sq-23377 and Leukemia-Lymphoma--Adult-T-Cell

Interferon-gamma (IFN-gamma), also known as type II interferon, is an important immunoregulatory gene that has multiple effects on the development, maturation, and function of the immune system. IFN-gamma mRNA and protein are expressed predominantly by T cells and large granular lymphocytes. The IFN-gamma mRNA is induced/inhibited in these cell types by a wide variety of extracellular signals, thus implicating a number of diverse, yet convergent signal transduction pathways in its transcriptional control. In this review, I describe how DNA methylation and specific DNA binding proteins may regulate transcription of the IFN-gamma gene in response to extracellular signals.

Topics: Animals; Cyclosporine; DNA; DNA-Binding Proteins; Enhancer Elements, Genetic; Erythroid-Specific DNA-Binding Factors; Gene Expression Regulation; Glucocorticoids; Humans; Interferon-gamma; Ionomycin; Leukemia-Lymphoma, Adult T-Cell; Lymphocyte Activation; Lymphocyte Subsets; Methylation; Mice; NF-kappa B; Phytohemagglutinins; Promoter Regions, Genetic; RNA, Messenger; Signal Transduction; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

We have studied pim-1 proto-oncogene expression in human T cell responses to Ag receptor-generated signals. The pim-1 gene encodes a serine/threonine protein kinase that is expressed primarily in cells of hematopoietic lineage and is implicated in the intracellular signaling processes accompanying lymphocyte activation. We show here that pim-1 mRNA expression is rapidly induced after receptor cross-linking with anti-CD3 Abs. We examined the linkage of pim-1 expression to known signaling pathways generated through the T cell Ag receptor. pim-1 mRNA was not substantially induced after elevation of intracellular free Ca2+. In contrast, PMA, which directly activates PKC, induced rapid pim-1 expression. Further, anti-CD3- or PMA-induced pim-1 expression was markedly reduced by various PKC inhibitors and by deficiency of the PKC epsilon isoform in a mutant T cell line. Thus, T cell Ag receptor-linked pim-1 expression appears to be coupled to the PKC component of transmembrane signaling. Because the activation of protein kinase C has been shown to activate Raf-1 kinase activity, the involvement of Raf-1 in pim-1 expression was also investigated using a human T cell line stably transfected with an inducible Raf expression vector. Although the overexpression of activated Raf was shown to cause a substantial increase in IL-2 expression, no discernible effects on pim-1 were apparent. In addition, we examined transcriptional and post-transcriptional mechanisms involved in PKC-mediated pim-1 expression and observed that both transcriptional and post-transcriptional mechanisms are coordinately involved in the up-regulation of the pim-1 proto-oncogene.

Topics: Calcium; Cell Nucleus; Cytoplasm; Enzyme Activation; Enzyme Induction; Humans; Ionomycin; Isoenzymes; Leukemia-Lymphoma, Adult T-Cell; Lymphocyte Activation; Muromonab-CD3; Protein Kinase C; Protein Serine-Threonine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-pim-1; Proto-Oncogene Proteins c-raf; Proto-Oncogenes; Receptor-CD3 Complex, Antigen, T-Cell; Receptors, Antigen, T-Cell; RNA, Messenger; Sezary Syndrome; Signal Transduction; Skin Neoplasms; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The mechanism by which calcium-depleted intracellular stores may trigger an external calcium influx through a calcium release-activated channel was investigated by analyzing the effects of several protein tyrosine kinase inhibitors on calcium movements in Jurkat T cells. Tyrphostin A9, an inhibitor of the kinase activity of the platelet-derived growth factor (PDGF) receptor, dramatically impaired the sustained elevation of cytosolic calcium concentration, induced by either CD3 mAbs, thapsigargin, ionomycin at low (10(-7) M) concentration, or passive depletion of intracellular stores; other tested tyrphostins, lavendustin, genistein, and compound 5 lacked significant effect. Tyrphostin A9, added during the plateau phase, was able to return cytosolic calcium to resting concentration. Likewise, it abrogated manganese entry in cells stimulated by CD3 or thapsigargin, measured by the quenching of the fluorescence of Indo-1. However, it did not measurably modify kinetics of intracellular calcium releases monitored in the absence of extracellular calcium, nor did it reverse the inhibition of phosphatidylserine that occurs as a consequence of emptying intracellular stores. Analyses of tyrosine phosphorylations demonstrated that A9 inhibited the phosphorylation of proteins, which occurred every time that internal calcium stores were depleted. These phosphorylations were not impaired by chelation of external Ca2+, nor by La3+ that inhibits calcium release-induced calcium entry. We concluded that their inhibition was not a consequence, but may be a cause, of the blockade of calcium release-activated channel by tyrphostin A9.

Topics: Calcium; Calcium Channels; Depression, Chemical; Enzyme Inhibitors; Extracellular Space; Genistein; Humans; Intracellular Fluid; Ionomycin; Ionophores; Isoflavones; Leukemia-Lymphoma, Adult T-Cell; Muromonab-CD3; Neoplasm Proteins; Phenols; Phosphatidylserines; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Terpenes; Thapsigargin; Tumor Cells, Cultured

The calcium ionophore, ionomycin, promotes an increase of intracellular calcium and regulates mRNA expression of gamma/delta-TcR gene in human T lymphocytes. The mechanism of this regulation is not yet clear. Thus, the regulation by intracellular calcium requires elucidation. We studied the gamma-TcR gene expression in acute lymphoblastic leukemia cell line DND41 (CD4- CD8-) by Northern blot and flow cytometric analysis. The mRNA levels of gamma-TcR increased by ionomycin, anti-CD3, and with TPA. TPA had an antagonistic effect to both ionomycin and anti-CD3. Also, TPA inhibits the increased intracellular calcium promoted by ionomycin but not the increase promoted by anti-CD3 and ionomycin. Our results suggest that intracellular calcium induces mRNA and protein expression of gamma-TcR chain. This effect is antagonized by protein kinase C-activation. Thus, we conclude that the target cells of the differential regulation on gamma-TcR mRNA expression by intracellular calcium modulators are the CD4- CD8- cells, and this is due to cytosolic calcium mobilization.

Topics: Calcium; Enzyme Activation; Gene Expression Regulation, Leukemic; Humans; Ionomycin; Ionophores; Leukemia-Lymphoma, Adult T-Cell; Muromonab-CD3; Neoplasm Proteins; Protein Kinase C; Receptors, Antigen, T-Cell, gamma-delta; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Interleukin 2 (IL-2) and interleukin 4 (IL-4) secreted by activated but not by resting mature T cells are pleiotropic cytokines affecting growth and differentiation of diverse cell types, such as T cells, B cells, and mast cells. There is little information about the molecular basis for the constitutive repression of IL-2 and IL-4 gene expression in unstimulated T cells. We investigated the possibility that wild-type (wt) p53, a nuclear tumor suppressor protein, might serve to repress IL-2 and IL-4 gene expression in murine E14 T lymphoma and in human Jurkat cells. We transiently cotransfected these cells with constitutive simian virus 40 (SV 40) early promoter expression plasmids overproducing wt or mutant murine p53 and with appropriate luciferase (luc) reporter plasmids containing the promoter elements of murine IL-2 and IL-4 genes to evaluate the effect of various p53 species on these promoters. Murine wt p53 derived from pSG5p53cD strongly repressed the IL-2 and IL-4 promoters in both cell lines induced by the phorbol ester TPA and the Ca2+ ionophore ionomycin but not, however, in uninduced cells. In similar transient transfection experiments with lymphoma cells, overexpression of deletion mutant species of murine p53 revealed that the N-terminal and C-terminal domains are crucial for inhibition of both IL-2 and IL-4 gene expression. These parts of p53 comprise the transactivation domain at the amino terminal side, which has previously also been shown to interact with the TATA-box binding-protein TBP and the carboxy-terminal oligomerization domain. Additionally, it was shown that a previously described inhibitory protein, the high-mobility-group protein HMG-I/Y, does not functionally interact with p53. Cotransfection of expression plasmids for both p53 and HMG-I/Y did not alter the extent of inhibition by the individual proteins. These data suggest that p53 can downmodulate both IL-2 and IL-4 gene expression and that both the transactivation and oligomerization domains of the tumor suppressor protein are essential for this transcriptional repression.

Topics: Animals; Gene Expression Regulation, Neoplastic; Genes, p53; High Mobility Group Proteins; HMGA1a Protein; Humans; Interleukin-2; Interleukin-4; Ionomycin; Ionophores; Leukemia-Lymphoma, Adult T-Cell; Lymphoma, T-Cell; Mice; Promoter Regions, Genetic; Recombinant Fusion Proteins; Sequence Deletion; Tetradecanoylphorbol Acetate; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The lymphoid src kinase pp56(lck) has been shown to be essential for the induction of different T lymphocyte responses, including CD4-mediated enhancement of Ag-induced T cell activation, early T cell differentiation, induction of IL-2 production, and cytotoxicity. It is assumed that pp56(lck) acts on these processes by phosphorylating substrates. However, it has been recently reported that the NH2 regulatory domain is sufficient to mediate CD4 accessory function. In this report we address the contribution of the regulatory and catalytic domains of pp56(lck) to another function of this enzyme independent of CD4: TCR-induced IL-2 production. Two pp56(lck) mutants lacking either the entire catalytic domain or the entire NH2 regulatory domain were generated, and their abilities to trigger transactivation of the TCR-regulated nuclear factor of activated T cells (NF-AT) region of the IL-2 promoter were compared. Only the catalytic, but not the NH2 regulatory, domain of pp56(lck) was able to induce NF-AT region transactivation on its own and to cooperate with other intracellular signals to trigger this response. Moreover, the catalytic domain of pp56(lck) was able to induce IL-2 cytokine production to an extent similar to that of wild-type pp56(lck). We conclude that different domains of the pp56(lck) molecule contribute to regulate distinct biologic functions. In fact, while the NH2 regulatory domain is sufficient to mediate CD4 accessory function, we show here that the catalytic domain of pp56(lck) is sufficient for induction of IL-2 production, mimicking TCR ligation.

Topics: Allosteric Site; Binding Sites; Biological Transport; Calcium; Catalysis; DNA-Binding Proteins; Gene Expression Regulation, Leukemic; Humans; Interleukin-2; Ionomycin; Ionophores; Leukemia-Lymphoma, Adult T-Cell; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Neoplasm Proteins; NFATC Transcription Factors; Nuclear Proteins; Peptide Fragments; Promoter Regions, Genetic; Receptors, Antigen, T-Cell; Signal Transduction; src-Family Kinases; Structure-Activity Relationship; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

The human granulocyte-macrophage CSF (GM-CSF) 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 activator protein-1 (AP-1)-like site, as well as an upstream nuclear factor-kappa B (NF-kappa B) 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 NF-kappa B site result in reduced PMA/Ca2+ activation of a 620-bp human GM-CSF promoter-luciferase 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 AP-1-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 AP-1, NF-ATp, and a higher order NF-ATp/AP-1 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 calcineurin could substitute for Ca2+ ionophore and synergize with PMA to activate the GM-CSF promoter, and conversely that mutant-activated Ras could substitute for PMA and cooperate with Ca2+ ionophore. Co-expression of Ras and calcineurin, however, did not activate the GM-CSF promoter, but required the additional expression of NF-kappa B p65. These results imply that at least three signals are required to activate the GM-CSF proximal promoter, and that the signals impinge on distinct transcription factors that bind to the hCLEO and NF-kappa B regions of the promoter.

Topics: Base Sequence; Calcineurin; Calmodulin-Binding Proteins; Cyclosporine; DNA-Binding Proteins; Gene Expression Regulation; Genes, Reporter; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Ionomycin; Leukemia-Lymphoma, Adult T-Cell; Lymphocyte Activation; Molecular Sequence Data; NFATC Transcription Factors; Nuclear Proteins; Phosphoprotein Phosphatases; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription Factors; Tumor Cells, Cultured

The human T-cell leukemia virus type I Tax protein activates NF-kappa B transcription factors from preformed cytoplasmic pools, including those pools that are retained by the I kappa B-alpha inhibitory protein. Degradation of I kappa B-alpha is enhanced by Tax, resulting in the liberation of some NF-kappa B, which then translocates into the nucleus. Here we have investigated the mechanism by which Tax causes degradation of I kappa B-alpha. Two I kappa B-alpha mutants defective in extracellular signal-induced degradation of I kappa B-alpha also blocked Tax-mediated kappa B-dependent transactivation when cotransfected into Jurkat T cells. Cotransfected wild-type I kappa B-alpha or an irrelevant mutant did not significantly effect transactivation induced by Tax. The signal-defective I kappa B-alpha proteins are mutated at either of two closely spaced serines in the N terminus of the protein (Ser32 and Ser36). In wild-type I kappa B-alpha, one or both of these serines are inducibly phosphorylated with extracellular stimuli, and such phosphorylation appears necessary for subsequent degradation and thus activation of NF-kappa B. These results suggest that Tax triggers I kappa B-alpha degradation and thus NF-kappa B activation by a mechanism that converges with that induced by extracellular stimulation such as phorbol 12-myristate 13-acetate/ionomycin or tumor necrosis factor alpha. A role for Tax in activating signal transduction pathways upstream of I kappa B-alpha is implied.

Topics: Gene Expression Regulation; Gene Products, tax; Human T-lymphotropic virus 1; Humans; Ionomycin; Leukemia-Lymphoma, Adult T-Cell; Mutagenesis, Site-Directed; NF-kappa B; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Recombinant Fusion Proteins; Serine; Signal Transduction; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription Factor RelB; Transcription Factors; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha