG was also observed in human peripheral T cells. Further, like PMA, TG markedly induced <n>NF kappa B</n> in Jurkat T cells. However, TG and PMA exhibited a synergistic action on IL-2R alpha expression, suggesting that TG and PMA induce IL-2R alpha through distinct pathways. PMA- but not TG-induced IL-2R alpha is inhibited by the <n>PKC</n> inhibitor H7, whereas TG- but not PMA-induced IL-2R alpha was inhibited by cholera toxin, forskolin and 1,9-dideoxy forskolin. In toto, these results suggest that TG induces IL-2R alpha in human T cells through a <n>PKC</n>-independent pathway.
95311627>Physiological concentration of estradiol inhibits polymorphonuclear leukocyte chemotaxis via a receptor mediated system. Estrogen exhibits a variety of actions, including immuno-modulatory effects, in vivo and in vitro. The mechanism by which estrogen exerts its anti-inflammatory effect is not yet understood. We investigated the possible mechanisms of estradiol acting via the polymorphonuclear leukocytes (PMNs), which are important in the immune response. The agent, 17 beta-estradiol, but not 17 alpha-estradiol, significantly reduced PMNs chemotaxis to FMLP in a dose-dependent manner (control vs estrogen 10(-10)-(-6) M, P &lt; 0.05). Physiological concentrations of estradiol significantly reduced the chemotaxis of PMNs (10(-10) mol). Pre-incubation with clomiphene or tamoxifen which are estrogen receptor antagonists, eliminated the inhibitory effect of 17 beta-estradiol on the chemotaxis of PMNs, restoring it to the control level. These observations suggest that 17 beta-estradiol suppressed the chemotaxis of PMNs by a receptor-dependent mechanism. In addition, the level of estradiol in human plasma, which PMNs were drawn, showed a close, inverse correlation with the PMNs chemotaxis to FMLP (r = -0.821 p &lt; 0.001). Estrogen may modify the activity of neutrophils during the normal menstrual cycle, not only during pregnancy, and influence inflammation.
95107991>Identification of the TCL1 gene involved in T-cell malignancies. The TCL1 locus on chromosome 14q32.1 is frequently involved in chromosomal translocations and inversions with one of the T-cell receptor loci in human T-cell leukemias and lymphomas. The chromosome 14 region translocated or rearranged involves approximately 350 kb of DNA at chromosome band 14q32.1. Within this region we have identified a gene coding for a 1.3-kb transcript, expressed only in restricted subsets of cells within the lymphoid lineage and expressed at high levels in leukemic cells carrying a t(14;14)(q11;q32) chromosome translocation or a inv(14)(q11;q32) chromosome inversion. The cognate cDNA sequence reveals an open reading frame of 342 nt encoding a <n>protein of 14 kDa</n>. The TCL1 gene sequence, which, to our knowledge, shows no sequence homology with other human genes, is preferentially expressed early in T- and B-lymphocyte differentiation.
95059037>Identification of a region which directs the monocytic activity of the <n>colony-stimulating factor 1</n> (<n>macrophage colony-stimulating factor</n>) receptor promoter and binds <n>PEBP2/CBF</n> (<n>AML1</n>). The receptor for the <n>macrophage colony-stimulating factor</n> (or <n>colony- stimulating factor 1</n> [<n>CSF-1</n>]) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the <n>CSF-1</n> receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor <n>PU.1</n> (D.-E.Zhang, C.J.Hetherington, H.-M.Chen, and D.G.Tenen, Mol.Cell. Biol.14:373-381, 1994). Here we report that the tissue specificity of this promoter is also mediated by sequences in a region II (bp -88 to - 59), which lies 10 bp upstream from the <n>PU.1</n>-binding site. When analyzed by <n>DNase</n> footprinting, region II was protected preferentially in monocytic cells. Electrophoretic mobility shift assays confirmed that region II interacts specifically with nuclear proteins from monocytic cells. Two gel shift complexes (Mono A and Mono B) were formed with separate sequence elements within this region. Competition and supershift experiments indicate that Mono B contains a member of the polyomavirus enhancer-binding protein 2/core-binding factor (<n>PEBP2/CBF</n>) family, which includes the AML1 gene product, while <n>Mono A</n> is a distinct complex preferentially expressed in monocytic cells. Promoter constructs with mutations in these sequence elements were no longer expressed specifically in monocytes. Furthermore, multimerized region II sequence elements enhanced the activity of a heterologous thymidine kinase promoter in monocytic cells but not other cell types tested. These results indicate that the monocyte/B-cell-specific transcription factor <n>PU.1</n> and the Mono A and Mono B protein complexes act in concert to regulate monocyte-specific transcription of the <n>CSF-1</n> receptor.
95077537>Pyrrolidine dithiocarbamate, a potent inhibitor of <n>nuclear factor kappa B</n> (<n>NF-kappa B</n>) activation, prevents apoptosis in human promyelocytic leukemia HL-60 cells and thymocytes. We examined the effect of pyrrolidine dithiocarbamate (PDTC), which potently blocks the activation of <n>nuclear factor kappa B</n> (<n>NF-kappa B</n>), on the induction of apoptosis by a variety of agents. Treatment of a human promyelocytic leukemia cell line, HL-60, with 10 micrograms/mL etoposide or 2 microM 1-beta-D-arabinofuranosylcytosine induced <n>NF- kappa B</n> activation within 1 hr and subsequently caused apoptosis within 3-4 hr. The simultaneous addition of 50-500 microM PDTC with these agents blocked <n>NF-kappa B</n> activation and completely abrogated both morphologically apoptotic changes and internucleosomal DNA fragmentation for up to 6 hr. However, PDTC failed to inhibit the <n>endonuclease</n> activity contained in the whole cell lysates. The inhibitory effect of PDTC was also observed in etoposide- and dexamethasone-induced apoptosis in human thymocytes at a concentration of 1-10 microM. Since PDTC has both antioxidant and metal-ion chelating activities, we tested the effects of N-acetyl-L-cysteine (NAC) (antioxidant) or o-phenanthroline (OP) (metal-ion chelator) on the induction of apoptosis. Pretreatment of HL-60 cells or thymocytes with 100-500 microM OP for 2 hr, but not 10-60 mM NAC, suppressed subsequent occurrence of apoptosis induced by etoposide. These results suggest that the activation of <n>NF-kappa B</n> plays an important role in the apoptotic process of human hematopoietic cells.
95151640>Overexpression of <n>protein kinase C</n>-zeta stimulates leukemic cell differentiation. A function for <n>protein kinase C</n>-zeta (<n>PKC</n>-zeta), a member of the phorbol ester nonresponsive atypical <n>protein kinase C</n> subfamily, in modulating differentiation was examined in the leukemic U937 cell. Transfected U937 cells stably overexpressing <n>PKC-zeta</n> displayed a longer doubling time, lower saturation density at confluency, and an increase in adherence to plastic as compared to control cells. <n>PKC-zeta</n> cells expressed a more differentiated phenotype as assessed by changes in morphology, surface antigen expression, and lysosomal enzyme activities and were distinct from parental U937 cells stimulated to differentiate by exposure to phorbol esters. In contrast to parental U937 cells, <n>PKC-zeta</n> cells constitutively expressed mRNA transcripts for c-jun and a low mobility <n>AP-1</n> binding activity. Thus, <n>PKC-zeta</n> overexpression stimulates a type of phenotypic differentiation that differs significantly from maturation occurring upon activation of other <n>PKC</n> subfamilies induced by phorbol ester treatment. Increased expression of the c-jun protooncogene and an increase in <n>AP-1</n> binding activity in <n>PKC-zeta</n> cells provides a potential mechanism for explaining the altered differentiation status of this cell.
95016436>A family of serine proteases expressed exclusively in myelo-monocytic cells specifically processes the nuclear factor-kappa B subunit p65 in vitro and may impair human immunodeficiency virus replication in these cells. Two groups of U937 promonocytic cells were obtained by limiting dilution cloning which differed strikingly in their ability to support human immunodeficiency virus 1 (HIV-1) replication. "Plus" clones replicated the virus efficiently, whereas "minus" clones did not. We examined these clones for differences in nuclear factor (NF)-kappa B activity which might account for the observed phenomenon. Stimulation of plus clones liberated the classical p50-p65 complex from cytoplasmic pools, whereas minus clones produced an apparently novel, faster- migrating complex, as judged by electrophoretic mobility shift assays. It is surprising that the faster-migrating complex was composed also of p50 and p65. However, the p65 subunit was COOH-terminally truncated, as shown by immunoprecipitation. The truncation resulted from limited proteolysis of p65 during cellular extraction which released particular lysosomal serine proteases, such as elastase, <n>cathepsin G</n>, and <n>proteinase 3</n>. These specific proteases are coordinately expressed and were present exclusively in the minus U937 clones, but not in the plus clones, as demonstrated in the case of <n>cathepsin G</n>. In addition, these proteases were detected in certain subclones of THP-1 and HL-60 cells and in primary monocytes, in each case correlating with the truncated from of p65. We demonstrate in vitro cleavage of p65 by purified elastase and <n>cathepsin G</n>. It is possible that particular serine proteases may have inhibiting effects on the replication of HIV-1 in myelo-monocytic cells. The data also demonstrate that special precautions must be taken when making extracts from myelo-monocytic cells
95186362>A germline TaqI restriction fragment length polymorphism in the progesterone receptor gene in ovarian carcinoma [see comments] Clinical outcome in ovarian carcinoma is predicted by progesterone receptor status, indicating an endocrine aspect to this disease. Peripheral leucocyte genomic DNAs were obtained from 41 patients with primary ovarian carcinoma and 83 controls from Ireland, as well as from 26 primary ovarian carcinoma patients and 101 controls in Germany. Southern analysis using a human progesterone receptor (hPR) cDNA probe identified a germline TaqI restriction fragment length polymorphism (RFLP) defined by two alleles: T1, represented by a 2.7 kb fragment; and T2, represented by a 1.9 kb fragment and characterised by an additional TaqI restriction site with respect to T1. An over- representation of T2 in ovarian cancer patients compared with controls in the pooled Irish/German population (P &lt; 0.025) was observed. A difference (P &lt; 0.02) in the distribution of the RFLP genotypes between Irish and German control populations was also observed. The allele distributions could not be shown to differ significantly from Hardy- Weinberg distribution in any subgroup. Using hPR cDNA region-specific probes, the extra TaqI restriction site was mapped to intron G of the hPR gene.
95163103><n>OBF-1</n>, a novel B cell-specific coactivator that stimulates immunoglobulin promoter activity through association with octamer- binding proteins. Recent biochemical and genetic studies indicate that in addition to the octamer-binding proteins <n>Oct-1</n> and <n>Oct-2</n>, other B cell components are required for lymphoid-restricted, octamer site-mediated immunoglobulin gene promoter activity. Using a genetic screen in yeast, we have isolated B cell-derived cDNAs encoding <n>Oct-binding factor 1</n> (<n>OBF-1</n>), a novel protein that specifically associates with <n>Oct-1</n> and <n>Oct-2</n>. Biochemical studies demonstrate that <n>OBF-1</n> has no intrinsic DNA-binding activity and recognizes the POU domains of <n>Oct-1</n> and <n>Oct-2</n>, but not those of <n>Oct-4</n> and <n>Oct-6</n>. The <n>OBF-1</n> mRNA is expressed in a highly cell- specific manner, being most abundant in B cells and essentially absent in most of the other cells or tissues tested. Furthermore, expression of <n>OBF-1</n> in HeLa cells selectively stimulates the activity of a natural immunoglobulin promoter in an octamer site-dependent manner. Thus, <n>OBF- 1</n> has all the properties expected for a B cell-specific transcriptional coactivator protein.
95301128>Modulation of transcription factor <n>NF kappa B</n> activity by intracellular glutathione levels and by variations of the extracellular cysteine supply. HIV-infected individuals and SIV-infected rhesus macaques have, on the average, decreased plasma cysteine and cystine concentrations and decreased intracellular glutathione levels. We now show that a depletion of intracellular glutathione in a human T cell line (Molt-4) inhibits the activation and nuclear translocation of the transcription factor <n>NF kappa B</n>, whereas incubation with increasing extracellular concentrations of cysteine inhibits the DNA-binding and transactivating activity of <n>NF kappa B</n>. Because inhibition of DNA-binding activity is associated with increasing intracellular glutathione disulfide levels and GSSG can be shown to inhibit the DNA-binding activity directly in cell-free systems, our studies suggest that GSSG is a physiologically relevant inhibitor in intact cells also. <n>NF kappa B</n> controls many immunologically important genes, so our studies suggest that the immune system may be sensitive not only against a cysteine and glutathione deficiency but also against an excess of cysteine.
95138118>Two distinct signalling pathways are involved in the control of the biphasic junB transcription induced by <n>interleukin-6</n> in the B cell hybridoma 7TD1. We have measured the level of junB mRNA in the B hybridoma cell line 7TD1, under <n>interleukin-6</n> (<n>IL-6</n>) stimulation. <n>IL-6</n> increases junB mRNA in a biphasic fashion. The first early-induced peak was transient and likely corresponds to the well documented typical junB mRNA, stimulated in response to numerous growth factors, including <n>IL-6</n>. At variance, the second peak which has never been reported previously, lasted several hours. As a consequence of its effect on junB mRNA, <n>IL-6</n> stimulated, in a biphasic fashion, the nuclear accumulation of the <n>JunB protein</n>. In this study, we demonstrated that <n>IL-6</n> regulation occurred exclusively at the transcriptional level and that the bimodal increase of junB mRNA and <n>JunB protein</n> can be accounted for by a biphasic stimulation of junB transcription. Furthermore, our data point to two major differences between the mechanism of control of the early and the late <n>IL-6</n>-induced junB transcription waves. First, cycloheximide strongly potentiated the transcription of the second wave, whereas it failed to affect the early-induced burst. Second, tyrphostin, a tyrosine kinase inhibitor, impaired the expression of the first but not the second junB mRNA peak. Conversely, genistein, another tyrosine kinase inhibitor, totally abolished the expression of the second peak of junB mRNA whereas it did not affect the expression of the first peak. Altogether these data indicate that, in 7TD1 cells, <n>IL-6</n> controls junB transcription in a biphasic fashion by means of two separate transduction pathways.
95235452>A newly established megakaryoblastic/erythroid cell line that differentiates to red cells in the presence of <n>erythropoietin</n> and produces platelet-like particles. In August, 1992, we established a leukemic cell line (NS-Meg) from a patient in megakaryoblastic transformation of Philadelphia chromosome- positive chronic myeloid leukemia. The NS-Meg cells were positive for <n>alpha-naphthyl acetate esterase</n> and periodic acid-Schiff (PAS) staining and for surface <n>CD4</n>, <n>CD7</n>, <n>CD13</n>, <n>CD34</n>, <n>CD41a</n>, and <n>glycophorin A antigens</n>. Ultrastructurally, the cells had alpha-granules, demarcation membranes, and <n>platelet peroxidase</n> activity. The NS-Meg cells spontaneously produced platelet-like particles which contained alpha- granules, mitochondria and dense bodies, strongly suggesting platelet production. <n>Erythropoietin</n> (<n>Epo</n>), granulocyte/macrophage colony stimulating factor(<n>GM-CSF</n>), and <n>interleukin 3</n> (<n>IL-3</n>) promoted the growth of NS-Meg cells. Phorbol-12-myristate-13-acetate increased the expression of both <n>CD41a</n> and CD61 antigens. Ten-day exposure to <n>Epo</n> induced mature erythroblasts and red cells. These benzidine-positive cells were positive for hemoglobin F staining. Untreated NS-Meg cells expressed mRNA for the <n>Epo receptor</n> (<n>EpoR</n>), for <n>GATA-1</n>, and for alpha 1, alpha 2 and gamma globin genes. These results indicate that NS-Meg cells undergo terminal differentiation of both megakaryocytic and erythroid lineages. This cell line should be a very useful tool for the investigation of both megakaryocytic and erythroid maturation.
95081587>Differential regulation of proto-oncogenes c-jun and c-fos in T lymphocytes activated through <n>CD28</n>. The T cell surface molecule <n>CD28</n> binds to ligands on accessory cells and APCs, playing an important costimulatory role in the response of T cells to Ags. Our knowledge of the intracellular signaling pathways coupled to this receptor is incomplete. In addition to activation of <n>phospholipase C gamma 1</n>, ligation of this receptor also seems to activate a calcium-independent, <n>CD28</n>-specific pathway. In this paper, we report that cross-linking of <n>CD28</n> (but not <n>CD2</n>, <n>CD5</n>, <n>LFA-1</n>, or <n>CD7</n>) leads to an elevation of c-jun mRNA, with only minimal activation of c- fos expression. <n>CD28</n>-dependent induction of c-jun expression requires <n>protein tyrosine kinase</n> activity, but does not depend on activation of a <n>phorbol ester-responsive protein kinase C</n> or elevation of cytosolic calcium. Furthermore, <n>CD28</n>-dependent elevation of c-jun mRNA does not appear to be mediated at the level of mRNA stability. A mechanism is suggested whereby expression of c-jun and junB, in the absence of members of the fos family, can prevent inappropriate activation of T cells caused by ligation of <n>CD28</n> in the absence of a specific antigenic stimulus.
95056004>Enhanced responsiveness to <n>nuclear factor kappa B</n> contributes to the unique phenotype of simian immunodeficiency virus variant SIVsmmPBj14. Infection with a variant of simian immunodeficiency virus, SIVsmmPBj14, leads to severe acute disease in macaques. This study was designed to investigate the functional significance of previously described mutations in the viral long terminal repeat (LTR) and to elucidate their contribution to the unique phenotype of SIVsmmPBj14. LTR-directed transcription was measured by using luciferase reporter constructs that were transiently transfected into cultured cells. In a wide range of cell types, the basal transcriptional activity of the LTR from SIVsmmPBj14 was found to be 2- to 4.5-fold higher than that of an LTR from a non-acutely pathogenic strain. These LTRs differ by five point mutations and a 22-bp duplication in SIVsmmPBj14, which includes a <n>nuclear factor kappa B</n> (<n>NF kappa B</n>) site. Transcriptional differences between these LTRs were further enhanced by two- to threefold upon treatment of cells with phorbol ester or <n>tumor necrosis factor alpha</n> or by cotransfection with plasmids expressing <n>NF kappa B</n> subunits. Mutagenesis studies, and the use of a reporter construct containing an enhancerless promoter, indicate that these transcriptional effects are due principally to the 22-bp sequence duplication and the <n>NF kappa B</n> site contained within it. Finally, infectious virus stocks that were isogenic except for the LTR were generated. The LTR from SIVsmmPBj14 was found to confer an increase in the kinetics of virus replication in cultured cells. Inclusion of this LTR in recombinant SIVs also resulted in a two- to threefold rise in the extent of cellular proliferation that was induced in quiescent simian peripheral blood mononuclear cells. These studies are consistent with the hypothesis that LTR mutations assist SIVsmmPBj14 in responding efficiently to cellular stimulation and allow it to replicate to high titers during the acute phase of viral infection.
95091657>Constitutive <n>nuclear NF-kappa B</n> in cells of the monocyte lineage. In monocytes, the nuclear factor <n>NF-kappa B</n> has been invoked as an important transcription factor in the expression of cytokine genes, of cell-surface receptors and in the expression of human immunodeficiency virus. In such cells, DNA binding activity of <n>NF-kappa B</n> can be detected without intentional stimulation. In our studies, cells of the human monocytic line Mono Mac 6, cultured in medium containing fetal- calf serum and low levels of lipopolysaccharide (LPS), also exhibit such 'constitutive' <n>NF-kappa B</n>, as demonstrated by mobility-shift analysis of nuclear extracts. This nuclear <n>NF-kappa B</n> was still present when contaminant LPS was removed by ultrafiltration and when serum was omitted. Protein-DNA complexes of constitutive <n>NF-kappa B</n> are similar in mobility to the LPS-induced <n>NF-kappa B</n> and both are recognized by an antibody specific to the p50 subunit of <n>NF-kappa B</n>. By contrast, treatment of cells with pyrrolidine dithiocarbamate (PDTC) will only block LPS-induced <n>NF-kappa B</n>, but not the constitutive binding protein. Using LPS-free and serum-free conditions, constitutive <n>NF-kappa B</n> can be detected in different cell lines of the monocytic lineage (HL60, U937, THP-1, Mono Mac 1 and Mono Mac 6), but not in Molt 4 T cells or K562 stem cells. When ordered according to stage of maturation, the amount of constitutive <n>NF-kappa B</n> was not increased in more mature cell lines. Furthermore, when inducing differentiation in Mono Mac 6 cells, with vitamin D3, no change in constitutive or inducible <n>NF-kappa B</n> can be detected. Analysis of primary cells revealed substantial constitutive <n>NF-kappa B</n>-binding activity in blood monocytes, pleural macrophages and alveolar macrophages. The constitutive <n>NF-kappa B</n> appears to be functionally active, since a low level of tumour necrosis factor (TNF) transcript is detectable in monocytes, and this level can be increased by blocking transcript degradation using cycloheximide. The level of constitutive <n>NF-kappa B</n> in these cells is variable and is frequently found to be lower in the more mature macrophages. Constitutive <n>NF-kappa B</n> was not maintained by autocrine action of cytokines TNF, <n>interleukin 6</n>, <n>interleukin</n> 10, <n>granulocyte-macrophage colony-stimulating factor</n> or <n>macrophage colony-stimulating factor</n>, since neutralizing antibodies did not reduce constitutive DNA-binding activity. Furthermore, blockade of prostaglandin or leukotriene biosynthesis did not affect constitutive <n>NF-kappa B</n>. (ABSTRACT TRUNCATED AT 400 WORDS)
95036334><n>Steel factor</n> affects <n>SCL</n> expression during normal erythroid differentiation. <n>Steel factor</n> is one of the growth factors that controls the proliferation and differentiation of hematopoietic cells and <n>SCL</n>, also known as <n>Tcl-5</n> or <n>Tal-1</n>, is a transcription factor involved in erythropoiesis. In this report, we studied the role of SCL in the proliferation of human peripheral blood burst-forming unit-erythroid (BFU-E) and the effects of <n>Steel factor</n> on <n>SCL</n> expression in proliferating erythroid cells. BFU-E-derived colonies increase progressively in size, as determined by cell number, from day 7 to day 14 of culture, with the greatest increase in colony size (10-fold expansion) occurring between day 7 and day 10. <n>SCL protein</n> levels in BFU-E-derived cells were highest in day 7 cells and decreased progressively from day 7 to day 14 of culture, suggesting an association of <n>SCL</n> with erythroid proliferation. In contrast, <n>SCL</n> mRNA levels did not decrease significantly between day 7 and day 14 cells, suggesting that posttranscriptional mechanisms are largely responsible for the decrease in <n>SCL</n> protein observed. The role of <n>SCL</n> in <n>Steel factor</n>-induced erythroid proliferation was then examined. In BFU-E- derived colonies cultured with <n>Steel factor</n>, colony size was significantly increased compared to control. In day 7 and day 10 erythroid precursors cultured with <n>Steel factor</n>, <n>SCL</n> protein was increased significantly compared to control. The increase in <n>SCL</n> protein levels in early erythroid precursors stimulated with <n>Steel factor</n> suggests one mechanism through which <n>Steel factor</n> may enhance normal erythroid proliferation. <n>SCL</n> mRNA levels assessed by Northern blot in day 7 cells did not increase significantly in response to <n>Steel factor</n> stimulation, suggesting that posttranscriptional mechanisms may also be important in the increase in <n>SCL</n> protein observed in response to <n>Steel</n>.
95015010><n>C/EBP beta</n> regulation of the <n>tumor necrosis factor alpha</n> gene. Activated macrophages contribute to chronic inflammation by the secretion of cytokines and proteinases. <n>Tumor necrosis factor alpha</n> (<n>TNF alpha</n>) is particularly important in this process because of its ability to regulate other inflammatory mediators in an autocrine and paracrine fashion. The mechanism(s) responsible for the cell type- specific regulation of <n>TNF alpha</n> is not known. We present data to show that the expression of <n>TNF alpha</n> is regulated by the transcription factor <n>C/EBP beta</n> (<n>NF-IL6</n>). <n>C/EBP beta</n> activated the <n>TNF alpha</n> gene promoter in cotransfection assays and bound to it at a site which failed to bind the closely related protein <n>C/EBP alpha</n>. Finally, a dominant-negative version of <n>C/EBP beta</n> blocked <n>TNF alpha</n> promoter activation in myeloid cells. Our results implicate <n>C/EBP beta</n> as an important regulator of <n>TNF alpha</n> by myelomonocytic cells
95173590><n>Calcium/calmodulin-dependent protein kinase II</n> downregulates both calcineurin and protein kinase C-mediated pathways for cytokine gene transcription in human T cells. Engagement of the T cell receptor for antigen activates <n>phospholipase C</n> resulting in an increase in intracellular free calcium concentration ([Ca2+]i) and activation of <n>protein kinase C</n> (<n>PKC</n>). Increased [Ca2+]i activates Ca2+/calmodulin-dependent kinases including the multifunctional Ca2+/calmodulin-dependent protein kinase II (<n>CaM-K II</n>), as well as <n>calcineurin</n>, a type 2B protein phosphatase. Recent studies have identified <n>calcineurin</n> as a key enzyme for interleukin (IL)-2 and IL-4 promoter activation. However, the role of <n>CaM-K II</n> remains unknown. We have used mutants of these kinases and phosphatases (<n>gamma B*CaM-K</n> and <n>delta CaM-AI</n>, respectively) to explore their relative role in cytokine gene transcription and their interactions with <n>PKC</n>- dependent signaling systems. <n>gamma B*CaM-K</n> and <n>delta CaM-AI</n>, known to exhibit constitutive Ca(2+)-independent activity, were cotransfected (alone or in combination) in Jurkat T cells with a plasmid containing the intact IL-2 promoter driving the expression of the chloramphenicol acetyltransferase reporter gene. Cotransfection of <n>gamma B*CaM-K</n> with the IL-2 promoter construct downregulated its transcription in response to stimulation with ionomycin and phorbol myristate acetate (PMA). The inhibitory effect of <n>CaM-K II</n> on IL-2 promoter was associated with decreased transcription of its AP-1 and NF-AT transactivating pathways. Under the same conditions, <n>delta CaM-AI</n> superinduced IL-2 promoter activity (approximately twofold increase). When both mutants were used in combination, <n>gamma B*CaM-K</n> inhibited the induction of the IL-2 promoter by <n>delta CaM-AI</n>. Similar results were obtained when a construct containing the <n>IL-4</n> promoter also was used. <n>gamma B*CaM-K</n> also downregulated the activation of <n>AP-1</n> in response to transfection with a constitutively active mutant of <n>PKC</n> or stimulation with PMA. These results suggest that <n>CaM-K II</n> may exert negative influences on cytokine gene transcription in human T cells, and provide preliminary evidence for negative cross-talk with the calcineurin- and PKC- dependent signaling systems.
95130292>Induction of <n>ICAM-1</n> and <n>LFA-3</n> by <n>Tax1</n> of human T-cell leukemia virus type 1 and mechanism of down-regulation of <n>ICAM-1</n> or <n>LFA-1</n> in adult-T- cell-leukemia cell lines. The present study was undertaken to determine the role of <n>HTLV-I TaxI</n> in the up-regulation of <n>ICAM-I</n> and <n>LFA-3</n> in human T cells transformed with HTLV-I and the mechanism of down-regulation of <n>ICAM-I</n> and <n>LFA-I</n> in ATL-derived cell lines. Induction of <n>TaxI</n> in a human T-cell line Jurkat carrying the <n>TaxI</n> gene under the metallothionein promoter led to increases in mRNA and surface expression of <n>ICAM-I</n>. The response of <n>LFA- 3</n> to <n>TaxI</n> induction was, on the other hand, relatively slow and weak, and might be indirect. Transactivation of the <n>ICAM-I</n> promoter by <n>TaxI</n> was further shown by co-transfection of a CAT reporter construct with the <n>ICAM-I</n> promoter and a plasmid expressing <n>TaxI</n>. The mechanism of down-regulation of <n>ICAM-I</n> or <n>LFA-I</n> in 4 ATL cell lines was next examined. <n>ICAM-I</n> mRNA was quite low in MT-I, but no genomic changes were found. The CAT reporter with the <n>ICAM-I</n> promoter was inactive in MT-I. Finally, combined treatment of MT-I with 5-azacytidine and <n>IFN- gamma</n> induced re-expression of <n>ICAM-I</n>. Collectively, (a) transcriptional factor(s) necessary for expression of <n>ICAM-I</n> gene may be repressed in MT-I through DNA methylation. Three other ATL cell lines (TL-OmI, H582, HuT102) were found to have little mRNA for the LFA- I beta chain (CD18). H582 and HuT102 were also negative for the <n>LFA-I</n> alpha chain (CDIIa) mRNA. No genomic changes were found, and a CAT reporter gene with the CD18 promoter was inactive in the 3 of them, again suggesting lack of (a) transcriptional factor(s) necessary for <n>CD18</n> expression.
95287902>Infection with Theileria annulata induces expression of <n>matrix metalloproteinase 9</n> and transcription factor <n>AP-1</n> in bovine leucocytes. Theileria annulata infects bovine leucocytes and results in their reversible transformation such that they become immortalised and metastatic. The present study describes parasite-induced changes in host cell gene expression which have a direct bearing on this transformation process. T. annulata-infected leucocytes produce a number of novel <n>metalloproteinase</n> activities. One of these, previously called <n>B1</n>, is a <n>97-kDa protein</n> which is secreted in large amounts and has been purified from protein-free, conditioned medium. An antiserum to this enzyme was used to isolate a cDNA clone. The predicted protein sequence of <n>B1</n> is 81% identical to human <n>matrix metalloproteinase 9</n> (<n>MMP9</n>), demonstrating that it is the bovine homologue of this enzyme. <n>RNAase</n> protection assays demonstrated that the <n>MMP9</n> activity, unique to infected cells, is due to increased <n>MMP9</n> mRNA levels. We also assayed the levels of transcription factor <n>AP-1</n> and demonstrated that it was constitutively present in increased amounts in Theileria-infected cells. In addition we assayed the level of mRNA encoding <n>c-Fos</n>, a common component of <n>AP-1</n> and observed that it was indeed up-regulated in infected cells. Since <n>AP-1</n> is implicated in the control of the cell cycle, and <n>MMP9</n> can confer metastatic properties, these results are of considerable significance with respect to the transformed phenotype induced by Theileria infection.
95129555>B-cell proliferation and induction of early G1-regulating proteins by Epstein-Barr virus mutants conditional for <n>EBNA2</n>. Infection of primary B-lymphocytes by Epstein-Barr virus (EBV) leads to growth transformation of these B-cells in vitro. EBV nuclear antigen 2 (<n>EBNA2</n>), one of the first genes expressed after EBV infection of B- cells, is a transcriptional activator of viral and cellular genes and is essential for the transforming potential of the virus. We generated conditional EBV mutants by expressing <n>EBNA2</n> as chimeric fusion protein with the hormone binding domain of the <n>estrogen receptor</n> on the genetic background of the virus. Growth transformation of primary normal B- cells by mutant virus resulted in estrogen-dependent lymphoblastoid cell lines expressing the chimeric <n>EBNA2</n> protein. In the absence of estrogen about half of the cells enter a quiescent non-proliferative state whereas the others die by apoptosis. <n>EBNA2</n> is thus required not only for initiation but also for maintenance of transformation. Growth arrest occurred at G1 and G2 stages of the cell cycle, indicating that functional <n>EBNA2</n> is required at different restriction points of the cell cycle. Growth arrest is reversible for G1/G0 cells as indicated by the sequential accumulation and modification of cell cycle regulating proteins. EBV induces the same cell cycle regulating proteins as polyclonal stimuli in primary B-cells. These data suggest that EBV is using a common pathway for B-cell activation bypassing the requirement for antigen, T-cell signals and growth factors.
95234639><n>IL-1 receptor</n> and TCR signals synergize to activate <n>NF-kappa B</n>-mediated gene transcription. Previous studies have demonstrated that <n>IL-1 receptor</n> (<n>IL-1R</n>)- and TCR- initiated signals can interact synergistically to increase the rate of transcription of several lymphokine and lymphokine receptor genes during the competence phase of the activation program in T helper lymphocytes. In this report we describe how signals initiated through the type I <n>IL-1R</n> interact with signals from the antigen receptor to synergistically augment the transactivating properties of <n>NF-kappa B</n>. The synergistic antigen receptor initiated signals are mediated through <n>protein kinase C</n> because they can be mimicked by the phorbol ester, 12- O-tetradecanoylphorbol-13-acetate, but not with calcium ionophores; and are staurosporine sensitive but cyclosporine resistant. Gel shift analyses demonstrate that <n>NF-kappa B</n> nuclear translocation is stimulated primarily by <n>IL-1</n> rather than by antigen receptor signals. Western blot and phosphorylation analyses demonstrate that the synergistic effect on <n>NF-kappa B</n> functional activity is independent of <n>I kappa B alpha</n> (<n>MAD3</n>)-<n>NF-kappa B</n> dissociation in the cytosol and is not associated with <n>I kappa B</n> nuclear translocation. The <n>IL-1</n>-induced <n>NF-kappa B</n> DNA nuclear localization is transient and can be prolonged either by an antigen receptor-initiated signal or by inhibiting protein synthesis. These results suggest that <n>IL-1</n> induces both <n>NF-kappa B</n> nuclear translocation and the synthesis of a protein(s) responsible for terminating <n>NF-kappa B</n>-DNA interaction in the nucleus. Antigen receptor signals prolong <n>NF-kappa B</n>-DNA interaction, probably by functionally antagonizing the <n>IL-1</n>-induced synthesis of a protein(s) responsible for the transient <n>NF-kappa B</n>-DNA interaction and consequently synergistically enhance <n>IL-1</n>-induced <n>NF-kappa B</n>-dependent gene transcription.
95121921>Identification of the promoter region of chicken anemia virus (CAV) containing a novel enhancer-like element. The single promoter region in the cloned genome [Noteborn et al., J. Virol. 65 (1991) 3131-3139] of chicken anemia virus (CAV) in chicken T- cells was analysed via <n>CAT</n> assays. A unique region containing four or five near-perfect direct repeats (DR) of 21 bp with one 12-bp insert was proven to be the main transcription-activation element, with enhancer-like characteristics. PCR studies revealed that CAV isolates from across the world all contained this promoter sequence. Electrophoretic mobility-shift assays (EMSA) showed that individual DR units, as well as the 12-bp insert, can bind to nuclear factors of chicken T-cells. Competition assays revealed that the DR units bound to factors other than the 12-bp insert. A synthetic oligodeoxyribonucleotide containing an SP1-box (5'-GGGCGG) could compete with factors binding to the 12-bp insert. Purified <n>human SP1</n> was shown to have very strong affinity for the 12-bp insert.
95053781>Functional Myc-Max heterodimer is required for activation-induced apoptosis in T cell hybridomas. T cell hybridomas respond to activation signals by undergoing apoptotic cell death, and this is likely to represent comparable events related to tolerance induction in immature and mature T cells in vivo. Previous studies using antisense oligonucleotides implicated the <n>c-Myc protein</n> in the phenomenon of activation-induced apoptosis. This role for <n>c-Myc</n> in apoptosis is now confirmed in studies using a dominant negative form of its heterodimeric binding partner, <n>Max</n>, which we show here inhibits activation-induced apoptosis. Further, coexpression of a <n>reciprocally mutant Myc protein</n> capable of forming functional heterodimers with the mutant <n>Max</n> can compensate for the dominant negative activity and restore activation-induced apoptosis. These results imply that <n>Myc</n> promotes activation-induced apoptosis by obligatory heterodimerization with <n>Max</n>, and therefore, by regulating gene transcription.
95065655><n>Epstein-Barr virus SM protein</n>. The protein products of the Epstein-Barr virus (EBV) BMLF1 open reading frame have been characterized in the early productive cycle in B95-8 and Akata cells. The <n>SM protein</n> derived from the spliced RNA joining <n>BSLF2</n> to <n>BMLF1</n> is much the most abundant protein. <n>SM</n> is a phosphoprotein in EBV-infected cells and can be phosphorylated in vitro with <n>casein kinase II</n> (<n>CKII</n>). Computer analysis of the <n>SM</n> protein sequence showed a C terminal section of <n>SM</n> to be related to genome positional homologues of four other herpesviruses and revealed consensus <n>CKII</n> sites near the N termini of the EBV <n>SM protein</n>, the <n>herpes simplex virus (HSV) ICP27 protein</n> and the <n>herpesvirus saimiri (HVS) open reading frame 57 protein</n>. Site-directed mutagenesis of the consensus <n>CKII</n> site in EBV SM greatly reduced the in vitro phosphorylation of <n>SM</n> by <n>CKII</n>. The mechanism of transactivation by <n>BMLF1</n> proteins has been controversial but <n>SM</n> was shown to transactivate gene expression from a CAT reporter construct by increasing the amount of cytoplasmic CAT mRNA. Mutagenesis of the <n>CKII</n> site in <n>SM</n> made no difference to the transactivation in this transient transfection assay.
95015876>Inducible binding to the c-fos serum response element during T cell activation is regulated by a phosphotyrosine-containing protein. The proto-oncogene c-fos is an immediate-early gene, and one of the first genes transcribed after stimulation of most cells with a variety of ligands. Fos expression may be a pivotal event in converting ligand- receptor interactions at the membrane into functional modulation of cell phenotype. The serum response element (SRE) in the c-fos regulatory region participates in induction of transcription by various growth factors and by phorbol esters and subsequent squelching of transcription. We show that an inducible protein complex (<n>Band A</n>) binds to SRE DNA within 10 min after mitogenic stimulation of human PBL-T, and becomes nondetectable by 60 min. <n>Band A</n> contains the serum response factor plus additional factor(s). A protein that is phosphorylated on a tyrosine residue in resting PBL-T suppresses binding of a component of <n>Band A</n> to the SRE motif. Upon stimulation of the cells, this protein no longer prevents binding of DNA by <n>Band A</n>, and suppression of binding is restored within 30 min. The phosphorylated tyrosine residue itself is important for the protein-protein interaction.
95010257>In vivo modification of major histocompatibility complex class II DRA promoter occupancy mediated by the <n>AIR-1 trans-activator</n>. RJ 2.2.5 is a human B cell mutant derived from the Burkitt lymphoma Raji cell which is defective in the AIR-1 locus function. This locus encodes a transcriptional trans-activator required for the constitutive expression of major histocompatibility complex (MHC) class II genes. Here we show, by in vivo <n>DNase I</n> footprinting, that the AIR-1 locus defect correlates with changes in the DRA promoter occupancy. Interestingly, reexpression of human MHC class II genes in RJ 2.2.5 x mouse spleen cell hybrids is associated with partial reversion of DRA promoter occupancy to the Raji pattern. DRA promoter occupancy in other class II-negative B cell lines, derived from patients with bare lymphocyte syndrome, is drastically different from the one observed in RJ 2.2.5 and Raji cells. Moreover, the use of the <n>DNase I</n> as an in vivo footprinting agent reveals that the patients' cell lines do not display a completely "bare promoter" as previously reported using dimethyl sulfate as the footprinting agent. Thus, the use of <n>DNase I</n> allowed us, for the first time, to correlate the AIR-1 locus defect with class II promoter occupancy alterations and distinguish these alterations from the ones observed in phenotypically similar but genetically distinct MHC class II-negative cells
95166264>Regulation of cell-type-specific interleukin-2 receptor alpha-chain gene expression: potential role of physical interactions between <n>Elf-1</n>, <n>HMG-I(Y)</n>, and NF-kappa B family proteins. The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is rapidly and potently induced in T cells in response to mitogenic stimuli. Previously, an inducible enhancer between nucleotides -299 and -228 that contains NF-kappa B and CArG motifs was identified. We now report the characterization of a second essential positive regulatory element located between nucleotides -137 and -64 that binds <n>Elf-1</n> and <n>HMG-I(Y)</n>. This element had maximal activity in lymphoid cells, paralleling the cell type specificity of <n>Elf-1</n> expression. Transcription from the IL-2R alpha promoter was inhibited when either the Elf-1 or the HMG-I(Y) binding site was mutated. Coexpression of both proteins activated transcription of the -137 to -64 element in COS-7 cells. <n>Elf-1</n> physically associated with <n>HMG-I</n> and with <n>NF-kappa B</n> p50 and c-Rel in vitro, suggesting that protein-protein interactions might functionally coordinate the actions of the upstream and downstream positive regulatory elements. This is the first report of a physical interaction between an Ets family member and <n>NF-kappa B</n> family proteins. These findings provide significant new insights into the protein-protein and protein-DNA interactions that regulate cell-type-specific and inducible IL-2R alpha gene expression and also have implications for other genes regulated by <n>Elf-1</n> and <n>NF-kappa B</n> family proteins.
95169271>Isolation of cDNA clones for 42 different Kruppel-related zinc finger proteins expressed in the human monoblast cell line U-937. To study the complexity and structural characteristics of zinc finger proteins expressed during human hematopoiesis and to isolate novel regulators of blood cell development, a degenerate oligonucleotide probe specific for a consensus zinc finger peptide domain was used to isolate 63 cDNA clones for Kruppel-related zinc finger genes from the human monoblast cell line U-937. By extensive nucleotide sequence and Northern blot analysis, these cDNA clones were found to originate from approximately 42 different genes (HZF 1-42) of which only 8 have previously been described. Northern blot analysis showed that a majority of these genes were expressed at comparable levels in U-937 and HeLa cells. The large number of individual genes represented among the 63 clones and their apparent non-cell-type-specific expression suggest that the majority of the Kruppel-related zinc finger genes are likely to be expressed in most human tissues. In contrast, some of the genes displayed a restricted expression pattern, indicating that they represent potential regulators of monocyte differentiation or proliferation. Detailed structural analysis of the first 12 cDNAs (HZF 1-10) and a partial characterization of HZF 11-42 revealed that a common feature of human Kruppel-related zinc finger proteins is the presence of tandem arrays of zinc fingers ranging in number from 3 to over 20 that are preferentially located in the carboxy-terminal regions of the proteins. In addition, several novel KRAB-containing zinc finger genes and a novel conserved sequence element were identified.
95276747>CAG repeat length variation in sperm from a patient with Kennedy's disease. Using a modified sperm typing protocol, the mutation frequency of the CAG repeat region at the androgen receptor locus has been measured using a rare semen sample from an individual with spinal and bulbar muscular atrophy (SBMA). Among 258 X chromosome-containing sperm, 19% had a repeat number equal to the donor's somatic DNA (47 repeats), 66% were expansions and 15% were contractions. The average expansion was 2.7 repeats. More than half of the expansions involved one or two repeats; the largest was 11 repeats. 68% of the contractions were also one or two repeats but six (16%) were very large (12-25 repeats). One contraction generated an allele in an intermediate size range (33-39 repeats). Such alleles have not been observed among more than 900 normal and SBMA X-chromosomes that have been examined. Comparison of the SBMA sperm typing results with mutation frequency data on normal alleles supports the hypothesis that trinucleotide repeat expansions may have a different molecular origin than contractions.
96146856>Aspirin-like drugs can protect human T lymphocytes against benzoquinone cytotoxicity: evidence for a <n>NAD(P)H:quinone reductase</n>-dependent mechanism. Benzene toxicity towards lymphocytes is thought to be mediated by metabolites of benzene including benzoquinone (BQ). <n>NAD(P)H:quinone reductase</n> (<n>QR</n>) is known to protect against BQ toxicity. The expression of the <n>QR</n> gene is regulated by the transcription factor <n>AP-1</n>. We had previously found that aspirin-like drugs (ALD) induce <n>AP-1</n> in human T lymphocytes. It was therefore hypothesized that ALD would protect lymphocytes against BQ toxicity by inducing <n>QR</n>. Molt-4 cells (M4), a human T lymphocyte cell line, were incubated with different concentrations of two ALD, flurbiprofen and sodium diclofenac, and then exposed to BQ. Toxicity was measured by viability (trypan blue exclusion). Both drugs protected the cells against BQ cytotoxicity in a dose-dependent manner, e.g., sodium diclofenac at 15 microM reduced the fraction of BQ-treated dead cells by 70%. ALDs induced <n>QR</n> activity in the M4 cells in the same range of concentrations that protected the cells against BQ toxicity. The protective effect of ALD was significantly reduced by dicoumarol, a <n>QR</n>-specific inhibitor. Since human T cells and T cell lines do not metabolize arachidonic acid, our data suggest that ALD can protect human T lymphocytes against a metabolite of benzene by induction of <n>QR</n> activity.
96071057>Correlation of differentiation-inducing activity of retinoids on human leukemia cell lines HL-60 and NB4. Retinoids, including all-trans-retinoic acid, its isomers, and fifty synthetic retinoids (retinobenzoic acids), were tested for differentiation-inducing activity on human leukemia cell lines HL-60 and NB4. A good linear correlation, with an r value of 0.91, between the ED50 values for the differentiation-inducing activity towards HL-60 cells and that towards NB4 cells was found.
95100937>Platelet-activating factor (PAF) positively auto-regulates the expression of <n>human PAF receptor transcript 1</n> (leukocyte-type) through <n>NF-kappa B</n>. The <n>human platelet-activating factor receptor</n> (<n>PAFR</n>) gene is transcribed by two distinct promoters (promoter 1 and promoter 2) to generate two transcripts (designated as <n>PAFR</n> transcript 1 and <n>PAFR</n> transcript 2), though their open reading frames are identical. By primer extension analysis to discriminate two transcripts, we found that the levels of <n>PAFR</n> transcript 1 (leukocyte-type), but not <n>PAFR</n> transcript 2 (tissue-type), are upregulated by <n>PAF</n> as well as by 12-O- tetradecanoylphorbol-13-acetate (TPA) in the human stomach cancer cell line (JR-St cells) which expresses both functional <n>PAFR</n> transcript 1 and <n>PAFR</n> transcript 2 endogenously. Functional analysis of the promoter 1 with a transient expression assay using chloramphenicol acetyltransferase (CAT) gene as a reporter showed that both <n>PAF</n> and TPA activated the promoter 1 but not the deleted promoter lacking the three consensus binding sites for <n>NF-kappa B</n> located from -571 bp to -459 bp. These findings suggest a molecular mechanism of positive regulation of <n>PAFR</n> gene expression by <n>PAF</n> through <n>NF-kappa B</n>, possibly by a phosphorylation reaction involving <n>protein kinase C</n> by <n>PAF</n>.
95053740>One gene, two transcripts: isolation of an alternative transcript encoding for the <n>autoantigen La/SS-B</n> from a cDNA library of a patient with primary Sjogrens' syndrome. A cDNA library was prepared from peripheral blood lymphocytes of an autoimmune patient with primary Sjogrens' syndrome. The cDNA library was screened with the patients own autoimmune serum being monospecific for the nuclear <n>autoantigen La/SS-B</n>. Thereby an alternative type of La mRNA was identified that differed from the known La mRNA due to an exchange of the exon 1. Sequencing of the genomic region between the exons 1 and 2 showed that the alternative 5'-end is a part of the intron. In addition, the presence of an alternative promoter site, which exists within the intron downstream of the exon 1, became evident. In consequence, the alternative La mRNA is the result of a promoter switching combined with an alternative splicing mechanism. In the intron, further transcription factor binding sites, including a NF- kappa B element, were identified leading to the suggestion that the expression of the gene encoding for the nuclear <n>autoantigen La/SS-B</n> alters in dependence on disease conditions.
95052601>Cross-linking <n>CD40</n> on B cells rapidly activates <n>nuclear factor-kappa B</n>. The B cell-associated surface molecule <n>CD40</n> functions to regulate B cell responses. Cross-linking <n>CD40</n> on B cells can lead to homotypic cell adhesion, <n>IL-6</n> production, and, in combination with cytokines, to Ig isotype switching. Tyrosine kinase activity is increased shortly after engagement of this receptor. Little is known about how the very early events induced by <n>CD40</n> cross-linking link to cellular responses. In this study, we demonstrate that nuclear factor (NF)-kappa B and NF- kappa B-like transcription factors are activated after cross-linking <n>CD40</n> on resting human tonsillar B cells and on B cell lines. The activation is rapid and is mediated through a tyrosine kinase-dependent pathway. The complexes detected in electrophoretic mobility shift assays contain p50, p65 (<n>RelA</n>), c-Rel, and most likely other components. By using transient transfection assays, we found that cross- linking <n>CD40</n> supports <n>NF-kappa B</n>-dependent gene expression. Our results define the <n>NF-kappa B</n> system as an intermediate event in <n>CD40</n> signaling and suggest that the <n>CD40</n> pathway can influence the expression of B cell-associated genes with <n>NF-kappa B</n> consensus sites.
95014473>Protease inhibitors block lipopolysaccharide induction of tissue factor gene expression in human monocytic cells by preventing activation of c- Rel/p65 heterodimers. Tissue factor (TF) is expressed rapidly by human monocytes exposed to bacterial endotoxin (lipopolysaccharide, or LPS). Transcriptional regulation is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site in the TF promoter. Nuclear translocation of cytosolic c- Rel/p65 heterodimers and other members of the <n>NF-kappa B</n>/Rel family requires dissociation and proteolytic degradation of the inhibitor protein, <n>I kappa B alpha</n>. The protease inhibitors N alpha- tosylphenylalanyl chloromethyl ketone (TPCK) and N alpha-tosyl-L-lysine chloromethyl ketone (TLCK) block activation of <n>NF-kappa B</n>/Rel proteins by preventing degradation of <n>I kappa B alpha</n>. To determine if TPCK and TLCK inhibited LPS induction of TF expression, freshly isolated human monocytes and monocytic THP-1 cells were pretreated with these inhibitors for 30 min before LPS stimulation. Both TPCK and TLCK inhibited LPS induction of <n>TF protein</n>, TF mRNA and TF promoter activity in a dose-dependent manner. These inhibitors specifically prevented degradation of <n>I kappa B alpha</n> and nuclear translocation of c-Rel/p65 heterodimers. In contrast, TPCK and TLCK did not block induction of an immediate-early gene encoding the transcription factor, <n>Egr-1</n>. Taken together, these data indicated that inhibiting nuclear translocation of c-Rel/p65 heterodimers prevented LPS induction of TF gene transcription in monocytic cells.
95152321>1,25-Dihydroxyvitamin D3 receptors in peripheral blood mononuclear cells from patients with primary and secondary hyperparathyroidism. A decreased number of calcitriol (1,25(OH)2D3) receptors has been observed in parathyroid glands of uremic animals. In humans, studies carried out in surgically removed parathyroid glands have shown that calcitriol binding is higher in primary than in secondary hyperparathyroidism. Since specific receptors for calcitriol have been described in peripheral blood mononuclear cells (PBMC), we have investigated the specific uptake of 3H-labelled 1,25(OH)2D3 in PBMC of 12 women with primary hyperparathyroidism (PHP), 8 women with hyperparathyroidism secondary to chronic renal failure (SH), 9 women with renal transplant (RT), and 23 healthy women. The median dissociation constant (Kd) was similar in all three groups of patients and in healthy women (mean +/- S.D. (range): PHP, 1.2 +/- 1.0 (0.2-4) x 10(-10) M; SH, 0.6 +/- 0.4 (0.2-1.2) x 10(-10) M; RT, 1.1 +/- 0.5 (0.4- 1.9) x 10(-10) M; controls, 1.0 +/- 0.6 (0.3-2.6) x 10(-10) M). However, the maximal binding capacity (Nmax) was significantly enhanced in PHP (3.9 +/- 1.9 (1.3-7.6) fmol/10(7) cells vs. 2.3 +/- 0.9 (1.1- 4.4) fmol/10(7) cells in controls; P = 0.0006) and decreased in SH (0.8 +/- 0.5 (0.2-1.6) fmol/10(7) cells vs. 2.3 +/- 0.9 (1.1-4.4) fmol/10(7) cells in controls; P = 0.0001), whereas no changes were seen in RT (2.3 +/- 0.7 (1.2-3.3) fmol/10(7) cells vs. 2.3 +/- 0.9 (1.1-4.4) fmol/10(7) cells in controls). In three patients with PHP who were subjected to parathyroidectomy, the calcitriol number came down to normal. Changes of <n>calcitriol receptors</n> in primary and secondary hyperparathyroidism could magnify the consequences of disturbances in serum concentration of calcitriol itself and might play an important role in the development of secondary hyperparathyroidism in uremia
94361146>The severe phenotype of females with tiny ring X chromosomes is associated with inability of these chromosomes to undergo X inactivation. Mental retardation and a constellation of congenital malformations not usually associated with Turner syndrome are seen in some females with a mosaic 45,X/46,X,r(X) karyotype. Studies of these females show that the XIST locus on their tiny ring X chromosomes is either not present or not expressed. As XIST transcription is well correlated with inactivation of the X chromosome in female somatic cells and spermatogonia, nonexpression of the locus even when it is present suggests that these chromosomes are transcriptionally active. We examined the transcriptional activity of ring X chromosomes lacking XIST expression (XISTE-), from three females with severe phenotypes. The two tiny ring X chromosomes studied with an antibody specific for the acetylated isoforms of <n>histone H4</n> marking transcribed chromatin domains were labeled at a level consistent with their being active. We also examined tow of the XISTE- ring chromosomes to determine whether genes that are normally silent on an inactive X are expressed from these chromosomes. Analyses of hybrid cells show that TIMP, ZXDA, and ZXDB loci on the proximal short arm, and AR and PHKA1 loci on the long arm, are well expressed from the tiny ring X chromosome lacking XIST DNA. Studies of the ring chromosome that has XIST DNA but does not transcribe it show that its AR allele is transcribed along with the one on the normal X allele. (ABSTRACT TRUNCATED AT 250 WORDS)
94347089>Inhibition of activation of transcription factor <n>AP-1</n> by <n>CD28</n> signalling in human T-cells. Co-stimulation of T-lymphocytes by T-cell receptor (TcR) occupancy and activation of the <n>CD28</n> surface molecule results in enhanced proliferation and <n>interleukin 2</n> (<n>IL-2</n>) production. The increase in <n>IL-2</n> gene expression triggered by <n>CD28</n> involves a kappa B-like sequence in the 5'-regulatory region of the <n>IL-2</n> promoter, called <n>CD28</n>-responsive element. Stimulation of T-cells by agonistic anti-<n>CD28</n> antibodies in conjunction with phorbol 12-myristate 13-acetate (PMA)- or TcR-derived signals induces the enhanced activation of the transcription factor <n>NF- kappa B</n>. Here we report that <n>CD28</n> engagement, however, exerts opposite effects on the transcription factor <n>AP-1</n>. Whereas anti-<n>CD28</n> together with PMA increased the DNA binding and trans-activation activity of <n>NF- kappa B</n>, PMA-induced activation of <n>AP-1</n> was significantly suppressed. The inhibitory effect exerted by anti-<n>CD28</n> was observed at the level of DNA binding as well as in functional reporter-gene assays. These results suggest that the two transcription factors are independently regulated and may perform different functions during T-cell activation.
95187990><n>HIV-1 Nef</n> leads to inhibition or activation of T cells depending on its intracellular localization. <n>Nef</n> of primate lentiviruses is required for viremia and progression to AIDS in monkeys. Negative, positive, and no effects of <n>Nef</n> have also been reported on viral replication in cells. To reconcile these observations, we expressed a hybrid CD8-<n>Nef</n> protein in Jurkat cells. Two opposite phenotypes were found, which depended on the intracellular localization of <n>Nef</n>. Expressed in the cytoplasm or on the cell surface, the chimera inhibited or activated early signaling events from the T cell antigen receptor. Activated Jurkat cells died by apoptosis, and only cells with mutated nef genes expressing truncated <n>Nefs</n> survived, which rendered <n>Nef</n> nonfunctional. These mutations paralleled those in other viral strains passaged in vitro. Not only do these positional effects of <n>Nef</n> reconcile diverse phenotypes of <n>Nef</n> and suggest a role for its N-terminal myristylation, but they also explain effects of <n>Nef</n> in HIV infection and progression to AIDS.
94253152>An intricate arrangement of binding sites for the Ets family of transcription factors regulates activity of the alpha 4 integrin gene promoter. alpha 4 integrins mediate cell-cell and cell-extracellular matrix interactions that are critical for maturation and function of the immune system as well as differentiation of skeletal muscle. Here we examine molecular mechanisms controlling the pattern of <n>alpha 4</n> expression. The activity of constructs containing 5' deletion mutants of the <n>alpha 4</n> gene promoter was compared in transfection assays into cell lines that express <n>alpha 4</n> and cell lines that do not. The sequence between position -42 and -76 base pairs (bp) was required for efficient transcription in cells that express <n>alpha 4</n>, but it showed no activity in HeLa cells, which do not express <n>alpha 4</n>. Three binding sites for the Ets family of transcription factors are found in this region: two adjacent sites at positions -50 and -54 bp and a more 5' site at position -67 bp. Using a series of constructs containing deletions and mutations in this region, we found that the 3'-most site alone was sufficient for binding <n>GA-binding protein alpha</n> (<n>GABP alpha</n>)/<n>GABP beta</n> and for a low level of transcriptional activation. When all three sites were present, a second complex "a" was detected, which contains an unknown member of the Ets family. Formation of complex a was cell-type specific and correlated with a high level of transcription. Deletion of the 5'-most Ets site had no effect on binding to GABP alpha/GABP beta, but it eliminated a. Concomitant with this loss of a, a new Ets-1-containing complex "c" appeared. Complex c substituted efficiently for complex a in transcriptional activation. We conclude that although neither of the two 5'-most Ets sites alone binds nuclear protein, they appear to act as modulators which control the pattern of Ets proteins that bind the <n>alpha 4</n> gene promoter. This arrangement of Ets sites, coupled with the tissue- and developmental- specific expression of Ets members, likely play a key role in defining the pattern of <n>alpha 4 integrin</n>.
95011022>Mechanism of antiandrogen action: conformational changes of the receptor. Androgen receptor mRNA was translated in vitro, and androgen- and antiandrogen-bound receptor complexes were studied using limited proteolytic digestion by <n>trypsin</n>. Partial proteolysis of <n>androgen-bound receptor protein</n> resulted in a <n>29-kDa proteolysis-resisting fragment</n>, whereas antiandrogen binding stabilised a <n>35-kDa fragment</n>. Both fragments contain the entire ligand binding domain, and the <n>35-kDa fragment</n> extended into the hinge region of the receptor. Several antiandrogens show agonistic properties for a <n>mutated androgen receptor</n> (LNCaP cell variant); <n>trypsin</n> digestion of <n>antiandrogen-bound mutated receptor</n> also resulted in a <n>29-kDa fragment</n>. Our results point to an important difference between antiandrogens and antagonists of other steroid hormone receptors. Antiandrogens result in protection of both the hinge region and C-terminus of the androgen receptor agonist proteolytic attack, whereas other studies showed that antiestrogens and antiprogestagens expose the C-terminal end of the ligand binding domain of their respective receptors to <n>protease</n>. Differences in conformation of the hinge region distinguish androgen-bound from antiandrogen-bound receptor complexes, which represents an important feature of antiandrogen action.
94209877>Activation of <n>nuclear factor kappa B</n> in human neuroblastoma cell lines. The <n>nuclear factor kappa B</n> (<n>NF-kappa B</n>) is a eukaryotic transcription factor. In B cells and macrophages it is constitutively present in cell nuclei, whereas in many other cell types, <n>NF-kappa B</n> translocates from cytosol to nucleus as a result of transduction by <n>tumor necrosis factor alpha</n> (<n>TNF alpha</n>), phorbol ester, and other polyclonal signals. Using neuroblastoma cell lines as models, we have shown that in neural cells <n>NF-kappa B</n> was present in the cytosol and translocated into nuclei as a result of <n>TNF alpha</n> treatment. The <n>TNF alpha</n>-activated <n>NF-kappa B</n> was transcriptionally functional. <n>NF-kappa B</n> activation by <n>TNF alpha</n> was not correlated with cell differentiation or proliferation. However, reagents such as nerve growth factor (NGF) and the phorbol ester phorbol 12-myristate 13-acetate (PMA), which induce phenotypical differentiation of the SH-SY5Y neuroblastoma cell line, activated <n>NF- kappa B</n>, but only in that particular cell line. In a NGF-responsive rat pheochromocytoma cell line, PC12, PMA activated <n>NF-kappa B</n>, whereas NGF did not. In other neuroblastoma cell lines, such as SK-N-Be(2), the lack of PMA induction of differentiation was correlated with the lack of <n>NF-kappa B</n> activation. We found, moreover, that in SK-N-Be(2) cells <n>protein kinase C</n> (<n>PKC</n>) enzymatic activity was much lower compared with that in a control cell line and that the low <n>PKC</n> enzymatic activity was due to low <n>PKC</n> protein expression. <n>NF-kappa B</n> was not activated by retinoic acid, which induced morphological differentiation of all the neuroblastoma cell lines used in the present study. Thus, <n>NF-kappa B</n> activation was not required for neuroblastoma cell differentiation. Furthermore, the results obtained with <n>TNF alpha</n> proved that <n>NF-kappa B</n> activation was not sufficient for induction of neuroblastoma differentiation.
94217726><n>ERP</n>, a new member of the ets transcription factor/oncoprotein family: cloning, characterization, and differential expression during B- lymphocyte development. The ets gene family encodes a group of proteins which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy- chain (IgH) enhancer, pi and microB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the pi or the microB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in all members of the ets family. We have cloned the gene for a new ets- related transcription factor, <n>ERP</n> (<n>ets-related protein</n>), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The <n>ERP</n> protein contains a region of high homology with the ETS DNA-binding domain common to all members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and <n>SAP-1</n> genes, a subgroup of the ets gene family that interacts with the serum response factor. Full-length <n>ERP</n> expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables <n>ERP</n> to interact with a variety of ets- binding sites including the E74 site, the IgH enhancer pi site, and the lck promoter ets site, suggesting a carboxy-terminal negative regulatory domain. At least three <n>ERP</n>-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, <n>ERP</n> is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH pi site. These data suggest that <n>ERP</n> might play a role in B-cell development and in IgH gene regulation.
94281746>Gene for a tissue-specific transcriptional activator (EBF or Olf-1), expressed in early B lymphocytes, adipocytes, and olfactory neurons, is located on human chromosome 5, band q34, and proximal mouse chromosome 11. Murine B lymphocytes, adipocytes, and olfactory neurons contain a DNA- binding protein that participates in the regulation of genes encoding tissue-specific components of signal transduction. Purification and cloning of this protein, termed early <n>B-cell factor</n> (EBF), from murine B lymphocytes and independent cloning of a protein, termed Olf-1, from olfactory neuronal cells revealed virtual complete amino acid sequence identity between these proteins. As a first step towards identifying a human genetic disorder or mouse mutation for which EBF could be a candidate gene, we have chromosomally mapped the corresponding locus in both species. By Southern hybridization analyses of somatic cell hybrid panels with murine cDNA probe, fluorescence chromosomal in situ hybridization (FISH) of human genomic clones, and analysis of recombinant inbred mouse strains, we have found single sites for EBF homologous sequences on human Chromosome (Chr) 5, band q34, and on proximal mouse Chr 11, in an evolutionarily conserved region.
94183204><n>Calcineurin</n> activates transcription from the GM-CSF promoter in synergy with either <n>protein kinase C</n> or <n>NF-kappa B</n>/<n>AP-1</n> in T cells. Two cis-acting elements GM-kappa B/GC-box and CLE0, of the <n>granulocyte- macrophage colony-stimulating factor</n> (<n>GM-CSF</n>) gene are required for maximal induction in Jurkat T cells by costimulation with phorbol-12- myristate acetate (PMA) and Ca2+ ionophore (A23187). The GM-kappa B sequence is recognized by <n>NF-kappa B</n>, which is mainly induced by PMA. The CLE0 sequence interacts with factors, related to a PMA-induced <n>AP-1</n> and a <n>PMA/A23187-induced NF-AT</n>. We examined whether signal transducing components in T cells can activate transcription of the GM-CSF gene. Cotransfection of NF-kappa B (p50/p65)- or AP-1 (c-Jun/c-Fos)- expression vectors into Jurkat cells with a luciferase reporter containing the GM-CSF promoter did not stimulate transcription from the GM-CSF promoter. In contrast, cotransfection with a combination of <n>NF- kappa B</n> and <n>AP-1</n> significantly augmented transcription from the GM-CSF promoter containing the GM-kappa B/GC-box and the CLE0 (<n>AP-1</n>/NF-AT). Expression of a constitutively active <n>calcineurin</n> (<n>CN</n>), a Ca2+/calmodulin-dependent protein phosphatase, potentiated by two fold the transcriptional activation by <n>NF-kappa B</n>/<n>AP-1</n>. Both constitutively active forms of <n>CN</n> and <n>protein kinase C</n> (<n>PKC</n>) synergistically activated transcription from the GM-CSF promoter. These results suggest that cooperation among NF-kappa B-, AP-1- and NF-AT-binding sequences is required for induction of the GM-CSF gene through PKC- and Ca2+- signaling pathways downstream of T cell activation.
94285937>Nonpituitary human prolactin gene transcription is independent of <n>Pit-1</n> and differentially controlled in lymphocytes and in endometrial stroma. Expression of the human PRL (hPRL) gene in extrapituitary sites such as the uterus (decidualized endometrial stroma and myometrium) and cells of the hematopoietic lineage is directed by an alternative promoter which is located approximately 6 kilobases (kb) upstream of the pituitary-specific start site. In order to delineate the tissue- specific mechanisms governing the control of nonpituitary PRL gene expression, we have cloned and sequenced 3 kb 5'-flanking DNA of the upstream decidual/lymphoid (dPRL) promoter. Based on sequence homology we identified two binding motifs for <n>Pit-1</n> and seven half-sites for glucocorticoid receptor/progesterone receptor (PR) binding. We focused our studies on the role of <n>Pit-1</n> and of PR as potential transcriptional regulators, since the POU domain protein <n>Pit-1</n> is essential in the control of pituitary <n>PRL</n> expression, and progesterone induces decidual transformation of the endometrial stroma, a differentiation process during which the decidual PRL gene is activated. We demonstrate in a variety of cell types, including lymphocytes and endometrial stroma, that <n>Pit-1</n> is not involved in the regulation of dPRL promoter/reporter gene constructs carrying 3 kb 5'-flanking DNA. Our experiments also show that activated PR does not confer direct transcriptional control on the dPRL promoter. When we compared the activity of the transfected dPRL promoter in PRL-secreting and nonsecreting lymphoid cells, we found that the 3 kb 5'-flanking region of the dPRL promoter did not contain elements restricting expression to only those lymphocytes that produce PRL but allowed expression of fusion reporter genes irrespective of the status of the endogenous PRL gene. This was in sharp contrast to endometrial cells where 3 kb 5'-flanking DNA conferred strong transcriptional activation on the dPRL promoter in decidualized endometrial stromal cells actively secreting <n>PRL</n>, but did not allow transcription in undifferentiated non-PRL-secreting endometrial stromal cells. Activation of the dPRL promoter construct in these undifferentiated cells could however be induced by the addition of cAMP, in the absence of progesterone, suggesting that a signal transduced through the cAMP signaling pathway is a primary inducer of decidual PRL gene expression
94365323>Signals and nuclear factors that regulate the expression of interleukin- 4 and interleukin-5 genes in helper T cells. Mouse thymoma line EL-4 cells produce cytokines such as <n>interleukin (IL)-2</n>, <n>IL-3</n>, <n>IL-4</n>, <n>IL-10</n>, and <n>granulocyte-macrophage colony- stimulating factor</n> in response to phorbol 12-myristate 13-acetate (PMA). EL-4 cells also produce low levels of <n>IL-5</n> when stimulated by PMA alone; however, cAMP greatly augments PMA-dependent <n>IL-5</n> production. A transient transfection assay revealed that two signals, PMA and cAMP, are required for optimal activation of the <n>IL-5</n> promoter. In contrast, cAMP almost completely inhibited the PMA-dependent activation of the endogenous <n>IL-2</n> gene, as well as the transfected <n>IL-2</n> promoter. These results indicate that the <n>IL-5</n> gene is positively regulated by cAMP in a manner opposite to that for the <n>IL-2</n> gene. One of the nuclear factors (NFs) that regulates the response of the <n>IL-5</n> promoter to cAMP and PMA has properties similar to NF for activated t cell. The P sequence of the <n>IL-4</n> gene, defined as a responsive element for PMA and calcium ionophore (A23187), shares sequence similarity with the NF kappa B and the NF-activated T cell binding sites. We attempted to determine whether <n>NF(P)</n>, a nuclear factor specific for the P sequence, is related to <n>NF-kappa B</n> and <n>nuclear factor for activated T cell</n> (<n>NF-AT</n>). In electromobility shift assays both <n>NF-kappa B</n> (P65 or P65/P50 heterodimer) and <n>NF-AT</n> bound to the P sequence. However, sequence specificity of <n>NF-AT</n> was more similar to that of <n>NF(P)</n>, and only a small amount of P65 was detected in <n>NF(P)</n>. These results indicate that a component or components of <n>NF-AT</n> have the potential to reconstitute <n>NF(P)</n>, whereas <n>NF-kappa B</n> alone does not account for <n>NF(P)</n> in Jurkat crude extract. Taken together, these results suggest that NF- AT-like factors are involved in the regulation of IL-4 and IL-5 genes.
94339116>Solution structure of a POU-specific homeodomain: 3D-NMR studies of human B-cell transcription factor <n>Oct-2</n>. The POU DNA-binding motif defines a conserved family of eukaryotic transcription factors involved in regulation of gene expression. This bipartite motif consists of an N-terminal POU-specific domain (POUs), a flexible linker, and a C-terminal POU-specific homeodomain (POUHD). Here we describe the solution structure of a POU-specific homeodomain. An NMR model is obtained from <n>Oct-2</n>, a human B-cell specific transcription factor which participates in the regulation of immunoglobulin genes. A fragment of <n>Oct-2</n> containing POUHD and an adjoining linker was expressed in Escherichia coli and characterized by three-dimensional nuclear magnetic resonance (3D-NMR) spectroscopy. Complete 1H and 15N resonance assignment of the POUHD moiety is presented. The POUHD solution structure, as calculated by distance geometry and simulated annealing (DG/SA), is similar to that of canonical homeodomains. A salient difference between solution and crystal structures is observed in the C-terminal segment of alpha-helix 3 (the HTH recognition helix), which is not well ordered in solution. Because this segment presumably folds upon specific DNA binding, its flexibility in solution may reduce the intrinsic DNA affinity of POUHD in the absence of POUs.
94303183>NF-kappa B-dependent and -independent pathways of HIV activation in a chronically infected T cell line. J delta K cells were isolated as a chronically infected survivor cell line, following infection of Jurkat CD4+ T cells with dl-NF, a mutated strain of human immunodeficiency virus type 1 (HIV-1) containing a deletion of the long terminal repeat (LTR) <n>NF-kappa B</n> sites. J delta K cells exhibited very low levels of constitutive HIV production. HIV-1 expression was activated from J delta K cells by treatment with phorbol myristate acetate (PMA), sodium butyrate (NaB), or hexamethylene bisacetamide (HMBA), but not <n>tumor necrosis factor alpha</n> (<n>TNF-alpha</n>), confirming the role of <n>NF-kappa B</n> in mediating <n>TNF-alpha</n> induction of HIV transcription. The strong induction of HIV expression by NaB or HMBA in J delta K cells clearly demonstrates the existence of <n>NF-kappa B</n>-independent mechanisms of HIV activation in chronically infected cells. J delta K cells may provide a useful model for characterizing <n>NF- kappa B</n>-independent transcriptional activation of the HIV LTR.
94265262>JNK is involved in signal integration during costimulation of T lymphocytes. T lymphocyte activation and <n>interleukin-2</n> (<n>IL-2</n>) production require at least two signals, generated by phorbol ester (TPA) and Ca2+ ionophore or costimulation of the T cell receptor (TCR) and the <n>CD28 auxiliary receptor</n>. We investigated how these stimuli affect mitogen activated protein (MAP) kinases. Full activation of the MAP kinases that phosphorylate the Jun activation domain, <n>JNK1</n> and <n>JNK2</n>, required costimulation of T cells with either TPA and Ca2+ ionophore or antibodies to TCR and <n>CD28</n>. Alone, each stimulus resulted in little or no activation. Similar to its effect on <n>IL-2</n> induction, cyclosporin A (CsA) inhibited the synergistic activation of <n>JNK</n>, and a competitive inhibitor of <n>Jun</n> phosphorylation by <n>JNK</n> inhibited <n>IL-2</n> promoter activation. By contrast, the MAP kinases <n>ERK1</n> and <n>ERK2</n> were fully activated by TPA or TCR stimulation and were not affected by Ca2+, CD28, or CsA. Hence, integration of signals that lead to T cell activation occurs at the level of <n>JNK</n> activation.
95023587>Inhibition of rat splenocyte proliferation with methylprednisolone: in vivo effect of liposomal formulation. The effect of a liposomal formulation of methylprednisolone (MPL) on the inhibition of lymphocyte proliferation in spleen cells was investigated following IV dosing in rats. Liposomes do not alter the suppressive action of MPL when placed in lymphocyte culture. Rat splenocytes were found to have greater sensitivity to MPL (EC50 = 7.9 nM) than do human peripheral blood lymphocytes (EC50 = 28 nM). In vivo studies in rats utilized 2 mg/kg IV bolus doses of liposomal MPL compared to drug in solution. Animals were sacrificed at various times post-dosing until 120 h, spleen was excised and, after incubation of lymphocytes with <n>PHA</n>, splenocyte blastogenic responses were assessed by measuring cellular incorporation of 3H-thymidine. The suppressive effect of liposomal MPL in comparison with free drug was significantly prolonged (&gt; 120 h vs &lt; 18 h). Inhibition effects versus time were described by a pharmacodynamic model using MPL concentrations in plasma as an input function. A nonlinear relationship was found between suppression of splenocyte proliferation and the concentration of bound glucocorticoid receptors in spleen. Only partial receptor occupancy accompanied complete lymphocyte suppression. The suppression of endogenous corticosterone in plasma for both treatments was similar with values from L-MPL rats returning to baseline after 24 h. These results demonstrate enhanced efficacy of local immunosuppression by targeting spleen with liposomal MPL.
94217690>Function of <n>NF-kappa B</n>/Rel binding sites in the major histocompatibility complex class II invariant chain promoter is dependent on cell-specific binding of different <n>NF-kappa B</n>/Rel subunits. The promoter of the human major histocompatibility complex class II- associated invariant-chain gene (Ii) contains two <n>NF-kappa B</n>/Rel binding sites located at -109 to -118 (Ii kappa B-1) and -163 to -172 (Ii kappa B-2) from the transcription start site. We report here that the differential function of each of these <n>NF-kappa B</n>/Rel sites in several distinct cell types depends on cell-specific binding of <n>NF- kappa B</n>/Rel transcription factors. Ii kappa B-1 is a positive regulatory element in B-cell lines and in the Ii-expressing T-cell line, H9, but acts as a negative regulatory element in myelomonocytic and glia cell lines. In vivo protein-DNA contacts are detectable at Ii kappa B-1 in cell lines in which this site is functional as either a positive or negative regulator. Electrophoretic mobility supershift assays determine that members of the <n>NF-kappa B</n>/Rel family of transcription factors can bind to this site in vitro and that DNA- binding complexes that contain p50, p52, p65, and cRel correlate with positive regulation whereas the presence of p50 correlates with negative regulation. Ii kappa B-2 is a site of positive regulation in B- cell lines and a site of negative regulation in H9 T cells, myelomonocytic, and glial cell lines. In vivo occupancy of this site is observed only in the H9 T-cell line. Again, in vitro supershift studies indicate that the presence of p50, p52, p65, and cRel correlates with positive function whereas the presence of only p50 and p52 correlates with negative function. This differential binding of specific <n>NF-kappa B</n>/Rel subunits is likely to mediate the disparate functions of these two <n>NF-kappa B</n>/Rel binding sites.
94338593>Epstein-Barr virus (EBV) replicative gene expression in tumour cells of AIDS-related non-Hodgkin's lymphoma in relation to <n>CD4</n> cell number and antibody titres to EBV. OBJECTIVE: To determine whether activation of Epstein-Barr virus (EBV) replication in tumour cells of AIDS-related non-Hodgkin's lymphoma (ARNHL) is correlated with <n>CD4+</n> cell counts and influences antibody response to EBV [<n>anti-Z Epstein-Barr replicative activator</n> (<n>ZEBRA</n>), anti-early antigen (EA), anti-viral capsid antigen (VCA)]. DESIGN: Retrospective study based on immunohistochemistry and in situ hybridization to detect EBV replicative gene products in tissue samples from patients affected by ARNHL and correlation with <n>CD4+</n> cell counts and results of EBV serology (including anti-<n>ZEBRA</n> activity) in sera from the same patients. METHODS: Seventeen out of 22 cases of ARNHL were selected for the presence of EBV [Epstein-Barr early region (EBER) RNA-positive]. Immunohistochemistry was performed with anti-<n>ZEBRA</n>, anti- EA-restricted, anti-VCA antibodies and in situ hybridization with BHLF1/NotI oligoprobes on tumour samples. Results were statistically correlated with those of <n>CD4+</n> cell counts (17 out of 17) and with anti- EBV antibody titres (13 out of 17) assessed using standard immunofluorescence method and enzyme-linked immunosorbent assay procedure using recombinant <n>ZEBRA</n> protein and synthetic peptides as antigens. RESULTS: <n>BZLF1</n> (<n>ZEBRA</n>) or early gene products (<n>EA-R</n> and <n>EA- D/BHLF1/NotI</n>) were detected in a small proportion (&lt; 0.01-5%) of tumour cells in eight of these 17 cases by immunohistochemistry and in situ hybridization. Demonstration of replicative gene expression did not correlate with either low <n>CD4+</n> cell counts (P &gt; 0.05) or anti-EBV antibody titres (P &gt; 0.05). Anti-<n>ZEBRA</n> activity was not significantly increased in patients affected with ARNHL, the cells of which expressed replicative gene products (P &gt; 0.05). CONCLUSION: The degree of immunodeficiency does not clearly enhance replicative gene expression in tumour cells of ARNHL. EBV serology, including anti-<n>ZEBRA</n> activity, is not a reliable tool for predicting the occurrence of such proliferations.
94289333>Effects of <n>prostaglandin E2</n> on Th0-type human T cell clones: modulation of functions of nuclear proteins involved in cytokine production. The effects of <n>prostaglandin E2</n> (<n>PGE2</n>) on cytokine production and proliferation of the CD4+ human helper T cell clone SP-B21 were investigated. In cells stimulated with <n>anti-CD3 mAb</n>, <n>PGE2</n> inhibited cell proliferation and the production of all the cytokines examined. Addition of <n>rIL-2</n> fully restored the proliferative response and partially restored the production of <n>IL-4</n> and <n>IL-5</n>, but not that of other cytokines. In contrast, in cells stimulated with phorbol myristate acetate (PMA)/A23187, <n>PGE2</n> enhanced the production of <n>IL-4</n> and <n>IL-5</n>, and only partially inhibited the production of other cytokines. Therefore, the effects of <n>PGE2</n> vary depending on the mode of T cell activation, and the <n>IL-4</n> and <n>IL-5</n> are regulated differently from other cytokines. In a mobility shift assay, only the <n>NF-kappa B</n> (p50/p50) homodimer was observed in a complex formed with the kappa B sequence in unstimulated SP-B21 cells. When cells were stimulated with <n>anti-CD3 mAb</n> or PMA/A23187, a complex formation of <n>NF-kappa B</n> (p50/p65) heterodimer with the kappa B sequence was induced. Interestingly, <n>PGE2</n> or di-butyryl (Bt2)cAMP abolished the binding of <n>NF-kappa B</n> (p50/p65) heterodimer to the kappa B sequence in cells stimulated with <n>anti-CD3 mAb</n> but not with PMA/A23187. Our results suggest that the target of <n>PGE2</n> action is a component in the signal transduction pathway leading to the activation of <n>protein kinase C</n>. However, the inhibition of the T cell activation signals by <n>PGE2</n> is selective. <n>PGE2</n> enhanced the complex formation with NF-AT, AP-1 and CLE0 sequences when the cells were activated by either <n>anti-CD3 mAb</n> or PMA/A23187 stimulation. It seems therefore that <n>PGE2</n>, by elevating cAMP levels, interferes with the activation pathway for <n>NF-kappa B</n> but not for <n>NF-AT</n>, <n>AP-1</n> or <n>CLE0 binding protein</n>.
94183209>Characterization of <n>NF(P)</n>, the nuclear factor that interacts with the regulatory P sequence (5'-CGAAAATTTCC-3') of the human interleukin-4 gene: relationship to <n>NF-kappa B</n> and <n>NF-AT</n>. The P sequence of the human interleukin-4 (<n>IL-4</n>) gene, which was defined as a responsive element for phorbol 12-myristate 13-acetate and calcium ionophore (A23187) in Jurkat T cells, shares sequence similarity with the <n>NF-kappa B</n> and the <n>NF-AT</n> binding sites. We examined whether <n>NF(P)</n>, a nuclear factor specific for the P sequence, is related to <n>NF-kappa B</n> and <n>NF-AT</n>. <n>NF-kappa B</n> (P65 or P65/P50 heterodimer) bound to the P sequence in electrophoretic mobility shift assays (EMSA) and activated transcription through the P sequence when expression plasmids were cotransfected with P sequence-driven reporter plasmids in Jurkat T cells. In EMSAs, <n>NF(P)</n> binding was inhibited by the unlabeled <n>NF-AT</n> binding site but not by the unlabeled AP1 binding site and purified NF- AT contained an activity that bound to the P sequence. Both mobility shift and sequence specificity of <n>NF-AT</n> were similar to those of <n>NF(P)</n> and only a small amount of P65 was detected in <n>NF(P)</n> in crude nuclear extracts. These results indicate that the component(s) of <n>NF-AT</n> has the potential to reconstitute <n>NF(P)</n> whereas <n>NF-kappa B</n> alone cannot account for <n>NF(P)</n> in crude extracts. Unlike <n>NF-AT</n>, <n>NF(P)</n> does not contain <n>AP1</n> as its DNA binding component.
94289294>Activation of early growth response 1 gene transcription and <n>pp90rsk</n> during induction of monocytic differentiation. The present work has studied mechanisms responsible for induction of early growth response 1 (EGR-1) gene expression during monocytic differentiation of U-937 myeloid leukemia cells. Differentiation of U- 937 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of the serine/threonine <n>protein kinase C</n>, was associated with transcriptional activation of EGR-1 promoter-reporter constructs. The EGR-1 promoter contains six CC(A/T)6GG (CArG) motifs. The two 5'-most distal CArG sequences conferred TPA inducibility. In contrast, there was little effect of TPA on EGR-1 transcription in a TPA-resistant U- 937 cell variant, designated TUR. Treatment of both U-937 and TUR cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases 1 and 2A, was associated with induction of monocytic differentiation and EGR-1 transcription through the 5'-most CArG element. Since these findings supported the involvement of serine/threonine protein phosphorylation in the regulation of EGR-1 expression, we studied activation of the 40S ribosomal protein S6 serine/threonine kinases, pp70S6K and <n>pp90rsk</n>. Although both kinases participate in regulating cell growth, there was no detectable activation of pp70S6K during TPA- or okadaic acid-induced monocytic differentiation. Moreover, rapamycin, an inhibitor of pp70S6K activation, had no effect on induction of EGR-1 expression. In contrast, analysis of <n>pp90rsk</n> activity by phosphorylation of a peptide derived from <n>S6 protein</n> demonstrated stimulation of this kinase in TPA- treated U-937, and not TUR, cells. Okadaic acid treatment of both cell types was associated with activation of <n>pp90rsk</n>
95001805>Antioxidants inhibit monocyte adhesion by suppressing <n>nuclear factor- kappa B</n> mobilization and induction of <n>vascular cell adhesion molecule-1</n> in endothelial cells stimulated to generate radicals. Cell adhesion to endothelial cells <n>stimulated by tumor necrosis factor- alpha</n> (<n>TNF</n>) is due to induction of surface receptors, such as <n>vascular cell adhesion molecule-1</n> (<n>VCAM-1</n>). The antioxidant pyrrolidine dithiocarbamate (PDTC) specifically inhibits activation of <n>nuclear factor-kappa B</n> (<n>NF-kappa B</n>). Since kappa B motifs are present in <n>VCAM-1</n> and <n>intercellular adhesion molecule-1</n> (<n>ICAM-1</n>) promoters, we used PDTC to study the regulatory mechanisms of <n>VCAM-1</n> and <n>ICAM-1</n> induction and subsequent monocyte adhesion in TNF-treated human umbilical vein endothelial cells (HUVECs). PDTC or N-acetylcysteine dose dependently reduced TNF-induced <n>VCAM-1</n> but not <n>ICAM-1</n> surface protein (also in human umbilical arterial endothelial cells) and mRNA expression (by 70% at 100 mumol/L PDTC) in HUVECs as assessed by flow cytometry and polymerase chain reaction. Gel-shift analysis in HUVECs demonstrated that PDTC prevented <n>NF-kappa B</n> mobilization by TNF, suggesting that only <n>VCAM-1</n> induction was controlled by <n>NF-kappa B</n>. Since HUVECs released superoxide anions in response to TNF, and H2O2 induces <n>VCAM-1</n>, PDTC may act as a radical scavenger. Although <n>ICAM-1</n> induction was unaffected, inhibitors of <n>NADPH oxidase</n> (apocynin) or <n>cytochrome P-450</n> (SKF525a) suppressed <n>VCAM-1</n> induction by TNF, revealing that several radical-generating systems are involved in its regulation. PDTC, apocynin, or SKF525a decreased adhesion of monocytic U937 cells to TNF- treated HUVECs (by 75% at 100 mumol/L PDTC). Inhibition by anti-<n>VCAM-1</n> monoclonal antibody <n>1G11</n> indicated that U937 adhesion was <n>VCAM-1</n> dependent and suppression by antioxidants was due to reduced <n>VCAM-1</n> induction. (ABSTRACT TRUNCATED AT 250 WORDS)
94344126>Displacement of an E-box-binding repressor by basic helix-loop-helix proteins: implications for B-cell specificity of the immunoglobulin heavy-chain enhancer. The activity of the immunoglobulin heavy-chain (IgH) enhancer is restricted to B cells, although it binds both B-cell-restricted and ubiquitous transcription factors. Activation of the enhancer in non-B cells upon overexpression of the basic helix-loop-helix (bHLH) protein <n>E2A</n> appears to be mediated not only by the binding of <n>E2A</n> to its cognate E box but also by the resulting displacement of a repressor from that same site. We have identified a "two-handed" zinc finger protein, denoted <n>ZEB</n>, the DNA-binding specificity of which mimics that of the cellular repressor. By employing a derivative E box that binds <n>ZEB</n> but not <n>E2A</n>, we have shown that the repressor is active in B cells and the IgH enhancer is silenced in the absence of binding competition by bHLH proteins. Hence, we propose that a necessary prerequisite of enhancer activity is the B-cell-specific displacement of a <n>ZEB</n>-like repressor by bHLH proteins.
94329809>Inhibition of <n>NF-kappa B</n> by sodium salicylate and aspirin [see comments] The transcription factor <n>nuclear factor-kappa B</n> (<n>NF-kappa B</n>) is critical for the inducible expression of multiple cellular and viral genes involved in inflammation and infection including <n>interleukin-1</n> (<n>IL-1</n>), <n>IL-6</n>, and adhesion molecules. The anti-inflammatory drugs sodium salicylate and aspirin inhibited the activation of <n>NF-kappa B</n>, which further explains the mechanism of action of these drugs. This inhibition prevented the degradation of the <n>NF-kappa B</n> inhibitor, <n>I kappa B</n>, and therefore <n>NF-kappa B</n> was retained in the cytosol. Sodium salicylate and aspirin also inhibited <n>NF-kappa B</n>-dependent transcription from the Ig kappa enhancer and the human immunodeficiency virus (HIV) long terminal repeat (LTR) in transfected T cells.
95152420>Effects of the antisense myb expression on hemin- and erythropoietin- induced erythroid differentiation of K562 cells. In order to elucidate the role of c-myb gene in erythroid differentiation of K562 cell induced by <n>hemin</n> (<n>Hm</n>) and <n>erythropoietin</n> (<n>Epo</n>), we constructed recombinant plasmid that could produce antisense myb RNA after induction with dexamethasone. During treatment with <n>Hm</n>, K562 cells constitutively expressed c-myb mRNA, and 50% of them began to synthesize <n>hemoglobin</n> (<n>Hb</n>). Expression of antisense myb RNA reduced the amount of c-myb mRNA, and the percentage of <n>Hb</n>-synthesizing cells was decreased to 20%. In the presence of <n>Epo</n>, c-myb mRNA declined and 20% of K562 cells synthesized <n>Hb</n> regardless of antisense myb RNA expression. It is suggested that constitutive expression of c-myb mRNA is necessary for <n>Hm</n>-induced differentiation, and that a decrease in the amount of c-myb mRNA induced by antisense myb RNA expression suppresses <n>Hm</n>-induced differentiation. The amount of c-myb mRNA in K562 cells was reduced during the differentiation induced by <n>Epo</n>. Expression of GATA-1 mRNA was almost constant during <n>Hm</n>-induced differentiation, but increased during <n>Epo</n> treatment. It is supposed that the mechanism of <n>Hm</n>- induced differentiation is distinguished from that of <n>Epo</n>-induced differentiation in K562 cells.
94259822>Prenatal immune challenge alters the hypothalamic-pituitary- adrenocortical axis in adult rats. We investigated whether non-abortive maternal infections would compromise fetal brain development and alter hypothalamic-pituitary- adrenocortical (HPA) axis functioning when adult. To study putative teratogenic effects of a T cell-mediated immune response versus an endotoxic challenge, 10-d-pregnant rats received a single intraperitoneal injection of 5 x 10(8) human red blood cells (HRBC) or gram-negative bacterial endotoxin (Escherichia coli LPS: 30 micrograms/kg). The adult male progeny (3 mo old) of both experimental groups showed increased basal plasma corticosterone levels. In addition, after novelty stress the HRBC group, but not the LPS group, showed increased ACTH and corticosterone levels. Both groups showed substantial decreases in mineralocorticoid (MR) and glucocorticoid receptor (GR) levels in the hippocampus, a limbic brain structure critical for HPA axis regulation, whereas GR concentrations in the hypothalamus were unchanged and in anterior pituitary were slightly increased. HRBC and LPS indeed stimulated the maternal immune system as revealed by specific anti-HRBC antibody production and enhanced IL-1 beta mRNA expression in splenocytes, respectively. This study demonstrates that a T cell-mediated immune response as well as an endotoxic challenge during pregnancy can induce anomalies in HPA axis function in adulthood. Clinically, it may be postulated that disturbed fetal brain development due to prenatal immune challenge increases the vulnerability to develop mental illness involving inadequate responses to stress.
95003681>A low NM23.H1 gene expression identifying high malignancy human melanomas. The NM23 gene has been proposed as a metastasis-suppressor gene, and its use has been suggested as prognostic factor. <n>NM23</n> was identified in a system of murine melanoma cell lines, in which an inverse relationship was found between <n>NM23</n> expression and metastatic ability. In a human malignant melanoma study <n>NM23</n> expression was found to be significantly lower in metastases that developed less than 24 months after diagnosis of the primary tumours. The present paper studies the expression of the <n>NM23</n>.H1 gene in cell lines which derive from primary or metastatic human malignant melanomas in relation to staging, infiltration degree, lymphocytic infiltration, cell morphology, cell pigmentation, karyotype, and disease-free survival. The level of mRNA expression of the <n>NM23</n> gene is significantly lower in cell lines that derive from more infiltrating primary melanomas than in cell lines obtained from less infiltrating tumours. Moreover, cell lines derived from tumours of patients with a disease-free survival of more than 24 months (24-58 months) express the <n>NM23</n> gene at higher levels than cell lines obtained from melanomas of patients with a disease-free survival of less than 24 months (6-15 months).
94209654>Activation of a novel serine/threonine kinase that phosphorylates <n>c-Fos</n> upon stimulation of T and B lymphocytes via antigen and cytokine receptors. Ligation of <n>Ag receptors</n> in T and B lymphocytes initiates signal transduction cascades which alter the expression of genes that regulate cellular proliferation and differentiation. The transmission of signals from the membrane to the nucleus is mediated principally through the action of protein tyrosine and serine/threonine kinases. We have identified and characterized a novel serine/threonine kinase that phosphorylated the proto-oncogene product, <n>c-Fos</n>, and is termed <n>Fos kinase</n>. <n>Fos kinase</n> was rapidly activated after ligation of the CD3 and CD2 receptors in Jurkat and normal human T lymphocytes and in response to <n>IL-6</n> and anti-IgM in the human B cell lines AF10 and Ramos, respectively. The phorbol ester, PMA, was also a potent inducer of <n>Fos kinase</n> activity in all of the above populations, suggesting that <n>PKC</n> plays a role in the regulation of this enzyme. <n>Fos kinase</n> phosphorylates <n>c-Fos</n> at a site near the C-terminus, as well as a peptide derived from this region (residues 359-370, RKGSSSNEPSSD), and Fos peptide competitively inhibited <n>c-Fos</n> phosphorylation. <n>Fos kinase</n> was shown to be distinct from other identified serine/threonine kinases, including <n>protein kinase A</n>, <n>protein kinase C</n>, <n>casein kinase II</n>, MAP kinases, <n>p70S6K</n> and <n>p90RSK</n>. <n>Fos kinase</n> was purified by anion exchange chromatography and exhibited an apparent M(r) = 65,000 and isoelectric point = 6.1. <n>Fos kinase</n> may play a role in transcriptional regulation through its capacity to phosphorylate <n>c-Fos</n> at a site required for expression of the transcriptional transrepressive activity of this molecule. Moreover, its rapid activation suggests it may have a wider role within signal transduction cascades in lymphocytes.
94202265>Antigenic specificities of human CD4+ T-cell clones recovered from recurrent genital herpes simplex virus type 2 lesions. Lesions resulting from recurrent genital herpes simplex virus (HSV) infection are characterized by infiltration of CD4+ lymphocytes. We have investigated the antigenic specificity of 47 HSV-specific CD4+ T- cell clones recovered from the HSV-2 buttock and thigh lesions of five patients. Clones with proliferative responses to <n>recombinant truncated glycoprotein B</n> (<n>gB</n>) or <n>gD</n> of HSV-2 or purified natural gC of HSV-2 comprised a minority of the total number of HSV-specific clones isolated from lesions. The gC2- and gD2-specific CD4+ clones had cytotoxic activity. The approximate locations of the HSV-2 genes encoding HSV-2 type-specific <n>CD4+ antigens</n> have been determined by using HSV-1 x HSV-2 intertypic recombinant virus and include the approximate map regions 0.30 to 0.46, 0.59 to 0.67, 0.67 to 0.73, and 0.82 to 1.0 units. The antigenic specificity of an HLA DQ2-restricted, HSV-2 type-specific T-cell clone was mapped to amino acids 425 to 444 of <n>VP16</n> of HSV-2 by sequential use of an intertypic recombinant virus containing <n>VP16</n> of HSV-2 in an HSV-1 background, recombinant <n>VP16</n> fusion proteins, and synthetic peptides. Each of the remaining four patients also yielded at least one type-specific T-cell clone reactive with an HSV-2 epitope mapping to approximately 0.67 to 0.73 map units. The antigenic specificities of lesion-derived CD4+ T-cell clones are quite diverse and include at least 10 epitopes. Human T-cell clones reactive with <n>gC</n> and <n>VP16</n> are reported here for the first time.
95001542>Marked basophilia in acute promyelocytic leukaemia treated with all- trans retinoic acid: molecular analysis of the cell origin of the basophils. We report a patient with acute promyelocytic leukaemia who developed marked basophilia during all-trans retinoic acid treatment. We studied genomic DNA and RNA extracted from the patient's peripheral leucocytes in order to determine the origin of the basophils. The <n>RAR alpha</n> rearranged band in the Southern blot analysis and a chimaeric product of <n>PML-RAR alpha</n> by polymerase chain reaction were strongly visible before ATRA treatment, but at the time of maximal basophilia both of them were markedly diminished. These findings suggest that the basophils which appeared during the ATRA treatment are reactive in nature rather than a leukaemic clone.
94181565>Activation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by <n>nuclear factors NF-IL6</n> and <n>NF-kappa B</n> [published erratum appears in Proc Natl Acad Sci U S A 1995 Apr 11;92(8):3632] The host response to Mycobacterium tuberculosis includes granuloma formation at sites of infection and systemic symptoms. Cytokines have been identified by immunohistochemistry in granulomas in animal models of bacillus Calmette-Guerin (BCG) infection and are released by mononuclear phagocytes upon stimulation by mycobacterial proteins. In this regard, the cytokine <n>interleukin 6</n> (<n>IL-6</n>) may play a role in the clinical manifestations and pathological events of tuberculosis infection. We have demonstrated that <n>lipoarabinomannan</n> (<n>LAM</n>) from the mycobacterial cell wall, which was virtually devoid of lipopolysaccharide (LPS), stimulated mononuclear phagocytes to release <n>IL-6</n> in a dose-response manner. <n>LAM</n> and LPS were potent inducers of IL- 6 gene expression in peripheral blood monocytes. Both <n>LAM</n>- and LPS- inducible <n>IL-6</n> promoter activity was localized to a DNA fragment, positions -158 to -49, by deletion analysis and <n>chloramphenicol acetyltransferase</n> assay. Two <n>nuclear factor NF-IL6</n> (positions -153 to - 145 and -83 to -75) and one nuclear factor <n>NF-kappa B</n> (positions -72 to -63) motifs are present within this fragment. Site-directed mutagenesis of one or more of these motifs within the <n>IL-6</n> promoter demonstrated that each has positive regulatory activity and that they could act in a function- and orientation-independent manner. Deletion of all three elements abolished inducibility of <n>IL-6</n> promoter activity by both <n>LAM</n> and LPS. We conclude that the NF-IL6 and NF-kappa B sites mediate <n>IL-6</n> induction in response to both LPS and <n>LAM</n>, acting as bacterial or mycobacterial response elements
94375892>Regulation of <n>CD14</n> expression during monocytic differentiation induced with 1 alpha,25-dihydroxyvitamin D3. <n>CD14</n>, a monocyte/macrophage receptor for the complex of LPS and LPS binding protein, is a differentiation marker for the monocyte/macrophage lineage. We have analyzed the regulation of <n>CD14</n> expression during 1 alpha,25-dihydroxyvitamin D3 (VitD3)-induced monocytic differentiation. Using FACS, Northern blotting, and nuclear run-on analyses, we demonstrate that the up-regulation of <n>CD14</n> expression during monocytic cell maturation is regulated mainly at the level of gene transcription, and that new protein synthesis is required for <n>CD14</n> induction. We have recently cloned the <n>CD14</n> 5' upstream sequence and demonstrated its tissue-specific promoter activity. Using stable transfection of the monocytoid U937 cell line with a series of deletion mutants of the <n>CD14</n> 5' upstream sequence coupled to a reporter gene construct, we show that bp -128 to -70 is the critical region for the induction of <n>CD14</n> expression. This region contains two binding sites for the <n>Sp1 transcription factor</n>. A 3-bp mutation at the distal Sp1-binding site not only eliminates <n>Sp1</n> interaction, but also abolishes most of the VitD3 induction of <n>CD14</n> expression. Electrophoretic mobility shift analysis does not detect a direct interaction of the <n>CD14</n> distal Sp1-binding site with the vitamin D3 receptor and its partner, the <n>retinoid X receptor</n>. These data demonstrate that VitD3 induces <n>CD14</n> indirectly through some intermediary factor, and suggest a critical role for <n>Sp1</n> in this process.
94344108>DNA-binding and transcriptional regulatory properties of <n>hepatic leukemia factor</n> (<n>HLF</n>) and the t(17;19) acute lymphoblastic leukemia chimera E2A-<n>HLF</n>. The t(17;19) translocation in acute lymphoblastic leukemias results in creation of E2A-<n>hepatic leukemia factor</n> (<n>HLF</n>) chimeric proteins that contain the DNA-binding and protein dimerization domains of the basic leucine zipper (bZIP) protein <n>HLF</n> fused to a portion of E2A proteins with transcriptional activation properties. An in vitro binding site selection procedure was used to determine DNA sequences preferentially bound by wild-type <n>HLF</n> and chimeric E2A-<n>HLF</n> proteins isolated from various t(17;19)-bearing leukemias. All were found to selectively bind the consensus sequence 5'-GTTACGTAAT-3' with high affinity. Wild-type and chimeric <n>HLF</n> proteins also bound closely related sites identified previously for bZIP proteins of both the proline- and acidic amino acid- rich (PAR) and C/EBP subfamilies; however, E2A-<n>HLF</n> proteins were significantly less tolerant of certain deviations from the <n>HLF</n> consensus binding site. These differences were directly attributable to loss of an <n>HLF</n> ancillary DNA-binding domain in all E2A-<n>HLF</n> chimeras and were further exacerbated by a zipper mutation in one isolate. Both <n>wild- type</n> and chimeric <n>HLF</n> proteins displayed transcriptional activator properties in lymphoid and nonlymphoid cells on reporter genes containing HLF or <n>C/EBP</n> consensus binding sites. But on reporter genes with nonoptimal binding sites, their transcriptional properties diverged and E2A-<n>HLF</n> competitively inhibited activation by wild-type PAR proteins. These findings establish a spectrum of binding site- specific transcriptional properties for E2A-<n>HLF</n> which may preferentially activate expression of select subordinate genes as a homodimer and potentially antagonize expression of others through heteromeric interactions.
94327655><n>ZAP-70 tyrosine kinase</n>, <n>CD45</n>, and T cell receptor involvement in UV- and H2O2-induced T cell signal transduction. Several mammalian responses to UV irradiation, including the activation of <n>NF-kappa B</n>, are believed to involve tyrosine phosphorylation. UV irradiation and H2O2 treatment of T lymphocytes induce protein tyrosine phosphorylation and Ca2+ signals similar to those observed following biological stimulation. We have examined the role of cell surface molecules in these responses. Normal T lymphocytes whose surface expression of <n>CD3</n> was depleted showed impaired UV-induced tyrosine phosphorylation and Ca2+ signals. Similarly, Jurkat T cell lines deficient in CD3 or CD45 expression also gave impaired UV responses. However, all these cell types still gave strong Ca2+ and tyrosine phosphorylation responses to H2O2. The T cell tyrosine <n>kinase ZAP-70</n> was found to be highly responsive to UV and H2O2 treatment. <n>ZAP-70</n> responsiveness to UV required expression of both <n>CD3</n> and <n>CD45</n>, whereas only <n>CD3</n> was required for the response to H2O2. UV-induced activation of <n>NF-kappa B</n> was blocked by <n>CD3</n> depletion, indicating the importance of such cell surface molecules in biological responses to UV. In nonlymphoid cells, the epidermal growth factor receptor displayed increased tyrosine phosphorylation within seconds of UV irradiation. These results suggest that UV-induced signal transduction is mediated via cell surface receptors that normally respond to biological stimulation, whereas H2O2 is able to partially bypass this requirement.
94330935>Inhibition of the differentiation of human myeloid cell lines by redox changes induced through glutathione depletion. We have investigated the effect of redox changes in vivo on the differentiation of two human myeloid cell lines, HL-60 and KG-1. The glutathione-depleting agent diethyl maleate (DEM) prevented the development of differentiated features in response to phorbol esters, including adherence of the cells to plastic surfaces and repression of the myeloperoxidase and CD34 genes. Moreover, DEM abolished phorbol 12- myristate 13-acetate-induced activation of the transcription factors <n>AP- 1</n> and <n>Egr-1</n>, suggesting that inhibition of differentiation may be due, at least in part, to redox modifications of these proteins.
94254835>Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site. Exposure of monocytic cells to bacterial lipopolysaccharide (LPS) activates the <n>NF-kappa B</n>/Rel family of proteins and leads to the rapid induction of inflammatory gene products, including tissue factor (TF). TF is the primary cellular initiator of the coagulation protease cascades. Here we report the characterization of a nuclear complex from human monocytic cells that bound to a kappa B-like site, 5'-CGGAGTTTCC- 3', in the 5'-flanking region of the human TF gene. This nuclear complex was activated by LPS with kinetics that preceded induction of the TF gene. In vitro binding studies demonstrated that the TF site bound translated c-Rel and p65 homodimers but not p50/p65 heterodimers or p50 homodimers. Base-pair substitutions in the TF site indicated that the presence of a cytosine at position 1 precluded binding of <n>NF- kappa B</n>. In fact, under low-ionic-strength conditions, the TF complex did not migrate with translated p50/p65 dimers but instead comigrated with <n>c-Rel</n>/p65 dimers. Antibodies against the <n>NF-kappa B</n> and Rel proteins and UV cross-linking studies revealed the presence of <n>c-Rel</n> and p65 and the absence of p50 in the TF complex and further showed that c-Rel/p65 heterodimers selectively bound to the TF kappa B-like site. Functional studies indicated that the TF site conferred LPS inducibility on a heterologous promoter and was transactivated by <n>c-Rel</n> or p65. Taken together, our results demonstrated that binding of c- Rel/p65 heterodimers to a novel kappa B-like site mediated LPS induction of TF gene expression in monocytic cells.
94246160>Inhibition of T cell activation by the extracellular matrix protein <n>tenascin</n>. <n>Tenascin</n> (<n>TN</n>) is an extracellular matrix protein that is expressed widely in the fetus and sparingly in the adult, but reappears at high levels in certain areas of tissue insult such as tumor matrices and sites of wound healing. We show here that soluble <n>TN</n> inhibits proliferation of human T cells in response to alpha <n>CD3</n> Ab co- immobilized with the extracellular matrix protein <n>fibronectin</n> (<n>FN</n>). <n>TN</n> also inhibits proliferation driven by alpha <n>CD3</n>/IL-2 or by <n>phorbol ester/IL-2,</n> and it prevents high level induction of <n>IL-2R</n>. The presence of <n>TN</n> in culture medium does not detectably alter the pattern of tyrosine phosphorylation resulting from T cell triggering with alpha <n>CD3</n>, but at later time points prevents the appearance of functional NF- AT1 transcription factor complexes in T cell nuclear extracts. These findings are consistent with the postulated role for <n>TN</n> as a natural antagonist to <n>FN</n> action, and suggest that T cell responses occurring at tissue sites in which <n>TN</n> is expressed could be influenced by its presence.
94202312>Human immunodeficiency virus type 1 Nef protein down-regulates transcription factors <n>NF-kappa B</n> and <n>AP-1</n> in human T cells in vitro after T-cell receptor stimulation. Human immunodeficiency virus type 1 (HIV-1) <n>negative factor</n> (<n>Nef</n>) has been shown to down-regulate the transcription factors <n>NF-kappa B</n> and <n>AP- 1</n> in vitro. To define the mechanism of action of the <n>Nef</n> protein, the signal transduction pathways which may be affected in T cells by constitutive expression of the nef gene were examined. Stimulation of T cells with tumor necrosis factor, interleukin-1, or lipopolysaccharide resulted in the recruitment of transcriptional factors to a similar level whether or not the cells expressed the nef gene. On the other hand, stimulation of T cells by mitogens or antibodies to the T-cell receptor (TCR)-<n>CD3</n> complex resulted in the down-regulation of transcriptional factors <n>NF-kappa B</n> and <n>AP-1</n> in cells expressing the nef gene compared with cells not expressing the nef gene. Because the <n>Nef</n> protein does not affect the surface expression of the <n>CD3</n>-TCR complex, we conclude that the <n>Nef</n> protein down-regulates the transcriptional factors <n>NF-kappa B</n> and <n>AP-1</n> in T cells in vitro through an effect on the TCR-dependent signal transduction pathway.
94235006>Effects of alpha-lipoic acid and dihydrolipoic acid on expression of proto-oncogene c-fos. The transcription factor <n>AP-1</n> is an important human mediator of the cellular response to serum, growth factors, and phorbol esters such as 12-O-tetradecanoyl-phorbol-13 acetate (TPA). The <n>AP-1</n> complex consists of distinct protein heterodimers encoded by the proto-oncogene c-fos and c-jun mRNA whose gene expression can be induced by TPA, cyclic AMP and growth factors. Recent findings suggest an involvement of reactive oxygen species in the pathway of TPA and <n>protein kinase C</n> leading to expression of c-fos and c-jun mRNA. To investigate the role of reactive oxygen species we studied the effects of alpha-lipoic acid and dihydrolipoic acid (natural thiol antioxidants) on the expression of c- fos mRNA in human Jurkat T cells. When cells were preincubated with dihydrolipoic acid (0.2 mM) the expression of c-fos mRNA was suppressed at 30 min after stimulation of TPA (0.5 microM) whereas in the case of preincubation of alpha-lipoic acid (0.2 microM), the expression was enhanced at 30 min. These studies support the idea that superoxide anion radical plays a role in the expression of c-fos mRNA.
94347465>Appraisal of potential therapeutic index of antioxidants on the basis of their in vitro effects on HIV replication in monocytes and <n>interleukin 2</n>-induced lymphocyte proliferation. Antioxidant molecules have been suggested to be of therapeutic value in the treatment of HIV-infected patients. To evaluate this possibility, we examined in vitro the effects of two types of antioxidant molecules in terms of inhibition of HIV replication in monocytes, one of the main reservoirs of HIV, and also in terms of modulation of the immune competence as measured by PBMC proliferation. We tested the effects of BHA, a phenolic, lipid-soluble, chain-breaking antioxidant, and NAC, a known glutathione precursor with some direct free-radical scavenging properties as well, on the regulation of HIV-1 expression in latently infected U1 cells and in productively and chronically infected U937 cells. Both antioxidants inhibited TNF- or PMA-induced <n>NF-kappa B</n> activity in U1 cells, as well as the sustained <n>NF-kappa B</n> activity permanently induced by the virus itself in chronically HIV-infected U937 cells. This resulted in only a partial inhibition of TNF- or PMA- induced HIV replication in U1 cells, and no detectable effect on HIV replication in chronically infected U937 cells. This may be the first limitation to potential antiviral effects of antioxidant therapies. Another limitation is that antioxidant concentrations high enough to block <n>NK-kappa B</n> activation were shown to have a suppressive effect on immune functions in vitro, because NAC and BHA blocked <n>IL-2</n>-induced PBMC proliferation. These data warrant prudence in the design of antioxidant-based therapies aimed at suppressing HIV replication.
94169380>Isolation and characterization of gelatinase granules from human neutrophils. We recently confirmed the existence of gelatinase granules as a subpopulation of peroxidase-negative granules by double-labeling immunogold electron microscopy on intact cells and by subcellular fractionation. Further characterization of gelatinase granules has been hampered by poor separation of specific and gelatinase granules on both two-layer Percoll gradients and sucrose gradients. We have developed a three-layer Percoll density gradient that allows separation of the different granules and vesicles from human neutrophils; in particular, it allows separation of specific and gelatinase granules. This allows us to characterize these two granule populations with regard to their content of membrane proteins, which become incorporated into the plasma membrane during exocytosis. We found that gelatinase granules, defined as peroxidase-negative granules containing <n>gelatinase</n> but lacking <n>lactoferrin</n>, contain 50% of total cell <n>gelatinase</n>, with the remaining residing in specific granules. Furthermore, we found that 20% to 25% of both the adhesion protein <n>Mac-1</n> and the <n>NADPH-oxidase component cytochrome</n> <n>b558</n> is localized in <n>gelatinase</n> granules. Although no qualitative difference was observed between specific granules and <n>gelatinase</n> granules with respect to <n>cytochrome b558</n> and <n>Mac-1</n>, stimulation of the neutrophil with FMLP resulted in a selective mobilization of the least dense peroxidase-negative granules, ie, <n>gelatinase</n> granules, which, in concert with secretory vesicles, furnish the plasma membrane with <n>Mac-1</n> and <n>cytochrome b558</n>. This shows that <n>gelatinase</n> granules are functionally important relative to specific granules in mediating early inflammatory responses
95014190>Regulation of interleukin-2 receptor alpha chain expression and <n>nuclear factor.kappa B</n> activation by <n>protein kinase C</n> in T lymphocytes. Autocrine role of <n>tumor necrosis factor alpha</n>. The regulation of interleukin-2 receptor alpha chain (IL-2R alpha) expression and nuclear factor (NF) activation by <n>protein kinase C</n> (<n>PKC</n>) in resting T cells, has been studied. Treatment of human resting T cells with phorbol esters strongly induced the expression of IL-2R alpha and the activation of <n>NF.kappa B</n>. This activation was due to the translocation of <n>p65</n> and <n>c-Rel</n> <n>NF.kappa B</n> proteins from cytoplasmic stores to the nucleus, where they bound the kappa B sequence of the IL- 2R alpha promoter either as p50.p65 or as p50.<n>c-Rel</n> heterodimers. Interestingly, all of those events were largely indirect and mediated by endogenously secreted <n>tumor necrosis factor alpha</n> (<n>TNF alpha</n>), as they were strongly inhibited by a neutralizing anti-<n>TNF alpha</n> monoclonal antibody. Furthermore, cyclosporin A, which blocked <n>TNF alpha</n> production induced by <n>PKC</n>, strongly inhibited <n>IL-2R</n> alpha and <n>NF.kappa B</n> activation. The addition of either <n>TNF alpha</n> or <n>IL-2</n> partially recovered cyclosporin A-induced IL-2R alpha inhibition, but only <n>TNF alpha</n> completely recovered <n>NF.kappa B</n> activation. Those results indicate that, in resting T cells, <n>PKC</n> activation has only a triggering role, whereas the endogenously secreted <n>TNF alpha</n> plays an essential role in the quantitative control of the expression of IL-2R alpha chain or <n>NF.kappa B</n> activation.
94340810>Superantigens activate HIV-1 gene expression in monocytic cells. Binding of superantigens to MHC class II molecules results in transduction of biochemical signals leading to cellular activation and gene expression. We demonstrate that the staphylococcal superantigens <n>toxic shock syndrome toxin-1</n> (<n>TSST-1</n>) and <n>staphylococcal enterotoxin A</n> (<n>SEA</n>) activate HIV-1-LTR-driven transcription of <n>chloramphenicol acetyl transferase</n> in the human monocytic cell line THP-1. Induction of HIV-1- LTR-driven transcription in THP-1 cells by superantigens was associated with the induction of <n>nuclear factor-kappa B</n> DNA-binding activity. Superantigens also increased viral protein secretion from the <n>granulocyte-macrophage colony-stimulating factor</n>-pretreated chronically infected human monocytic cell line U1. Induction of HIV-1 gene expression in monocytic cells by superantigens occurred via tumor necrosis factor-alpha-dependent and -independent mechanisms. Our results suggest that superantigens and other MHC class II ligands may activate HIV-1 gene expression in monocytes/macrophages.
94327512>Mitogen activation of human peripheral T lymphocytes induces the formation of new cyclic AMP response element-binding protein nuclear complexes. A large body of evidence indicates that experimental agents which raise cellular cAMP levels inhibit T cell growth and division. By contrast, many studies have reported that mitogen activation of T cells increases cAMP levels, implying a positive physiological role for cAMP in the activation process. In the present study we demonstrate that mitogen activation of human peripheral T lymphocytes induces nuclear factors that form complexes with <n>cyclic AMP response element-binding protein</n> (<n>CREB</n>). Four complexes are identified by the electrophoretic mobility shift assay, two of which are induced by mitogen activation. All four complexes contain <n>CREB</n> and are bound to the cAMP response element (CRE) core sequence (TGACGTCA), as indicated by antibody and oligonucleotide competition experiments. Binding of the four complexes to CRE is prevented by dephosphorylation of nuclear extracts and is restored by rephosphorylation with <n>cAMP-dependent protein kinase</n> or endogenous kinases. Similar complexes are detected in nuclear extracts of Jurkat cells. Mitogen induction of the electrophoretic mobility shift assay complexes is not accounted for by protein phosphorylation or by induction of <n>CREB</n>. Rather, the data indicate that mitogen increases the levels of a nuclear factor(s) that dimerizes with <n>CREB</n>. Induction of new <n>CREB</n> complexes implies a physiological role for cAMP in mitogen activation of T lymphocytes.
94314962>Alpha-tocopherol inhibits agonist-induced monocytic cell adhesion to cultured human endothelial cells. Antioxidants have been proposed to be anti-atherosclerotic agents; however, the mechanisms underlying their beneficial effects are poorly understood. We have examined the effect of alpha-tocopherol (alpha-tcp) on one cellular event in atherosclerotic plaque development, monocyte adhesion to stimulated endothelial cells (ECs). Human umbilical vein ECs were pretreated with alpha-tcp before stimulation with known agonists of monocyte adhesion: <n>IL-1</n> (10 ng/ml), LPS (10 ng/ml), <n>thrombin</n> (30 U/ml), or PMA (10 nM). Agonist-induced monocytic cell adhesion, but not basal adhesion, was inhibited in a time- and concentration-dependent manner by alpha-tcp. The IC50 of alpha-tcp on an <n>IL-1</n>-induced response was 45 microM. The inhibition correlated with a decrease in steady state levels of E-selectin mRNA and cell surface expression of <n>E-selectin</n> which is consistent with the ability of a monoclonal antibody to <n>E-selectin</n> to inhibit monocytic cell adhesion in this system. Probucol (50 microM) and N-acetylcysteine (20 mM) also inhibited agonist-induced monocytic cell adhesion; whereas, several other antioxidants had no significant effect. <n>Protein kinase C</n> (<n>PKC</n>) does not appear to play a role in the alpha-tcp effect since no suppression of phosphorylation of <n>PKC</n> substrates was observed. Activation of the transcription factor <n>NF-kappa B</n> is reported to be necessary but not sufficient for <n>E-selectin</n> expression in EC. Electrophoretic mobility shift assays failed to show an alpha-tcp- induced decrease in activation of this transcription factor after cytokine stimulation. It has been hypothesized that alpha-tcp acts as an anti-atherosclerotic molecule by inhibiting generation of oxidized LDL--a putative triggering molecule in the atherosclerotic process. Our results point to a novel alternative mechanism of action of alpha-tcp.
94246743>Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by <n>Tat</n>. The <n>Tat protein</n> of human immunodeficiency virus type 1 (HIV-1) is a potent activator of long terminal repeat-directed transcription. While in most cell types, activation requires interaction of Tat with the unusual transcription element TAR, astrocytic glial cells support TAR- independent transactivation of HIV-1 transcription by Tat. This alternative pathway of <n>Tat</n> activation is mediated by the viral enhancer, a kappa B domain capable of binding the prototypical form of the transcription factor nuclear factor kappa B (<n>NF-kappa B</n>) present in many cell types, including T lymphocytes. <n>Tat</n> transactivation mediated by the kappa B domain is sufficient to allow replication of TAR-deleted mutant HIV-1 in astrocytes. The present study demonstrates the existence of kappa B-specific binding factors present in human glial astrocytes that differ from prototypical <n>NF-kappa B</n>. The novel astrocyte-derived kappa B-binding activity is retained on an HIV-1 <n>Tat</n> affinity column, while prototypical <n>NF-kappa B</n> from Jurkat T cells is not. In vitro transcription studies demonstrate that astrocyte-derived kappa B-binding factors activate transcription of the HIV-1 long terminal repeat and that this activation is dependent on the kappa B domain. Moreover, TAR-independent transactivation of HIV-1 transcription is reproduced in vitro in an astrocyte factor-dependent manner which correlates with kappa B-binding activity. The importance of the central nervous system-enriched kappa B transcription factor in the regulation of HIV-1 expression is discussed.
94259178><n>Human interleukin-13</n> activates the interleukin-4-dependent transcription factor <n>NF-IL4</n> sharing a DNA binding motif with an <n>interferon-gamma-induced nuclear binding factor</n>. The effects of <n>interleukin-13</n> (<n>IL-13</n>) and <n>interleukin-4</n> (<n>IL-4</n>) on cellular functions were shown to be quite similar. We provide evidence that in monocytes as well as in T lymphocytes both <n>IL-4</n> and <n>IL-13</n> activate the same recently identified transcription factor <n>NF-IL4</n> which binds to the specific responsive element &gt;IL-4RE. In addition, we show that a nuclear factor activated by interferon-gamma also interacts with the IL-4RE. It differs from <n>NF-IL4</n> in the electrophoretic mobility of the complex with DNA, in its DNA-binding specificity and in the proteins interacting with the DNA sequence. Sensitivity against various enzyme inhibitors suggests that components of the signal transduction pathway are shared by all three cytokines.
94202306>Encephalomyocarditis virus internal ribosomal entry site RNA-protein interactions. Translational initiation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal entry into the 5' nontranslated region of the EMCV mRNA, rather than by ribosomal scanning. Internal ribosomal binding requires a cis-acting element termed the internal ribosomal entry site (IRES). IRES elements have been proposed to be involved in the translation of picornavirus mRNAs and some cellular mRNAs. Internal ribosome binding likely requires the interaction of trans-acting factors that recognize both the mRNA and the ribosomal complex. Five cellular proteins (<n>p52</n>, <n>p57</n>, <n>p70</n>, <n>p72</n>, and <n>p100</n>) cross-link the EMCV IRES or fragments of the IRES. For one of these proteins, <n>p57</n>, binding to the IRES correlates with translation. Recently, <n>p57</n> was identified to be very similar, if not identical, to <n>polypyrimidine tract-binding protein</n>. On the basis of cross-linking results with 21 different EMCV IRES fragments and cytoplasmic HeLa extract or rabbit reticulocyte lysate as the source of polypeptides, consensus binding sites for <n>p52</n>, <n>p57</n>, <n>p70</n>, and <n>p100</n> are proposed. It is suggested that each of these proteins recognizes primarily a structural feature of the RNA rather than a specific sequence.
94216261>A novel heterodimerization partner for thyroid hormone receptor. <n>Peroxisome proliferator-activated receptor</n>. Retinoid-like receptors play a central role in hormonal responses by forming heterodimers with other nuclear hormone receptors. In this study we have identified the <n>peroxisome proliferator-activated receptor</n> (<n>PPAR</n>) as a new <n>thyroid hormone receptor (THR) auxiliary nuclear protein</n>, heterodimerizing with THR in solution. Although these heterodimers do not recognize a classical thyroid hormone response element (TRE) characterized by direct repeat separated by four nucleotides (DR+4), <n>PPAR</n> behaves as a dominant negative regulator of thyroid hormone (TH) action. However, a TH-dependent positive effect is elicited by selective interaction of the THR beta-<n>PPAR</n> but not the THR alpha-<n>PPAR</n> heterodimer with a novel TRE (DR+2). The critical region of THR beta was mapped to 3 amino acids in the distal box of the DNA binding domain. Hence, <n>PPAR</n> can positively or negatively influence TH action depending on TRE structure and THR isotype.
94200898>Comparison of retinoic acid and phorbol myristate acetate as inducers of monocytic differentiation. Several human myeloid leukemia cell lines growing in vitro can be induced to differentiate to more mature monocyte/macrophage-like cells by treatment with <n>protein kinase C</n>-activating phorbol esters, such as PMA. In addition to PMA, cells of the THP-1 myeloid leukemia cell line acquire macrophage-like characteristics after treatment with all-trans retinoic acid (RA). To analyze the signal transduction mechanisms induced by RA, we first compared the effects of PMA and RA on the expression of genes which are known to be regulated during monocytic differentiation. Both RA and PMA effectively down-regulated c-myc expression, while c-myb expression decreased only after PMA treatment. Expression of the beta 2-integrin genes, CD11a and CD11b, was clearly increased after both of these treatments. Their effects on the src- family tyrosine kinase genes were different: hck expression was similarly induced by these agents but lyn expression was stronger and more rapid after RA treatment. RA also enhanced lyn mRNA production rapidly in HL-60, indicating that the activation of lyn gene expression is common in monocytic and granulocytic maturation of myeloid leukemia cells. To examine whether the AP-1 enhancer activity is involved in RA- induced monocytic differentiation, THP-1 cells were transiently transfected with a <n>chloramphenicol acetyl transferase</n> (<n>CAT</n>)-reporter gene containing 5 copies of the <n>AP-1</n> binding sites. In contrast to PMA, RA did not induce any <n>CAT</n> activity in these cells, thus suggesting that the RA-induced changes in the expression of those genes described above were not dependent on the <n>AP-1</n> enhancer activity.
95011572>An active <n>v-abl protein tyrosine kinase</n> blocks immunoglobulin light- chain gene rearrangement. Lymphoid cells transformed by Abelson murine leukemia virus have provided one of the classic models for study of early B-cell development and immunoglobulin rearrangement. Most of these cells have rearranged their heavy-chain locus but not their light chain genes, suggesting that an active <n>v-abl protein</n> interferes with this differentiation step. To test this hypothesis, light-chain gene structure was examined in pre-B cells transformed by temperature- sensitive mutants of the Abelson virus and in derivatives that survive at the nonpermissive temperature because they express a human BCL-2 gene. Our studies reveal that inactivation of the <n>v-abl protein</n> tyrosine kinase triggers high-frequency rearrangement of kappa and lambda light-chain genes. These events are accompanied by marked increases in the expression of RAG-1 and RAG-2 RNAs. These increases occur in the absence of protein synthesis but are dependent on inactivation of the <n>v-abl protein</n> tyrosine kinase. As documented in the accompanying paper (Klug et al., this issue), an active <n>v-abl protein</n> also suppresses the activity of <n>NF-kappa B</n>/rel and expression controlled by the kappa intron enhancer. Together these data demonstrate that the <n>v-abl protein</n> specifically interferes with light- chain gene rearrangement by suppressing at least two pathways essential for this stage of B-cell differentiation and suggest that tyrosine phosphorylation is important in regulating RAG gene expression
94376879>Calcium signalling in T cells stimulated by a cyclophilin B-binding protein. The immunosuppressant drug cyclosporin A blocks a calcium-dependent signal from the T-cell receptor (TCR) that normally leads to T-cell activation. When bound to <n>cyclophilin</n>, cyclosporin A binds and inactivates the key signalling intermediate <n>calcineurin</n>. To identify potential cellular homologues of cyclosporin A that might regulate calcium signalling, we have cloned human genes encoding <n>cyclophilin</n> B- binding-proteins using the yeast two-hybrid system. One gene product, when overexpressed in Jurkat T cells, specifically induced transcription from the interleukin-2 enhancer, by activating the T-cell- specific transcription factors <n>NF-AT</n> and <n>NF-IL2A</n>. This protein, termed calcium-signal modulating <n>cyclophilin</n> ligand (<n>CAML</n>), acts downstream of the TCR and upstream of <n>calcineurin</n> by causing an influx of calcium. <n>CAML</n> appears to be a new participant in the calcium-signal transduction pathway, implicating <n>cyclophilin</n> B in calcium signalling, even in the absence of cyclosporin.
95162017>Expression and genomic configuration of <n>GM-CSF</n>, <n>IL-3</n>, <n>M-CSF receptor</n> (<n>C- FMS</n>), early growth response gene-1 (EGR-1) and M-CSF genes in primary myelodysplastic syndromes. Peripheral blood mononuclear cells from seventeen patients with primary myelodysplastic syndromes (MDS) in advanced stage were enriched for blasts and tested for (1) karyotype, (2) genomic configuration and (3) expression of IL-3, GM-CSF, FMS and EGR-1 genes which are all located on the long arm of chromosome 5. The expression of the M-CSF gene, that has been recently reassigned to the short arm of chromosome 1 (lp), was also investigated. Aims of the study were to (1) assess the potential role of the expression of these genes in the maintenance and expansion of the neoplastic clones and (2) search for constitutional losses or rearrangements of one allele followed by a deletion of the second allele of the same genes in the leukemic cells. The latter issue was investigated by comparing, in 8 cases, constitutive DNA from skin fibroblasts with leukemic DNA. Eleven of the 17 patients had abnormal karyotypes. The M-CSF gene was expressed in 6 cases and the FMS and the EGR-1 genes were expressed in 2 of the latter cases. An autocrine mechanism of growth could be hypothesized only for the 2 patients whose cells expressed both the M-CSF and FMS genes. No germline changes or rearrangements were observed in any of the genes studied. Thus, deregulation of genes encoding for certain hemopoietic growth factors or receptors does not seem to represent a major mechanism of MDS progression.
94327547>A novel human homeobox gene distantly related to proboscipedia is expressed in lymphoid and pancreatic tissues. A novel human homeobox gene, HB9, was isolated from a cDNA library prepared from in vitro stimulated human tonsil B lymphocytes and from a human genomic library. The HB9 gene is composed of 3 exons spread over 6 kilobases of DNA. An open reading frame of 1206 nucleotides is in frame with a diverged homeodomain. The predicted <n>HB9 protein</n> has a molecular mass of 41 kilodaltons and is enriched for alanine, glycine, and leucine. The HB9 homeodomain is most similar to that of the Drosophila melanogaster homeobox gene proboscipedia. Northern blot analysis of poly(A) RNA purified from the human B cell line RPMI 8226 and from activated T cells revealed a major mRNA transcript of 2.2 kilobases. Similar analysis of poly(A) RNA from a variety of adult tissues demonstrated HB9 transcripts in pancreas, small intestine, and colon. Reverse transcriptase-polymerase chain reaction was used to examine HB9 RNA transcripts in hematopoietic cell lines. HB9 RNA transcripts were most prevalent in several human B cell lines and K562 cells. In addition, transcripts were detected in RNA prepared from tonsil B cells and in situ hybridization studies localized them in the germinal center region of adult tonsil. These findings suggest the involvement of HB9 in regulating gene transcription in lymphoid and pancreatic tissues.
94267876><n>Human immunodeficiency virus type 1 Tat</n> upregulates <n>interleukin-2</n> secretion in activated T cells. Dysregulation of cytokines secreted by T cells may play an important role in the pathogenesis of AIDS. To investigate the effects of <n>human immunodeficiency virus type 1 (HIV-1) Tat</n> on <n>interleukin-2</n> (<n>IL-2</n>) expression, we used <n>IL-2</n> promoter-<n>chloramphenicol acetyltransferase</n> constructs and <n>IL-2</n>-secreting Jurkat T cells as a model system. Transient expression of <n>HIV-1 Tat</n> induced a five- to eightfold increase in <n>IL-2</n> promoter activity in Jurkat T cells stimulated with <n>phytohemagglutinin</n> and phorbol myristate acetate. <n>IL-2</n> secretion was increased more than twofold in both Jurkat T cells and primary T cells stimulated by extracellular <n>HIV-1 Tat</n> protein. Analysis of mRNA suggested that Tat exerts its effect on <n>IL-2</n> primarily at the transcriptional level. The NF-kappa B site at positions -206 to -195 of the <n>IL-2</n> promoter was required but not sufficient for the <n>Tat</n> effect. The <n>Tat</n>-mediated increase in <n>IL-2</n> promoter activity could selectively be blocked by antisense tat or-unlike the analogous effect of <n>human T- cell lymphotropic virus type 1 Tax</n>-by cyclosporin A. The observed increase in <n>IL-2</n> levels might facilitate virus spread from or to T cells. Furthermore, it might contribute to the hypergammaglobulinemia or, together with other cytokines found to be dysregulated, the T- helper cell dysfunctions observed in AIDS patients.
94240241>Activation of <n>nuclear factor kappa B</n> in human lymphoblastoid cells by low-dose ionizing radiation. <n>Nuclear factor kappa B</n> (<n>NF-kappa B</n>) is a pleiotropic transcription factor which is involved in the transcriptional regulation of several specific genes. Recent reports demonstrated that ionizing radiation in the dose range of 2-50 Gy results in expression of <n>NF-kappa B</n> in human KG-1 myeloid leukemia cells and human B-lymphocyte precursor cells; the precise mechanism involved and the significance are not yet known. The present report demonstrates that even lower doses of ionizing radiation, 0.25-2.0 Gy, are capable of inducing expression of <n>NF-kappa B</n> in EBV-transformed 244B human lymphoblastoid cells. These results are in a dose range where the viability of the cells remains very high. After exposure to 137Cs gamma rays at a dose rate of 1.17 Gy/min, a maximum in expression of <n>NF-kappa B</n> was seen at 8 h after a 0.5-Gy exposure. Time-course studies revealed a biphasic time-dependent expression after 0.5-, 1- and 2-Gy exposures. However, for each time examined, the expression of <n>NF-kappa B</n> was maximum after the 0.5-Gy exposure. The expression of the p50 and p65 NF-kappa B subunits was also shown to be regulated differentially after exposures to 1.0 and 2.0 Gy.
94240138>Alternative splicing of RNA transcripts encoded by the murine p105 NF- kappa B gene generates I kappa B gamma isoforms with different inhibitory activities. The gene encoding the <n>105-kDa protein</n> (<n>p105</n>) precursor of the p50 subunit of transcription factor <n>NF-kappa B</n> also encodes a <n>p70 I kappa B protein</n>, <n>I kappa B gamma</n>, which is identical to the C-terminal 607 amino acids of <n>p105</n>. Here we show that alternative RNA splicing generates <n>I kappa B gamma</n> isoforms with properties different from those of p70. One <n>63-kDa isoform</n>, termed <n>I kappa B gamma-1</n>, which lacks 59 amino acids C-terminal to ankyrin repeat 7, has a novel 35-amino acid C terminus encoded by an alternative reading frame of the <n>p105</n> gene. A <n>55- kDa isoform</n>, <n>I kappa B gamma-2</n>, lacks the 190 C-terminal amino acids of p70<n>I kappa B gamma</n>. In contrast to p70<n>I kappa B gamma</n>, which is a cytoplasmic protein, <n>I kappa B gamma-1</n> is found in both the cytoplasm and nucleus, whereas <n>I kappa B gamma-2</n> is predominantly nuclear. The <n>I kappa B gamma</n> isoforms also display differences in specificity and affinity for Rel/<n>NF-kappa B</n> proteins. While p70<n>I kappa B gamma</n> inhibits p50-, p65-, and c-Rel-mediated transactivation and/or DNA binding, both <n>I kappa B gamma-1</n> and <n>I kappa B gamma-2</n> are specific for p50 and have different affinities for this subunit. The absence in <n>I kappa B gamma-1</n> and <n>I kappa B gamma-2</n> of a protein kinase A site whose phosphorylation modulates p70<n>I kappa B gamma</n> inhibitory activity suggests that alternative RNA splicing may be used to generate <n>I kappa B gamma</n> isoforms that respond differently to intracellular signals.
94375000>Structure and expression of the human GATA3 gene. <n>GATA3</n>, a member of the GATA family that is abundantly expressed in the T-lymphocyte lineage, is thought to participate in T-cell receptor gene activation through binding to enhancers. To understand <n>GATA3</n> gene regulation, we cloned the human gene and the 5' end of the mouse <n>GATA3</n> gene. We show that the human <n>GATA3</n> gene contains six exons distributed over 17 kb of DNA. The two human <n>GATA3</n> zinc fingers are encoded by two separate exons highly conserved with those of <n>GATA1</n>, but no other structural homologies between these two genes can be found. The human and mouse GATA3 transcription units start at a major initiation site. The promoter sequence analysis of these two genes revealed that they are embedded within a CpG island and share structural features often found in the promoters of housekeeping genes. Finally, we show that a DNA fragment containing the human <n>GATA3</n> transcription unit, 3 kb upstream from the initiation site and 4 kb downstream from the polyadenylation site, displays T-cell specificity.
94209308>Characterization of the human gene encoding <n>LBR</n>, an integral protein of the nuclear envelope inner membrane. We have characterized the human gene encoding <n>LBR</n>, an integral protein of the nuclear envelope inner membrane. Restriction mapping shows that the transcription unit spans approximately 35 kilobases. A transcription start site is located approximately 4 kilobases 5' to the translation initiation codon, and an RNA splice of 3863 bases occurs in the 5'-untranslated region to generate mature HeLa cell mRNA. 5' to the identified transcription start site are two CCAAT sequences and potential recognition sites for several transcription factors including <n>Sp1</n>, <n>AP-1</n>, <n>AP-2</n>, and <n>NF-kB</n>. There are 13 protein coding exons in the <n>LBR</n> gene. <n>LBR</n>'s nucleoplasmic domain is encoded by exons 1-4, and its hydrophobic domain, with eight putative transmembrane segments, is encoded by exons 5-13. The hydrophobic domain is homologous to three yeast polypeptides, suggesting that this higher eukaryotic gene could have evolved from recombination between a gene that encoded a soluble nuclear protein and a membrane protein gene similar to those in yeast. These results are the first to demonstrate the structural organization of a vertebrate gene encoding an integral membrane protein of the nuclear envelope that may be a member of a family of polypeptides conserved in evolution.
94185011>Retinoic acid-induced expression of <n>CD38 antigen</n> in myeloid cells is mediated through <n>retinoic acid receptor-alpha</n>. <n>CD38</n> is a leukocyte differentiation antigen that has been thought to be a phenotypic marker of different subpopulations of T- and B- lymphocytes. In myeloid cells, <n>CD38</n> is expressed during early stages of differentiation. Virtually no information is available on regulation and functions of <n>CD38</n>. Recently we reported that all-trans-retinoic acid (ATRA) is a potent and highly specific inducer of <n>CD38</n> expression in human promyelocytic leukemia cells. Here we report that ATRA-induced expression of <n>CD38</n> antigen in myeloid cells is mediated through <n>retinoic acid-alpha receptor</n> (<n>RAR alpha</n>). ATRA failed to induce <n>CD38</n> expression in a mutant subclone of the HL-60 myeloid leukemia cell line (designated HL-60R) that is relatively resistant to ATRA-induced granulocytic differentiation. Retroviral vector-mediated transduction of RA receptor (<n>RAR alpha</n>) into this HL-60R subclone completely restored the sensitivity of these cells to ATRA in terms of their ability to express <n>CD38</n>. In contrast, <n>CD38</n> expression was not inducible by ATRA in HL-60R cells, transfected with a functional <n>RAR beta</n>, <n>RAR gamma</n>, or <n>RXR alpha receptor</n>. Induction of <n>CD38</n> in acute promyelocytic and acute myeloblastic leukemia cells was independent of ATRA-induced cytodifferentiation. Following culture with ATRA, increased <n>CD38</n> protein levels were also observed in normal CD34+ bone marrow cells, but not on normal circulating granulocytes. From these results, we conclude that <n>CD38</n> is ATRA inducible in myeloid leukemia cells and normal CD34+ bone marrow cells. This effect is independent of differentiation and is mediated by <n>RAR alpha</n> in HL-60 cells, suggesting a similar role for <n>RAR alpha</n> in <n>CD38</n> expression in other hematopoietic cells.
94242719>Some antioxidants inhibit, in a co-ordinate fashion, the production of <n>tumor necrosis factor-alpha</n>, <n>IL-beta</n>, and <n>IL-6</n> by human peripheral blood mononuclear cells. Some antioxidants, including butylated hydroxyanisole (BHA), tetrahydropapaveroline (THP), nordihydroguiauretic acid, and 10,11- dihydroxyaporphine (DHA), were found to be potent inhibitors of the production of <n>tumor necrosis factor (TNF)-alpha</n>, <n>IL-1 beta</n>, and <n>IL-6</n> by human peripheral blood mononuclear cells (PBMC) stimulated by lipopolysaccharide (LPS) (IC50s in the low micromolar range). Inhibition of cytokine production was gene selective and not due to general effects on protein synthesis. Inhibition of cytokine production by PBMC was observed also when other inducers were used (staphylococci, silica, zymosan). Much higher concentrations of other antioxidants-- including ascorbic acid, trolox, alpha-tocopherol, butylated hydroxytoluene, and the 5-lipoxygenase inhibitor zileuton--did not affect the production of these cytokines. The active compounds did not inhibit <n>IL-1</n>-induced production of <n>IL-6</n> in fibroblasts, showing the cell selectivity of the effect. Antioxidant-mediated inhibition of cytokine production was correlated with low levels of the corresponding messenger RNAs. Nuclear run-on experiments showed that THP inhibited transcription of the <n>IL-1</n> beta gene. THP decreased the concentration of the transcription factors <n>NF-kappa B</n> and <n>AP-1</n> detected in nuclear extracts of PBMC cultured in the presence or absence of LPS. THP and DHA markedly decreased the levels of <n>TNF-alpha</n> and <n>IL-1</n> beta in the circulation of mice following LPS injection. Thus antioxidants vary widely in potency as inhibitors of the activation of transcription factors and of the transcription of genes for pro-inflammatory cytokines. Coordinate inhibition of the transcription of genes for inflammatory cytokines could provide a strategy for therapy of diseases with inflammatory pathogenesis and for septic shock
94367369>An interleukin-4-induced transcription factor: <n>IL-4 Stat</n>. <n>Interleukin-4</n> (<n>IL-4</n>) is an immunomodulatory cytokine secreted by activated T lymphocytes, basophils, and mast cells. It plays an important role in modulating the balance of T helper (Th) cell subsets, favoring expansion of the Th2 lineage relative to Th1. Imbalance of these T lymphocyte subsets has been implicated in immunological diseases including allergy, inflammation, and autoimmune disease. <n>IL-4</n> may mediate its biological effects, at least in part, by activating a <n>tyrosine-phosphorylated DNA binding protein</n>. This protein has now been purified and its encoding gene cloned. Examination of the primary amino acid sequence of this protein indicates that it is a member of the signal transducers and activators of transcription (Stat) family of DNA binding proteins, hereby designated <n>IL-4 Stat</n>. Study of the inhibitory activities of phosphotyrosine-containing peptides derived from the intracellular domain of the <n>IL-4</n> receptor provided evidence for direct coupling of receptor and transcription factor during the <n>IL-4 Stat</n> activation cycle. Such observations indicate that <n>IL-4 Stat</n> has the same functional domain for both receptor coupling and dimerization.
95134788>Evaluation of the respiratory epithelium of normals and individuals with cystic fibrosis for the presence of adenovirus E1a sequences relevant to the use of E1a- adenovirus vectors for gene therapy for the respiratory manifestations of cystic fibrosis. Lung disease associated with disorders such as cystic fibrosis (CF) may be amenable to somatic gene therapy in which there is delivery of the normal gene directly to the respiratory epithelium using E1a- adenovirus (Ad) type 2- or 5-based vectors. For safety reasons, the Ad vectors are rendered replication deficient by deletion of the E1a region. Because there is the theoretical possibility of an E1a- replication-deficient vector replicating as a result of recombination or complementation with Ad 2/5 E1a sequences present in the target cell, this study is directed toward evaluating respiratory epithelium of normals and individuals with CF for the presence of E1a sequences. Using Ad 2/5 E1a-specific primers and the polymerase chain reaction to evaluate DNA recovered from freshly isolated nasal and bronchial epithelium recovered by brushing, E1a sequences were detected in respiratory epithelium of 19 of 91 normals (21%). In the E1a-positive samples, the average of E1a copy number was 55 +/- 18/10(3) recovered cells. In CF individuals, 7 of 52 (13%) had detectable E1a sequences in the respiratory epithelium, with E1a copy number in the positive samples of 80 +/- 21/10(3) recovered cells. These results demonstrate that there are detectable Ad 2/5 E1a sequences in the respiratory epithelium of a small percentage of normals and individuals with CF. Because of the theoretical potential of such sequences supporting replication of E1a- Ad vectors, human gene therapy protocols for CF utilizing such vectors should consider evaluating study individuals for the presence of Ad 2/5 E1a sequences in the respiratory epithelium.
94341725>Leiomyosarcoma of the vulva: report of a case. A 52-year-old female presented with a progressively enlarging vulvar mass. Pathological evaluation revealed a high-grade vulvar leiomyosarcoma. Immunohistochemical and ultrastructural studies were performed to support the diagnosis. In an effort to better understand the biology of this tumor additional immunohistochemical studies for the protein product of p53 tumor suppressor gene and estrogen receptor expression by tumor cells, as well as the type of immune cells infiltrating the tumor were performed. Tumor cells showed an overexpression of <n>p53 protein</n> and were estrogen receptor-positive. Macrophages and T and B lymphocytes infiltrated the tumor in moderate numbers with occasional lymphoid aggregate formation. This study is the first attempt to better understand the biology of these tumors.
94267706>Stimulation of HIV replication in mononuclear phagocytes by <n>leukemia inhibitory factor</n>. This study examined the effects of <n>leukemia inhibitory factor</n> (<n>LIF</n>) on human immunodeficiency virus (HIV) replication in mononuclear phagocytes (MNP). <n>LIF</n> induced a dose-dependent increase in <n>p24 antigen</n> production in the chronically infected promonocytic cell line U1. The magnitude and time kinetics of the <n>LIF</n> effects were similar to <n>interleukin 1</n> (<n>IL-1</n>), <n>IL-6</n>, and tumor necrosis factor (TNF), other cytokines known to induce HIV replication in this cell line. To characterize mechanisms responsible for these <n>LIF</n> effects, levels of HIV mRNA, activation of the DNA binding protein nuclear factor (NF)-kB, signal transduction pathways, and potential interactions with other cytokines were analyzed. <n>LIF</n> increased steady-state levels of HIV mRNA at 2.0, 4.3, and 9.2 kB. This was detectable by 24 h and persisted until 72 h. The <n>DNA binding protein NF-kB</n> is a central mediator in cytokine activation of HIV transcription. <n>NF-kB</n> levels were higher in unstimulated U1 cells as compared to the parent cell line U937. In both cell lines <n>LIF</n> increased <n>NF-kB</n> activity. Induction of <n>NF-kB</n> and HIV replication by cytokines are at least in part dependent on reactive oxygen intermediates. The oxygen radical scavenger N-acetyl-L-cysteine, but not an inhibitor of <n>nitric oxide synthase</n>, inhibited <n>LIF</n>-induced HIV replication. <n>LIF</n> induces the production of other cytokines in monocytes but its effects on HIV replication were not inhibited by antibodies to <n>IL-1</n>, TNF, or <n>IL-6</n>. These results identify <n>LIF</n> as a stimulus of HIV replication. (ABSTRACT TRUNCATED AT 250 WORDS)
94375097>Mechanisms involved in the inhibition of growth of a human B lymphoma cell line, B104, by anti-MHC class II antibodies. The mechanisms involved in the inhibition of growth of a human B lymphoma cell line, B104, by anti-MHC class II antibodies (Ab) were compared with those in anti-IgM Ab-induced B104 growth inhibition. Two anti-MHC class II Ab, <n>L227</n> and <n>2.06</n>, inhibited the growth of B104 cells, although <n>2.06</n>, but not <n>L227</n>, needed to be further cross-linked with a goat anti-mouse IgG Ab (GAM) to show the effect. <n>L227</n> induced an increase in intracellular free Ca2+ concentration ([Ca2+]i) from the intracellular pool and little or no protein tyrosine phosphorylation, phosphatidyl inositol turnover, or expression of Egr-1 mRNA, whereas <n>2.06</n> plus GAM induced an increase in [Ca2+]i from both the intracellular and, in particular, the extracellular pools. The inhibition of B104 cell growth induced by anti-MHC class II Ab was Ca(2+)-independent and not inhibited by actinomycin D or cyclosporin A, and cell cycle arrest at the G2/M interphase was not observed. These features are very different from those observed in B104 cell death induced by anti-IgM Ab. Neither DNA fragmentation nor the morphology of apoptosis was observed. These findings demonstrate that cross-linking of MHC class II molecules transduced the negative signals through intracellular mechanisms different from those present in the cross- linking of surface IgM.
94230451>Functional block for 1 alpha,25-dihydroxyvitamin D3-mediated gene regulation in human B lymphocytes. Elements necessary for the steroid hormone 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) to induce a biological response include the presence of specific intracellular receptors (vitamin D3 receptors (VDR)) and modulation of gene expression via hormone-activated receptor binding to regulatory regions of target genes. These parameters were examined in normal and Epstein-Barr virus-immortalized human B cells and compared with 1 alpha,25-(OH)2D3-responsive cells of the T and monocytic lineages. Although resting tonsillar B cells did not express VDR mRNA, activation of these cells with <n>interleukin-4</n> induced VDR in the absence of exogenously supplemented 1 alpha,25-(OH)2D3. As indicators of hormone-mediated gene regulation we analyzed modulation of <n>CD23</n>, a common B cell/monocyte surface antigen, and <n>24-hydroxylase</n>. 1 alpha,25-(OH)2D3 inhibited <n>CD23</n> expression in U937 cells, yet failed to modulate <n>CD23</n> expression in B cells. Furthermore, 1 alpha,25-(OH)2D3 induced <n>24-hydroxylase</n> mRNA expression and metabolic activity in both U937 cells and lectin-activated T cells, yet failed to induce 24- hydroxylase mRNA or its metabolic activity in B cells. These findings suggest that although human B lymphocytes can express VDR mRNA and protein, they exhibit a functional block for vitamin D-dependent gene regulation.
94331553>Positive and negative regulation of <n>IL-2</n> gene expression: role of multiple regulatory sites. Interleukin 2 (<n>IL-2</n>) is an important lymphokine required in the process of T cell activation, proliferation, clonal expansion and differentiation. The <n>IL-2</n> gene displays both T cell specific and inducible expression: it is only expressed in CD4+ T cells after antigenic or mitogenic stimulation. Several cis-acting regulatory sites are required for induction of the <n>IL-2</n> gene after stimulation. In this study, we have analysed the function of these cis-acting regulatory sites in the context of the native <n>IL-2</n> enhancer and promoter sequence. The results of this study suggest that the <n>NFAT</n> (-276 to -261), the distal octamer (-256 to -248) and the proximal octamer (-75 to -66) sites not only act as enhancers of <n>IL-2</n> gene transcription in the presence of cellular stimulation, but also have a silencing effect on <n>IL-2</n> gene expression in resting cells. Two other sites display disparate effects on <n>IL-2</n> gene expression in different T leukemia cell lines: the distal purine box (-291 to -277) and the proximal purine box sites (-145 to -128). Finally, the AP-1 (-186 to -176) and the kappa B sites (-206 to -195) respond to different cellular activation in EL4 cells. The AP-1 site mediated the response to PMA stimulation while the kappa B site responded to <n>IL-1</n> stimulation. These data suggest that the regulation of <n>IL-2</n> gene expression is a complex process and multiple cis-acting regulatory sites interact to exert different effects in T cells representative of alternative stages of differentiation.
94209322><n>Sp1</n> is a critical factor for the monocytic specific expression of <n>human CD14</n>. <n>CD14</n> is a membrane glycoprotein expressed specifically on monocytes and macrophages, and its expression is markedly increased during the process of monocyte differentiation. In order to study <n>CD14</n> gene regulation, the human <n>CD14</n> gene was cloned from a partial <n>EcoRI</n> digested chromosome 5 library. A 5.5-kilobase genomic clone contained the full-length <n>CD14</n> coding sequence and 4.2 kilobases of 5'-upstream sequence. One major and one minor transcription start site were identified 101 and 130 base pairs (bp) upstream, respectively, from the protein translation start ATG. A DNA fragment containing 128 bp of upstream sequence had strong, monocyte-specific promoter activity in the <n>CD14</n> positive monocytic cell line Mono Mac 6 as compared to the nonmonocytic cell lines HeLa and REX. Four regions in this DNA fragment interact with nuclear proteins isolated from monocytic cells. The <n>Sp1</n> transcription factor bound to three different regions in the <n>CD14</n> promoter. Mutation of the major <n>Sp1</n> binding site (-110 bp) decreased tissue-specific promoter activity, and these results, together with transactivation experiments, demonstrate that <n>Sp1</n> plays a critical role in the tissue-specific expression of <n>CD14</n> in monocytic cells. <n>CD14</n> <n>Sp1</n> site oligonucleotides bound preferentially to a 105-kDa <n>Sp1</n> species, which is present in higher relative levels in monocytic than non- monocytic cells, suggesting that modification of <n>Sp1</n>, such as phosphorylation, may explain how the <n>Sp1</n> site mediates monocytic specific promoter activity.
94179250>The interleukin-8 AP-1 and kappa B-like sites are genetic end targets of FK506-sensitive pathway accompanied by calcium mobilization. FK506, an immunosuppressant, inhibits the production of several cytokines in T lymphocytes. We observed that FK506 suppressed the transcription of a chemotactic cytokine, <n>interleukin-8</n> (<n>IL-8</n>) in a human T cell line, Jurkat cells, activated by phorbol 12-myristate 13- acetate (PMA) and calcium (Ca2+) ionophore (ionomycin). By deleted and mutated analysis of the <n>IL-8</n> promoters, the AP-1 and kappa B-like sites were identified as the responsive elements for PMA and ionomycin. FK506 suppressed the transcriptions through the AP-1 or kappa B-like sites induced by PMA plus Ca(2+)-mobilizing agents, but not those induced by Ca(2+)-independent stimuli. In gel retardation analysis, FK506 had little effect on the binding to the AP-1 site of PMA/ionomycin-induced nuclear factors, which were recognized with anti-JunD or c-Fos antibody. In contrast, FK506 or EGTA (Ca2+ chelator) similarly affected the formation of kappa B-like site binding complexes, which were not recognized by any antibodies against the human Rel family proteins (<n>c- Rel</n>, <n>p65</n>, <n>p50</n>, and <n>p49</n>). Furthermore, we confirmed the previous report that FK506 suppressed the PMA/ionomycin-induced activation through authentic kappa B site of immunoglobulin (Ig) gene, to which <n>NF-kappa B</n> binding was also decreased by FK506, indicating that both <n>IL-8</n> kappa B- like site and Ig kappa B site are FK506-sensitive in spite of the difference of binding factors. Our results indicate that not only the reported IL-2 NF-AT and NFIL-2A sites and Ig kappa B site, but also the <n>IL-8</n> AP-1 and kappa B-like sites are terminals of FK506-sensitive pathway involving Ca2+ mobilization.
94190839>Androgen binding sites in peripheral human mononuclear leukocytes of healthy males and females. Androgen binding sites have been identified in circulating human mononuclear leukocytes of healthy donors of both sexes. Cells were separated from blood samples on a Ficoll gradient and incubated with different concentrations of [3H]testosterone in the presence or absence of a 400-fold excess of unlabelled testosterone. Binding data were derived from Scatchard analysis. The binding sites fulfil the required criteria for specific steroid binding sites however differ somewhat from the classic androgen receptors from genital skin fibroblast: in fertile adult males (n = 20) the binding sites showed (1) a high affinity for testosterone (1.32 +/- 0.49 nM; mean +/- SD), (2) a saturable capacity (184 +/- 52 binding sites per cell; mean +/- SD), and (3) a characteristic competitive binding profile for other steroid hormones (relative binding affinities: testosterone = dihydrotestosterone &gt; 17 beta-estradiol &gt; progesterone, whereas aldosterone, 17-hydroxy-progesterone and cortisol did not compete appreciably). Furthermore the number of binding sites determined using [3H]dihydrotestosterone, [3H]RU-1881, or [3H]testosterone were comparable. This raises the possibility that androgen receptors in peripheral mononuclear leukocytes differ from those in genital skin fibroblasts. There was no apparent correlation between serum testosterone concentrations and androgen binding sites. In fertile women remarkable changes in androgen binding sites were seen in the course of the menstrual cycle, with a significant increase in the immediate preovulatory period. The presence of androgen receptors in peripheral mononuclear leukocytes provides for the first time the experimental basis for an hypothesis of direct, receptor-mediated effects of androgens on mature immunocompetent cells. The immunological implications
94358417>Induction of <n>IL-8</n> expression in T cells uses the <n>CD28</n> costimulatory pathway. <n>IL-8</n>, a potent chemotactic factor for neutrophil granulocytes and lymphocytes, is a proinflammatory cytokine secreted by a variety of cell types, including T cells. Stimulation of the <n>CD28</n> cell surface molecule delivers costimulatory signals essential for lymphokine production in activated T cells via a conserved sequence element found in the promoter of several lymphokine genes. Anti-<n>CD28</n>-stimulated T cells produced significant amounts of <n>IL-8</n>; additionally, costimulation with anti-CD3 and anti-<n>CD28</n> Abs resulted in a synergistic induction of <n>IL-8</n> secretion. Sequence homology, single nucleotide mutations, and anti-<n>CD28</n> Ab stimulation studies established that the <n>NF-kappa B</n>-like sequence in the promoter of the <n>IL-8</n> gene functioned as a <n>CD28</n> response element. Furthermore, cyclosporin A, but not rapamycin, blocked the synergistic induction of <n>IL-8</n> expression achieved with anti-CD3 and anti-<n>CD28</n> costimulation. The involvement of a <n>CD28</n> response element in the induction of <n>IL-8</n> expression in activated T cells may provide new insights into the pathogenesis and persistence of immune disorders characterized by increased levels of <n>IL-8</n>, such as psoriasis and rheumatoid arthritis.
95032566>MHC class II signaling in B-cell activation [see comments] The cognate interaction between T cells and antigen-presenting cells (APCs), mediated by major histocompatibility complex (MHC) class II molecules, results in the delivery of activation signals to the APC. These signals contribute to the expression of co-stimulatory activity by APCs and have important consequences for cell effector function. MHC class II molecules also serve as receptors for B-cell stimulation by microbial superantigens. In this review, Paul Scholl and Raif Geha discuss recent advances in our understanding of mechanisms of MHC class II signaling and analyse their role in human B-cell activation.
94331016>Effects of glucocorticoids in rheumatoid arthritis. Diminished glucocorticoid receptors do not result in glucocorticoid resistance. OBJECTIVE. Lymphocytes of patients with rheumatoid arthritis (RA) have diminished receptor density; thus, patients with RA should show partial resistance to glucocorticoids. We investigated the glucocorticoid sensitivity of lymphocytes in RA patients compared with healthy subjects. METHODS. We determined the effects of glucocorticoids on lymphocyte proliferation and cytokine release. RESULTS. Proliferation and cytokine release were inhibited in RA patients to the same extent as in healthy controls. CONCLUSION. Diminished receptor density in RA patients does not result in glucocorticoid resistance.
95031992>Arrested development: understanding v-abl. The protein tyrosine kinase activity of the v-abl oncogene has been demonstrated to subvert the normal second messenger systems used by lymphoid cells for growth and differentiation. Transformation of bone marrow with the Abelson murine leukemia virus results in the appearance of B cell lineage cells arrested at the pre-B cell stage. Recent reports have characterized these cells expressing high v-abl kinase activity as deficient in detectable <n>NF-kappaB</n> DNA binding activity and low level RAG gene expression. These observations suggest that v-abl may be inhibiting the differentiation of B cells by blocking these two crucial elements in the maturation pathway.
94318550>Corticosteroid receptors in lymphocytes: a possible marker of brain involution? A similarity has recently been found between the regulation of corticosteroid receptors in brain and in lymphoid tissue. We have studied the regulation of corticosteroid receptors in human mononuclear leukocytes as a possible marker of brain involution. Type I corticosteroid receptors are down regulated by excess of mineralocorticoids (primary and secondary hyperaldosteronism, pseudohyperaldosteronism) and of glucocorticoids (Cushing's syndrome). Type II corticosteroid receptors are not reduced by excess of endogenous corticosteroids (Cushing's syndrome). In normal adults there is a direct significant correlation between plasma cortisol and Type I and between plasma cortisol and Type II receptors in mononuclear leukocytes, while in Cushing's syndrome the correlation is inverse between plasma cortisol at 8 a.m. and Type II receptors. In an aged population the mean numbers of Type I and of Type II receptors are lower and plasma cortisol is higher than in adult controls, but the increase of plasma cortisol is not followed by a clinical picture of hypercorticism. Corticosteroid Type I and Type II receptors are inversely correlated with age. After dexamethasone suppression (1 mg at 11 p.m.) Type I receptors always decrease in controls while the response of Type II is not homogeneous. In an aged group of patients, both receptors are reduced by dexamethasone. We conclude that the decrease with age of corticosteroid receptors is possibly related to a physiological involution of corticosteroid receptors and that this reduction does increase plasma cortisol concentration, without affecting the glucocorticoid effector mechanism.
94238904>All-trans retinoic acid and 1 alpha,25-dihydroxyvitamin D3 co-operate to promote differentiation of the human promyeloid leukemia cell line HL60 to monocytes. A basis for differentiation therapy of leukemias is provided by knowledge of agents which induce specific lineage maturation. All-trans retinoic acid (RA) induces differentiation of HL60 cells to neutrophils and is used to treat acute promyelocytic leukemia. We observed that RA did not induced neutrophil differentiation in serum-free grown HL60 cells whereas 50 nM 1 alpha,25-dihydroxyvitamin D3 (D3) induced maximal monocyte differentiation. Increasing RA concentrations reduced the D3 concentration required for monocyte differentiation. Cells treated with 5 nM D3 showed little response, but differentiated maximally with 5 nM D3 and 10 nM RA. The D3 analogs MC903, EB1089 and KH1060 were more potent inducers of monocyte differentiation. The extent to which analog activity was increased after cotreatment with RA was inversely related to potency. Twenty-four hour treatment with 10 nM RA primed cells for response to 5 nM D3; the reverse sequence being ineffective. Priming with 10 nM RA, or subsequent treatment with D3 (5 nM), did not alter expression of mRNAs encoding receptors for D3 (VDR), RA (<n>RAR alpha</n>) or 9-CIS RA (RXR alpha, beta, gamma). That RA promotes both neutrophil and monocyte differentiation has implications for the use of RA and D3 in treatment of leukemias and provides insight into mechanisms whereby RAR, VDR and RXR facilitate monocyte differentiation.
95012031>[An overexpression of <n>retinoic acid receptor alpha</n> blocks myeloid cell differentiation at the promyelocyte stage] Retinoic acid (RA), a vitamin A derivative, exerts a wide range of biological effects related to cell proliferation and differentiation. The pleiotropic effects of RA are thought to be mediated through specific nuclear RA receptors (RARs). RARs are members of the steroid/thyroid hormone receptor superfamily and exhibit a molecular structure that possess discrete DNA-binding and RA (ligand)-binding domains. In hematopoietic system, RA and RARs, predominantly <n>RAR alpha</n> may play key roles for the proliferation and differentiation of hematopoietic progenitors. However, it is currently unknown how RA and RARs are involved in regulating normal hematopoietic differentiation. To make clear the roles of RA and <n>RAR alpha</n> in the normal hematopoiesis, I have introduced the construct of human <n>RAR alpha</n> (<n>hRAR alpha</n>) into murine bone marrow cells with retroviral vector, and selected infected cells with drug resistant marker (Neo(r)) cultured on the stroma cell line (PA6-neo), and analyzed the behavior of infected cells. All of procedure were done in vitro. Most cells infected with <n>hRAR alpha</n> exhibited promyelocytic morphology and were thought to be blocked at the promyelocytic stage in their myeloid differentiation. Furthermore, these immature cells differentiated terminally into mature granulocytes by adding with RA (10(-6) M). <n>RAR alpha</n> infected cells were also able to differentiate into mature macrophages in the both of long term culture and IL3 colony. These observations suggest that an overexpression of <n>RAR alpha</n> alone is effective to suppress myeloid cell differentiation and <n>RAR alpha</n> plays a crucial role in the terminal differentiation of myeloid precursors. The system described here may serve as a model for studying the the essential genes for differentiation of normal bone marrow cells.
94216500>Hypoxic induction of <n>interleukin-8</n> gene expression in human endothelial cells. Because leukocyte-mediated tissue damage is an important component of the pathologic picture in ischemia/reperfusion, we have sought mechanisms by which PMNs are directed into hypoxic tissue. Incubation of human endothelial cells (ECs) in hypoxia, PO2 approximately 14-18 Torr, led to time-dependent release of <n>IL-8</n> antigen into the conditioned medium; this was accompanied by increased chemotactic activity for PMNs, blocked by antibody to <n>IL-8</n>. Production of <n>IL-8</n> by hypoxic ECs occurred concomitantly with both increased levels of <n>IL-8</n> mRNA, based on polymerase chain reaction analysis, and increased <n>IL-8</n> transcription, based on nuclear run-on assays. Northern analysis of mRNA from hypoxic ECs also demonstrated increased levels of mRNA for <n>macrophage chemotactic protein-1</n>, another member of the chemokine superfamily of proinflammatory cytokines. <n>IL-8</n> gene induction was associated with the presence of increased binding activity in nuclear extracts from hypoxic ECs for the NF-kB site. Studies with human umbilical vein segments exposed to hypoxia also demonstrated increased elaboration of <n>IL-8</n> antigen compared with normoxic controls. In mice exposed to hypoxia (PO2 approximately 30-40 Torr), there was increased pulmonary leukostasis, as evidenced by increased <n>myeloperoxidase</n> activity in tissue homogenates. In parallel, increased levels of transcripts for <n>IP-10</n>, a murine homologue in the chemokine family related to <n>IL-8</n>, were observed in hypoxic lung tissue. Taken together, these data suggest that hypoxia constitutes a stimulus for leukocyte chemotaxis and tissue leukostasis.
94179241><n>Thrombin</n> and thrombin receptor agonist peptide induce early events of T cell activation and synergize with TCR cross-linking for <n>CD69</n> expression and interleukin 2 production. <n>Thrombin</n> stimulation of the T leukemic cell line Jurkat induced a transient increase in [Ca2+]i. Proteolytic activity of the enzyme was required for this effect since <n>diisopropyl fluorophosphate-thrombin</n> failed to increase [Ca2+]i. Furthermore, hirudin and anti-thrombin III inhibited the thrombin-induced [Ca2+]i rise in Jurkat T cells. A synthetic thrombin receptor agonist peptide (TRP) of 7 residues (SFLLRNP) was found to be as effective as <n>thrombin</n> for [Ca2+]i mobilization, and both agonists induced Ca2+ release exclusively from internal stores. <n>Thrombin</n> stimulated tyrosine phosphorylation of several proteins of molecular mass 40, 42, 70, 120, and 130 kDa. There was a good correlation between <n>thrombin</n>-induced tyrosine phosphorylation of the latter three proteins and Ca2+ mobilization. <n>Thrombin</n> and TRP also caused translocation of <n>protein kinase C</n> from the cytosol to the plasma membrane. As a likely consequence of these events, <n>thrombin</n> activated the <n>nuclear factor NF-kB</n>. Several cell lines of hematopoietic origin including the leukemic T cell line HPB.ALL and the erythroleukemic cell line K562 were responsive to <n>thrombin</n>, whereas others such as THP1, a myelomonocytic cell line, and BL2, a Burkitt lymphoma were refractory to <n>thrombin</n> or TRP stimulation. The magnitude of the <n>thrombin</n> response in the different cell types paralleled the expression of the <n>thrombin</n> receptor mRNA. We found that activation of Jurkat T cells by a combination of <n>phytohemagglutinin</n> and phorbol 12- myristate 13-acetate led to a dramatic inhibition of <n>thrombin</n> receptor mRNA expression and to a concomitant loss of the <n>thrombin</n> response. Finally, we demonstrate that <n>thrombin</n> and TRP enhanced <n>CD69</n> expression and <n>interleukin 2</n> production induced by T cell receptor cross-linking in both Jurkat T cells and peripheral blood lymphocytes. These findings highlight the role of <n>thrombin</n> as a potential regulator of T lymphocyte activation.
94172207>Stress response of senescent T lymphocytes: reduced <n>hsp70</n> is independent of the proliferative block. Senescent human T lymphocyte cultures are unable to undergo proliferation, but show no difference from early passage cells in cytotoxic function or surface antigenic profile. A second feature of senescent T cells is the dramatic reduction in <n>hsp70</n> production in response to heat shock. This decline is associated with a decrease in binding of nuclear extracts to the consensus heat shock element. Interestingly, the progressive decline in the heat shock response of cultured T cells correlates with the percent proliferative life span completed rather than with the actual proliferative activity at the time of heat shock. This suggests that for senescent T cells the reduced ability to respond to heat shock by producing <n>hsp70</n>, although possibly lying at the level of transcriptional control, may nevertheless be unrelated to the reduced DNA synthesis or the diminished proliferative activity
94358411>Involvement of <n>phospholipase D</n> in the activation of transcription factor <n>AP-1</n> in human T lymphoid Jurkat cells. The induction of the <n>AP-1</n> transcription factor has been ascribed to the early events leading to T lymphocyte activation. We have examined the possibility that stimulation of <n>phospholipase D</n> (<n>PLD</n>) may regulate activation of transcription factor <n>AP-1</n> in human T cells by transfecting human T lymphocyte Jurkat cells with a plasmid containing an AP-1 enhancer element and a <n>chloramphenicol acetyltransferase</n> reporter gene. We have detected activatable <n>PLD</n> in Jurkat cells, and we have found that addition of phosphatidic acid (PA), the physiologic product of <n>PLD</n> action on phospholipids, is rapidly incorporated into Jurkat cells and leads to activation of transcription factor <n>AP-1</n>. Treatment of Jurkat cells with anti-CD3 mAb activated both <n>PLD</n> and transcription factor <n>AP-1</n>. Wortmannin, an inhibitor of receptor-coupled <n>PLD</n> activation, blocked the <n>anti-CD3</n>-induced increases in both <n>PLD</n> activity and AP-1 enhancer activity. We found a good correlation in the transfected cells between <n>PLD</n> activation and induction of AP-1 enhancer activity under different experimental conditions. Furthermore, ethanol, an inhibitor of the <n>PLD</n> pathway, blocked the anti-CD3-stimulated AP-1 enhancer activity. However, this <n>anti-CD3</n>-mediated response was not inhibited by neomycin, an inhibitor of phosphoinositide hydrolysis. The increases in AP-1 enhancer activity induced by PA or anti-CD3 mAb were efficiently abrogated by the presence of propranolol, an inhibitor of PA <n>phosphohydrolase</n> and <n>protein kinase C</n> (<n>PKC</n>). Furthermore, the PA- and the <n>anti-CD3</n>-induced increases in AP-1 enhancer activity were blocked by the presence of <n>PKC</n> inhibitors or by <n>PKC</n> down-regulation. These data indicate that <n>PLD</n> stimulation can activate the transcription factor <n>AP-1</n> in T lymphocytes, and suggest that the induction of AP-1 enhancer factor activity by PA is mediated via <n>PKC</n> stimulation, either through a direct activating effect of PA or through PA-derived diacylglycerol formation. These data also provide evidence for a role of <n>PLD</n>-derived lipids in the induction of AP-1 enhancer activity resulting from stimulation of the TCR/CD3 complex, suggesting that increased <n>PLD</n> activity can play an important role in T lymphocyte activation.
94335073>Upregulation of bcl-2 by the <n>Epstein-Barr virus latent membrane protein</n> <n>LMP1</n>: a B-cell-specific response that is delayed relative to <n>NF-kappa B</n> activation and to induction of cell surface markers. An ability of the <n>Epstein-Barr virus latent membrane protein</n> <n>LMP1</n> to enhance the survival of infected B cells through upregulation of the bcl-2 oncogene was first suggested by experiments involving gene transfection and the selection of stable <n>LMP1</n>+ clones (S.Henderson, M. Rowe, C.Gregory, F.Wang, E.Kieff, and A.Rickinson, Cell 65:1107- 1115, 1991). However, it was not possible to ascertain whether <n>Bcl-2</n> upregulation was a specific consequence of <n>LMP1</n> expression or an artifact of the selection procedure whereby rare <n>Bcl-2</n>+ cells already present in the starting population might best be able to tolerate the potentially toxic effects of <n>LMP1</n>. We therefore reexamined this issue by using two different experimental approaches that allowed <n>LMP1</n>- induced effects to be monitored immediately following expression of the viral protein and in the absence of selective pressures; activation of the <n>NF-kappa B</n> transcription factor and upregulation of the cell adhesion molecule <n>ICAM-1</n> were used as early indices of <n>LMP1</n> function. In the first approach, stable clones of two B-cell lines carrying an <n>LMP1</n> gene under the control of an inducible metallothionein promoter were induced to express <n>LMP1</n> in all cells. Activation of <n>NK-kappa B</n> and upregulation of <n>ICAM-1</n> occurred within 24 h and were followed at 48 to 72 h by upregulation of <n>Bcl-2</n>. In the second approach, we tested the generality of this phenomenon by transiently expressing <n>LMP1</n> from a strong constitutively active promoter in a range of different cell types. All six B-cell lines tested showed <n>NF-kappa B</n> activation in response to <n>LMP1</n> expression, and this was followed in five of six lines by expression of <n>ICAM-1</n> and <n>Bcl-2</n>. In the same experiments, all three non-B-cell lines showed <n>NF-kappa B</n> activation and <n>ICAM-1</n> upregulation but never any effect upon <n>Bcl-2</n>. We therefore conclude that <n>Bcl-2</n> upregulation is part of the panoply of cellular changes induced by <n>LMP1</n> but that the effect is cell type specific. Our data also suggest that whilst <n>NF-kappa B</n> may be an essential component of <n>LMP1</n> signal transduction, other cell-specific factors may be required to effect some functions of the viral protein.
94313679>Long-term inositol phosphate release, but not <n>tyrosine kinase</n> activity, correlates with <n>IL-2</n> secretion and <n>NF-AT</n> induction in anti-CD3- activated peripheral human T lymphocytes. The cascade of events within the first few minutes of T cell stimulation has been well characterized. Although many second messengers have been shown to be necessary and sufficient for T cell activation in a number of model systems, the rate-limiting step in peripheral T cells has not been demonstrated. To model effective versus ineffective <n>CD3</n>-mediated stimulation in peripheral T cells, we used two anti-CD3 mAbs that differ in their ability to stimulate purified T cells: <n>OKT3</n>, which causes early second messenger generation but is unable to activate T cells without a second signal, and <n>64.1</n>, which stimulates T cell proliferation on its own. We found that <n>tyrosine kinase</n> activity was similar for both mAbs over a period of hours. However, the inositol phosphate response was stronger for <n>64.1</n> than for <n>OKT3</n>. To tie these events to gene activation, we measured <n>NF-kappa B</n> and <n>NF-AT</n> activity in the nucleus after <n>anti-CD3</n> stimulation. Both stimuli induced the appearance of the <n>NF-kappa B</n> components (c-Rel, p65 (RelA), and p50 (NF-kappa B1)) and <n>NF-kappa B</n> DNA binding activity in the nucleus. However, only <n>64.1</n> induced <n>NF-AT</n> in the nucleus, correlating with its ability to activate T cells. Thus, <n>NF-AT</n> induction and <n>IL-2</n> secretion were correlated with the levels of inositol phosphate release but not with gross levels of <n>tyrosine kinase</n> activity induced late following the response. On the other hand, <n>NF-kappa B</n> induction and <n>IL-2</n> receptor expression occurred even with the smaller second messenger response generated by <n>OKT3</n>.
94289707>HLA-DR-, CD33+, CD56+, CD16- myeloid/natural killer cell acute leukemia: a previously unrecognized form of acute leukemia potentially misdiagnosed as French-American-British acute myeloid leukemia-M3 [see comments] We have identified and characterized a previously unrecognized form of acute leukemia that shares features of both myeloid and natural killer (NK) cells. From a consecutive series of 350 cases of adult de novo acute myeloid leukemia (AML), we identified 20 cases (6%) with a unique immunophenotype: CD33+, CD56+, CD11a+, CD13lo, CD15lo, CD34+/-, <n>HLA-DR</n>- , <n>CD16</n>-. Multicolor flow cytometric assays confirmed the coexpression of myeloid (CD33, CD13, CD15) and NK cell-associated (CD56) antigens in each case, whereas reverse transcription polymerase chain reaction (RT- PCR) assays confirmed the identity of <n>CD56</n> (neural cell adhesion molecule) in leukemic blasts. Although two cases expressed <n>CD4</n>, no case expressed <n>CD2</n>, <n>CD3</n>, or <n>CD8</n> and no case showed clonal rearrangement of genes encoding the T-cell receptor (TCR beta, gamma, delta). Leukemic blasts in the majority of cases shared unique morphologic features (deeply invaginated nuclear membranes, scant cytoplasm with fine azurophilic granularity, and finely granular Sudan black B and <n>myeloperoxidase</n> cytochemical reactivity) that were remarkably similar to those of acute promyelocytic leukemia (APL); particularly the microgranular variant (<n>FAB AML-M3v</n>). However, all 20 cases lacked the t(15;17) and 17 cases tested lacked the <n>promyelocytic/retinoic acid receptor alpha</n> (<n>RAR alpha</n>) fusion transcript in RT-PCR assays; 12 cases had 46,XX or 46,XY karyotypes, whereas 2 cases had abnormalities of chromosome 17q: 1 with del(17)(q25) and the other with t(11;17)(q23;q21) and the promyelocytic leukemia zinc finger/<n>RAR alpha</n> fusion transcript. All cases tested (6/20), including the case with t(11;17), failed to differentiate in vitro in response to all-trans retinoic acid (ATRA), suggesting that these cases may account for some APLs that have not shown a clinical response to ATRA. Four of 6 cases tested showed functional NK cell-mediated cytotoxicity, suggesting a relationship between these unique CD33+, CD56+, <n>CD16</n>- acute leukemias and normal CD56+, CD16- NK precursor cells. Using a combination of panning and multiparameter flow cytometric sorting, we identified a normal CD56+, CD33+, CD16- counterpart cell at a frequency of 1% to 2% in the peripheral blood of healthy individuals. Our studies suggest that this form of acute leukemia may arise from transformation of a precursor cell common to both the myeloid and NK cell lineages; thus we propose the designation myeloid/NK acute leukemia. Recognition of this new leukemic entity will be important in distinguishing these ATRA- nonresponsive cases from ATRA-responsive true APL.
94312466><n>IL-4</n> down-regulates <n>IL-2</n>-, <n>IL-3</n>-, and <n>GM-CSF</n>-induced cytokine gene expression in peripheral blood monocytes. <n>IL-4</n>, a product of the T-helper 0 (Th0) and 2 (Th2) subset, was originally described as a B-cell stimulatory factor and has subsequently been found to suppress IL-1 alpha, IL-1 beta, IL-6, IL-8, and TNF-alpha gene expression in monocytes stimulated with LPS, and to upregulate IL-1 receptor antagonist (IL1-RA) gene expression. In this study we investigated the effect of <n>IL-4</n> on the expression of cytokine genes in monocytes evoked by other T-helper cell cytokines: <n>IL-2</n>, <n>IL-3</n>, and <n>GM-CSF</n>. <n>IL-4</n> down-regulated mRNA accumulation of the proinflammatory cytokines <n>IL-1 beta</n>, <n>IL-8</n>, and <n>TNF-alpha</n> in monocytes stimulated with <n>IL-2</n>, <n>IL-3</n>, and <n>GM-CSF</n>. <n>IL-4</n> also suppressed the <n>IL-2</n>- induced <n>IL-6</n> mRNA expression. Temporal analysis of the <n>IL-4</n> down- regulatory effect on the IL-2-, IL-3-, or GM-CSF-induced proinflammatory cytokine gene expression in monocytes provided evidence that <n>IL-4</n> acts predominantly on the post-transcriptional level. This was supported by the observation that the down-regulatory capacity of <n>IL-4</n> appeared to be dependent on de novo protein synthesis. <n>IL-4</n> did not exert significant influence on the induction of expression of <n>IL-1- RA</n> or various CSFs by <n>IL-2</n>, <n>IL-3</n>, and <n>GM-CSF</n>. (ABSTRACT TRUNCATED AT 250 WORDS)
94238882>Induction of proto-oncogene and cytokine expression in human peripheral blood monocytes and the monocytic cell line THP-1 after stimulation with mycoplasma-derived material MDHM. Mycoplasma fermentans-derived high-molecular-weight material (MDHM) was originally described to induce differentiation of murine thymocytes to cytolytic effector T-cells by stimulating <n>IL-6</n> release from adherent cells. This study shows that human peripheral blood monocytes (PBMo) also respond to MDHM with increases in <n>IL-1 beta</n>, <n>IL-6</n> and <n>TNF alpha</n> expression, both at the mRNA and protein level. The induced expression of IL-1 beta and TNF alpha mRNA in the monocytic THP-1 cell line increased as quickly as in primary cells. In contrast to PBMo, THP-1 and 14 other monocytic/myeloid leukemia-derived cell lines did not secrete measurable amounts of the cytokines upon treatment with MDHM. <n>IL-1 beta</n> and <n>IL-6</n> genes contain <n>AP-1</n> binding sites as regulatory elements, the <n>AP-1</n> protein being composed of c-jun and c-fos gene products. In THP-1 cells c-jun mRNA expression increased after incubation with MDHM while positive c-fos expression remained unaffected. Although these data suggest <n>AP-1</n> regulated cytokine mRNA expression, results from PBMo are not in accordance with this notion. In the primary cells MDHM-induced elevation of cytokine mRNA levels was preceded by a downregulation of c-fos expression while positive c-jun expression was not modulated. c-myc mRNA expression, constitutively high in THP-1 cells, was induced in MDHM-stimulated PBMo. In conclusion, MDHM-stimulated induction of cytokine mRNA expression was accompanied by different proto-oncogene responses in PBMo and THP-1 cells. These differences may represent different regulatory pathways of the two cell systems. Alternatively, these data support the notion that neither <n>AP-1</n> nor the <n>c-myc protein</n> are involved in the MDHM-induced increase in IL-1 beta, IL-6 or TNF alpha mRNA levels. Furthermore, the present results demonstrate clearly that mycoplasma products can have a profound impact on the activation status of eukaryotic cells.
95003956>Novel membrane receptors for aldosterone in human lymphocytes: a <n>50 kDa protein</n> on SDS-PAGE. Fast in vitro effects of aldosterone on the <n>Na+/H(+)-exchanger</n>, inositoltrisphosphate generation and corresponding specific binding to plasma membranes at Kd-values of approximately 0.1 nM have been found in human mononuclear leukocytes and vascular smooth muscle cells. The novel aldosterone membrane receptor was analyzed on SDS-PAGE after labeling of microsomal membranes from human mononuclear leukocytes with a [125I]-aldosterone-derivative by use of BASED as a photoactivatable crosslinker. Binding of 1 nM [125I]-aldosterone was found at a molecular weight of approximately 50 kDa which was absent with 1 microM cold aldosterone, but not cortisol in the binding media. This aldosterone-selectivity is typical and discriminatory for the new <n>aldosterone membrane receptor</n>. Solubilization of the receptor protein from membranes by high salt concentrations (1 M NaCl, 1 mM EDTA) was not achieved. It, thus, appears as an integral membrane protein. Dithiothreitol, a sulfhydryl agent, does not reduce specific aldosterone binding indicating the absence of SH-groups in the binding domain or sensitive structures of the receptors. The results are the first to characterize the novel membrane receptor for aldosterone with regard to molecular weight and basic properties. These findings and other related results are reviewed here.
94187105>A transcriptional regulatory element is associated with a nuclease- hypersensitive site in the pol gene of human immunodeficiency virus type 1. Analysis of the chromatin organization of the integrated human immunodeficiency virus type 1 (HIV-1) genome has previously revealed a major constitutive DNase I-hypersensitive site associated with the pol gene (E. Verdin, J. Virol. 65:6790-6799, 1991). In the present report, high-resolution mapping of this site with <n>DNase I</n> and micrococcal nuclease identified a nucleosome-free region centered around nucleotides (nt) 4490 to 4766. A 500-bp fragment encompassing this hypersensitive site (nt 4481 to 4982) exhibited transcription-enhancing activity (two- to threefold) when it was cloned in its natural position with respect to the HIV-1 promoter after transient transfection in U937 and CEM cells. Using in vitro footprinting and gel shift assays, we have identified four distinct binding sites for nuclear proteins within this positive regulatory element. Site B (nt 4519 to 4545) specifically bound four distinct nuclear protein complexes: a ubiquitous factor, a T- cell-specific factor, a <n>B-cell-specific factor</n>, and the monocyte/macrophage- and B-cell-specific transcription factor <n>PU.1/Spi- 1</n>. In most HIV-1 isolates in which this PU box was not conserved, it was replaced by a binding site for the related factor <n>Ets1</n>. Factors binding to site C (nt 4681 to 4701) had a DNA-binding specificity similar to that of factors binding to site B, except for <n>PU.1/Spi-1</n>. A GC box containing a binding site for <n>Sp1</n> was identified (nt 4623 to 4631). Site D (nt 4816 to 4851) specifically bound a ubiquitously expressed factor. These results identify a transcriptional regulatory element associated with a nuclease-hypersensitive site in the pol gene of HIV-1 and suggest that its activity may be controlled by a complex interplay of cis-regulatory elements.
94194029>Expression of v-src in T cells correlates with nuclear expression of <n>NF- kappa B</n>. <n>NF-kappa B</n> is a rapidly inducible transcriptional activator that responds to a variety of signals and influences the expression of many genes involved in the immune response. Protein <n>tyrosine kinase</n>s transmit signals from cytokine and immune receptors. Very little information exists linking these two important classes of signaling molecules. We now demonstrate that v-src expression correlates with nuclear expression of a kappa B binding complex similar to that induced by phorbol ester and ionomycin, as detected by electrophoretic mobility shift assay using a variety of kappa B sites. This complex was blocked by the <n>tyrosine kinase</n> inhibitor, herbimycin A. The v-src-induced complex comprised the p50 and p65 components of <n>NF-kappa B</n>, as determined by supershift and immunoblot analysis. As a functional correlate of this finding, transient co-transfection of HIV-1 LTR reporter constructs in a different T cell line demonstrated that v-src activated this promoter in a kappa B-dependent manner. We found that transactivation of the HIV-1 LTR by v-src was more sensitive to mutations of the proximal, rather than the distal, kappa B element. The implications for T cell receptor signaling and HIV-1 gene expression are considered.
94167865>trans-activation of the HIV promoter by a cDNA and its genomic clones of human herpesvirus-6. Human herpesvirus 6 (HHV-6) is a lymphotropic herpesvirus, and in vitro, it can productively infect human CD4+ T cells as HIV-1. Co- infection of T cells by HIV-1 and HHV-6 can lead to both activation of the HIV-1 promoter and acceleration of the cytopathic effects. An HHV-6 (GS) cDNA clone, pCD41, encoding for a <n>41-kDa nuclear protein</n> was identified and characterized previously (Chang and Balachandran, J. Virol. 65, 2884-2894 and 7085, 1991). Sequence analyses show that this protein has significant homology with the human cytomegalovirus UL44 gene coding for the ICP36 family of early-late-class phosphoprotein. Using this cDNA as the probe, a 3.8-kb EcoRI genomic fragment encoding the <n>HHV-6(GS)P41</n> was cloned and designated as pGD41. When cotransfected with the HIV LTR CAT into CV-1 cells, both the pCD41 and pGD41 clones trans-activated the HIV LTR. Sequence analyses of pCD41 indicate that there are two potential open reading frames (ORFs), A and B, which are homologous to the ORFs found in the genomic clone pGD41. Deletion constructs of the pCD41 clone demonstrated that <n>ORF-A</n> was critical for the HIV LTR activation. Deletion analyses of the pCD41 <n>ORF-A</n> and the use of promoter constructs further mapped an internal functional promoter within the pCD41 sequence that can direct the synthesis of the trans-activating protein. By using HIV LTR deletion mutants, the <n>NF- kappa B</n> binding sites were found to be critical for response to the pCD41 trans-activation
94350980><n>CD14</n>-mediated translocation of <n>nuclear factor-kappa B</n> induced by lipopolysaccharide does not require <n>tyrosine kinase</n> activity. During the course of serious bacterial infections, lipopolysaccharide (LPS) is believed to interact with macrophage receptors, resulting in the generation of inflammatory mediators and systemic symptoms including hemodynamic instability and shock. <n>CD14</n>, a glycosylphosphatidylinositol-linked antigen, functions as an LPS signaling receptor. A critical issue concerns the mechanism by which <n>CD14</n>, which has no transmembrane domain, transduces its signal following LPS binding. Recently, investigators have hypothesized that <n>CD14</n>-mediated signaling is effected through a receptor-associated <n>tyrosine kinase</n> (TK), suggesting a multicomponent receptor model of LPS signaling. Wild-type Chinese hamster ovary (CHO)-K1 cells can be activated by endotoxin to release arachidonate following transfection with human <n>CD14</n> (CHO/<n>CD14</n>). Nuclear translocation of cytosolic NF-kappa B is correlated with a number of LPS-inducible responses. We sought to determine if this pathway were present in CHO/<n>CD14</n> cells and to elucidate the relationship of <n>NF-kappa B</n> activation to the <n>CD14</n> receptor system. LPS-stimulated translocation of <n>NF-kappa B</n> in CHO/<n>CD14</n> cells resembled the same response in the murine macrophage-like cell line RAW 264.7. Protein synthesis inhibitors and corticosteroids, which suppress arachidonate release and the synthesis of proinflammatory cytokines, had no effect on translocation of <n>NF-kappa B</n> in CHO/<n>CD14</n> or RAW 264.7 cells, demonstrating that <n>NF-kappa B</n> translocation is an early event. Although TK activity was consistently observed by immunoblotting extracts from activated RAW 264.7 cells, LPS-induced phosphotyrosine residues were not observed from similarly treated CHO/CD14 cells. Furthermore, the TK inhibitors herbimycin A and genistein failed to inhibit translocation of <n>NF-kappa B</n> in CHO/CD14 or RAW 264.7 cells, although both of these agents inhibited LPS-induced TK activity in RAW 264.7 cells. These results imply that TK activity is not obligatory for <n>CD14</n>-mediated signal transduction to occur in response to LPS.
94354848>Signals transduced through the <n>CD4 molecule</n> on T lymphocytes activate <n>NF-kappa B</n>. We have demonstrated that native envelope glycoproteins of HIV-1, gp160 can induce activation of the transcription factor, <n>NF-kappa B</n>. The stimulatory effects of gp160 are mediated through the <n>CD4 molecule</n>, since pretreatment with soluble <n>CD4</n> abrogates its activity. The gp160- induced <n>NF-kappa B</n> complex consists of p65, p50 and c-rel proteins. The stimulatory effect of gp160 on <n>NF-kappa B</n> activation is protein synthesis independent, is dependent upon protein tyrosine phosphorylation, and abrogated by inhibitors of <n>protein kinase C</n>. The gp160-mediated activation of <n>NF-kappa B</n> in <n>CD4</n> positive T cells may be involved in biological effects, e.g., enhanced HIV replication, hypergammaglobulinemia, increased cytokine secretion, hypercellularity in bone marrow and apoptosis.
95081342>No evidence for the expression of the progesterone receptor on peripheral blood lymphocytes during pregnancy [see comments] The expression of the progesterone receptor in human peripheral blood lymphocytes was analysed, using an enzyme linked immunosorbent assay (Abbott PgR-EIA monoclonal), in order to evaluate its prognostic character in the context of spontaneous abortion. Cytosols were prepared from lymphocytes of 24 healthy pregnant women (11 first, 10 second and three third trimester), seven healthy non-pregnant women, nine women with recurrent spontaneous abortion, and six healthy men. In addition, a human breast carcinoma cell line (ZR-75-1), which expresses the progesterone receptor, was analysed throughout. The ZR-75-1 cell line showed an expression of 642 fmol/mg whereas lymphocytes of pregnant women showed an expression &lt; or = 4 fmol/mg. Lymphocytes of non-pregnant women, women with threatened pre-term delivery, and men showed equivalent levels: 3 +/- 1, 3 +/- 2 and 5 +/- 4 fmol/mg respectively. These results show that there is no evidence of specific expression of the progesterone receptor in pregnancy and exclude any prognostic character in spontaneous abortion. A role for the progesterone receptor in the mechanism of the known effect of progesterone on peripheral blood lymphocytes is also excluded.
94274683>Tolerance to lipopolysaccharide involves mobilization of <n>nuclear factor kappa B</n> with predominance of p50 homodimers. Stimulation of the human monocytic cell line Mono Mac 6 with lipopolysaccharide (LPS) leads to rapid and transient expression of cytokines like tumor necrosis factor (TNF). When such cells are precultured for 2 days with a low dose of LPS (20 ng/ml) followed by stimulation with a high dose of LPS (1 microgram/ml), expression of the TNF gene is minimal, i.e. the cells are tolerant. In nuclear run-on analysis, such tolerant cells show only a low degree of transcription, indicating that tolerance operates at or upstream of the transcription level. The <n>CD14</n> LPS receptor is, however, up-regulated (not down- regulated) in tolerant cells, and LPS can, in fact, still lead to activation of tolerant cells as evidenced by mobilization of the transcription factor <n>nuclear factor kappa B</n> (<n>NF-kappa B</n>). Resolution of the <n>NF-kappa B</n> complex in gel shift analysis shows that the binding protein, mobilized in naive Mono Mac 6 cells, consists mainly of p50- p65 heterodimers, while in tolerant cells, the p50 homodimer is predominant. This increase in p50 homodimers coincides with an increase in p105 mRNA, suggestive of a transcriptional up-regulation of p50. Reporter gene analysis reveals that the <n>NF-kappa B</n> complex mobilized in tolerant cells is functionally inactive in that <n>NF-kappa B</n>-dependent luciferase constructs containing the human immunodeficiency virus long terminal repeat or the TNF 5'-region show only minimal transactivation after LPS stimulation. Similar to Mono Mac 6 cells, primary blood monocytes, when precultured with a low dose of LPS, also become tolerant and produce little TNF after LPS stimulation. The tolerant blood monocytes also up-regulate <n>CD14</n>, and they mobilize <n>NF-kappa B</n> with a predominance of p50 homodimers. Taken together, these results demonstrate that tolerance to LPS is determined by post-receptor mechanisms that involve an altered composition of the <n>NF-kappa B</n> complex.
94278430>Analysis of Oct2-isoform expression in lipopolysaccharide-stimulated B lymphocytes. Oct2-isoform expression in splenic B cells stimulated with lipopolysaccharide or lipopolysaccharide plus phorbol-di-butyrate was analysed by cDNA cloning. The frequency of Oct2-positive clones was 1/15,000 in both libraries. Two new isoforms were found that generate novel amino- or carboxy-terminal sequences. An isoform lacking exon 11 destroyed the carboxy-terminal leucin-zipper region and introduced a frame shift creating a novel, proline-rich carboxy terminus. A new exon containing a highly basic region (4c) was characterized, between exons 4 and 5. This exon was inserted between glutamine-rich regions 2 and 3, carboxy terminal of a tentative leucine-zipper structure. In addition, a new combination isoform containing <n>Oct2a</n>'s amino terminal insert (exon 7a) and <n>Oct2b</n>'s carboxy terminal insert (exon 13) was found that created a novel large isoform, <n>Oct2ab</n>. More frequent use of the classical <n>Oct2a</n> and <n>Oct2b</n> isoforms was observed in the lipopolysaccharide-stimulated B cells, while a preference for the <n>Oct2ab</n> and <n>Oct2ba</n> isoforms was observed in lipopolysaccharide plus phorbol-di-butyrate-treated cells.
94217708>Positive regulators of the lineage-specific transcription factor <n>GATA-1</n> in differentiating erythroid cells. The zinc finger transcription factor <n>GATA-1</n> is a major regulator of gene expression in erythroid, megakaryocyte, and mast cell lineages. <n>GATA-1</n> binds to WGATAR consensus motifs in the regulatory regions of virtually all erythroid cell-specific genes. Analyses with cultured cells and cell-free systems have provided strong evidence that <n>GATA-1</n> is involved in control of globin gene expression during erythroid differentiation. Targeted mutagenesis of the <n>GATA-1</n> gene in embryonic stem cells has demonstrated its requirement in normal erythroid development. Efficient rescue of the defect requires an intact GATA element in the distal promoter, suggesting autoregulatory control of <n>GATA-1</n> transcription. To examine whether <n>GATA-1</n> expression involves additional regulatory factors or is maintained entirely by an autoregulatory loop, we have used a transient heterokaryon system to test the ability of erythroid factors to activate the <n>GATA-1</n> gene in nonerythroid nuclei. We show here that proerythroblasts and mature erythroid cells contain a diffusible activity (TAG) capable of transcriptional activation of <n>GATA-1</n> and that this activity decreases during the terminal differentiation of erythroid cells. Nuclei from <n>GATA-1</n>- mutant embryonic stem cells can still be reprogrammed to express their globin genes in erythroid heterokaryons, indicating that de novo induction of <n>GATA-1</n> is not required for globin gene activation following cell fusion.
95000924>Role of <n>HIV-1 Nef</n> expression in activation pathways in <n>CD4</n>+ T cells. The role of the human immunodeficiency virus (HIV-1) <n>Nef protein</n> in T cell activation pathways was investigated using a Jurkat <n>CD4</n>+ cell line stably transfected with a Nef expression vector. Secretion of <n>IL-2</n> and <n>TNF-alpha</n>, surface expression of <n>IL-2R</n>, and DNA-binding activity of <n>NF- kappa B</n> and AP-1 (Fos/Jun) complex in response to phorbol myristate acetate, <n>TNF-alpha</n>, or immobilized antibodies to <n>CD3</n> were monitored. These parameters were not modified by <n>Nef</n> expression in Jurkat cells, whereas stimulation with the same stimuli resulted in partial inhibition of LTR activation in <n>Nef</n>+ Jurkat cells. This inhibition was not mediated through <n>Nef</n> phosphorylation on Thr-15 or GTP-binding activity because mutations in critical sites did not alter this inhibition. Analysis of truncated LTRs confirmed that inhibition of LTR activation was not mediated through <n>NF-kappa B</n>-binding activity but through the region containing the negative responding elements (NREs). These results suggest that <n>Nef</n> downmodulates LTR activation without significantly inhibiting the capacity of T cells to respond to immunological activations.
94338582><n>Tat-binding protein 7</n> is a subunit of the 26S protease. Subunit 6 (S6), an integral component of the 26S protease from human erythrocytes, has been studied by SDS-PAGE, peptide mapping and sequence analysis. S6 was cleaved with <n>CNBr</n> and three internal peptides were sequenced. A comparison with known proteins in Genbank revealed that all three S6 peptides match the predicted sequence of <n>TBP7</n>, <n>Tat- binding protein 7</n>. Based on peptide matches covering more than 10% of the <n>TBP7</n> sequence, and the fact that the migration of S6 on SDS-PAGE is consistent with the estimated molecular mass for <n>TBP7</n>, we conclude that subunit 6 of the 26S protease is <n>TBP7</n>.
94184956>Hypoxia causes the activation of <n>nuclear factor kappa B</n> through the phosphorylation of <n>I kappa B alpha</n> on tyrosine residues. The response of mammalian cells to stress is controlled by transcriptional regulatory proteins such as <n>nuclear factor kappa B</n> (<n>NF- kappa B</n>) to induce a wide variety of early response genes. In this report, we show that exposure of cells to hypoxia (0.02% O2) results in <n>I kappa B alpha</n> degradation, increased <n>NF-kappa B</n> DNA binding activity, and transactivation of a reporter gene construct containing two <n>NF- kappa B</n> DNA binding sites. Pretreatment of cells with protein <n>tyrosine kinase</n> inhibitors and the dominant negative allele of c-Raf-1 (<n>Raf 301</n>) inhibited <n>I kappa B alpha</n> degradation, <n>NF-kappa B</n> binding, and transactivation of kappa B reporter constructs by hypoxia. To demonstrate a direct link between changes in the phosphorylation pattern of <n>I kappa B alpha</n> with <n>NF-kappa B</n> activation, we immunoprecipitated <n>I kappa B alpha</n> after varying times of hypoxic exposure and found that its tyrosine phosphorylation status increased during hypoxic exposure. Inhibition of the transfer of tyrosine phosphoryl groups onto <n>I kappa B alpha</n> prevented <n>I kappa B alpha</n> degradation and <n>NF-kappa B</n> binding. In comparison to other activators of <n>NF-kappa B</n> such as phorbol myristate acetate or tumor necrosis factor, we did not detect changes in the tyrosine phosphorylation status of <n>I kappa B alpha</n> following treatment with either of these agents. These results suggest that tyrosine phosphorylation of <n>I kappa B alpha</n> during hypoxia is an important proximal step which precedes its dissociation and degradation from <n>NF-kappa B</n>.
94151007>Overproduction of <n>NFKB2</n> (<n>lyt-10</n>) and c-Rel: a mechanism for <n>HTLV-I Tax</n>- mediated trans-activation via the <n>NF-kappa B</n> signalling pathway. Molecular, biochemical and epidemiological evidence implicate HTLV-I as an etiologic agent of adult T cell leukemia (ATL). The <n>Tax protein</n> of HTLV-I, a positive transcriptional activator of HTLV-I gene expression, is a viral oncogene that also increases transcription of cellular genes including <n>GM-CSF</n>, <n>IL-2R alpha</n> and <n>IL-2</n>. One of the cellular targets of the trans-activating effects of Tax is the <n>NF-kappa B</n>/Rel family of transcription factors, pleiotropic regulators of immunoregulatory, cytokine and viral gene expression. In this report, we demonstrate that <n>NFKB2</n> (<n>lyt-10</n>) and c-Rel are overexpressed in HTLV-I infected and Tax- expressing cells and, together, account for the majority of the constitutive <n>NF-kappa B</n> binding activity in these cells before and after PMA stimulation. Most importantly, we show a <n>Tax</n>-dependent correlation between expression of <n>NFKB2</n>(<n>p100</n>) and processing to the DNA binding <n>NFKB2</n>(<n>p52</n>) form, induction of c-Rel, and trans-activation of <n>NF- kappa B</n>-mediated gene expression. Furthermore, the <n>NFKB2</n> precursor is physically associated with c-Rel and with <n>Tax</n> in HTLV-I infected cells. We propose that <n>NFKB2</n> synthesis and processing allows continuous nuclear expression of an otherwise cytoplasmic protein and, in conjunction with overexpression of c-Rel, <n>NFKB2</n> alters the <n>NF-kappa B</n> signalling pathway and contributes to leukemic transformation of T cells by HTLV-I
94129004>Retinoic acid downmodulates erythroid differentiation and <n>GATA1</n> expression in purified adult-progenitor culture. All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of <n>interleukin-3</n> (<n>IL-3</n>) <n>granulocyte- macrophage colony-stimulating factor</n> (<n>GM-CSF</n>) and <n>erythropoietin</n> (<n>Ep</n>) (combined with <n>c-kit</n> ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid- suspension culture supplemented with saturating <n>Ep</n> level and low-dose <n>IL-3</n>/<n>GM-CSF</n>, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of <n>GATA1</n> mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of <n>GATA1</n> induction in the early stages of erythropoietic differentiation.
94134077>Induction of phosphatidylinositol turnover and EGR-1 mRNA expression by crosslinking of surface IgM and IgD in the human B cell line B104. We have previously shown that a human B lymphoma cell line, B104, expressed <n>surface IgM</n> (<n>sIgM</n>) and <n>surface IgD</n> (<n>sIgD</n>), and that crosslinking of <n>sIgM</n> and <n>sIgD</n> by <n>anti-IgM antibody (Ab)</n> and <n>anti-IgD Ab</n>, respectively, induced Ca2+ influx to almost the same degree, whereas only <n>sIgM</n>-crosslinking caused B104 cell death. Here, we investigated the accumulation of cyclic AMP (cAMP), the hydrolysis of inositol phosphates, <n>protein kinase C</n> (<n>PKC</n>) activity and the induction of Egr-1 and c-fos mRNA expression by sIgM- and sIgD-crosslinking to examine differences in the signals mediated through <n>sIgM</n> and <n>sIgD</n> in B104 cells. Both sIgM- and sIgD-crosslinking with antibodies induced elevation of cAMP levels, phosphatidylinositol turnover, <n>PKC</n> activation and expression of Egr-1 and c-fos mRNA, although <n>sIgM</n>-crosslinking was more effective than <n>sIgD</n>-crosslinking, presumably due to the higher expression of <n>sIgM</n> than of <n>sIgD</n>. Egr-1 mRNA expression induced by sIgM- and sIgD-crosslinking was inhibited by <n>H7</n>, erbstatin and genistein, but not by HA1004. Erbstatin and genistein inhibited the sIg-crosslinking- induced Egr-1 mRNA expression in a dose-dependent manner parallel to that observed in the inhibition of sIg-crosslinking-induced protein tyrosine phosphorylation. Phorbol myristate acetate induced Egr-1 mRNA expression but forskolin and dibutyryl cyclic AMP did not. These findings suggest that the Egr-1 mRNA activating signals through <n>sIgM</n> and <n>sIgD</n> are protein tyrosine kinase- and PKC-dependent, but <n>protein kinase A</n>-independent. Cyclosporin A (CsA) and FK506 rescued B104 cells from death induced by <n>anti-IgM Ab</n>, but did not affect the expression of <n>Egr-1</n> and c-fos mRNA, showing that CsA and FK506 affect signal transducers differently from or downstream to these molecules. The difference in signals transduced through <n>sIgM</n> and <n>sIgD</n> in B104 cells is discussed.
94166578>Direct exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases infectivity of human erythrocytes to a malarial parasite. Direct exposure to 10 nM 2,3,7,8-TCDD caused a 75% increase and a 2- fold increase in the infectivity of isolated human erythrocytes to P. falciparum after 48 hours when the parasites were in an unsynchronized or synchronized state of growth, respectively. Treatment of human erythrocytes with 10 microM sodium orthovanadate (NaOV), an inhibitor of plasma membrane Ca-ATPase and <n>phosphotyrosine phosphatase</n>, decreased parasitemia by 30%. Co-treatment of RBCs with TCDD and NaOV completely blocked the TCDD-induced increase in parasitemia. Because erythrocytes are anucleated, these results are discussed as evidence for biochemical changes by TCDD without requiring the activation of gene products.
94314370>Evidence for a trans-acting activator function regulating the expression of the human <n>CD5</n> antigen. Interspecies somatic cell hybrids were generated by fusing the mouse T- lymphoma cell line, BW5147, with normal human T lymphocytes at different stages of differentiation. Thymocytes, activated peripheral T lymphocytes, or an activated T-cell clone were used as human partners, respectively, in three independent fusions. Irrespective of the human cell partner used for fusion, a certain number of hybrids lost <n>CD5</n> surface expression over a period of time in culture. Analysis at the phenotype and genetic level showed that lack of <n>CD5</n> expression was due neither to segregation of human autosome 11, on which the <n>CD5</n> gene has been mapped, nor to deletion of the <n>CD5</n> structural gene. Furthermore, loss of <n>CD5</n> surface expression correlated with the absence of specific mRNA. Since these hybrids preferentially segregate human chromosomes, these results indicate the existence of a non-syntenic trans-active locus, or loci, positively controlling the expression of the human <n>CD5</n> gene.
97113352>Induction of the CD11b gene during activation of the monocytic cell line U937 requires a novel nuclear factor <n>MS-2</n> [published erratum appears in J Immunol 1999 Jul 15;163(2):1091] The differentiation of myeloid precursors into mature myelomonocytic cells is characterized by the induction of the gene encoding the beta2 integrin <n>CD11b</n>. The transcription factors <n>Sp1</n> and <n>PU.1 prime</n> the <n>CD11b</n> promoter, but the nature of the factors responsible for its inducible expression are unknown. In addition to the <n>CD11b</n> gene, the homologous genes encoding <n>CD11a</n> and <n>CD11c</n> also exhibit inducible expression during myeloid differentiation. Therefore, we compared the nucleotide sequences of the CD11a, CD11b, and CD11c gene promoters to identify common elements that might contribute to inducible expression. This analysis identified one such element repeated four times within the <n>CD11b</n> promoter. Mutation of these elements indicated that two, <n>MS-2beta</n> and <n>MS-2gamma</n>, are critical to the induction of the <n>CD11b</n> gene during differentiation of the pro-monocytic cell line U937. Electrophoretic mobility shift assays indicate that <n>MS-2beta</n> and <n>MS-2gamma</n> interact with <n>nuclear factors</n> that are induced during U937 differentiation. These factors are detected at the time the <n>CD11b</n> promoter is activated. The molecular mass of these factors is approximately 28 kDa, and their DNA binding characteristics are indistinguishable from those of the novel nuclear factor <n>MS-2</n>. Taken together, our data indicate that <n>MS-2</n> mediates induction of the <n>CD11b</n> gene as cells of the monocytic lineage mature. The presence of multiple potential binding sites for <n>MS-2</n> in the promoter regions of a wide range of genes expressed in mature myeloid cells suggests this factor plays a general role in myeloid differentiation.
97127169>Acetylsalicylic acid and sodium salicylate inhibit LPS-induced NF-kappa B/c-Rel nuclear translocation, and synthesis of tissue factor (TF) and <n>tumor necrosis factor alfa</n> (<n>TNF-alpha</n>) in human monocytes. We have investigated the effects of acetylsalicylic acid and sodium salicylate on the LPS-induced synthesis of the pro-coagulant protein tissue factor (TF) and the pro-inflammatory protein tumor necrosis factor-alpha (<n>TNF-alpha</n>), as well as the <n>prostaglandin PGE2</n> in human monocytes. Both drugs dose-dependently inhibited <n>LPS-induced TF</n> and <n>TNF- alpha</n> synthesis at the mRNA and the protein level, and reduced <n>PGE2</n> production. As evidenced by electro mobility shift assay (EMSA) and the use of a NF-kappa B prototypic probe, these drugs probably exert their inhibitory effects by interference with the nuclear translocation of NF- kappa B/c-Rel proteins. These data may expand the understanding of the anti-thrombotic and anti-inflammatory effects of these drugs when activation of monocytes occurs.
97074532>Interferon augments <n>PML</n> and PML/RAR alpha expression in normal myeloid and acute promyelocytic cells and cooperates with all-trans retinoic acid to induce maturation of a retinoid-resistant promyelocytic cell line. The <n>PML</n> gene is fused to the retinoic acid receptor alpha gene (RAR alpha) in the acute promyelocytic leukemia (APL) 15; 17 translocation. <n>PML</n> is expressed in diverse tissues and cell lines and localized in the nucleus with a typical speckled pattern. In the bone marrow, it is preferentially expressed in myeloid cells. <n>PML</n> appears to be transcriptionally regulated by class I and II interferons, which raises the possibility that interferons modulate the function and growth and differentiation potential of normal myeloid cells and precursors by activating <n>PML</n>-dependent pathways. Similarly, interferons could act on APL cells, alone or in combination with all-trans retinoic acid (RA), especially if the PML/RAR alpha fusion transcript that results from the t(15; 17) is induced by interferon. We report here that <n>PML</n> is expressed at low levels or not expressed in normal circulating human monocytes, lymphocytes, and polymorphonucleate cells, but is markedly induced by interferon; that <n>PML</n> and PML/RAR alpha expression is augmented by interferon in the NB4 APL cell line, which carries the t(15; 17), and in APL blasts from patients; that interferon inhibits growth and survival of NB4 APL cells in cooperation with RA; that interferons alone have minimal maturation effect on NB4 cells; and, finally, that interferon gamma, but not alpha or beta, induces maturation and growth suppression of NB4 cells with de novo retinoid resistance, and partially restores RA response.
97107281>Effects of Ara-C on <n>neutral sphingomyelinase</n> and mitogen- and stress- activated protein kinases in T-lymphocyte cell lines. <n>Neutral sphingomyelinase</n> (<n>SMase</n>) can be activated by extracellular signals to produce ceramide, which may affect mitogen-activated protein kinase (MAPK) activities. Neutral <n>SMase</n> activity was assessed in membranes from Jurkat, a human T-cell line, and EL4, a murine T-cell line. Ara-C activated <n>SMase</n> with 10 minutes in both Jurkat and EL4 cells, while phorbol ester (PMA) had no effect. PMA, but not Ara-C or ceramides, activated <n>ERK MAPKS</n>, in Jurkat and EL4. PMA acted synergistically with ionomycin to activate JNK MAPKs in Jurkat and EL4 within 10 minutes. Ara-C activated JNKs only after prolonged incubation (90-120 minutes). Thus, ceramide is not a positive signal for <n>ERK</n> activation in T-cell lines. The effects of Ara-C on <n>JNK</n> activity may be mediated through secondary response pathways.
97048062>Comparative analysis identifies conserved <n>tumor necrosis factor receptor-associated factor 3</n> binding sites in the human and simian Epstein-Barr virus oncogene LMP1. Nonhuman primates are naturally infected with a B-lymphotropic herpesvirus closely related to Epstein-Barr virus (EBV). These simian EBV share considerable genetic, biologic, and epidemiologic features with human EBV, including virus-induced tumorigenesis. However, latent, transformation-associated viral genes demonstrate marked sequence divergence among species despite the conserved functions. We have cloned the <n>latent membrane protein 1</n> (<n>LMP1</n>) homologs from the simian EBV naturally infecting baboons (cercopithicine herpesvirus 12, herpesvirus papio) and rhesus monkeys (cercopithicine herpesvirus 15) for a comparative study with the human EBV oncogene. The transmembrane domains are well conserved, but there is striking sequence divergence of the carboxy-terminal cytoplasmic domain essential for B-cell immortalization and interaction with the tumor necrosis factor receptor signaling pathway. Nevertheless, the simian EBV <n>LMP1s</n> retain most functions in common with EBV <n>LMP1</n>, including the ability to induce <n>NF- (kappa)B</n> activity in human cells, to bind the <n>tumor necrosis factor- associated factor 3</n> (<n>TRAF3</n>) in vitro, and to induce expression of tumor necrosis factor-responsive genes, such as ICAM1, in human B lymphocytes. Multiple <n>TRAF3</n> binding sites containing a PXQXT/S core sequence can be identified in the simian EBV <n>LMP1s</n> by an in vitro binding assay. A PXQXT/S-containing sequence is also present in the cytoplasmic domain of the Hodgkin's disease marker, <n>CD30</n>, and binds <n>TRAF3</n> in vitro. The last 13 amino acids containing a PXQXT/S sequence are highly conserved in human and simian EBV <n>LMP1</n> but do not bind <n>TRAF3</n>, suggesting a distinct role for this conserved region of <n>LMP1</n>. The conserved <n>TRAF3</n> binding sites in <n>LMP1</n> and the <n>CD30</n> Hodgkin's disease marker provides further evidence that a <n>TRAF3</n>-mediated signal transduction pathway may be important in malignant transformation.
97056026>Chromosome 1 aneusomy with 1p36 under-representation is related to histologic grade, DNA aneuploidy, high <n>c-erb B-2</n> and loss of bcl-2 expression in ductal breast carcinoma. Chromosome 1 abnormalities with loss of 1p36 have been investigated in 95 breast-cancer samples by means of a dual-target fluorescence in-situ hybridization (FISH) technique using the pUC 1.77 and p1-79 probes, specific for the 1q12 and 1p36 regions, respectively. Abnormalities for one or both probes were detected in 83/95 samples. Relative 1p36 under- representation was found in 79/95. The clinical relevance of these alterations was studied by comparing the FISH results with several parameters currently used in breast-cancer pathology. Distinct patterns of chromosome 1 abnormalities were found among the histologic types of breast carcinoma. Lobular or mucinous samples showed few or no alterations, whereas most ductal samples had high chromosome 1 polysomy with under-representation of 1p36. In ductal carcinomas, chromosome 1 alterations increased with histologic grade, DNA aneuploidy, loss of bcl-2 and high <n>c-erb B-2</n> expression. These associations were found to be statistically significant. No correlation between chromosome 1 alterations and nuclear grade, age, size, lymph-node involvement, hormonal receptor presence, proliferation activity or <n>p53</n> protein expression was detected. These results indicate the utility of this FISH technique for a better definition of the biological characteristics of ductal carcinomas
94140841>G(Anh)MTetra, a natural bacterial cell wall breakdown product, induces <n>interleukin-1 beta</n> and <n>interleukin-6</n> expression in human monocytes. A study of the molecular mechanisms involved in inflammatory cytokine expression [published erratum appears in J Biol Chem 1994 Jun 17;269(24):16983] It is believed that induction of cytokine expression by bacterial cell wall components plays a role in the development and course of sepsis. However, most attention has been focused on lipopolysaccharide (LPS). We studied the ability of N-acetylglucosaminyl-1,6-anhydro-N- acetylmuramyl-L-alanyl-D- isoglutamyl-m-diaminopimelyl-D-alanine (G(Anh)MTetra), a naturally occurring breakdown product of peptidoglycan that is produced by soluble <n>lytic transglycosylase</n> of Escherichia coli, to induce cytokine expression in human monocytes. G(Anh)MTetra was found to strongly induce <n>interleukin (IL)-1 beta</n> and <n>IL-6</n> mRNA expression after 2 h and <n>IL-1 beta</n> and <n>IL-6 protein</n> secretion after 48 h of activation. The increase in mRNA accumulation was at least partly due to an increase in the transcription rates of the respective genes and was accompanied by a strong induction of nuclear factor-kappa B and activator protein-1 transcription factor expression. Experiments using inhibitors of <n>protein kinase C</n>, <n>protein kinase A</n>, and tyrosine kinase-dependent pathways revealed that G(Anh)MTetra-induced <n>IL-1 beta</n> and <n>IL-6</n> mRNA expression involves activation of an H7- inhibitable pathway. By using the protein synthesis inhibitor cycloheximide, it was shown that G(Anh)MTetra-induced <n>IL-6</n> mRNA expression depends on the synthesis of new protein, whereas G(Anh)MTetra-induced <n>IL-1 beta</n> mRNA accumulation does not. When responses to G(Anh)MTetra were compared with those to LPS and muramyldipeptide (MDP), it was found that the optimal response to G(Anh)MTetra induction was similar to that of LPS but significantly higher than the response to MDP. Furthermore, maximal G(Anh)MTetra- induced <n>IL-1 beta</n> and <n>IL-6</n> mRNA expression could be enhanced by co- stimulation with LPS or MDP, suggesting that different receptors and/or transduction pathways were involved. These results indicate that G(Anh)MTetra induces <n>IL-1 beta</n> and <n>IL-6</n> expression in human monocytes suggesting a possible role for G(Anh)MTetra in the release of cytokines during sepsis.
94110603>Increased proliferation, cytotoxicity, and gene expression after stimulation of human peripheral blood T lymphocytes through a surface ganglioside (GD3) [published erratum appears in J Immunol 1994 Jul 15;153(2):910] Previous studies have suggested that gangliosides have an important role in cell signaling and recognition. However, their specific function in these processes has not been clearly defined. A mAb, <n>R24</n>, that reacts specifically with a cell surface ganglioside (GD3) has been demonstrated to stimulate proliferation of T cells derived from human peripheral blood. In this study, we have investigated the mechanisms by which the <n>R24</n> mAb affects T cell functions. We have observed that the <n>R24</n> mAb stimulates GD3+ T cell proliferation, cytotoxicity, and surface marker expression of IL-2R alpha-chain, IL-2R beta-chain, <n>HLA-DR</n>, <n>CD11a</n>, and <n>CD11c</n>. Additionally, <n>IFN-gamma</n> activity but not IL-1, IL-2, or IL-4 activity was present in culture supernatants 72 h after <n>R24</n> stimulation. In some donors, increased <n>IL-6</n> and <n>TNF-alpha</n> activity also was detected after <n>R24</n> treatment. Furthermore, <n>R24</n> treatment resulted in translocation of <n>c-rel</n>, but little or no NF kappa B p50 or p65, from the cytoplasm to the nucleus and an increase of <n>NF kappa B</n> binding complexes containing <n>c-rel</n> and p50. This treatment also caused increased tyrosine phosphorylation of specific protein substrates. <n>R24</n>- stimulated increases in proliferation, cytotoxicity, and cell surface protein expression could be blocked by cyclosporin and staurosporin, indicating that cyclophilin/calcineurin and <n>protein kinase C</n> may be involved in the <n>R24</n> signaling pathway. Additionally, herbimycin A, a tyrosine kinase inhibitor, blocked the <n>R24</n>-stimulated increase in proliferation but not cytotoxicity at concentrations consistent with specificity for tyrosine kinases. These results suggest that multiple biochemical pathways are involved in the activation of human T cells by <n>R24</n>.
94214477>Genes encoding general initiation factors for <n>RNA polymerase II</n> transcription are dispersed in the human genome. General transcription factors are required for accurate initiation of transcription by RNA polymerase II. Human cDNAs encoding subunits of these factors have been cloned and sequenced. Using fluorescence in situ hybridization (FISH), we show here that the genes encoding the TATA-box binding protein (TBP), TFIIB, TFIIE alpha, TFIIE beta, RAP30, RAP74 and the 62 kDa subunit, of <n>TFIIH</n> are located at the human chromosomal bands 6q26-27, 1p21-22, 3q21-24, 8p12, 13q14, 19p13.3 and 11p14-15.1, respectively. This dispersed localization of a group of functionally related gene provides insights into the molecular mechanism of human genome evolution and their possible involvement in human diseases.
96063079><n>BCL-6</n> and the molecular pathogenesis of B-cell lymphoma. The results presented identify the first genetic lesion associated with DLCL, the most clinically relevant form of NHL. Although no proof yet exists of a role for these lesions in DLCL pathogenesis, the feature of the <n>BCL-6</n> gene product, its specific pattern of expression in B cells, and the clustering of lesions disrupting its regulatory domain strongly suggest that deregulation of <n>BCL-6</n> expression may contribute to DLCL development. A more precise definition of the role of <n>BCL-6</n> in normal and neoplastic B-cell development is the goal of ongoing study of transgenic mice engineered either to express <n>BCL-6</n> under heterologous promoters or lacking <n>BCL-6</n> function due to targeted deletions. In addition to contributing to the understanding of DLCL pathogenesis, the identification of <n>BCL-6</n> lesions may have relevant clinical implications. DLCL represent a heterogeneous group of neoplasms which are treated homogeneously despite the fact that only 50% of patients experience long-term disease-free survival (Schneider et al. 1990). The fact that <n>BCL-6</n> rearrangements identify biologically and clinically distinct subsets of DLCL suggests that these lesions may be useful as markers in selection of differential therapeutic strategies based on different risk groups. Furthermore, the <n>BCL-6</n> rearrangements can be used to identify and monitor the malignant clone with sensitive PCR- based techniques. Since clinical remission has been observed in a significant fraction of DLCL cases, these markers may serve as critical tools for sensitive monitoring of minimal residual disease and early diagnosis of relapse (Gribben et al. 1993).
97175006>Pancreatic development and maturation of the islet B cell. Studies of pluripotent islet cultures. Pancreas organogenesis is a highly regulated process, in which two anlage evaginate from the primitive gut. They later fuse, and, under the influence of the surrounding mesenchyme, the mature organ develops, being mainly composed of ductal, exocrine and endocrine compartments. Early buds are characterized by a branching morphogenesis of the ductal epithelium from which endocrine and exocrine precursor cells bud to eventually form the two other compartments. The three compartments are thought to be of common endodermal origin; in contrast to earlier hypotheses, which suggested that the endocrine compartment was of neuroectodermal origin. It is thus generally believed that the pancreatic endocrine-lineage possesses the ability to mature along a differentiation pathway that shares many characteristics with those of neuronal differentiation. During recent years, studies of insulin-gene regulation and, in particular, the tissue-specific transcriptional control of insulin-gene activity have provided information on pancreas development in general. The present review summarizes these findings, with a special focus on our own studies on pluripotent endocrine cultures of rat pancreas.
97128276>Octamer independent activation of transcription from the kappa immunoglobulin germline promoter. Previous analyses of immunoglobulin V region promoters has led to the discovery of a common octamer motif which is functionally important in the tissue-specific and developmentally regulated transcriptional activation of immunoglobulin genes. The germline promoters (Ko) located upstream of the J region gene segments of the kappa locus also contain an octamer motif (containing a single base pair mutation and referred to as the variant octamer) which has been shown previously to bind <n>Oct- 1</n> and <n>Oct-2</n> transcription factors in vitro. To further elucidate the role of this variant octamer motif in the regulation of germline transcription from the unrearranged kappa locus, we have quantitated the relative binding affinity of <n>Oct-1</n> and <n>Oct-2</n> for the variant octamer motif and determined the functional role of this octamer motif in transcriptional activation. We find that, although the variant octamer motif binds <n>Oct-1</n> and <n>Oct-2</n> in vitro with 5-fold lower affinity than the consensus octamer motif, mutation of the variant octamer motif to either a consensus octamer or non-octamer motif has no effect on transcriptional activation from the germline promoter. We also find significant differences in activation of germline and V region promoters by kappa enhancers. Our results suggest that the germline promoters and V region promoters differ in their dependence on octamer for activation and respond differently to enhancer activation. These findings have important implications in regulation of germline transcription as well as concomitant activation of the V-J recombination of the kappa light chain locus.
97074543>Generation of CD1+RelB+ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated giant cells from human monocytes. We previously showed that <n>granulocyte-macrophage colony-stimulating factor</n> (<n>GM-CSF</n>) and macrophage colony-stimulating factor (<n>M-CSF</n>) stimulate the differentiation of human monocytes into two phenotypically distinct types of macrophages. However, in vivo, not only <n>CSF</n> but also many other cytokines are produced under various conditions. Those cytokines may modulate the differentiation of monocytes by CSFs. In the present study, we showed that CD14+ adherent human monocytes can differentiate into CD1+relB+ dendritic cells (DC) by the combination of <n>GM-CSF</n> plus <n>interleukin-4</n> (<n>IL-4</n>) and that they differentiate into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleated giant cells (MGC) by the combination of <n>M-CSF</n> plus <n>IL-4</n>. However, the monocyte-derived DC were not terminally differentiated cells; they could still convert to macrophages in response to <n>M-CSF</n>. <n>Tumor necrosis factor-alpha</n> (<n>TNF-alpha</n>) stimulated the terminal differentiation of the DC by downregulating the expression of the <n>M-CSF</n> receptor, cfms mRNA, and aborting the potential to convert to macrophages. In contrast to <n>IL-4</n>, <n>interferon-gamma</n> (<n>IFN-gamma</n>) had no demonstrable effect on the differentiation of monocytes. Rather, <n>IFN- gamma</n> antagonized the effect of <n>IL-4</n> and suppressed the DC and MGC formation induced by <n>GM-CSF</n> + <n>IL-4</n> and <n>M-CSF</n> + <n>IL-4</n>, respectively. Taken together, these results provide a new aspect to our knowledge of monocyte differentiation and provide evidence that human monocytes are flexible in their differentiation potential and are precursors not only of macrophages but also of CD1+relB+DC and TRAP-positive MGC. Such a diverse pathway of monocyte differentiation may constitute one of the basic mechanisms of immune regulation.
97177471>The NF-kappa B inhibitor, tepoxalin, suppresses surface expression of the cell adhesion molecules <n>CD62E</n>, <n>CD11b/CD18</n> and <n>CD106</n>. Tepoxalin, a dual enzyme inhibitor of <n>cyclooxygenase</n> and <n>5-lipoxygenase</n> has been shown to inhibit T-cell activation. Its immunosuppressive property is distinct from cyclosporin because only tepoxalin, but not cyclosporin, suppresses NF-kappa B activation. Here we report that tepoxalin selectively inhibits <n>intercellular adhesion molecule-1</n> (<n>ICAM- 1</n>, <n>CD54</n>)/MAC-1 (<n>CD11b/CD18</n>) dependent adhesion of polymorphonuclear cells to <n>IL-1</n> activated human umbilical vein endothelial cells. The mechanism of inhibition is related to the surface expression of several cell adhesion molecules. Flow cytometry analyses on cultured cells that were treated with tepoxalin or antisense oligonucleotides to the P65/p50 subunit of NF-kappa B, and then stimulated with PMA, revealed a reduced expression of <n>CD11b/CD18</n> on monocytic HL60 cells, and <n>endothelial adhesion molecule-1</n> (<n>CD62E</n>) and <n>vascular adhesion molecule- 1</n> (<n>CD106</n>) on human umbilical vein endothelial cells. Expression of other adhesion molecules such as <n>lymphocyte function associated-antigen- 1</n> (<n>CD11a</n>/CD18) and <n>CD54</n> were unaffected. Tepoxalin also inhibited the secretion of a NF-kappa B regulated chemokine, <n>IL-8</n>, a known inducer of <n>CD11b/CD18</n> expression. Thus the suppression of <n>CD11b/CD18</n> expression by tepoxalin may involve <n>IL-8</n>. Our results suggest that by inhibiting NF- kappa B activation, surface expression of several adhesion molecules can be modulated and that tepoxalin may be useful in treating selected adhesion mediated events such as leukocyte migration or atherosclerotic plaque formation.
97066958>Characterization of a <n>CD43/leukosialin</n>-mediated pathway for inducing apoptosis in human T-lymphoblastoid cells. The monoclonal antibody (mAb) <n>J393</n> induces apoptosis in Jurkat T-cells. <n>NH2</n>-terminal amino acid sequence analysis identified the <n>140-kDa surface antigen</n> for <n>mAb J393</n> as <n>CD43/leukosialin</n>, the major sialoglycoprotein of leukocytes. While Jurkat cells co-expressed two discrete cell-surface isoforms of <n>CD43</n>, recognized by <n>mAb J393</n> and <n>mAb G10-2</n>, respectively, only J393/<n>CD43</n> signaled apoptosis. J393/<n>CD43</n> was found to be hyposialylated, bearing predominantly O-linked monosaccharide glycans, whereas G10-2/<n>CD43</n> bore complex sialylated tetra- and hexasaccharide chains. Treatment with soluble, bivalent <n>mAb J393</n> killed 25-50% of the cell population, while concomitant engagement of either the CD3.TcR complex or the integrins <n>CD18</n> and <n>CD29</n> significantly potentiated this effect. Treatment of Jurkat cells with <n>mAb J393</n> induced tyrosine phosphorylation of specific protein substrates that underwent hyperphosphorylation upon antigen receptor costimulation. Tyrosine kinase inhibition by herbimycin A diminished J393/<n>CD43</n>-mediated apoptosis, whereas inhibition of phosphotyrosine phosphatase activity by bis(maltolato)oxovanadium-IV enhanced cell death. Signal transduction through tyrosine kinase activation may lead to altered gene expression, as J393/<n>CD43</n> ligation prompted decreases in the nuclear localization of the transcriptional regulatory protein NF- kappaB and proteins binding the interferon-inducible regulatory element. Since peripheral blood T-lymphocytes express cryptic epitopes for <n>mAb J393</n>, these findings demonstrate the existence of a tightly regulated <n>CD43</n>-mediated pathway for inducing apoptosis in human T-cell lineages.
97103140>Sterol dependent LDL-receptor gene transcription in lymphocytes from normal and CML patients. Sterol regulatory element (SRE) has been recognized to regulate various key genes coding for especially <n>low density lipoprotein</n> (<n>LDL</n>)-receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase known to play a crucial role in the cholesterol feedback mechanism. The deranged cholesterol feedback mechanism has been widely recognised in initiation as well as progression of various types of cancers including chronic myeloid leukaemia (CML). Consequently, the present study was addressed to understand this phenomenon and revealed the existence of a <n>unique 47 kDa protein factor</n> having affinity for this SRE sequence in lymphocytes from normal subjects as well as its absence in lymphocytes from untreated CML patients. However, this factor appeared when the CML patients achieved complete haematological remission (CHR) through <n>alpha-interferon</n> therapy. Further, an inverse relationship was also observed between sterol modulated <n>LDL</n>-receptor gene transcription and the binding affinity of this <n>47 kDa factor</n> to the SRE sequence. Based upon these results we propose that <n>alpha- interferon</n> through its receptor initiates phosphatidic acid dependent signalling which in turn regulates the affinity of <n>47 kDa sterol regulatory element binding factor</n> as well as <n>LDL</n>-receptor gene transcription in lymphocytes from CML patients
94285936>Multiple prolactin-responsive elements mediate G1 and S phase expression of the interferon regulatory factor-1 gene. The interferon regulatory factor-1 (<n>IRF-1</n>) gene is both an immediate- early G1 phase gene and an S phase gene inducible by <n>PRL</n> in rat Nb2 T lymphocytes. To understand the mechanism by which <n>PRL</n> regulates the biphasic expression of <n>IRF-1</n>, we cloned the rat <n>IRF-1</n> gene and functionally characterized the <n>IRF-1</n> promoter. Upon transfection into Nb2 T cells, 1.7 kilobases (kb) of <n>IRF-1</n> 5'-flanking DNA linked to a <n>chloramphenicol acetyl transferase</n> (<n>CAT</n>) reporter gene mediated a 30- fold induction of <n>CAT</n> enzyme activity in response to 24 h of <n>PRL</n> stimulation. Deletion mutants containing 1.3, 0.6, and 0.2 kb 5'- flanking DNA were incrementally less transcriptionally active, although 0.2 kb still mediated a 12-fold induction by <n>PRL</n>. The sequence between - 1.7 and -0.2 kb linked to a heterologous thymidine kinase promoter failed to respond to <n>PRL</n> stimulation, suggesting that the activity of upstream <n>PRL</n> response elements may require an interaction with promoter- proximal elements. By assaying <n>CAT</n> enzyme activity across a 24-h <n>PRL</n> induction time course, we were able to assign G1 vs. S phase <n>PRL</n> responses of the <n>IRF-1</n> gene to different regions of the <n>IRF-1</n> 5'- flanking and promoter DNA. The 0.2-kb IRF-<n>CAT</n> construct was induced by <n>PRL</n> stimulation during the G1 phase of the cell cycle. In contrast, the 1.7-kb IRF-<n>CAT</n> construct was inducible by <n>PRL</n> during both G1 and S phase of the cell cycle. Hence, the <n>PRL</n>-induced biphasic expression of the <n>IRF-1</n> gene appears to be controlled by separate <n>PRL</n>-responsive elements: elements in the first 0.2 kb of the <n>IRF-1</n> promoter region act during early activation, and elements between 0.2 and 1.7 kb act in concert with the proximal 0.2-kb region during S phase progression.
94120719>Regulation of the BZLF1 promoter of Epstein-Barr virus by second messengers in anti-immunoglobulin-treated B cells. Initiation of the Epstein-Barr virus (EBV) lytic cycle is dependent on the transcription of the BZLF1 gene. The BZLF1 gene promoter (Zp) was activated by crosslinking of cell surface immunoglobulin (Ig) with anti- Ig antibody in B cells, even in the absence of other viral genes. We identified several anti-Ig response elements within Zp, which were originally defined as 12-O-tetradecanoylphorbol-13-acetate (TPA) response elements (ZI repeats and ZII, an AP-1-like domain). Since anti- Ig crosslinking leads to activation of <n>protein kinase C</n> (<n>PKC</n>) and an increase in intracellular calcium level, Zp was tested for the response to these cellular factors. Treatment with calcium ionophore A23187 increased Zp activity. When the calcium ionophore was used in conjunction with TPA, a <n>PKC</n> activator, the Zp induction was synergistically enhanced. 1-(5-Isoquinolinyl sulfonyl)-2- methylpiperazine, an inhibitor of <n>PKC</n>, inhibited the anti-Ig inducibility of Zp. Calmodulin antagonists, compound R24571 and trifluoperazine, blocked the Zp activation with anti-Ig. These findings suggest that Zp responds directly to changes in the activity of both <n>PKC</n> and calcium/calmodulin-dependent protein kinase. Requirement of tyrosine kinase activation for the anti-Ig-mediated Zp activation was also demonstrated through the use of the tyrosine kinase inhibitor herbimycin. These cellular gene regulatory molecules induced with anti- Ig may cooperatively play an important part in achieving efficient EBV activation as seen with anti-Ig treatment in B cells.
94118533>The <n>SCL protein</n> displays cell-specific heterogeneity in size. <n>SCL protein</n> production was examined in a variety of hemopoietic cell lines by immunoblotting using specific polyclonal antisera. <n>SCL protein</n> was detected in erythroid, megakaryocyte, mast and early myeloid cell lines, as well as in several lymphoid leukemia cell lines which are known to harbor SCL gene rearrangements. In most cell lines, proteins of molecular weight 49 and 44 kDa were found, however two myeloid cell lines expressed only lower molecular weight species of 24 and 22 kDa. This size discrepancy appeared to be due to cell-specific translational regulation, since overexpression of a retrovirally transfected SCL gene yielded the higher molecular weight forms in most cell lines (GP+E-86, AT2.5, M1) but only the <n>22 kDa form</n> in the myeloid cell line, WEHI- 3B/D+. Overexpression of full-length <n>SCL protein</n> in the lymphoid cell lines, SupT1 and Raji, did not alter cell phenotype and there was no evidence for autoregulation of <n>SCL</n> transcription. The restricted pattern of <n>SCL protein</n> synthesis is consistent with the restricted expression of SCL mRNA documented previously. In addition, the present results indicate that <n>SCL protein</n> size was determined by regulation of translation in a cell-specific manner.
94290489>Description and functional implications of a novel mutation in the sex- determining gene SRY. The sex-determining gene SRY was screened for molecular alteration in an XY sex-reversed female by single-strand conformation polymorphism (SSCP) technique. An A-to-G transition was detected which leads to an exchange of a tyrosine by a cysteine in the <n>SRY protein</n>. The affected tyrosine residue located at the C terminus of the DNA binding protein is evolutionarily strongly conserved among the members of the HMG box containing proteins. Using gel shift assay and peptide synthesis such a mutation is shown to abolish the SRY protein DNA binding ability. The involvement of this particular amino acid in the binding specificity is also discussed.
94107268>Induction of tyrosine phosphorylation and T-cell activation by vanadate peroxide, an inhibitor of protein tyrosine phosphatases. Rapid tyrosine phosphorylation of key cellular proteins is a crucial event in the transduction of activation signals to T-lymphocytes. The regulatory role of protein tyrosine phosphatases (PTPases) in this process was explored by studying the effects of a powerful <n>PTPase</n> inhibitor, vanadate peroxide (pervanadate), on the activation cascade of Jurkat human leukaemic T-cells. Pervanadate induced activation of the tyrosine kinases <n>lck</n> and <n>fyn</n> (4- and 3-fold respectively) and a dramatic increase in tyrosine phosphorylation of cellular proteins, notably <n>phospholipase C gamma 1</n>. After this event, we observed a rise in intracellular Ca2+ concentration, corresponding to an influx. This effect required surface expression of the <n>CD45 PTPase</n> and was not observed in CD45-deficient variants of Jurkat cells. In the CD45- negative variant, the effect of pervanadate on tyrosine phosphorylation was globally decreased and some phosphorylated substrates were specifically missing. Pervanadate also stimulated transcription of the c-fos gene and accumulation of its mRNA as well as several other hallmarks of T-lymphocyte activation such as surface expression of the CD69 antigen and the interleukin 2 receptor alpha-chain (CD25). Pervanadate synergized with signals delivered by T-cell antigen receptor engagement or by a phorbol ester to induce interleukin 2 production. <n>Pervanadate activated NF-kappa B</n>, as shown by an increase in DNA-binding activity of this transcription factor. We thus conclude that PTPases play a crucial role in the negative regulation of signal transduction culminating in T-lymphocyte activation. Moreover, induction of tyrosine phosphorylation appears sufficient per se to initiate a complete activation programme.
97113313>Activation of <n>nuclear factor-kappaB</n> via T cell receptor requires a Raf kinase and Ca2+ influx. Functional synergy between Raf and <n>calcineurin</n>. Signals transduced via the TCR activate the transcription factor <n>nuclear factor-kappaB</n> (<n>NF-kappaB</n>), which, in turn, is critical to the transcriptional induction of many genes important for the proliferation and expression of a differentiated phenotype. Treatment of T cells with the <n>protein kinase C</n> activator PMA in combination with Ca2+ ionophores mimics this process, and the two agents are often substituted for TCR stimulation, bypassing the TCR. Here we identify intracellular signaling components involved in activation of <n>NF-kappaB</n> following TCR stimulation. TCR signaling was triggered by treating Jurkat T cells with <n>PHA</n> or anti-CD3 Abs, and <n>NF-kappaB</n> activation was monitored by electrophoretic mobility shift assays and/or by <n>kappaB</n>-dependent reporter assays. Contrary to the idea that <n>protein kinase C</n> is involved in TCR-mediated activation of <n>NF-kappaB</n>, high doses of staurosporine did not interfere with activation of <n>NF-kappaB</n> by <n>PHA</n>, while the same dose of staurosporine completely blocked activation by PMA. <n>PHA</n>-induced kappaB-dependent reporter activity was, however, effectively blocked by a dominant negative form of <n>Raf-1</n>, suggesting a critical role for a Raf kinase. The TCR-mediated activation of <n>NF-kappaB</n> was also dependent on a Ca2+ influx, because the Ca2+ channel blocker, SK&amp;F 96365, as well as other agents that prevented the Ca2+ influx, inhibited <n>NF-kappaB</n> activation. Cotransfection of a constitutively active form of <n>calcineurin</n> largely substituted for the Ca2+ requirement and reversed the blockade by SK&amp;F 96365. Consistent with these observations, coexpression of constitutively active forms of <n>Raf-1</n> and <n>calcineurin</n> synergistically induced <n>kappaB</n>-dependent reporter activity, suggesting a physiologically relevant functional interaction between the kinase and the phosphatase.
97126060>Synergistic interactions between overlapping binding sites for the serum response factor and <n>ELK-1 proteins</n> mediate both basal enhancement and phorbol ester responsiveness of primate cytomegalovirus major immediate-early promoters in monocyte and T-lymphocyte cell types. Cytomegalovirus (CMV) infection is nonpermissive or persistent in many lymphoid and myeloid cell types but can be activated in differentiated macrophages. We have shown elsewhere that both the major immediate- early gene (MIE) and lytic cycle infectious progeny virus expression can be induced in otherwise nonpermissive monocyte-like U-937 cell cultures infected with either human CMV (HCMV) or simian CMV (SCMV) by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Two multicopy basal enhancer motifs within the SCMV MIE enhancer, namely, 11 copies of the 16-bp cyclic AMP response element (CRE) and 3 copies of novel 17-bp serum response factor (SRF) binding sites referred to as the SNE (SRF/NFkappaB-like element), as well as four classical NFkappaB sites within the HCMV version, contribute to TPA responsiveness in transient assays in monocyte and T-cell types. The SCMV SNE sites contain potential overlapping core recognition binding motifs for SRF, Rel/NFkappaB, ETS, and YY1 class transcription factors but fail to respond to either serum or tumor necrosis factor alpha. Therefore, to evaluate the mechanism of TPA responsiveness of the SNE motifs and of a related 16-bp SEE (SRF/ETS element) motif found in the HCMV and chimpanzee CMV MIE enhancers, we have examined the functional responses and protein binding properties of multimerized wild-type and mutant elements added upstream to the SCMV MIE or simian virus 40 minimal promoter regions in the U-937, K-562, HL-60, THP-1, and Jurkat cell lines. Unlike classical NFkappaB sites, neither the SNE nor the SEE motif responded to phosphatase inhibition by okadaic acid. However, the TPA responsiveness of both CMV elements proved to involve synergistic interactions between the core SRF binding site (CCATATATGG) and the adjacent inverted ETS binding motifs (TTCC), which correlated directly with formation of a bound tripartite complex containing both the cellular SRF and <n>ELK-1 proteins</n>. This protein complex was more abundant in U-937, K-562, and HeLa cell extracts than in Raji, HF, BALB/c 3T3, or HL-60 cells, but the binding activity was altered only twofold after TPA treatment. A 40-fold stimulation of <n>chloramphenicol acetyltransferase</n> activity mediated by four tandem repeats of the SNE could be induced within 2 h (and up to 250-fold within 6 h) after addition of TPA in DNA-transfected U-937 cells, indicating that the stimulation appeared likely to be a true <n>protein kinase C</n>-mediated signal transduction event rather than a differentiation response. Slight differences in the sequence of the core SRF binding site compared with that of the classical c-Fos promoter serum response element, together with differences in the spacing between the SRF and ETS motifs, appear to account for the inability of the SCMV SNEs to respond to serum induction.
97069862>T-cell-directed <n>TAL-1</n> expression induces T-cell malignancies in transgenic mice. The TAL-1 gene specifies for a basic domain-helix-loop-helix protein, which is involved in the control of normal hematopoiesis. In human pathology, the <n>TAL-1 gene product</n> is expressed in a high percentage of T-cell acute lymphoblastic leukemias in the pediatric age range; however, it has not been established whether the expression has a causal role in oncogenesis. In this report, we describe the phenotype of mouse transgenic lines obtained by inducing <n>tal-1 protein</n> expression in lymphoid tissues using the LCK promoter. The survival rate of tal-1 transgenic animals was much lower as compared with control mice. Histopathological analysis revealed lymphomas of T-cell type, often comprising a minor B-cell component. Some mice showed marked splenic lymphocyte depletion. Primary lymphocyte cultures showed partial independence from exogenous growth stimuli and increased resistance to low-serum apoptosis. To further unravel the tal-1 oncogenic potential, a strain of tal-1 transgenic mice was crossbred with p53-/- mice; the survival rate in these animals was reduced by more than one-half when compared with that of tal-1 mice, and histopathological analysis revealed exclusively T-cell lymphomas. These data indicate that <n>TAL-1</n>, expressed in T cells, is per se a potent oncogene, which may exert a key leukemogenetic role in the majority of T-cell acute lymphoblastic leukemias.
97087515>Tissue and cell-type specific expression of the tuberous sclerosis gene, TSC2, in human tissues. TSC2 is a gene on chromosome 16p13.3 associated with the autosomal dominant neurocutaneous disorder, tuberous sclerosis complex (TSC). By using a partial nucleotide sequence from the cloned TSC2 and polymerase chain reaction methodology, we constructed a digoxigenin-labeled complementary DNA probe to examine TSC2 gene expression in autopsy- or biopsy-derived human tissues by in situ hybridization. TSC2 messenger RNA was widely expressed in various cell types throughout the body, including epithelia, lymphocytes, and cells with endocrine functions, e.g., adrenal cortex and anterior pituitary. It was prominently and selectively (within the central nervous system) expressed in pyramidal cells of the cerebral cortex and other motor neurons, e.g., in spinal cord and brainstem nuclei. Visceral TSC2 expression was comparable in autopsy tissues from patients with and without TSC; TSC2 messenger RNA expression was most prominent in cells with a rapid mitotic rate and turnover, e.g., epithelia and lymphocytes, with central nervous system pyramidal cells and other neurons being an obvious exception, and/or in cells with important secretory/transport functions. This widespread expression of the TSC2 gene supports the view that it encodes a protein vital to cell growth and metabolism or one that functions as a tumor/growth suppressor.
97047773>Apoptosis signaling pathways in normal T cells: differential activity of <n>Bcl-2</n> and <n>IL-1beta</n>-converting enzyme family protease inhibitors on glucocorticoid- and Fas-mediated cytotoxicity. <n>Fas</n>-mediated apoptosis plays an important role in regulating the immune response in peripheral T cells. Restimulation of T cell blasts up- regulates <n>Fas</n> and <n>Fas</n> ligand expression, with subsequent interaction leading to cell death. Overexpression of <n>Bcl-2</n> in tumor cells blocks apoptosis induced by many stimuli, but inhibition of <n>Fas</n>-mediated killing has not been consistently observed. To examine the behavior of <n>Bcl-2</n> in normal cells, T cell blasts were transiently transfected with <n>Bcl-2</n> and related gene products to determine the effect on apoptotic signaling. Transient overexpression of <n>Bcl-2</n> in mouse and human T cell blasts did not block <n>Fas</n>-mediated apoptosis, whereas etoposide- and glucocorticoid-induced cytotoxicity was potently inhibited. Expression of <n>Bcl-xL</n> and <n>adenovirus E1B 19K</n> did not interfere with anti-<n>Fas</n> killing. In contrast, interleukin-1beta-converting enzyme family protease inhibitors Ac-DEVD-CHO and CrmA blocked <n>Fas</n>-mediated apoptosis. These results suggest that peripheral T cells use distinct apoptosis signaling pathways with differential sensitivity to <n>Bcl-2</n> and interleukin-1beta-converting enzyme family protease inhibitors. Since T cells normally express <n>Bcl-2</n> and <n>Bcl-xL</n> following activation, their inability to block <n>Fas</n>-mediated apoptosis may allow for the elimination of self-reactive cells and the appropriate regulation of immune responses
94284067>Increased <n>interleukin 2</n> transcription in murine lymphocytes by ciprofloxacin. The fluoroquinolone antibiotic, ciprofloxacin (cipro), induces hyperproduction of <n>interleukin 2</n> (<n>IL-2</n>) and <n>interferon-gamma</n> (<n>IFN- gamma</n>) in stimulated human peripheral blood lymphocytes. In this investigation an enhanced and prolonged <n>IL-2</n> and IL-2 mRNA response was also detected in both stimulated (T cell mitogens or alloantigens) murine splenocytes and in the stimulated murine T cell line EL-4 in the presence of ciprofloxacin (5-80 micrograms/ml) as compared to control cells without antibiotics. However, in contrast to human lymphocytes, <n>IFN-gamma</n> production was inhibited and <n>IFN-gamma</n> mRNA levels were unaffected at 24 h and only slightly upregulated at 48 and 72 h of culture in murine splenocytes incubated with cipro (20 micrograms/ml). EL-4 cells were transfected with a plasmid containing the IL-2 promoter and enhancer region linked to the <n>chloramphenicol acetyltransferase</n> (<n>CAT</n>) reporter gene. Analysis of <n>CAT</n> activity revealed that cipro enhanced IL-2 gene induction. In addition, EL-4 cells incubated with ciprofloxacin showed an early peak and more activated nuclear factor of activated T cells (<n>NFAT-1</n>) as compared to control cells without antibiotics. Cipro did not affect the nuclear transcription factors <n>AP- 1</n> or <n>NFIL-2A</n>. Taken together, cipro inhibited <n>IFN-gamma</n> synthesis, but enhanced <n>IL-2</n> production in murine lymphocytes by means of influencing <n>NFAT-1</n> and causing an increased <n>IL-2</n> transcription.
94119088>Human T-cell leukemia virus type I <n>Tax</n> associates with and is negatively regulated by the <n>NF-kappa B2 p100 gene product</n>: implications for viral latency. Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of the adult T-cell leukemia, an aggressive and often fatal malignancy of activated human CD4 T cells. HTLV-I encodes an essential <n>40-kDa protein</n> termed <n>Tax</n> that not only transactivates the long terminal repeat of this retrovirus but also induces an array of cellular genes. <n>Tax</n>- mediated transformation of T cells likely involves the deregulated expression of various cellular genes that normally regulate lymphocyte growth produced by altered activity of various endogenous host transcription factors. In particular, <n>Tax</n> is capable of modulating the expression or activity of various host transcription factors, including members of the NF-kappa B/Rel and CREB/ATF families, as well as the cellular factors <n>HEB-1</n> and <n>p67SRF</n>. An additional distinguishing characteristic of HTLV-I infection is the profound state of viral latency that is present in circulating primary leukemic T cells. In this study, we demonstrate that HTLV-I <n>Tax</n> can physically associate with <n>p100</n>, the product of the Rel-related NF-kappa B2 gene, both in transfected cells and in HTLV-I-infected leukemic T-cell lines. Furthermore, the physical interaction of <n>Tax</n> with <n>p100</n> leads to the inhibition of <n>Tax</n>-induced activation of the HTLV-I and human immunodeficiency virus type 1 long terminal repeats, reflecting <n>p100</n>- mediated cytoplasmic sequestration of the normally nuclearly expressed <n>Tax</n> protein. In contrast, a mutant of <n>Tax</n> that selectively fails to activate nuclear <n>NF-kappa B</n> expression does not associate with <n>p100</n>. (ABSTRACT TRUNCATED AT 250 WORDS)
94110361>Rhabdomyosarcomas do not contain mutations in the DNA binding domains of myogenic transcription factors. Skeletal myogenesis is regulated by a group of transcription factors (<n>MyoD</n>, <n>myogenin</n>, <n>myf5</n>, and <n>myf6</n>) that are "basic helix-loop-helix" proteins that bind to the promoters of muscle-specific genes and promote their expression. We have previously shown that after a mutation of Leu122 to Arg the DNA binding basic domain of <n>MyoD</n> confers c-myc-like functional characteristics to the protein. In this study we used single-strand conformation polymorphism analysis to determine whether such mutations occur naturally in rhabdomyosarcomas. We have found that the basic domains of all the myogenic factors remain unaltered in rhabdomyosarcomas. Selection against such mutations may be the result of functional redundancy of these myogenic transcription factors.
94280766>Function and activation of <n>NF-kappa B</n> in the immune system. <n>NF-kappa B</n> is a ubiquitous transcription factor. Nevertheless, its properties seem to be most extensively exploited in cells of the immune system. Among these properties are <n>NF-kappa B</n>'s rapid posttranslational activation in response to many pathogenic signals, its direct participation in cytoplasmic/nuclear signaling, and its potency to activate transcription of a great variety of genes encoding immunologically relevant proteins. In vertebrates, five distinct DNA binding subunits are currently known which might extensively heterodimerize, thereby forming complexes with distinct transcriptional activity, DNA sequence specificity, and cell type- and cell stage- specific distribution. The activity of DNA binding <n>NF-kappa B</n> dimers is tightly controlled by accessory proteins called I kappa B subunits of which there are also five different species currently known in vertebrates. I kappa B proteins inhibit DNA binding and prevent nuclear uptake of NF-kappa B complexes. An exception is the <n>Bcl-3 protein</n> which in addition can function as a transcription activating subunit in th nucleus. Other I kappa B proteins are rather involved in terminating NF- kappa B's activity in the nucleus. The intracellular events that lead to the inactivation of <n>I kappa B</n>, i.e. the activation of NF-kappa B, are complex. They involve phosphorylation and proteolytic reactions and seem to be controlled by the cells' redox status. Interference with the activation or activity of NF-kappa B may be beneficial in suppressing toxic/septic shock, graft-vs-host reactions, acute inflammatory reactions, acute phase response, and radiation damage. The inhibition of NF-kappa B activation by antioxidants and specific protease inhibitors may provide a pharmacological basis for interfering with these acute processes.
99035056>Lack of T-cell-mediated recognition of the fusion region of the <n>pml/RAR- alpha hybrid protein</n> by lymphocytes of acute promyelocytic leukemia patients. In previous studies, it was shown that the fusion region of the <n>pml/RAR- alpha protein</n>, expressed by acute promyelocytic leukemia (APL) cells, can be specifically recognized in vitro by donor (D. E.) CD4 T cells in a HLA class II DR11-restricted fashion. We present here the results on the recognition of several pml/RAR-alpha peptides by APL patients expressing <n>HLA DR11</n>. The in vitro immunization of peripheral blood lymphocytes from four patients in remission (S.R., F.R., M.M., P. G.) with BCR1/25, a 25-mer pml/<n>RAR-alpha</n>, did not elicit either a polyclonal or a clonal immune response specific to the peptide. We then generated new donor anti-pml/<n>RAR-alpha</n> CD4(+) T-cell clones. These clones were tested for their recognition of BCR1/25. One clone (C3/5, CD3(+), CD4(+), CD8(-)) was selected for further analysis. Clone C3/5 showed specific proliferation, cytotoxicity, and cytokine (<n>tumor necrosis factor alpha</n>, <n>granulocyte-macrophage colony-stimulating factor</n>) production when challenged with autologous lymphoblastic cell lines pulsed with peptide BCR1/25. C3/5 cells developed specific proliferation and cytotoxicity when challenged with peptide-pulsed lymphoblastic cell lines and peripheral blood lymphocytes from the four DR11(+) APL patients. APL blasts, available only from patients F.R. and P.G., were not lysed by C3/5 and were unable to present peptide BCR1/25. Incubation of APL cells with <n>IFN-gamma</n> failed to induce HLA class II molecules and recognition by the C3/5 clone. Since APL cells do not express HLA class II molecules, we tested in two donors (D.E. and C.H.R.) and in patients S.R.and P.G.whether the use of 9-mer peptides (BCR1/9) would generate a <n>CD8</n>/HLA class I-restricted response. No peptide-specific T-cell line or clone could be generated from both donors and patients. These findings are discussed in relation to possible therapeutic approaches to the immunotherapy of APL.
97112990>The proximal regulatory element of the <n>interferon-gamma</n> promoter mediates selective expression in T cells. <n>Interferon-gamma</n> (<n>IFN-gamma</n>) is produced by natural killer cells and certain subsets of T cells, but the basis for its selective expression is unknown. Within the region between -108 and -40 base pairs of the <n>IFN-gamma</n> promoter are two conserved and essential regulatory elements, which confer activation-specific expression in T cells. This report describes studies indicating that the most proximal of these two regulatory elements is an important determinant of its restricted expression. The proximal element is a composite site that binds members of the CREB/ATF, AP-1, and octamer families of transcription factors. Jun is essential for activation-induced transcription and binds preferably as a heterodimer with <n>ATF-2</n>. In contrast, CREB appears to dampen transcription from this element. The CpG dinucleotide in this element is selectively methylated in Th2 T cells and other cells that do not express <n>IFN-gamma</n>, and methylation markedly reduces transcription factor binding. As a target for DNA methylation and for binding of transcription factors that mediate or impede transcription, this element appears to play a central role in controlling <n>IFN-gamma</n> expression.
97098712><n>CD40</n>, but not lipopolysaccharide and anti-IgM stimulation of primary B lymphocytes, leads to a persistent nuclear accumulation of <n>RelB</n>. In this study we analyzed the effect of <n>CD40</n> stimulation on the activity and nuclear appearance of Rel/nuclear factor kappaB (<n>NF- kappaB</n>) factors in primary murine B lymphocytes. We show that triggering of <n>CD40</n> signaling pathway(s) by <n>CD40</n> ligands expressed on L cells led to strong activation of an NF-kappaB-controlled beta-globin reporter gene in primary B lymphocytes from transgenic mice. Analyses of nuclear translocation of individual members of Rel proteins after <n>CD40</n> induction of primary B cells showed a strong and long-lasting accumulation of <n>RelB</n> and, less pronounced, of <n>c-Rel</n>. LPS stimulation did not give rise to a persistent nuclear accumulation of <n>RelB</n> and <n>c- Rel</n>, whereas nuclear <n>c-Rel</n>, but not <n>RelB</n>, accumulated after B cell receptor stimulation. <n>CD40</n> induced not only nuclear translocation but also de novo synthesis of <n>RelB</n> RNA and protein. S107 plasmacytoma cells, which express <n>CD40</n> but are defective for the nuclear appearance of p50/p65-NF-kappaB, do not express <n>RelB</n> after <n>CD40</n> stimulation. In S107 cells stably transfected with relB genes, stimulation of nuclear <n>RelB</n> translocation by <n>CD40</n> was observed. These results indicate that stimulation of <n>CD40</n> signaling pathways exerts a long-lasting stimulatory effect on both the transcription and nuclear translocation of <n>RelB</n>. Since LPS and anti-IgM were unable to activate <n>RelB</n>, <n>CD40</n> appears to trigger a special program of gene expression involved in the proliferation and/or differentiation of B lymphocytes.
97067175>Identification and characterization of a leukocyte-specific component of the nuclear body. The nuclear body (NB) is a cellular organelle that is involved in the pathogenesis of acute promyelocytic leukemia and viral infection. The NB is also a target of antibodies in the serum of patients with the autoimmune disease primary biliary cirrhosis. In this study, serum from a patient with primary biliary cirrhosis was used to identify a cDNA encoding a novel component of the NB, a <n>140-kDa protein</n> designated <n>Sp140</n>. The predicted amino acid sequence of the amino-terminal portion of <n>Sp140</n> was similar to <n>Sp100</n>, a previously identified NB protein. The carboxyl portion of <n>Sp140</n> contained a zinc-finger domain and a bromodomain, motifs that are present in proteins regulating gene transcription. High levels of <n>Sp140</n> mRNA were detected in human spleen and peripheral blood leukocytes, but not other human tissues. The level of SP140 mRNA in myeloid precursor cell lines HL60 and NB4 markedly increased in response to chemically induced cellular differentiation. Immunohistochemical techniques were used to demonstrate that <n>SP140</n> localized to the NB in differentiated HL60 and NB4 cells. The location of <n>Sp140</n> in the NB, and expression of this gene in cells involved in host defense, suggest that <n>Sp140</n> may be involved in the pathogenesis of acute promyelocytic leukemia and viral infection.
97083588>Regulation of cytokine and cytokine receptor expression by glucocorticoids. Glucocorticoids (GCS) profoundly inhibit several aspects of T cell immunity largely through inhibition of cytokine expression at the transcriptional and posttranscriptional levels. GCS were also reported to act indirectly by inducing transforming <n>growth factor-beta</n> expression, which in turn blocks T cell immunity. In exerting their antiproliferative effects, GCS diffuse into target cells where they bind their cytoplasmic receptor, which in turn translocates to the nucleus where it inhibits transcription of cytokine genes through direct binding to the glucocorticoid response elements (GRE), which are located in the promoter region of cytokine genes or, alternatively, through antagonism of the action of transcription factors required for optimal transcriptional activation. In contrast to their inhibitory effects on cytokine expression, GCS up-regulate cytokine receptor expression that correlates with enhanced cytokine effects on target cells. In this review, we summarize the current state of knowledge of the mechanism of action of GCS, including the phenomenon of steroid- induced rebound, which ensues upon GCS withdrawal.
97047769>Isolation and characterization of murine fra-1: induction mediated by <n>CD40</n> and surface Ig is <n>protein kinase C</n> dependent. The murine fra-1 gene, encoding <n>Fos-related Ag 1</n>, was isolated from a splenic cDNA library and sequenced. Murine fra-1 was highly homologous to rat and human fra-1. Oligonucleotide primers based on the murine sequence were used to construct a quantitative reverse transcription- PCR assay for gene expression. B lymphocyte stimulation via both <n>CD40</n> and surface Ig (sIg) receptors substantially induced fra-1 expression, and for both receptors, induction was <n>protein kinase C</n> (<n>PKC</n>) dependent. This contrasts with induction of c-fos by both <n>CD40</n> and sIg, which is <n>PKC</n> independent and indicates that <n>CD40</n> is capable of signaling through <n>PKC</n> or a closely related kinase. Induction of fra-1 following engagement of <n>CD40</n> did not require protein synthesis, suggesting that the <n>PKC</n>-dependent linkage between <n>CD40</n> and fra-1 is direct. <n>CD40</n>- mediated fra-1 induction did require tyrosine kinase activity. These results demonstrate that <n>CD40</n>, like sIg, may employ <n>PKC</n> in producing select outcomes, that individual B cell receptors may signal downstream events via both PKC-dependent and PKC-independent pathways, and that multiple signal transduction pathways may be used to activate the expression of closely related genes
94281402>Pentoxifylline for the treatment of infection with human immunodeficiency virus. Cytokine dysregulation in human immunodeficiency virus type 1 (HIV-1) infection has been documented in numerous studies and has been cited as an important component in the pathogenesis of this retroviral infection. Pharmacological modification of cytokine dysregulation, therefore, has been suggested as a therapeutic modality for HIV-1 infection. Dr. Dezube of Beth Israel Hospital (Boston) concisely reviews the state of our knowledge regarding the effects of pentoxifylline on expression of <n>tumor necrosis factor-alpha</n>, a cytokine known to influence HIV-1 replication and to play a possible role in the clinical manifestations of advanced infection with this virus. Pentoxifylline, a trisubstituted xanthine derivative, has been used to decrease blood viscosity and is reasonably well tolerated by most recipients of the drug. Results of preliminary studies, many of which were conducted by Dr. Dezube, suggest that use of this agent in combination with antiretroviral compounds may prove useful in the treatment of patients with HIV-1 infection.
94119067>Activation of the <n>granulocyte-macrophage colony-stimulating factor</n> promoter in T cells requires cooperative binding of <n>Elf-1</n> and <n>AP-1</n> transcription factors. The <n>granulocyte-macrophage colony-stimulating factor</n> (<n>GM-CSF</n>) gene has been studied extensively as a model system of transcriptional induction during T-lymphocyte activation. The <n>GM-CSF</n> gene is not expressed in resting peripheral blood T cells but is rapidly induced at the transcriptional level following activation through the cell surface T- cell receptor. A highly conserved 19-bp element located immediately 5' of the human <n>GM-CSF</n> TATA box (bp -34 to -52), herein called purine box 1 (PB1), has been shown to bind a T-cell nuclear protein complex and to be required for transcriptional induction of the <n>GM-CSF</n> gene following T-cell activation. The PB1 sequence motif is highly conserved in both human and murine GM-CSF genes. In this report, we demonstrate that the PB1 element alone confers inducibility on a heterologous promoter following transfection into human Jurkat T cells. In addition, we identify a major PB1 nuclear protein-binding complex that is not present in resting peripheral blood T cells but is rapidly induced following T-cell activation. Sequence analysis revealed that PB1 is composed of adjacent binding sites for <n>Ets</n> and <n>AP-1</n> transcription factors. In vitro mutagenesis experiments demonstrated that both the Ets and AP-1 sites are required for binding of the inducible PB1 nuclear protein complex and for the transcriptional activity of this element and the <n>GM-CSF</n> promoter in activated T cells. (ABSTRACT TRUNCATED AT 250 WORDS)
94110342>Steroid-resistant asthma. Cellular mechanisms contributing to inadequate response to glucocorticoid therapy. The current study examined whether alterations in <n>glucocorticoid receptor</n> (GR) binding contribute to poor response to glucocorticoid therapy in asthma. 29 asthma patients with forced expiratory volume in 1 s (FEV1) &lt; 70% predicted were studied. Patients were classified as steroid sensitive (SS) if their morning FEV1 increased &gt; 30% after a 1- wk course of oral prednisone 20 mg twice daily and steroid resistant (SR) if they failed to increase &gt; 15%. PBMC obtained from these two groups, 17 SR and 12 SS, as well as 12 normal controls were analyzed. SR patients had two distinguishable GR binding abnormalities: 15 of the 17 SR patients demonstrated a significantly reduced GR binding affinity, as compared with SS patients (P = 0.0001) and normal controls (P = 0.0001). This defect was localized to T cells and reverted to normal after 48 h in culture media. However, incubation with a combination of <n>IL-2</n> and <n>IL-4</n> sustained this abnormality. The other two SR patients had an abnormally low GR number with normal binding affinity that was not limited to T cells. Furthermore, GR number failed to normalize after incubation in media alone or <n>IL-2</n> and <n>IL-4</n>. Therefore, SR asthma may be due to more than one abnormality, the majority related to a reversible cytokine-induced reduction in GR binding affinity and the second related to an irreversible reduction in GR number. These findings may have important implications for the design of alternative treatment approaches for recalcitrant asthma.
95072277>Prevalence of aneuploidy, overexpressed ER, and overexpressed EGFR in random breast aspirates of women at high and low risk for breast cancer. Breast tissue biomarkers which accurately predict breast cancer development within a 10 year period in high risk women are needed but currently not available. We initiated this study to determine 1) the prevalence of one or more breast tissue abnormalities in a group of women at high risk for breast cancer, and 2) if the prevalence of biomarker abnormalities is greater in high risk than in low risk women. Eligible high risk women were those with a first degree relative with breast cancer, prior breast cancer, or precancerous mastopathy. Low risk women were those without these or other major identifiable risk factors. Ductal cells were obtained via random fine needle aspirations and cytologically classified. Biomarkers included DNA ploidy, estrogen receptor (ER), and epidermal growth factor receptor (EGFR). The prevalence of DNA aneuploidy was 30%, overexpression of ER 10%, and overexpression of EGFR 35%, in the 206 high risk women whose median 10 year Gail risk (projected probability) of developing breast cancer was 4.5%. The prevalence of aneuploidy and overexpressed EGFR was significantly higher in the high risk women than in the 25 low risk controls (p &lt; 0.002), whose median 10 year Gail risk was 0.7%. The difference in the prevalence of ER overexpression between high and low risk groups was not statistically significant (p = 0.095). This may be due to the low prevalence of overexpressed ER and the small number of controls. A significant difference was noted in the prevalence of one or more abnormal biomarkers between the high risk and low risk women (p &lt; 0.001). A large prospective trial is needed to determine if one or more of these biomarkers, is predictive of breast cancer development.
98128482>Inhibition of <n>nuclear factor kappa B</n> subunit p65 mRNA accumulation in lipopolysaccharide-stimulated human monocytic cells treated with sodium salicylate. Lipopolysaccharide is one of the most potent trigger substances for monocytes and macrophages causing secretion of inflammatory mediators such as tumor necrosis factor and <n>interleukin-1</n>. The nature of the nuclear factors involved in regulation of these cytokine genes is still unknown. Nuclear factor kappa B (NF-kappa B; heterodimer of p50 and p65) proteins have been suggested to play an important role in gene transcription of inflammatory mediators when monocytes are stimulated with lipopolysaccharide. Nonsteroidal anti-inflammatory drugs such as salicylates have been used to treat symptoms of inflammation, and a new mechanism of drug action was suggested recently. Salicylates have been shown to inhibit lipopolysaccharide-induced gene transcription via inhibition of NF-kappa B activation by preventing the degradation of <n>NF- kappa B</n> inhibitor "<n>I kappa B</n>", blocking the translocation of NF-kappa B into the nuclear compartment. However, the nature of the subunit involved in this mechanism has not been defined. To examine the mechanisms by which salicylates affect cytokine gene transcription, the amount of active and inactive NF-kappa B and NF-kappa B mRNA, in Porphyromonas gingivalis lipopolysaccharide-stimulated human monocytic cells was assessed. High doses of sodium salicylate suppressed NF-kappa B p65 mRNA accumulation, resulting in suppression of total NF-kappa B, p50 on tissue oligonucleotide had no effects on lipopolysaccharide- induced NF-kappa B activation. The data demonstrate that the p65 subunit of NF-kappa B is inhibited by salicylate treatment and highlight the role of salicylate in the control of gene expression of inflammatory mediators.
97131617>Glucocorticoid-mediated inhibition of <n>RANTES</n> expression in human T lymphocytes. The chemokine <n>RANTES</n> has been implicated in the pathogenesis of allergic inflammatory diseases including asthma and rhinitis which are frequently treated with glucocorticoids. We observed that dexamethasone dramatically inhibited <n>RANTES</n> mRNA expression dose dependently in anti- CD3 activated Hut-78 T cells and human PBMCs. Inhibition of <n>RANTES</n> expression did not appear to be secondary to <n>IL-2</n> inhibition and required binding to the intracellular <n>glucocorticoid receptor</n>. The down- regulation of <n>RANTES</n> expression by glucocorticoids in T cells may directly contribute to the efficacy of these agents in suppressing cellular infiltration and to their anti-inflammatory properties.
97098688>Association of <n>TRAF1</n>, <n>TRAF2</n>, and <n>TRAF3</n> with an Epstein-Barr virus LMP1 domai