calcimycin and Leukemia--Promyelocytic--Acute

Direct differentiation of myeloid leukemia blasts into antigen-presenting dendritic cells (DCs) for use as cellular vaccines is unique in that identification of tumor-specific antigens may not be necessary because the antigens should already be endogenously expressed. We hypothesized that signaling through protein kinase C (PKC) is required for differentiation of HL-60 promyeloblasts into DCs upon stimulation with calcium ionophore A23187. To demonstrate the inhibitory effect of PKC blockade, we pretreated HL-60 myeloid blasts with the protein kinase inhibitor bisindolylmaleimide I (Bis-1) for 24 hours and then treated the cells with calcium ionophore A23187 for an additional 24 hours. Controls consisted of HL-60 blasts treated with A23187, Bis-1 alone, or media. We noted that blasts cultured in media, Bis-1, or Bis-1 then A23187 did not develop the morphologic and phenotypic DC characteristics, up-regulate Rel B, or activate allogeneic T-cells. Our findings suggested that PKC blockade inhibits morphologic, phenotypic, and functional differentiation of HL-60 promyeloblasts into antigen-presenting DCs. Our findings supported the role of PKC as an obligatory pathway for calcium ionophore A23187-induced differentiation of HL-60 myeloblasts into antigen-presenting DCs.

Topics: Antigen-Presenting Cells; Calcimycin; Cell Differentiation; Dendritic Cells; HL-60 Cells; Humans; Immunotherapy, Adoptive; Indoles; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Maleimides; Protein Kinase C; Proto-Oncogene Proteins; T-Lymphocytes; Transcription Factor RelB; Transcription Factors; Up-Regulation

We have investigated whether the increases in ceramide levels that occur during apoptosis in SKW 6.4 cells induced by anti-Fas antibody depend on the activation of caspases. Using cells prelabelled to equilibrium with [14C]acetate, it was shown that the amount of ceramide approximately doubled after 24 h incubation with anti-Fas, but the time course of ceramide changes was slower than that of anti-Fas-induced cell death. Complete inhibition of the effects of anti-Fas on cell death and on ceramide production was observed when the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethane (zVAD.fmk) was added together with anti-Fas, but N-benzyloxycarbonyl-Phe-Ala-fluoromethane (a structurally similar cathepsin B inhibitor) had no effect. Treatment of cells with the Ca2+-ionophore A23187 also doubled ceramide levels, but in this case the effect was complete within 2 h, was not blocked by zVAD.fmk and was not associated with increases in nuclear fragmentation. These results suggest that ceramide is not an upstream messenger in Fas-mediated apoptosis and may instead be produced as a consequence of processes downstream of the activation of caspases and increases in cytosolic calcium concentration.

Topics: Acetates; Amino Acid Chloromethyl Ketones; Antibodies; Apoptosis; Calcimycin; Calcium; Ceramides; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; fas Receptor; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Lymphoma, B-Cell; Signal Transduction; Time Factors; Tumor Cells, Cultured

Transglutaminase is a calcium-dependent enzyme that catalyzes an amine incorporation and a cross-linking of proteins. Intracellular transglutaminase is induced when human promyelocytic leukemia HL-60 cells are treated with retinoic acid and human hepatoblastoma HepG2 cells, with interleukin-6. To find whether the intracellular reaction catalyzed by transglutaminase increased when the enzyme is induced in these cells, the transglutaminase-catalyzed incorporation of 14C-labeled methylamine into cellular proteins was measured. The incorporation level of the labeled methylamine into proteins of HL-60 and HepG2 cells did not increase after the transglutaminase had been induced. The presence of the calcium ionophore A23187 did not affect these results. These findings suggested that even after the enzyme induction the catalytic action of intracellular transglutaminase is maintained at a constant level in these cells by unknown regulatory mechanism(s).

Topics: Calcimycin; Catalysis; Enzyme Induction; Hepatoblastoma; HL-60 Cells; Humans; Interleukin-6; Ionophores; Leukemia, Promyelocytic, Acute; Liver Neoplasms; Transglutaminases; Tretinoin

We have selected calcium ionophore (A23187)-resistant cells (AR-7) in a human promyelocytic leukemia cell line, HL-60. AR-7 showed approximately 8.5-fold resistance to A23187 compared with the wild type cells after continuous exposure for 3 days. AR-7 had cross resistance to ionomycin (4.6-fold) and thapsigargin (340-fold) which can also increase intracellular Ca++. Similar magnitude of resistance to apoptosis, as defined by characteristic morphology and internucleosomal DNA fragmentation, induced by these agents was observed after 4 hr of incubation. However, both the elevation of intracellular Ca++ following stimulation with various concentrations of A23187 and the sensitivity to anti-cancer agents including etoposide, 1-beta-D arabinofuranosylcytosine, and hydroxyurea were comparable between the two cell types. This cell line is considered to be useful for exploring the mechanism(s) of cell death, especially apoptosis, induced by calcium ionophore.

Topics: Antineoplastic Agents; Apoptosis; Calcimycin; Calcium; Cell Division; Cytarabine; DNA; Drug Resistance; Etoposide; Flow Cytometry; Humans; Ionomycin; Leukemia, Promyelocytic, Acute; Terpenes; Thapsigargin; Tumor Cells, Cultured; Ultraviolet Rays

Calcium ionophore (A23187)-induced high molecular weight (HMW) and internucleosomal DNA fragmentation were investigated in human leukemia cell lines. An apoptosis-sensitive cell line, HL-60, showed HMW, internucleosomal DNA fragmentation and morphological changes of apoptosis by A23187. MOLT-4, which is resistant to apoptosis, exhibited only HMW DNA fragmentation and died of necrosis under the same conditions. Autodigestion experiments suggested the endonucleolytic activity to cause HMW fragmentation in the cytoplasm of both cell lines. The activity was more dependent on Mg2+ than Ca2+ in HL-60, whereas it was Ca(2+)-dependent in MOLT-4. These results suggest that HMW DNA fragmentation is not specific to apoptosis.

Topics: Apoptosis; Calcimycin; Calcium; Cell Nucleus; Cytoplasm; DNA; Endonucleases; Humans; Leukemia, Promyelocytic, Acute; Magnesium; Molecular Weight; Nucleosomes; Terpenes; Thapsigargin; Tumor Cells, Cultured

Urinary trypsin inhibitor (UTI) is one of the Kunitz-type protease inhibitors in human. Little is known about its anti-inflammatory functions other than protease inhibition. We studied the effect of UTI on gene expression of interleukin-8 (IL-8), an inflammatory cytokine. UTI inhibited IL-8 gene expression induced by lipopolysaccharide (LPS) in HL60 cells. The IL-8 concentrations in the cells and medium after LPS stimulation increased time-dependently in the absence of UTI, but did not increase in the presence of UTI. On the other hand, UTI did not inhibit either the synthesis or the release of IL-8 induced by the calcium ionophore A23187. UTI inhibited increase of cytosolic Ca2+ stimulated by LPS but not by A23187. Our results suggest that the inhibition by UTI is due to its effect on the cell membrane involved in regulating Ca2+ influx.

Topics: Calcimycin; Calcium; Cell Membrane; Culture Media, Conditioned; Cytosol; Gene Expression Regulation; Humans; Interleukin-8; Leukemia, Promyelocytic, Acute; Lipopolysaccharides; RNA, Messenger; Trypsin Inhibitor, Kunitz Soybean; Tumor Cells, Cultured

Tissue Factor (TF) is a transmembrane glycoprotein that serves as cofactor for Factor VII (FVII) in the initiation of blood coagulation and that is differentially expressed in a number of cell types, being constitutively expressed in some and inducible in others. We studied the localization and the functional activity of TF in monocytic leukemia U937 cells at different time intervals after lipopolysaccharides (LPS) stimulation, and the effect of calcium ionophore on the surface expressed TF. Exposure of U937 cells to 10 micrograms/ml LPS resulted in a time dependent increase of TF expression that reached a maximum at 12 h for TF antigen and at 24 h for TF activity. Blocking of surface TF with inhibitory anti-TF antibody abolished > 93% of the activity of lysed cells stimulated for 24 h, while it blocked only 80% of the activity in lysed cells stimulated for 12 h suggesting that at that time about 20% of TF is not accessible for the antibody. Even at 24 h when the specific activity of surface expressed TF is 5.5 times higher than at 12 h, this specific activity is still 10 fold lower than that of TF in lysed cells. Addition of Ca++ ionophore A23187 to LPS stimulated cells resulted in a fast increase of TF activity that was dependent on the dose of ionophore, on the extracellular Ca++ concentration and on the time that the cells had been incubated with LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antigens; Calcimycin; Calcium; Humans; Ionophores; Kinetics; Leukemia, Promyelocytic, Acute; Lipopolysaccharides; Thromboplastin; Tumor Cells, Cultured

An eosinophilic substrain of HL-60 cells (HL-60#7) predominantly synthesized cysteinyl leukotrienes after stimulation with the calcium ionophore A23187. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) specifically attenuated cysteinyl leukotriene production without affecting the biosynthesis of non-cysteinyl leukotrienes. The inhibition of cysteinyl leukotriene biosynthesis was prevented only by specific PKC inhibitors (staurosporine and bisindolylmaleimide) but not by inhibitors of tyrosine kinases (genistein, tyrphostin 47, and herbimycin A), protein kinase A (KT5720), or the oxidative burst (apocynin). Similar results were obtained when LTC4 synthase enzymatic activity was measured directly in the presence of saturating concentrations of exogenously added substrates. Therefore, the inhibitory effects of PKC activation on cysteinyl leukotriene formation in intact cells was attributable to effects on the LTC4 synthase enzyme. The mechanism of inhibition of LTC4 synthase by PKC activation was determined by kinetic analysis to be noncompetitive in both eosinophil-like HL-60#7 cells and monocytic THP-1 cells. Contrary to the effect of PKC activation on cysteinyl leukotriene biosynthesis, the formation of prostaglandin E2 and thromboxane B2 was elevated twofold to threefold after PMA treatment, which was prevented by the PKC inhibitor, staurosporine. We propose a regulatory model in which PKC activation shifts the profile of eicosanoid mediators produced by eosinophils from cysteinyl leukotrienes to prostanoids.

Topics: Alkaloids; Calcimycin; Cysteine; Down-Regulation; Eosinophils; Glutathione Transferase; Humans; Leukemia, Promyelocytic, Acute; Leukotrienes; Nitriles; Phenols; Prostaglandins; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tyrphostins

We recently reported that treatment with calcium ionophore, A23187, induces apoptosis in human myelogenous leukemia cells but causes necrotic cell death in T-lymphoblastic leukemia cells. To better understand the underlying mechanisms of such different modes of cell death, we established hybridomas between HL-60 promyelocytic and CEM T-lymphoblastic leukemia cells. The resulting hybridomas were divided into three groups in terms of their susceptibility to apoptosis following exposure to A23187: (1) hybridomas highly sensitive to apoptosis, (2) hybridomas with intermediate sensitivity to apoptosis which occurs later and to a lesser extent, and (3) hybridomas resistant to apoptosis. However, growth inhibition after 72 h of incubation and an initial rise in intracellular free calcium concentrations induced by A23187 were similar in the three groups. Expression of Ca(2+)-independent/Mg(2+)-dependent endonuclease, which had an optimal pH of 7.5-8.5 and was inhibited by Zn2+, was correlated with the susceptibility of the hybridomas to A23187-induced apoptosis. Thus, this endonuclease may play, at least in part, an important role in the induction of apoptosis in leukemia cell lines. Analysis of hybridomas between apoptosis-sensitive and apoptosis-resistant cells is useful in the elucidation of genetic factors which regulate cell death.

Topics: Apoptosis; Calcimycin; Endonucleases; Humans; Hybridomas; Leukemia-Lymphoma, Adult T-Cell; Leukemia, Promyelocytic, Acute; Magnesium; Tumor Cells, Cultured

Monocyte Chemotactic Protein-1 (MCP-1), a member of the Cys-Cys branch of the chemokine superfamily, induced a mepacrine- and manoalide-sensitive increase in the release of [3H]arachidonic acid from prelabeled human monocytes and monocytic THP-1 leukemic cells. The effect was rapid (<30 s), reached maximum at optimal chemotactic concentrations, and was completely blocked by pretreatment of monocytes with Bordetella pertussis toxin. A specific antiserum and heat inactivation blocked the induction of arachidonic release by MCP-1. No [3H]arachidonic acid release was observed in the absence of Ca2+ influx (5 mM EGTA or 5 mM Ni2+) or in monocytes loaded with a Ca(2+)-buffering agent. However, using ionophore-permeabilized monocytes and controlled intracellular Ca2+ concentration it was possible to dissociate MCP-1-induced Ca2+ influx from [3H]arachidonic acid release. Thus, the MCP-1-induced increase in [Ca2+]i is necessary but not sufficient for arachidonic acid accumulation. Phospholipase A2 inhibitors (mepacrine, p-bromophenacyl bromide, and manoalide) blocked monocyte polarization and chemotaxis induced by MCP-1. The related Cys-Cys chemokines RANTES and LD78/MIP1 alpha also induced a rapid release of [3H]arachidonic acid, and their chemotactic activity was blocked by phospholipase A2 inhibitors. Brief (5 min) pretreatment of monocytes with platelet-activating factor amplified MCP-1-induced arachidonic acid release and, at MCP-1 suboptimal concentrations, synergized in inducing monocyte migration. Since MCP-1 and platelet-activating factor are induced concomitantly by inflammatory cytokines in monocytes and endothelial cells, we speculate that the observed synergism may have in vivo relevance. The results presented here show that the Cys-Cys chemokines MCP-1, LD78/MIP1 alpha, and RANTES cause rapid release of arachidonic acid in monocytes and that this may be important in inducing monocyte chemotaxis.

Topics: Arachidonic Acid; Calcimycin; Calcium; Cell Line; Chemokine CCL2; Chemotactic Factors; Chemotaxis, Leukocyte; Cytokines; Egtazic Acid; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Pertussis Toxin; Platelet Activating Factor; Recombinant Proteins; Structure-Activity Relationship; Tumor Cells, Cultured; Virulence Factors, Bordetella

We compared the regulation of cytosolic phospholipase A2 (cPLA2) activity in undifferentiated and neutrophil-like HL60 cells. Although Ca(2+)-mobilizing P2-purinergic receptors are expressed in both cell types, arachidonic acid (AA) release stimulated by P2-purinergic agonists was 5-7-fold higher in the differentiated cells. Similarly, the stimulation of AA release by AlF4- in intact cells or by ATP and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in electropermeabilized cells was significantly higher in the differentiated cells. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced A23187-stimulated AA release in intact HL60 granulocytes with minimal effects in the undifferentiated cells. Immunoblotting experiments showed similar levels of cPLA2 and of agonist-mediated activation of mitogen-activated protein kinase in both cell types. Experiments measuring stimulation of AA release by either melittin, using endogenously labeled intact cells, or Ca2+, using homogenates and exogenous substrate, indicated that undifferentiated cells do not lack an activatable PLA2. The stimulatory effects of GTP gamma S and Ca2+ on AA release in homogenates from endogenously labeled cells suggested that undifferentiated cells display G protein-cPLA2 coupling. Basal and PMA-stimulated phosphorylation of cPLA2 was detected in differentiated, but not in undifferentiated cells. However, the two cell types displayed only subtle differences in the time courses of phosphorylation of mitogen-activated protein kinase triggered by agonists and PMA. The observed defect in cPLA2 phosphorylation may represent the alteration preventing agonist-mediated stimulation of AA release in undifferentiated HL60 cells.

Topics: Adenosine Triphosphate; Aluminum Compounds; Arachidonic Acid; Blotting, Western; Bucladesine; Calcimycin; Calcium; Cell Differentiation; Cell Line; Cytosol; Electrophoresis, Polyacrylamide Gel; Fluorides; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Kinetics; Leukemia, Promyelocytic, Acute; Neutrophils; Phospholipases A; Phospholipases A2; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The macrolide, FK-506, is a potent and effective inhibitor of lymphocyte activation. We studied the effects of FK-506 on human neutrophil activation induced by chemoattractants and by various substances which circumvent receptor stimulation. After preincubation for 5 min at 37 degrees C, FK-506 (1 microM) inhibited N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe)- or platelet-activating factor-induced superoxide production in neutrophils by about 30%. At therapeutic concentrations (0.1-1 nM) FK-506 was ineffective. FK-506 did not inhibit exocytosis and rises in cytosolic Ca2+ concentration [Ca2+]i mediated by these stimuli, and it did not at all inhibit neutrophil activation induced by C5a, leukotriene B4 and 4 beta-phorbol 12-myristate 13-acetate. FK-506 (1 microM) inhibited A23187-induced exocytosis by about 35%, but A23187-induced superoxide production was unaffected. After preincubation for 5 min at 37 degrees C, FK-506 inhibited fMet-Leu-Phe-induced superoxide production in dibutyryl cAMP-differentiated HL-60 cells by about 20%; preincubation with the drug for 24 h did not result in inhibition of superoxide production. FK-506 did not inhibit agonist-binding to formyl peptide receptors and fMet-Leu-Phe-stimulated GTP hydrolysis of heterotrimeric regulatory guanine nucleotide-binding proteins (G-proteins) in membranes from dibutyryl cAMP-differentiated HL-60 cells. FK-506 did not change steady-state and differential polarized phase fluorescence in HL-60 membranes using 1,6-diphenylhexa-1,3,5-triene and 12-(9-anthroyloxy)-stearate as probes. Our results show that FK-506 at supratherapeutic concentrations partially inhibits neutrophil activation. Inhibition by FK-506 of fMet-Leu-Phe-induced superoxide production is rapid in onset and is not due to inhibition of agonist-binding to receptors, interference with G-proteins or protein kinase C, reduction of rises in [Ca2+]i or alteration in physical membrane state.

Topics: Adult; Calcimycin; Calcium; Cyclosporine; Dose-Response Relationship, Drug; Female; Humans; Leukemia, Promyelocytic, Acute; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Superoxides; Tacrolimus; Tumor Cells, Cultured

One of the factors regulating the population size of a clone of proliferating cells is the induction of a physiological suicide mechanism known as apoptosis. We studied apoptosis in the HL-60 human promyelocytic leukemia cell line which differentiates when exposed to phorbol ester (S-cell), and in the PET-cell mutant of HL-60 which is defective in its response to phorbol ester. Exposing S-cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) (3 nM and above) induced morphological changes characteristic of apoptosis (visualized by light microscopy), and induced fragmentation of chromatin DNA to oligonucleosomal lengths. These changes were obvious in 48 h. In contrast, 1000 nM TPA for five days did not induce apoptosis in the PET-cell. DNA fragmentation was induced in both cell lines by A23187 (0.25 microM) and etoposide (7 microM). Novobiocin (600 and 900 microM) induced DNA fragmentation in S-cells, but higher concentrations inhibited fragmentation. Novobiocin is believed to induce DNA fragmentation by a direct action on DNA. In the case of PET-cells, novobiocin did not induce DNA fragmentation at any concentration, and prior treatment of PET-cells with novobiocin (300-1200 microM for 30 min) inhibited DNA fragmentation induced by A23187. Novobiocin inhibited cell growth equally in S-cell and PET-cells. It is concluded that the promyelocytes have the capacity to undergo apoptosis in response to agents which activate protein kinase C, and that the PET-cell has a mutation which disables both protein-kinase C-induced and novobiocin-induced DNA fragmentation, leaving intact the ability of novobiocin to protect DNA from calcium-entry-initiated fragmentation. The elucidation of the lesion responsible for the PET phenotype is likely to increase our understanding of this important pathway for regulating cellular proliferation and how it bears on leukemogenesis and chemotherapy.

Topics: Apoptosis; Calcimycin; DNA Damage; DNA, Neoplasm; Drug Tolerance; Etoposide; Humans; Leukemia, Promyelocytic, Acute; Mutation; Novobiocin; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

It has been demonstrated that perturbation of oxidative balance plays an important role in numerous pathological states as well as in physiological modifications leading to aging. In order to evaluate the role of the oxidative state in cells, biochemical and ultrastructural studies were carried out on K562 and HL-60 cell cultures. Particular attention was given to the transferrin receptor, which plays an important role in cellular iron metabolism. In order to evaluate if oxidative stress influences the transferrin receptor regulation process, the free-radical inducer menadione was used. The results obtained seem to indicate that oxidative stress is capable of inducing a rapid and specific down-modulation of the membrane transferrin receptor due to a block of receptor recycling on the cell surface, without affecting ligand-binding affinity. These effects were observed in the early stages of menadione treatment and before any typical signs of subcellular damage, including surface blebbing, a well-known cytopathological marker of menadione-induced injury. The mechanisms underlying such phenomena appear to be related to cytoskeletal protein thiol group oxidation as well as to the perturbation of calcium homeostasis, both induced by menadione. It is thus hypothesized that the data reported here represent a specific example of a general mechanism by which cell surface receptor expression and recycling can be influenced by oxidative balance.

Topics: Actin Cytoskeleton; Calcimycin; Calcium Channel Blockers; Cytochalasin B; Edetic Acid; Endocytosis; Humans; Iron; Leukemia, Erythroblastic, Acute; Leukemia, Promyelocytic, Acute; Microscopy, Electron; Microtubules; Oxidation-Reduction; Phalloidine; Reactive Oxygen Species; Receptors, Transferrin; Stress, Physiological; Tumor Cells, Cultured; Vitamin K

Previous studies have indicated that Ca2+ is a trigger for apoptosis (programmed cell death) in thymocytes and related cell lines. Recently we have shown that levels of apoptosis in leukaemic cells are diminished in Ca(2+)-deficient conditions, indicating that Ca2+ may be important in the mechanism of apoptosis in these cells. In the present study we investigated the possibility that Ca2+ serves as a trigger for apoptosis in the human leukaemic cell line, HL-60. Using fura-2 to measure cytosolic free Ca2+ concentrations, [Ca2+]i, in cell suspensions, and by using ratio imaging of fura-2 in single cells, we did not observe an early significant increase in [Ca2+]i in HL-60 cells undergoing apoptosis. The latter stages of apoptosis were, however, accompanied by increasing [Ca2+]i; these increases were apparently a result of, rather than a cause of, apoptosis. Furthermore, apoptosis could be induced in HL-60 cells under conditions of vastly reduced [Ca2+]i achieved by loading these cells with fura-2 in the presence of EGTA. These results indicate that elevation of [Ca2+]i is not a prerequisite for apoptosis in HL-60 cells and that apoptosis can occur in these cells in the presence of low [Ca2+]i.

Topics: Calcimycin; Calcium; Cell Death; Cytosol; DNA, Neoplasm; Electrophoresis, Agar Gel; Ethanol; Fura-2; Humans; Hydrogen Peroxide; Leukemia, Promyelocytic, Acute; Tumor Cells, Cultured; Ultraviolet Rays

Topics: Calcimycin; Calcium; Cell Death; Cell Line; Dose-Response Relationship, Drug; Humans; Kinetics; Leukemia, Promyelocytic, Acute

We have demonstrated translocation of HL-60 cell 5-lipoxygenase to a membrane compartment in response to both the calcium ionophore A23187 and the receptor-mediated stimulus, N-formyl-methionyl-leucyl-phenylalanine (fMLP). In addition, we have shown inhibition of A23187- and fMLP-induced 5-lipoxygenase translocation by an indole and a quinoline leukotriene synthesis inhibitor, MK-886 and L-674,573, respectively. Selectivity of inhibition of 5-lipoxygenase translocation in both fMLP- or A23187-challenged cells is shown using the indole L-583,916 and quinoline L-671,480, which neither inhibit leukotriene synthesis nor inhibit 5-lipoxygenase translocation. The present study in HL-60 cells is the first demonstration of the selective inhibition of 5-lipoxygenase translocation by quinoline leukotriene synthesis inhibitors, exemplified by L-674,573. Also described here is the first demonstration of 5-lipoxygenase translocation and inhibition in response to a stimulus other than A23187, namely the receptor-mediated stimulus, fMLP.

Topics: Arachidonate 5-Lipoxygenase; Calcimycin; Cell Differentiation; Cell Line; Cell Membrane; Dimethyl Sulfoxide; Humans; Indoles; Kinetics; Leukemia, Promyelocytic, Acute; Leukotriene Antagonists; Leukotrienes; N-Formylmethionine Leucyl-Phenylalanine; Quinolines; Subcellular Fractions

Topics: Animals; Calcimycin; Cell Differentiation; Dexamethasone; Endotoxins; Fibroblasts; Humans; Inflammation; Interleukins; Leukemia, Promyelocytic, Acute; Leukocytes, Mononuclear; Lipopolysaccharides; Macrophages; Mice; Neoplasm Proteins; Prostaglandin-Endoperoxide Synthases; Tumor Cells, Cultured

Treatment of human promyelocytic leukemia cells (HL-60) with the calcium ionophore A23187 induced a biphasic change in the expression of the c-myc protooncogene. Within 1 h of exposure to a single 2.5-microM dose of A23187, steady state levels of c-myc mRNA increased to 170% of control. After this early increase, c-myc mRNA expression underwent an abrupt decline over the next 2 to 4 h. Between 6 to 18 h of treatment with A23107, steady state levels of c-myc mRNA fell gradually toward zero. The early increase in expression of c-myc mRNA was mediated by both enhanced initiation of c-myc transcription and posttranscriptional stabilization of c-myc mRNA. Stimulation of HL-60 cells with A23187 for 30 min increased transcription of both exons 1 and 3 of c-myc by 125%. After 1 h of treatment with A23187, the half-life of c-myc mRNA increased to 150% of control. The late decrease in c-myc expression induced by A23187 was primarily mediated by interruption of elongation of c-myc nascent mRNA beyond exon 1. The combination of calcium ionophore A23187 and phorbol 12,13-dibutyrate acted synergistically to inhibit expression of c-myc mRNA.

Topics: Calcimycin; Calcium; Dactinomycin; Drug Synergism; Gene Expression Regulation; Humans; In Vitro Techniques; Leukemia, Promyelocytic, Acute; Phorbol 12,13-Dibutyrate; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

Activation of NADPH oxidase in undifferentiated HL-60 leukemic cells and in HL-60 cells differentiated along the myeloid pathway with dibutyryl cyclic AMP (dbcAMP) or dimethyl sulfoxide (Me2SO) was studied. Upon stimulation with a calcium ionophore, a phorbol ester, arachidonic acid or gamma-hexachlorocyclohexane, Me2SO-differentiated HL-60 cells generated superoxide (O2-) at higher rates than dbcAMP-differentiated cells. Undifferentiated cells generated O2- only at low rates upon stimulation with the above agents. In cell-free systems, NADPH oxidase activity was reconstituted by combining membranes of undifferentiated or dbcAMP- or Me2SO-differentiated HL-60 cells, cytosol of Me2SO-differentiated cells and arachidonic acid. This basal O2- formation was enhanced several-fold by guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]), a potent activator of guanine nucleotide-binding proteins. In contrast, cytosol of dbcAMP-differentiated cells reconstituted O2- formation only in the presence of GTP[gamma S], and cytosol of undifferentiated cells was inactive. Submaximally stimulatory amounts of cytosolic protein of Me2SO- and dbcAMP-differentiated cells synergistically stimulated O2- formation in the presence but not in the absence of GTP[gamma S]. We conclude that differentiations of HL-60 cells with Me2SO and dbcAMP are not equivalent with respect to activation of NADPH oxidase and that two cytosolic activation factors are involved in the regulation of this effector system.

Topics: Arachidonic Acid; Arachidonic Acids; Bucladesine; Calcimycin; Cell Differentiation; Cell Line; Cytosol; Dimethyl Sulfoxide; Enzyme Activation; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hexachlorocyclohexane; Humans; Kinetics; Leukemia, Promyelocytic, Acute; N-Formylmethionine Leucyl-Phenylalanine; NADH, NADPH Oxidoreductases; NADPH Oxidases; Superoxides; Tetradecanoylphorbol Acetate; Thionucleotides

It has recently been demonstrated that the chemotactic peptide N-formyl-Met-Leu-Phe activates phospholipase D (PLD) in dimethyl sulfoxide-differentiated HL-60 granulocytes to produce phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) (Pai, J.-K., Siegel, M. I., Egan, R. W., and Billah, M. M. (1988) J. Biol. Chem. 263, 12472-12477). We now report that biologically active phorbol esters, a cell-permeable diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), and calcium ionophore A23187 are also potent inducers of PLD in these HL-60 granulocytes. HL-60 granulocytes have been selectively labeled in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P by incubating the cells with alkyl-[32P]lyso-phosphatidylcholine (PC). When these labeled cells are treated with phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate, OAG, or A23187, alkyl-[32P]PA is formed. Because cellular ATP has not been labeled with 32P, the formation of alkyl-[32P]PA conclusively demonstrates PLD activation by these agents. In the presence of 0.5% ethanol, phorbol esters, OAG, and A23187 also induce formation of alkyl-[32P]PEt, demonstrating that the activated PLD catalyzes transphosphatidylation between the phosphatidyl moiety of the alkyl-[32P]PC and ethanol. Formation of alkyl-[32P]PA and alkyl-[32P]PEt in response to these various agents occurs in a time- and dose-dependent manner and exhibits differential Ca2+ requirements. Based on experiments with both [3H]alkyl-PC and alkyl-[32P]PC, it is concluded that alkyl-PA and alkyl-PEt formed in response to PMA, OAG, or A23187 are derived exclusively from PLD action on alkyl-PC. Furthermore, subthreshold concentrations of PMA (0.5-2.0 nM) or OAG (1.0-25 microM) combined with subthreshold levels of A23187 (15-60 nM) induce the formation of alkyl-[32P]PA and alkyl-[32P]PEt, suggesting that receptor-mediated activation of PLD might involve cooperative interactions between Ca2+ and diglyceride. Although PLD is activated by agents that also activate protein kinase C, the protein kinase C inhibitor, K252a, inhibits PMA-induced protein phosphorylation but causes only partial inhibition of PLD activation. We conclude that phorbol esters, OAG, and A23187 activate PLD in HL-60 granulocytes via protein kinase-independent as well as protein kinase-dependent mechanisms.

Topics: Calcimycin; Cell Line; Diglycerides; Enzyme Activation; Glycerides; Granulocytes; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Phorbol 12,13-Dibutyrate; Phospholipase D; Phospholipases; Protein Kinase C; Tetradecanoylphorbol Acetate

HL-60 cells differentiate into mature granulocytes in response to treatment with a variety of chemical agents. Such HL-60 cell derived granulocytes display many of the properties associated with their peripheral blood counterpart. In this study we have investigated the development of the degranulation response in dimethylsulfoxide (DMSO) or retinoic acid differentiated HL-60 cells over a six day period. The release of a number of enzymes in response to stimulation by a variety of agents was examined. Soluble aggregated IgG (SAIgG) stimulated the release primarily of elastase from HL-60 derived granulocytes with little or no release of other granule enzymes, in particular myeloperoxidase. This contrasted to what was seen when peripheral blood granulocytes were used. The lack of myeloperoxidase release was not due to the parallel release of enzyme inhibitors or failure of the stimulus to bind to the cells. Neither was it due to variations in the kinetics of enzyme release or the presence of myeloperoxidase and elastase in discrete sub-populations of HL-60 cells. When other stimuli such as fMet-Leu-Phe, A23187, or phorbol myristate acetate (PMA) were used a relatively normal degranulation response was seen. Thus, the degranulation response in granulocytes derived from HL-60 cells appears relatively normal when a range of commonly used stimuli are used but is impaired when aggregates of IgG are used.

Topics: Calcimycin; Cell Differentiation; Cell Line; Cytoplasmic Granules; Granulocytes; Humans; Immunoglobulin G; Leukemia, Promyelocytic, Acute; N-Formylmethionine Leucyl-Phenylalanine; Pancreatic Elastase; Peroxidase; Tetradecanoylphorbol Acetate

Metallothionein (MT) gene transcription is regulated in a developmental and tissue-specific manner by metal ions and other agents. We examined MT expression in the human promyelocytic leukemia cell line HL-60 during differentiation along macrophage and neutrophil lineages. All HL-60 phenotypes showed similar basal levels of MT RNA with significant induction following exposure to Cd2+ but not activators of PKC. MT RNA did not correlate with growth state or with known levels of PKC activity, thus our data do not support a role for MT in HL-60 differentiation or for PKC in MT expression.

Topics: Amino Acid Sequence; Base Sequence; Cadmium; Calcimycin; Calcitriol; Cell Differentiation; Cell Line; Enzyme Activation; Humans; Leukemia, Promyelocytic, Acute; Metallothionein; Molecular Sequence Data; Oligonucleotide Probes; Protein Kinase C; RNA, Messenger; Tetradecanoylphorbol Acetate

Topics: Calcimycin; Cell Differentiation; Humans; Leukemia, Promyelocytic, Acute; Macrophages; Protein Kinase C; Tumor Cells, Cultured