sq-23377 and Leukemia

MHC-multimers are reagents used for the detection and enumeration of antigen-specific T cells (ASTs). These reagents exploit the mechanism by which T cell receptors (TCR) on cytotoxic CD8 T cells recognize specific antigens in the context of a major histocompatibility complex (MHC) molecule during antigen presentation. MHC-multimers are fluorescently-labeled dextran polymers that carry MHC Class I molecules and peptide sequences that can be modified to represent specific cognate sequences of the antigen of interest with dextramers having a 10-fold multiplicity of the MHC/peptide combination within a single multimer. Since the binding of antigen-specific dextramers mimics antigen presentation to the TCR, the present study sought to determine whether this TCR engagement on the AST was sufficient to elicit a functional T cell response. The effect of binding of CMV specific dextramers on the activation of the NFAT signal transduction cascade was assessed in peripheral blood from bone marrow transplant recipients previously determined to be positive for CMV-ASTs (CASTs). NFAT activation was quantified by measuring nuclear translocation of NFAT1 in CD8+ CASTs and CD8+ non-CASTs by imaging flow cytometry. Our results demonstrate that an increase in the nuclear localization of NFAT1 was detectable in the CASTs following the CMV-dextramer binding and could be observed as early as 10min post-exposure. The NFAT1 activation correlated with a downstream functional response in the form of interferon gamma production. Sample preparation, temperature, and duration of dextramer exposure were important parameters affecting the dextramer-induced NFAT activation with 2h exposure in whole blood at room temperature being the optimal of the conditions tested. Intra- and inter-individual heterogeneity was observed with regards to the NFAT activation in the CASTs. Importantly, no effect of the dextramers was observed in the CD8+ non-CASTs, and therefore dextramer negative cell populations. Exposure to PMA/ionomycin following dextramer exposure resulted in a homogeneous NFAT activation in both the dextramer-positive but NFAT1 nonresponsive CAST and non-CAST cells. Thus, the data demonstrate that binding of antigen-specific dextramers to ASTs specifically results in activation of NFAT, that the NFAT activation correlates with a downstream functional response and that the response can be heterogeneous. This functional parameter may provide insight to the issue whether enumeration al

Topics: Antigen Presentation; Cytomegalovirus; Flow Cytometry; Fluorescent Dyes; Gene Expression Regulation; Hematopoietic Stem Cell Transplantation; Humans; Image Cytometry; Interferon-gamma; Ionomycin; Leukemia; Lymphocyte Activation; Major Histocompatibility Complex; NFATC Transcription Factors; Phycoerythrin; Primary Cell Culture; Receptors, Antigen, T-Cell; Staining and Labeling; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; Transplant Recipients

To address the role of different intracellular signals in prolactin (PRL) expression in leukocytes, we have investigated the effects of chlorophenylthio-cAMP (cptcAMP), phorbol myristate acetate (PMA) and ionomycin on the activation of the upstream PRL promoter in several leukemic cell lines. All three stimulators, alone or in synergism with each other, were able to modulate promoter activity, but their actions were cell-type dependent. In freshly isolated peripheral blood mononuclear cells (PBMC), PRL expression could only be stimulated by cptcAMP. The physiological importance of cAMP in the regulation of PRL expression in leukocytes is suggested by the finding that in PBMC, PRL expression is enhanced by prostaglandin-E(2) and the beta(2)-adrenergic agonist terbutaline, which both signal through cAMP.

Topics: Adult; Cyclic AMP; Dinoprostone; Gene Expression Regulation; Humans; Ionomycin; Leukemia; Leukocytes, Mononuclear; Middle Aged; Prolactin; Promoter Regions, Genetic; Protein Kinase C; RNA, Messenger; Terbutaline; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Changes in the cytoplasmic calcium concentration ([Ca(2+)](i)) regulate a wide variety of cellular processes. Here we demonstrate that increased [Ca(2+)](i) was able to induce hormone-independent survival and proliferation, as well as to evoke apoptosis in human myelo-erythroid GM-CSF/IL-3 dependent leukemia cells (TF-1). Cellular responses induced by elevated [Ca(2+)](i) depended on the duration and amplitude of the calcium-signal. Moderate or high, but transient, elevation of [Ca(2+)](i) caused a transient, biphasic activation of ERK1/2 and protected cells from hormone withdrawal-induced apoptosis.(1) In contrast, high and long-lasting elevation of [Ca(2+)](i) led to sustained activation of the ERK1/2 kinases and apoptosis of TF-1 cells. Our data suggest that a time-dependent action of the MAPK pathway works as a decision-point between cell proliferation and apoptosis.

Topics: Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Division; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Humans; Indoles; Ionomycin; Leukemia; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Tetradecanoylphorbol Acetate

The exact mechanism of immunosuppression by thalidomide is poorly understood. A common denominator in the pathogenesis of graft-vs.-host disease, graft rejection, reactional lepromatous leprosy, and autoimmune disorders modulated by thalidomide is the activation of T lymphocytes culminating in the synthesis of interleukin-2 (IL-2), the expression of high-affinity IL-2 receptors, and the induction of proliferation. We investigated the effect of thalidomide on the production of IL-2 by the human leukemia cell line Jurkat through induction of IL-2 gene enhancer activity and through the presence of IL-2 in supernatants. beta-galactosidase activity, encoded by a reporter lac z construct and controlled by a transcription factor in thalidomide-treated PMA- and ionomycin-stimulated Jurkat cells, was similar (97 +/- 1.33%; p > 0.1) to non-thalidomide-treated controls at all drug concentrations tested. IL-2 enhancer-driven beta-galactose activity of thalidomide-treated and stimulated cells was also similar to that of untreated controls (p > 0.2). The IL-2 production of activated nontransfected Jurkat cells was gauged by using the IL-2-dependent cell line HT-2 as a readout and by ELISA. Jurkat cells were subcloned by limiting dilution. Bulk cultures and three subclones (J.5.2.5., J.5.2.9., and J.5.3.8.) were assayed at 6, 12, and 24 hours after PHA/PMA-induced stimulation. No inhibitory effect on the IL-2 production by thalidomide could be detected at any of the drug concentrations tested (5-30 micrograms/mL), whereas 10 to 100 ng/mL of cyclosporine inhibited the IL-2 production by 95 to 100%. In addition, we observed neither inhibition of IL-2-dependent proliferation of HT-2 nor inhibition of PHA-induced proliferation of peripheral mononuclear cells by thalidomide at all drug concentrations used (5-30 micrograms/mL). These results do not support the possibility of a modulatory effect on the immune response by thalidomide via IL-2 production and IL-2 response.

Topics: beta-Galactosidase; Cells, Cultured; Clone Cells; Dose-Response Relationship, Drug; Enhancer Elements, Genetic; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-2; Ionomycin; Kinetics; Leukemia; Lymphocyte Activation; Lymphocytes; Tetradecanoylphorbol Acetate; Thalidomide; Transfection; Tumor Cells, Cultured

Although protein kinase C (PKC) activation has been shown to inhibit Ca2+ influx in T lymphocytes, the role of PKC on Ca2+ sequestration or extrusion processes has not been fully explored. We examined the effect of CD3 stimulation and PKC activators on cytosolic Ca2+ (Ca2+i) extrusion and 45Ca2+ efflux in human leukemic Jurkat T cells. Treatment of Fura-2 loaded cells with phorbol 12-myristate 13-acetate (PMA) or thymeleatoxin (THYM) resulted in a decrease in Ca2+i both in the presence and absence of extracellular Ca2+, whereas inactive phorbol esters had no effect. PKC activators added at the peak of a Ca2+i transient induced by anti-CD3 mAb, ionomycin or thapsigargin (TG) stimulated the rate and extent of return of Ca2+i to basal levels by 17-53%. PKC stimulation of the Ca2+i decline was not enhanced by the presence of Na+, indicating that PKC activators increase Ca2+ pump activity rather than a Na+/Ca2+ exchange mechanism. As CD3 receptor activation enhanced the Ca2+i decline in TG-treated cells, antigen-mediated activation of phospholipase C (PLC) signaling includes enhanced Ca2+ extrusion at the plasma membrane. The effect of PKC activators on parameters of Ca2+i extrusion were further explored. PMA significantly increased the rate of Ca2+ extrusion in TG-treated cells from 0.28 +/- 0.02 to 0.35 +/- 0.03 s-1 (mean +/- SEM) and stimulated the initial rate of 45Ca2+ efflux by 69% compared to inactive phorbol ester treated cells. The effects of PKC activation on the Ca2+i decline were eliminated by PKC inhibitors, PKC down regulation (24 h PMA pretreatment), ATP-depletion and conditions that inhibited the Ca2+ pump. In contrast, pretreatment of cells with okadaic acid enhanced the PMA-stimulated response. We suggest that Jurkat T cells contain a PKC-sensitive Ca2+ extrusion mechanism likely to be the Ca2+ pump. In lymphocytes, receptor/PLC-linked PKC activation modulates Ca2+i not only by inhibiting Ca2+ influx but also by stimulating plasma membrane Ca2+i extrusion.

Topics: Alkaloids; Calcium; Calcium-Transporting ATPases; Cytosol; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Humans; Ionomycin; Ionophores; Lanthanum; Leukemia; Naphthalenes; Phorbol Esters; Protein Kinase C; Sodium; Staurosporine; T-Lymphocytes; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

Histamine, ATP, and two microsomal Ca(2+)-pump inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), were able to release intracellular Ca2+ in human leukaemic HL-60 cells. The relationships between the agonist-, TG- and CPA-sensitive Ca2+ pools were investigated with optimal concentrations of these agents in Ca(2+)-free medium. CPA failed to release Ca2+ after the Ca2+ stores of the cells had been discharged by TG, and vice versa, suggesting that the TG- and CPA-sensitive pools exactly overlap. Using this protocol, it was further demonstrated that (a) histamine and ATP utilized the same agonist-sensitive pool, and (b) the CPA- or TG-sensitive pool was much larger than, and encompassed, the agonist-sensitive pool. Although optimal (30 microM) CPA treatment for 5 min totally emptied the agonist-sensitive pool, a brief exposure (1.5 min) to a sub-optimal concentration (3 microM) of CPA, which only slightly raised cytosolic free Ca2+ concentration ([Ca2+]i), substantially enhanced subsequent agonist-induced Ca2+ release. Brief pretreatments with sub-optimal concentrations of TG or ionomycin, which caused moderate [Ca2+]i elevation, also caused such enhancement. However, sub-optimal CPA pretreatment had no prominent effect on Ca2+ release, which was InsP3-independent: it did not enhance TG-induced Ca2+ release, and only relatively weakly augmented ionomycin-induced Ca2+ release. Our results represent a novel observation showing that low concentrations of CPA, TG and ionomycin can potentiate subsequent agonist-induced Ca2+ release, and suggest that a 'priming' moderate [Ca2+]i elevation can amplify subsequent InsP3-dependent Ca2+ release in HL-60 cells.

Topics: Adenosine Triphosphate; Calcium; Calcium-Transporting ATPases; Cytosol; Histamine; Humans; Indoles; Inositol 1,4,5-Trisphosphate; Ionomycin; Leukemia; Terpenes; Thapsigargin; Tumor Cells, Cultured

Differentiation of HL60 cells by treatment with dimethyl sulphoxide induces the expression of membrane receptors for N-formylmethionyl-leucyl-phenylalanine (fMLP) and for platelet-activating factor (PAF). In these cells both agonists produced an increase in the cytosolic Ca2+ concentration ([Ca2+]i) by release of Ca2+ from the intracellular stores, followed shortly by an acceleration of the entry of Ca2+ or Mn2+, used here as a Ca2+ surrogate for Ca2+ channels. Cytochrome P-450 inhibitors blocked the agonist-induced entry of Ca2+ or Mn2+ with no modification of Ca2+ release from the stores. Emptying the intracellular Ca2+ stores either by treatments inducing no inositol phosphate production, such as prolonged incubation in Ca(2+)-free medium or treatment with the Ca2+ ionophore ionomycin, increased the plasma-membrane permeability to Ca2+ and Mn2+. This Ca(2+)-store-regulated Mn2+ entry was inhibited by Ni2+ and by cytochrome P-450 inhibitors. Refilling of the Ca2+ stores by incubation in Ca(2+)-containing medium restored low Mn2+ permeability. The same mechanism is present and functional in non-differentiated cells, before expression of membrane receptors for fMLP and PAF. These results suggest that agonist-induced Ca2+ (Mn2+) entry is secondary to the emptying of the intracellular Ca2+ stores, which in turn activates plasma-membrane channels by a mechanism involving cytochrome P-450.

Topics: Antifungal Agents; Biological Transport; Calcium; Cell Differentiation; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Cytosol; Dimethyl Sulfoxide; Econazole; Humans; Imidazoles; Ionomycin; Leukemia; Manganese; N-Formylmethionine Leucyl-Phenylalanine; Nickel; Platelet Activating Factor; Platelet Membrane Glycoproteins; Receptors, Cell Surface; Receptors, Formyl Peptide; Receptors, G-Protein-Coupled; Receptors, Immunologic; Tumor Cells, Cultured

Apoptosis is a form of cell death in which the cell "participates," such that metabolic energy and often protein synthesis are required for the death to occur. Once begun, the process of apoptosis proceeds in an ordered fashion. In the earliest phase DNA fragmentation occurs, accompanied by cell shrinkage and dilation of the endoplasmic reticulum. This is followed by cell fragmentation with the formation of sealed membrane vesicles, termed apoptotic bodies. In the present study we have demonstrated that the fungal metabolite cytochalasin B inhibits cell fragmentation and the formation of apoptotic bodies, probably by its ability to interfere with actin polymerization. This effect was seen when HL-60 cells were pretreated with cytochalasin B and then exposed to one of a number of apoptosis-inducing agents, including UV irradiation, camptothecin, aphidocholin, or PMA plus ionomycin. The observed effect was not peculiar to HL-60 cells, inasmuch as it was also seen for both Molt-4 and U-937 cell lines. Cytochalasin B had no effect on DNA fragmentation occurring in the earliest stage of apoptosis, and it appeared to have no inhibitory effects on nuclear fragmentation. Staurosporin had an effect similar to that seen with cytochalasin B, probably due to its ability to inhibit protein kinase C, which is a known potentiator of microfilament assembly. These data demonstrate that microfilament assembly is necessary for the formation of apoptotic bodies in the later stages of the apoptotic process.

Topics: Actin Cytoskeleton; Aphidicolin; Camptothecin; Cell Death; Cell Line; Cell Survival; Cytochalasin B; Humans; Ionomycin; Kinetics; Leukemia; Tetradecanoylphorbol Acetate; Ultraviolet Rays

Phorbol esters exert diverse effects on cellular activation and differentiation. CD 7, a differentiation antigen appearing early in T cell ontogeny, may be involved in the activation and differentiation processes. CD 7 was found to be rapidly down-regulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) from mature T cell surface. The time course of CD 7 down-regulation was similar to that of other functionally important T cell antigens, CD 3 and CD 4. Within 2 h, TPA at 10 to 30 ng/ml induced a complete down-regulation of CD 7. Twenty-four hours later, the reappearance of CD 7 on TPA-treated cells was observed. This phenomenon was monocyte independent. In contrast, CD 7 expression on thymocytes was resistant to the effect of TPA. In addition, certain leukemic T cells were also resistant to TPA-induced CD 7 down-regulation. The mechanism underlying TPA-induced CD 7 down-regulation was investigated further. Synthetic diacylglycerol, sn-1,2-dioctanoylglycerol, which activates protein kinase C, did not induce down-regulation of CD 7 on mature T cells. Ionomycin, a calcium ionophore, did not down-regulate this antigen either. Thus, it is concluded that the processes of protein kinase C activation and/or cytosolic calcium influx are not sufficient for TPA-induced CD 7 down-regulation; other pathways induced by TPA may be responsible.

Topics: Antibodies, Monoclonal; Antigens, Differentiation, T-Lymphocyte; Depression, Chemical; Diglycerides; Ethers; Gene Expression Regulation; Humans; Ionomycin; Leukemia; Phosphatidylinositols; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

We have previously established subclones from human leukemia-derived HSB.2 cell line that produced high levels of interleukin (IL) 2 when stimulated with phytohemagglutinin (PHA) and IL-1. Herein, we investigated the signal requirement for IL-2 production, particularly concerning the role of IL-1 in this system. PHA but not IL-1 rendered marked protein kinase C (PKC) activation and IL-2 production induced by PHA plus IL-1 was totally abrogated by a potent PKC inhibitor, H-7. Concomitantly, PHA alone caused marked Ca2+ influx, whereas IL-1 neither induced Ca2+ influx nor augmented PHA-induced Ca2+ influx. As expected, a signal delivered by PHA could be substituted by phorbol 12-myristate 13-acetate (PMA) and ionomycin while IL-1 was still indispensable, indicating that at least three signals, i.e., those delivered by IL-1 as well as PKC activation and Ca2+ influx were required for optimal IL-2 production. Kinetic study indicated that while PMA and ionomycin should be added at the initiation of culture, delayed addition of IL-1 up to 4 hr later induced even higher levels of IL-2 production, demonstrating the requirement for IL-1 after PKC activation and Ca2+ influx. In this system, it was revealed that IL-1 was not involved in PKC activation, Ca2+ influx, and breakdown of phosphatidylinositols. Whereas PMA, ionomycin, and IL-1 stimulated high levels of IL-2 production, those combinations of signals did not induce breakdown of phosphatidylinositols. It should be noted that IL-2 production induced by these three signals seemed to bypass hydrolysis of phosphatidylinositols in contrast to PHA plus IL-1 stimulation that was accompanied with a marked breakdown of phosphatidylinositols.

Topics: Calcium; Enzyme Activation; Ethers; Humans; Interleukin-1; Interleukin-2; Ionomycin; Leukemia; Lymphocyte Activation; Phosphatidylinositols; Phytohemagglutinins; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured