calcimycin and Leukemia--T-Cell

Accumulated evidence demonstrates that adult T-cell leukemia (ATL) is frequently associated with eosinophilia, and human T-lymphotropic virus type 1 (HTLV-1)-infected cells frequently express interleukin-5 (IL-5). However, the molecular mechanism of constitutive IL-5 expression in HTLV-1-infected cells remains unclear. To clarify the mechanism of aberrant IL-5 expression in HTLV-1-infected cells, we investigated the response of the human IL-5 promoter to the HTLV-1-encoded protein Tax. Cotransfection experiments using Jurkat cells revealed that Tax is incapable of activating the IL-5 promoter by itself but that it synergistically transactivates the promoter with GATA-binding protein (GATA-4) and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation. By introducing a series of mutations within the IL-5 promoter, we found that conserved lymphokine element 0 (CLE0) is responsible for mediating the signal induced by Tax-TPA. A deletion construct of the promoter indicated that the -75 GATA element and CLE0 are sufficient to mediate synergistic activation of the IL-5 promoter. Electrophoretic mobility shift assays using Jurkat cell nuclear extracts demonstrated that TPA induces a transcription factor to bind CLE0, and an experiment using JPX-9 cell nuclear extracts showed that Tax enhances this binding activity. An antibody supershift experiment revealed that this band consists of c-Jun and JunD. However, among the Jun family members, only c-Jun is able to cooperate with Tax and GATA-4 to activate the IL-5 promoter. We have determined the minimum factors required for IL-5 gene activation by reconstituting the IL-5 promoter activity in F9 cells. This is the first report to demonstrate the functional involvement of Tax protein in IL-5 gene regulation and to suggest the functional triple synergism among Tax, GATA-4, and AP-1, which disrupts regulated control of the gene and leads to constitutive expression of the IL-5 gene.

Topics: Calcimycin; Cyclosporine; DNA-Binding Proteins; GATA4 Transcription Factor; Gene Expression Regulation, Viral; Gene Products, tax; Human T-lymphotropic virus 1; Humans; Immunosuppressive Agents; Interleukin-5; Ionophores; Leukemia, T-Cell; Mutation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription Factors; Transcriptional Activation; Transfection; Tumor Cells, Cultured

The SUP-T13 cell line, a human T leukemia, is susceptible to apoptosis by various inducers, including anti-TCR mAb, calcium ionophores, and anti-fas mAb. Induction of apoptosis by these three agents was investigated, and several differences were found. All three agents induced DNA fragmentation with a similar time course, but the kinetics of cell death were different for the three agents. Anti-TCR mAb-induced apoptosis, but not A23187- or anti-fas-induced apoptosis, was rescued by anti-CD3 mAb treatment. In contrast, only anti-fas mAb-mediated apoptosis was rescued by PKC activators such as PMA. These differences suggest that each of these three agents mediate apoptosis by unique signaling pathways. Nevertheless, two variant subclones of SUP-T13 were found to be resistant to all three apoptosis-inducing agents, suggesting a point(s) of common regulation between the different pathways. To determine whether this regulation occurred through bcl-2, p53, or c-myc, their expression in the parental and variant cells was determined. The three clones expressed approximately equal amounts of these proteins, and their levels did not change significantly upon treatment with anti-TCR or anti-TCR plus anti-CD3 mAb. Thus, although the proximal signaling by various apoptosis inducers were quite different, a common mediator(s) (as yet unknown) may still regulate apoptosis induced by these multiple agents.

Topics: Antibodies, Monoclonal; Apoptosis; Calcimycin; Calcium; CD3 Complex; Cytokines; fas Receptor; Humans; Ionophores; Leukemia, T-Cell; Protein Kinase C; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Receptors, Antigen, T-Cell; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We have isolated a novel cDNA clone encoding interferon (IFN) consensus sequence-binding protein in adult T-cell leukemia cell line or activated T cells (ICSAT); this protein is the human homolog of the recently cloned Pip/LSIRF. ICSAT is structurally most closely related to the previously cloned ICSBP, a member of the IFN regulatory factor (IRF) family of proteins that binds to interferon consensus sequences (ICSs) found in many promoters of the IFN-regulated genes. Among T-cell lines investigated, ICSAT was abundantly expressed in human T-cell leukemia virus type 1 (HTLV-1)-infected T cells. When the HTLV-1 tax gene was expressed or phorbol myristake acetate-A23187 stimulation was used, ICSAT expression was induced in Jurkat cells which otherwise do not express ICSAT. When the binding of ICSAT to four different ICSs was tested, the relative differences in binding affinities for those ICSs were determined. To study the functional role of ICSAT, we performed cotransfection experiments with the human embryonal carcinoma cell line N-Tera2. ICSAT was demonstrated to possess repressive function over the gene activation induced by IFN stimulation or by IRF-1 cotransfection. Such repressive function is similar to that seen in IRF-2 or ICSBP. However, we have found that ICSAT has a different repressive effect from that of IRF-2 or ICSBP in some IFN-responsive reporter constructs. These results suggest that a novel mechanism of gene regulation by "differential repression" is used by multiple members of repressor proteins with different repressive effects on the IFN-responsive genes.

Topics: Amino Acid Sequence; Animals; Base Sequence; Calcimycin; Carrier Proteins; Consensus Sequence; DNA-Binding Proteins; DNA, Complementary; Gene Expression Regulation; Genes, pX; Humans; Interferon Regulatory Factors; Interferons; Leukemia, T-Cell; Lymphocyte Activation; Molecular Sequence Data; Repressor Proteins; Sequence Homology, Amino Acid; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

Two cis-acting elements GM-kappa B/GC-box and CLE0, of the granulocyte-macrophage colony-stimulating factor (GM-CSF) 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 NF-kappa B, which is mainly induced by PMA. The CLE0 sequence interacts with factors, related to a PMA-induced AP-1 and a PMA/A23187-induced NF-AT. 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 NF-kappa B and AP-1 significantly augmented transcription from the GM-CSF promoter containing the GM-kappa B/GC-box and the CLE0 (AP-1/NF-AT). Expression of a constitutively active calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, potentiated by two fold the transcriptional activation by NF-kappa B/AP-1. Both constitutively active forms of CN and protein kinase C (PKC) 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.

Topics: Animals; Base Sequence; Calcimycin; Calcineurin; Calmodulin-Binding Proteins; Cell Line; Coleoptera; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, T-Cell; Luciferases; Mice; Models, Genetic; Molecular Sequence Data; NF-kappa B; Oligodeoxyribonucleotides; Phosphoprotein Phosphatases; Plasmids; Promoter Regions, Genetic; Protein Kinases; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; T-Lymphocytes; TATA Box; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) are produced by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in human T cell leukemia Jurkat cells. The expression of GM-CSF and IL-2 is inhibited by immunosuppressive drugs such as cyclosporin A (CsA) and FK506. Earlier studies on the IL-2 gene expression showed that overexpression of calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, can stimulate transcription from the IL-2 promoter through the NF-AT-binding site. In this study, we obtained evidence that transfection of the cDNAs for CN A (catalytic) and CN B (regulatory) subunits also augments transcription from the GM-CSF promoter and recovers the transcription inhibited by CsA. The constitutively active type of the CN A subunit, which lacks the auto-inhibitory and calmodulin-binding domains, acts in synergy with PMA to activate transcription from the GM-CSF promoter. We also found that the active CN partially replaces calcium ionophore in synergy with PMA to induce expression of endogenous GM-CSF and IL-2. By multimerizing the regulatory elements of the GM-CSF promoter, we found that one of the target sites for the CN action is the conserved lymphokine element 0 (CLE0), located at positions between -54 and -40. Mobility shift assays showed that the CLE0 sequence has an AP1-binding site and is associated with an NF-AT-like factor, termed NF-CLE0 gamma. NF-CLE0 gamma binding is induced by PMA/A23187 and is inhibited by treatment with CsA. These results suggest that CN is involved in the coordinated induction of the GM-CSF and IL-2 genes and that the CLE0 sequence of the GM-CSF gene is a functional analogue of the NF-AT-binding site in the IL-2 promoter, which mediates signals downstream of T cell activation.

Topics: Animals; Base Sequence; Calcimycin; Calcineurin; Calmodulin-Binding Proteins; Cyclosporine; Gene Expression Regulation; Genes; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-2; Leukemia, T-Cell; Lymphocyte Activation; Mice; Molecular Sequence Data; Neoplasm Proteins; Phosphoprotein Phosphatases; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Sequence Homology, Nucleic Acid; T-Lymphocytes; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

The inhibitory effects of azelastine hydrochloride on PAF-induced and fMLP-induced Ca2+ influx, actin polymerization and calcium ionophore A23187-induced and aggregated IgG-induced release of eosinophil cationic protein (ECP) of an eosinophilic leukaemia cell line, EoL-1, were examined. EoL-1 cells cultured with 0.2 mM dibutyryladenosine-cyclic monophosphate for 48 hours showed an increase in intracellular free Ca2+ concentration ([Ca2+]i) and actin polymerization when stimulated by PAF and fMLP. Azelastine hydrochloride inhibited PAF-induced and fMLP-induced Ca2+ influx ([Ca2+]i) in a dose-dependent manner with an IC50 of 1 x 10(-8) M and 1 x 10(-7) M, respectively. It also inhibited PAF-induced and fMLP-induced actin polymerization in a dose-dependent manner up to 40% and 30%, respectively. EoL-1 cells were differentiated to contain ECP in their eosinophilic granules when cultured for 9 days with supernatants of a human adult T cell leukaemia cell line, HIL-3 (HIL-3 sup). Calcium ionophore A23187 and aggregated IgG induced the secretion of ECP by EoL-1 cells. Azelastine hydrochloride inhibited the secretion of ECP in a dose-dependent manner. These inhibitory effects were seen even at therapeutic concentrations of 10(-8) M to 10(-9) M. These results indicate that the therapeutic effects of azelastine hydrochloride as an anti-allergic agent may include inhibition of the accumulation of eosinophils into the locus of allergic inflammation and of the release of cytotoxic granules from eosinophils.

Topics: Actins; Blood Proteins; Bucladesine; Calcimycin; Calcium-Transporting ATPases; Cell Differentiation; Culture Media; Dose-Response Relationship, Drug; Eosinophil Granule Proteins; Eosinophils; Humans; Hypersensitivity; Immunoglobulin G; Inflammation; Leukemia, Eosinophilic, Acute; Leukemia, T-Cell; Lipoxygenase Inhibitors; N-Formylmethionine Leucyl-Phenylalanine; Phthalazines; Platelet Activating Factor; Polymers; Ribonucleases; Tumor Cells, Cultured

The CEM-ON malignant T cell line and long-term cultured normal T cells can be induced to release CSF-1 in their culture supernatants. Chemical inducers (PMA + A23187) and, more interestingly, cytokines (tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha)), as well as physiological (antigen + IL-2) or specific (anti-CD3 + IL-2 or PMA) stimuli, lead to rapid transient colony-stimulating factor-1 (CSF-1) gene expression and production of biologically active CSF-1. These data suggest that CSF-1 may play a role in the early phases of immune response.

Topics: Antigens, Differentiation, T-Lymphocyte; Blotting, Northern; Calcimycin; Cells, Cultured; Colony-Forming Units Assay; Colony-Stimulating Factors; Humans; Interleukin-1; Leukemia, T-Cell; Macrophage Colony-Stimulating Factor; Monocytes; Phenotype; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The effect of calcium on the production of interleukin 1 (IL 1) and the expression of IL 1 receptors (R) of adult T-cell leukemia (ATL) cells was studied in vitro. ATL cells freshly obtained from patients and ATL cell lines produced limited amounts of IL 1 by culturing in a low-calcium concentration of medium (less than 0.01 mM). However, the production of IL 1 was enhanced by the addition of calcium chloride to the medium in a concentration-dependent manner and reached the maximum at the higher calcium concentration (3-4 mM) than at the standard calcium concentration of medium (1.26 mM). The production of IL 1 from ATL cells was further enhanced by calcium ionophore. Furthermore, the expression of IL 1R on ATL cells was augmented in proportion to the extracellular calcium concentration and calcium ionophore. In accordance with the change of the extracellular calcium concentration, the intracellular calcium concentration of ATL cells detected by Fura 2 was changed. However, this calcium dependency was not observed in the human T-cell leukemia virus I-negative acute T-cell leukemia cells. These results suggest that calcium plays a critical role in the regulation of the production of IL 1 and the expression of IL 1R on ATL cells.

Topics: Adult; Calcimycin; Calcium; Calcium Chloride; Humans; Interleukin-1; Kinetics; Leukemia, T-Cell; Receptors, Immunologic; Receptors, Interleukin-1; Tumor Cells, Cultured