sq-23377 and Leukemia--T-Cell

Tribbles 1 (TRB1), a member of the Tribbles family, is a pseudokinase that is conserved among species and implicated in various human diseases including leukemia, cardiovascular diseases, and metabolic disorders. However, the role of TRB1 in the immune response is not understood. To evaluate this role, we examined regulation of TRB1 expression and the function of TRB1 in interleukin-2 (IL-2) induction in Jurkat cells, a human acute T cell leukemia cell line. We found that TRB1 was strongly induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin in these cells. IL-2 expression was induced in Jurkat cells activated by PMA and ionomycin; however, knockdown of TRB1 resulted in decreased induction of IL-2. TRB1 null Jurkat cells established using the CRISPR/Cas9 system also showed reduction of IL-2 expression on PMA/ionomycin stimulation. TRB1 knockdown also markedly inhibited IL-2 promoter activation. To determine the mechanism of the stimulatory effect on IL-2 induction, we focused on histone deacetylases (HDACs), and found that HDAC1 preferentially interacts with TRB1. TRB1 suppressed the interaction of HDAC1 with nuclear factor of activated T cells 2 (NFAT2), which is a crucial transcription factor for IL-2 induction. These results indicate that TRB1 is a positive regulator of IL-2 induction in activated T cells.

Topics: Calcium Ionophores; Carcinogens; Histone Deacetylase 1; Humans; Interleukin-2; Intracellular Signaling Peptides and Proteins; Ionomycin; Jurkat Cells; Leukemia, T-Cell; Lymphocyte Activation; NFATC Transcription Factors; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; T-Lymphocytes; Tetradecanoylphorbol Acetate

Protein phosphatase 4 (PP4) is a protein phosphatase 2A (PP2A)-related, okadaic acid-sensitive, serine/threonine protein phosphatase that shares 65% amino acid identity with PP2A. Numerous studies have shown that protein phosphatase is involved in the regulation of T cell signaling and activation. In this study, we investigated the effect of overexpression of PP4 on the expression of members of the MAP kinase family in Jurkat leukemia T cells, which had previously been stimulated with UV, 12-O-tetradecanoylphorbol-13-acetate (TPA), ionomycin and okadaic acid. We found that the overexpression of PP4 expressed relatively low activity in the absence of any kind of stimulation. However, TPA, UV or ionomycin treatment strongly increased the activity of PP4. In addition, Jurkat T cells, transfected with various expression plasmids and/or stimulated with TPA, UV or ionomycin strongly induced the c-Jun N-terminal kinase (JNK) and p38, whereas the extracellular signal-regulated kinase (ERK)-1/2 kinase pathway was weekly activated. Treatment of Jurkat T cells with okadaic acid, an inhibitor of PP2, also inhibited the increase of JNK and p38 activity induced by PP4. The effect of okadaic acid on the activity of PP4 was similar to that observed in Jurkat T cells treated with a dominant negative c-Jun (dn-jun). These results indicate that the activation of JNK and p38, but not ERKs, is a target for the PP4 activity in Jurkat leukemia T cells.

Topics: Calcium Ionophores; Enzyme Activation; Enzyme Inhibitors; Humans; Ionomycin; JNK Mitogen-Activated Protein Kinases; Jurkat Cells; Leukemia, T-Cell; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Signal Transduction; Tetradecanoylphorbol Acetate; Ultraviolet Rays

The effects of the antioxidant alpha-lipoic acid (LA) on the proliferation of mitogen-stimulated human peripheral blood lymphocytes (HPBL) were investigated in comparison to its effects on the proliferation of two leukaemic T cell lines, Jurkat and CCRF-CEM. At low mM concentrations, LA inhibited in a dose-dependent manner DNA synthesis of HPBL stimulated with either phorbol myristate acetate (PMA) in combination with ionomycin (IoM), or phytohaemagglutinin (PHA). At similar concentrations, LA inhibited the proliferation of Jurkat and CCRF-CEM cells. However, LA was preferentially cytotoxic to the leukaemic cell lines. The selective toxicity of LA to Jurkat cells was shown by electron microscopy (EM) to be due to the induction of apoptosis. Furthermore, LA had different effects on the secretion of interleukin-2 (IL-2) and steady-state levels of IL-2 mRNA in mitogen-stimulated HPBL depending on the mitogens used. LA dramatically increased the induction of IL-2 mRNA and IL-2 protein secretion in PMA/IoM-stimulated HPBL, whereas it inhibited these in HPBL stimulated with PHA. The differential effects of LA on normal and leukaemic T lymphocytes may indicate a new route towards development of therapeutic agents.

Topics: Antioxidants; Cell Survival; Dose-Response Relationship, Drug; Humans; Ionomycin; Ionophores; Jurkat Cells; Leukemia, T-Cell; Lymphocyte Activation; Mitogens; Oxidation-Reduction; Phytohemagglutinins; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thioctic Acid

FK506, an immunosuppressant, inhibits the production of several cytokines in T lymphocytes. We observed that FK506 suppressed the transcription of a chemotactic cytokine, interleukin-8 (IL-8) 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 IL-8 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 (c-Rel, p65, p50, and p49). 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 NF-kappa B binding was also decreased by FK506, indicating that both IL-8 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 IL-8 AP-1 and kappa B-like sites are terminals of FK506-sensitive pathway involving Ca2+ mobilization.

Topics: Base Sequence; Binding Sites; Calcium; Cell Line; Chloramphenicol O-Acetyltransferase; Enzyme-Linked Immunosorbent Assay; Gene Expression; Humans; Interleukin-8; Ionomycin; Kinetics; Leukemia, T-Cell; Luciferases; Molecular Sequence Data; NF-kappa B; Polymerase Chain Reaction; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Sequence Deletion; Suppression, Genetic; T-Lymphocytes; Tacrolimus; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Non-sterol regulation of low density lipoprotein (LDL) receptor gene expression was examined in a mitogen-responsive human T cell line. Stimulation of the leukemic T cell line Jurkat with the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin rapidly and transiently increased LDL receptor mRNA levels. Inhibition of protein synthesis with cycloheximide (CHX) or puromycin resulted in superinduction of LDL receptor mRNA levels by mitogenic stimulation. The increase in LDL receptor mRNA levels resulted from increased gene transcription rather than stabilization of mRNA half-life. Thus, similar results were obtained when reporter gene expression was assessed in Jurkat cells transfected with LDL receptor promoter constructs and mRNA half-life was not significantly altered by the stimuli. Neither mitogenic induction nor superinduction of LDL receptor mRNA levels in Jurkat cells was prevented by sterol downregulation of LDL receptor gene expression. The protein synthesis inhibitors CHX and anisomycin, but not puromycin, also directly stimulated LDL receptor mRNA levels, suggesting that these compounds could provide a signal required for LDL receptor gene transcription. Taken together, these data indicate that various non-sterol stimuli, including activation of protein kinase C, increases in intracellular calcium, inhibition of protein synthesis, and signals generated by the protein synthesis inhibitors CHX and anisomycin, induce LDL receptor gene expression. Thus, transcription of the LDL receptor gene is not only regulated by ambient sterols but also by a variety of influences that govern the various primary response or immediate early genes. These stimuli may play an important role in normal regulation of LDL receptor gene expression.

Topics: Gene Expression Regulation, Neoplastic; Humans; Ionomycin; Leukemia, T-Cell; Mitogens; Receptors, LDL; Sterols; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Interleukin 2 (IL-2) is an important lymphokine required in the process of T cell activation, proliferation, clonal expansion and differentiation. The IL-2 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 IL-2 gene after stimulation. In this study, we have analysed the function of these cis-acting regulatory sites in the context of the native IL-2 enhancer and promoter sequence. The results of this study suggest that the NFAT (-276 to -261), the distal octamer (-256 to -248) and the proximal octamer (-75 to -66) sites not only act as enhancers of IL-2 gene transcription in the presence of cellular stimulation, but also have a silencing effect on IL-2 gene expression in resting cells. Two other sites display disparate effects on IL-2 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 IL-1 stimulation. These data suggest that the regulation of IL-2 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.

Topics: Animals; Base Sequence; Binding Sites; Cell Line; Chloramphenicol O-Acetyltransferase; Enhancer Elements, Genetic; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1; Interleukin-2; Ionomycin; Leukemia, T-Cell; Lymphocyte Activation; Molecular Sequence Data; Mutagenesis, Site-Directed; NF-kappa B; Oligodeoxyribonucleotides; Plasmids; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Purines; T-Lymphocytes; TATA Box; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

T-cell activation results in the production of multiple lymphokines. Efficient lymphokine gene expression appears to require both T-cell antigen receptor (TCR) signal transduction and an uncharacterized second or costimulatory signal. CD28 is a T-cell differentiation antigen that can generate intracellular signals that synergize with those of the TCR to increase T-cell activation and interleukin-2 (IL-2) gene expression. In these studies, we have examined the effect of CD28 signal transduction on granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and gamma interferon (IFN-gamma) promoter activity. Stimulation of CD28 in the presence of TCR-like signals increases the activity of the GM-CSF, IL-3, and IFN-gamma promoters by three- to sixfold. As previously demonstrated for the IL-2 promoter, the IL-3 and GM-CSF promoters contain distinct elements of similar sequence which specifically bind a CD28-induced nuclear complex. Mutation of the CD28 response elements in the IL-3 and GM-CSF promoters abrogates the CD28-induced activity without affecting phorbol ester- and calcium ionophore-induced activity. UV cross-linking indicates that the CD28-induced nuclear complex contains polypeptides of approximately 35, 36, and 44 kDa. These studies indicate that the TCR and CD28-regulated signal transduction pathways coordinately regulate the transcription of several lymphokines and that the influence of CD28 signals on transcription is mediated by a common complex.

Topics: Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Base Sequence; CD28 Antigens; DNA; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferon-gamma; Interleukin-2; Interleukin-3; Ionomycin; Leukemia, T-Cell; Lymphokines; Molecular Sequence Data; Promoter Regions, Genetic; Sequence Homology; Signal Transduction; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.

Topics: 4-Aminopyridine; Apamin; Calcium; Charybdotoxin; Humans; Ionomycin; Leukemia, T-Cell; Phytohemagglutinins; Potassium; Potassium Channels; Scorpion Venoms; Tumor Cells, Cultured

We have recently reported that methionine adenosyltransferase (MAT) in resting human peripheral blood T-cells is primarily present in the form of a precursor which we named lambda. This protein decreases upon cell stimulation, as both MAT activity and the amount of the catalytic alpha/alpha' subunits of the enzyme increase. When resting cells are activated by phytohemagglutinin, the decrease in lambda and increase in alpha/alpha' occurs after interleukin 2 (IL-2) production and before DNA synthesis. The human T-leukemia cell line, Jurkat, is unique in its ability to produce IL-2 in response to exogenous stimuli such as T-cell mitogens and therefore provides a convenient model for studying biochemical reactions involved in T-cell activation. In this study the regulation of MAT activity and S-adenosylmethionine (AdoMet) in resting and activated Jurkat cells was investigated. Here we report that MAT activity in unstimulated Jurkat cells is about 10- and 3-fold higher than the activity in resting and activated peripheral blood mononuclear cells, respectively. Activation of Jurkat cells with phytohemagglutinin resulted in increased IL-2-production, but not an increase in MAT activity. Identical results were obtained using freshly isolated cells from acute lymphoblastic leukemia patients. AdoMet utilization and pool size were approximately 3- and 10-fold higher, respectively, in Jurkat cells compared to peripheral blood mononuclear cells, and both parameters were unaffected by phytohemagglutinin stimulation. Jurkat MAT was determined to be structurally indistinguishable from enzyme from T- or B-leukemia cells but was different from resting, normal T-cells in that it lacked the lambda form. Furthermore, unlike MAT in resting T-cells, the relative amounts of the alpha, alpha', and beta subunits of the enzyme did not change throughout the course of IL-2 induction. We conclude that AdoMet metabolism and MAT activity in Jurkat cells are constitutively high and that induction of IL-2 synthesis in these cells is independent of changes in AdoMet synthesis or turnover. The lack of the lambda form and the difference in MAT regulation between leukemic T-cells and peripheral blood mononuclear cells may be exploited in the design of specific chemotherapeutic agents.

Topics: Enzyme Precursors; Humans; Interleukin-2; Ionomycin; Leukemia, T-Cell; Lymphocyte Activation; Methionine Adenosyltransferase; Phytohemagglutinins; S-Adenosylmethionine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured