guanosine-triphosphate and Leukemia--Promyelocytic--Acute

Gallic acid (GA) is a naturally occurring polyhydroxyphenolic compound and an excellent free radical scavenger. In this study, we examined its cytotoxic and biochemical effects on the human HL-60 promyelocytic leukemia cell line. GA caused a significant imbalance of deoxynucleosidetriphosphate (dNTP) pool sizes, indicating ribonucleotide reductase inhibition. Moreover, GA induced dose-dependent apoptosis in HL-60 cells (80microM GA led to the induction of apoptosis in 39% of cells) and attenuated progression from G0/G1 to the S phase of the cell cycle (60microM GA doubled the number of cells in G0/G1 phase from 22 to 44% when compared to untreated controls). We further determined IC(50) values of 3.5 and 4.4nM for the inhibition of cyclooxygenases I and II, respectively. When cells were simultaneously treated with GA and trimidox, another inhibitor of RR, highly synergistic growth inhibitory effects could be observed. Taken together, we identified novel biochemical effects of GA which could be the basis for further preclinical and in vivo studies.

Topics: Adenosine Triphosphate; Apoptosis; Benzamidines; Cell Cycle; Cell Proliferation; Cell Survival; Cyclooxygenase Inhibitors; Cytidine Triphosphate; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gallic Acid; Guanosine Triphosphate; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Molecular Structure; Prostaglandin-Endoperoxide Synthases; Ribonucleotide Reductases; Thymine Nucleotides

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors in both the plasma membrane and the cytosol. HL-60 cytosol was fractionated by ammonium sulfate and gel-permeation chromatography. Two cytosolic protein fractions were found to reconstitute the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-stimulated PLD in a reconstitution assay consisting of 3H-labeled HL-60 membranes and eluted column fractions. The major peak of reconstituting activity was in the region of 50 kDa, and a second discrete peak of PLD reconstitution activity was observed in the region of 18 kDa. Rho GDP/GTP exchange inhibitor, Rho GDI, comigrated with Rac2 and RhoA, but not Rac1. RhoA and Rac2 were entirely complexed with Rho GDI and eluted with an apparent molecular mass of 43 kDa by gel filtration chromatography. The partial overlap between cytosolic Rac2 and RhoA with the 50-kDa peak of reconstituting activity was not consistent with the participation of cytosolic Rho-related GTPases in the activation of PLD by guanine nucleotides. However, recombinant Rho GDI, which inhibits nucleotide exchange on the Rho family of small GTP-binding proteins, reduced GTP gamma S-stimulated PLD activity in HL-60 homogenates. The stimulatory exchange factor, Smg GDS, which is active on Rho and Rac, could be partially separated from the PLD-stimulating factor(s) by gel-permeation chromatography. Moreover, recombinant Smg GDS failed to stimulate GTP-dependent PLD activity. Cytosolic ADP-ribosylation factor (ARF) was exclusively located in the 18-kDa peak of reconstitution activity. Faint amounts of membrane-bound ARF were also detected using the monoclonal antibody 1D9. The effects of the 50-kDa and 18-kDa PLD-inducing factors on the salt-extracted PLD activity were synergistic. The weak stimulatory effect of ARF alone suggested that the GTP gamma S-stimulated PLD activity is dependent on the presence of another protein(s), presumably ARF-regulatory proteins. We propose that a membrane-bound GTP-binding protein, possibly ARF, may be involved in the activation of PLD when combined with the component(s) of the 50-kDa fraction.

Topics: ADP-Ribosylation Factors; Carrier Proteins; Cell Line; Chromatography, Gel; Cytosol; Electrophoresis, Polyacrylamide Gel; Granulocytes; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; Immunoblotting; Kinetics; Leukemia, Promyelocytic, Acute; Phospholipase D; Tumor Cells, Cultured

Differentiated HL-60 cells acquire responsiveness to fMet-Leu-Phe (fMLP), which activates phospholipase C and O2- generation in a pertussis toxin-sensitive manner. Addition of retinoic acid (RA) for the last 24 h during dimethyl sulfoxide (Me2SO)-induced differentiation enhanced fMLP-dependent signals and interaction between fMLP receptor and G(i). RA modifies both the function and subunit composition of G(i)2, the predominant G(i) of HL-60 membranes, as shown by comparing purified G(i)2 from membranes of Me2SO-treated cells (D-G(i)2) to G(i)2 from membranes of cells treated with both Me2SO and RA (DR-G(i)2). As compared to D-G(i)2, DR-G(i)2 induced more fMLP binding when added to membranes of pertussis toxin-treated HL-60 cells and, in the presence of GTP gamma S, stimulated beta gamma-sensitive phospholipase C in extracts of HL-60 cells to a much greater extent at a lower concentrations. Immunoblasts revealed that RA induced expression of the gamma 2 subunit, which was otherwise undetectable in G(i)2 purified from HL-60 cells or in HL-60 membranes. Possibly by inducing expression of gamma 2, RA alters two functions of the G(i) beta gamma subunit, modulation of fMLP receptor-G(i)2 coupling and activation of the effector, Phospholipase C.

Topics: Cell Differentiation; Cell Line; Cell Membrane; Chromatography, Affinity; Dimethyl Sulfoxide; Drug Synergism; Enzyme Activation; Gene Expression; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Immunoblotting; Kinetics; Leukemia, Promyelocytic, Acute; Macromolecular Substances; N-Formylmethionine Leucyl-Phenylalanine; Tretinoin; Tumor Cells, Cultured; Type C Phospholipases

Granulocyte colony-stimulating factor (G-CSF) can elicit responses that include proliferation, granulocytic differentiation, and activation of cellular functions in target cells. The biochemical pathways responsible for transduction of these signals from the G-CSF receptor (G-CSFR) have not been defined. In this report, we show that, in murine (NFS-60) and human (OCI-AML 1) myeloid leukemia cell lines and in murine pro-B-lymphocytic cells, BAF/B03, transfected with the murine G-CSFR, proliferative responses to G-CSF are associated with rapid activation of p42 and p44 MAP kinases and p21ras. Truncation of the cytoplasmic portion of the murine G-CSFR at residue 646 but not at residue 739 abolished G-CSF-induced stimulation of cellular proliferation as well as activation of MAP kinase and p21ras in transfected BAF/B03 cells. G-CSF-induced granulocytic differentiation of the murine leukemic cell line 32DC13(G) occurred in the absence of detectable activation of p42 MAP kinase. Nonproliferative responses to G-CSF in the human promyelocytic cell line HL-60 and in human neutrophils were similarly associated with no MAP kinase activation. These results imply that differing cellular effects of G-CSF may be involve the recruitment of differing signal transduction pathways with the p21ras/MAP kinase pathway being limited to proliferative responses.

Topics: Animals; B-Lymphocytes; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Cell Division; Cell Line; Enzyme Activation; Granulocyte Colony-Stimulating Factor; Guanosine Triphosphate; Humans; Interleukin-3; Kinetics; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Mice; Proto-Oncogene Proteins p21(ras); Receptors, Granulocyte Colony-Stimulating Factor; Recombinant Proteins; Signal Transduction; Transfection; Tumor Cells, Cultured

Topics: Adenosine Diphosphate Ribose; Cell Line; Cell Membrane; Cholera Toxin; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Hydrolysis; Indicators and Reagents; Kinetics; Leukemia, Promyelocytic, Acute; Macromolecular Substances; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Receptors, Cell Surface; Tumor Cells, Cultured; Virulence Factors, Bordetella

5,10-Dideazatetrahydrofolic acid (DDATHF) is an inhibitor of glycinamide ribonucleotide transformylase, the first of two tetrahydrofolate requiring enzymes in the de novo purine nucleotide biosynthetic pathway, and is a potent inducer of the maturation of HL-60 promyelocytic leukemia cells. The inhibition of cellular growth by DDATHF was effectively prevented by adenosine or deoxyadenosine, whereas guanosine or deoxyguanosine only partially prevented the growth inhibition produced by this folate antimetabolite, implying that the depletion of both ATP and GTP, which occurs with this agent, was responsible for its growth inhibitory effects. In contrast, the induction of differentiation by DDATHF was completely abolished by the presence of guanosine or deoxyguanosine, suggesting that the depletion of intracellular guanine nucleotides by DDATHF represents the event that is essential to the induction of differentiation by this folate analog. This possibility was supported by the observation that the concentration of dGTP was not decreased in cells treated with DDATHF under the conditions employed. Both guanine nucleosides selectively restored intracellular GTP pools depleted by the treatment with DDATHF to their normal level, whereas only adenine nucleosides completely restored the levels of both ATP and GTP to their normal intracellular concentrations. The relationship between guanine nucleotide pools and the induction of HL-60 differentiation by DDATHF was further supported by the finding that maturation and the depletion of intracellular GTP by DDATHF were not reversed by guanine nucleosides in HL-60 cells deficient in hypoxanthine-guanine phosphoribosyltransferase activity. The findings provide support for the hypothesis that the terminal differentiation of these leukemic cells by DDATHF is the result of the depletion of intracellular GTP pools.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Division; Guanosine Triphosphate; Humans; Intracellular Fluid; Leukemia, Promyelocytic, Acute; Purine Nucleotides; Tetrahydrofolates; Tumor Cells, Cultured

Cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3) recruit quiescent cells into the cell cycle and sensitize these cells towards cell cycle specific chemotherapeutic agents. We examined the in vitro effects of GM-CSF on HL-60 cells and tested its modulatory influence on biochemical and cytotoxic effects seen with tiazofurin, a potent and specific inhibitor of IMP dehydrogenase. Incubation of HL-60 cells with 500 U/ml GM-CSF for 4 d enhanced cell proliferation, which was accompanied by a significant increase in IMP dehydrogenase activity (from 2.22 in control cells to 3.70 nmol/mg/h in cells pretreated with GM-CSF). When HL-60 cells were incubated with 100 microM tiazofurin for 2 h, intracellular GTP decreased to 46% of untreated control cells. In HL-60 cells pretreated with GM-CSF, GTP pools decreased to 38% of control after incubation with tiazofurin which is 69% of the predicted value for additive effect. The MTT chemosensitivity assay yielded significantly decreased IC50 values for tiazofurin in HL-60 cells, preincubated with GM-CSF (IC50 decreased from 13 microM to 10 microM). Therefore our results suggest that combination therapy with GM-CSF and tiazofurin may be beneficial for the treatment of refractory leukaemia patients.

Topics: Antineoplastic Agents; Cell Division; Dose-Response Relationship, Drug; Drug Synergism; Granulocyte-Macrophage Colony-Stimulating Factor; Guanosine Triphosphate; Humans; IMP Dehydrogenase; In Vitro Techniques; Leukemia, Promyelocytic, Acute; Ribavirin; Tumor Cells, Cultured

Synthetic lipopeptides activate superoxide-anion (O2-) formation in human neutrophils in a pertussis-toxin (PTX)-sensitive manner, suggesting the involvement of G-proteins of the Gi family in the signal-transduction pathway. We compared G-protein activation by lipopeptides and the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in dibutyryl-cyclic-AMP-differentiated HL-60 cells. The lipopeptide (2S)-2-palmitoylamino-6-palmitoyloxymethyl-7-palmitoyloxy heptanoyl-SK4 (Pam3AhhSK4) and fMLP activated high-affinity GTPase, i.e. the enzymic activity of G-protein alpha-subunits, in HL-60 membranes in a time- and protein-dependent manner, but they had no effect on Mg(2+)-ATPase and Na+/K(+)-ATPase. Pam3AhhSK4 and fMLP increased Vmax. of GTP hydrolysis. Pam3AhhSK4 activated GTP hydrolysis with half-maximal and maximal effects at about 2 microM and 10 microM respectively. Other lipopeptides activated GTP hydrolysis as well. Lipopeptides were less effective than fMLP to activate GTPase. In membranes from PTX-treated cells, the stimulatory effects of lipopeptides and fMLP on GTPase were abolished. In N-ethylmaleimide-treated membranes, the relative stimulatory effect of Pam3AhhSK4 on GTP hydrolysis was enhanced, whereas that of fMLP was diminished. fMLP and Pam3AhhSK4 activated GTPase in an over-additive manner in N-ethylmaleimide-treated membranes. Unlike fMLP, Pam3AhhSK4 did not enhance incorporation of GTP azidoanilide into, and cholera-toxin-catalysed ADP-ribosylation of Gi-protein alpha-subunits in, HL-60 membranes and did not induce rises in cytosolic Ca2+ concentration. Pam3AhhSK4 and fMLP stimulated phosphatidic acid formation in a PTX-sensitive manner. Pam3AhhSK4 itself did not activate O2- formation, but potentiated the stimulatory effects of fMLP. Our data suggest that (i) lipopeptides activate the GTPase of Gi-proteins, (ii) lipopeptides and fMLP activate Gi-proteins differently, (iii) lipopeptides stimulate phospholipase D via Gi-proteins, and (iv) phosphatidic acid formation is not sufficient for activation of O2- formation.

Topics: Adenosine Diphosphate Ribose; Amino Acid Sequence; Bucladesine; Cell Differentiation; Cell Line; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Lipoproteins; Membrane Proteins; Molecular Sequence Data; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pertussis Toxin; Superoxides; Time Factors; Tumor Cells, Cultured; Virulence Factors, Bordetella

4-Hydroxynonenal (HNE), a major lipid peroxidation product, displays several biological actions. Among them, the differentiation of human HL-60 cells and the stimulation of neutrophil oriented migration occur at concentrations which can be actually found in normal tissues and in body fluids. In spite of its chemotactic activity, HNE fails to increase neutrophil oxidative metabolism. The action of the aldehyde on cell migration appears to be mediated by a phosphoinositide specific phospholipase C. The acceleration of phosphatidylinositol turnover induced by 10 pM 4-hydroxyoctenal, another lipid peroxidation product, is prevented by the pretreatment of neutrophils with pertussis toxin. The mechanism of action of these 4-hydroxyalkenals appears to follow pathways common to other chemoattractants, but some differences can be found too. In particular HNE seems unable to stimulate phospholipase D activity. The action of 4-hydroxyalkenals and other lipid peroxidation products on transmembrane signalling systems and on phospholipid metabolism might regulate several cell functions, such as motility, proliferation and differentiation.

Topics: Aldehydes; Animals; Guanosine Triphosphate; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Lipid Peroxidation; Male; Neutrophils; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases; Rats; Rats, Wistar; Tumor Cells, Cultured

One major substrate protein was phosphorylated with [gamma-32P]GTP in membranes of human leukemia (HL-60) cells. The phosphoprotein comigrated with beta-subunits of heterotrimeric GTP-binding proteins (G proteins) in different gel systems. Upon solubilization of the phosphorylated membranes, the phosphoprotein could be immunoprecipitated by a G protein beta-subunit-specific antiserum. The beta-subunit phosphorylation was transient and was found to be specific for GTP and its analog, guanosine 5'-O-(gamma-thio)triphosphate. When phosphorylated membranes were incubated with various nucleotides, the bound phosphate was specifically removed by GDP, suggesting that the phosphate can be retransferred onto GDP. Divalent cations, preferentially Mg2+ and Mn2+, were required for both phosphorylation and dephosphorylation. The phosphorylation was stable against treatment with NaOH but sensitive to treatment with heat, HCl, and hydroxylamine. Moreover, treatment of the membranes with the histidine-modifying agent, diethyl pyrocarbonate, resulted in a loss in phosphate incorporation. The data suggest that G protein beta-subunits are involved in a guanine nucleotide-specific enzymatic activity transferring the gamma-phosphate from GTP to GDP, presumably at G protein alpha-subunits, via a phosphohistidine intermediate.

Topics: Biological Transport; Cell Membrane; Diethyl Pyrocarbonate; Electrophoresis, Polyacrylamide Gel; GTP-Binding Proteins; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Histidine; Humans; Hydroxylamine; Hydroxylamines; Kinetics; Leukemia, Promyelocytic, Acute; Macromolecular Substances; Magnesium; Manganese; Membrane Proteins; Molecular Weight; Phosphorus Radioisotopes; Phosphorylation; Tumor Cells, Cultured

The major tyrosine protein kinase from HL60 (a human non-differentiated promyelocytic cell line) has been purified almost to homogeneity as judged by silver-stained SDS/PAGE. The procedure involved four chromatographic steps: DEAE-Sepharose, casein-agarose, cibacron-blue--agarose and hexyl-agarose. The purification resulted in more than 1000-fold enrichment in angiotensin II phosphorylation activity. A gel-sizing experiment, labeling with [35S]ATP[gamma s] and autophosphorylation of the enzyme in the presence of [gamma-32P]ATP, all led to the identification of a single protein species with a molecular mass of about 40 kDa. Western blot experiments showed that this protein does not belong to the src family and is not related to the abl and fes oncogene products. Phosphorylation of angiotensin II and casein by this 40-kDa human promyelocytic kinase was stimulated by high ionic strength especially from class IA metal salts. The Km for ATP was 2 microM and the Vmax 3.1 nmol.min-1.mg-1 using angiotensin II as a substrate. The kinase requires the presence of either Mn2+ or Mg2+ for full activity and utilizes ATP or dATP but not GTP as phosphate donor. Based on numerous biochemical observations, it was possible to demonstrate that kinase is different from any other tyrosine protein kinases described in the literature. This 40-kDa protein was used as a molecular tool for testing some tyrosine protein kinase inhibitors described in the literature. It is one of the rare tyrosine protein kinases purified from human cancer cells to date.

Topics: Adenosine Triphosphate; Angiotensin II; Binding, Competitive; Blotting, Western; Caseins; Guanosine Triphosphate; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Magnesium; Manganese; Metals; Osmolar Concentration; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured

In the regulation of GTP biosynthesis, complex interactions are observed. A major factor is the behavior of the activity of IMPDH, the rate-limiting enzyme of de novo GTP biosynthesis, and the activity of GPRT, the salvage enzyme of guanylate production. The activities of GMP synthase, GMP kinase and nucleoside-diphosphate kinase are also relevant. In neoplastic transformation, the activities and amounts of all these biosynthetic enzymes are elevated as shown by kinetic assays and by immunotitration for IMPDH. In cancer cells, the up-regulation of guanylate biosynthesis is amplified by the concurrent decrease in activities of the catabolic enzymes, nucleotidase, nucleoside phosphorylase, and the rate-limiting purine catabolic enzyme, xanthine oxidase. The up-regulation of the capacity for GTP biosynthesis is also manifested in the stepped-up capacity of the overall pathways of de novo and salvage guanylate production. The linking with neoplasia is also seen in the elevation of the activities of IMPDH and GMP synthase and de novo and salvage pathways as the proliferative program is expressed as cancer cells enter log phase in tissue culture. The activity of GMP reductase showed no linkage with neoplastic or normal cell proliferation; however, in induced differentiation in HL-60 cells the activity increased concurrently with the decline in the activity of IMPDH. This reciprocal regulation of the two enzymes is observed in differentiation induced by retinoic acid, DMSO or TPA in HL-60 cells. In support of enzyme-pattern-targeted chemotherapy, evidence was provided for synergistic chemotherapy with tiazofurin (inhibitor of IMPDH) and hypoxanthine (competitive inhibitor of GPRT and guanine salvage activity) in patients and in tissue culture cell lines. These investigations should contribute to the clarification of the controlling factors of GMP biosynthesis, the role of the various enzymes, the behavior of GMP reductase in mammalian cells and the application of the approaches of enzyme-pattern-targeted chemotherapy in patients.

Topics: Animals; Cell Differentiation; Cell Division; Colonic Neoplasms; Evaluation Studies as Topic; GMP Reductase; Guanosine Monophosphate; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthines; IMP Dehydrogenase; Inosine Monophosphate; Leukemia, Promyelocytic, Acute; Liver Neoplasms, Experimental; NADH, NADPH Oxidoreductases; Ribavirin; Tumor Cells, Cultured

The activity of IMP dehydrogenase (IMP DH), the rate-limiting enzyme of de novo GTP biosynthesis, was shown to be increased in cancer cells. Tiazofurin, an inhibitor of IMP dehydrogenase, proved to be an effective agent in the treatment of refractory granulocytic leukemia. To examine the cell cycle dependent alterations of GTP synthesis and sensitivities to tiazofurin, we measured IMP DH activities and GTP pools, as well as the effects of tiazofurin on cell cycle phase enriched HL-60 cells. We now show that IMP DH activities and GTP concentrations are increased in S-phase enriched fractions of HL-60 cells. Moreover, the depletion of GTP concentrations by tiazofurin is most effective in S-phase enriched HL-60 cells. These results may be utilized in cancer chemotherapy to combine tiazofurin with biologic response modifiers which recruit quiescent leukemic cells into the cell cycle.

Topics: Antimetabolites, Antineoplastic; Cell Cycle; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Ribavirin; Tumor Cells, Cultured

rap1/Krev-1 is a p21ras-related GTP-binding protein that has been implicated in the reversion of the ras-transformed cell phenotype. We have identified a GTPase-activating protein (GAP) specific for rap in plasma membranes isolated from differentiated HL60 cells. The rap GAP activity remained quantitatively associated with the membrane following washes with buffered 1 M LiCl containing 20 mM EDTA but was solubilized with the detergents Nonidet P-40 and deoxycholate. On the basis of size-exclusion chromatography, the membrane-associated rap GAP (rap GAPm) appeared distinct from the rap GAP detected in the cytosolic fraction from HL60 cells. The molecular sizes of the membrane and cytosolic forms were estimated to be 36 and 54 A, respectively. rap GAPm was solubilized and purified to near homogeneity by successive column chromatographies in the presence of detergent. The rap GAPm activity corresponded to a single polypeptide that migrated with a molecular mass of approximately 88 kDa on SDS/polyacrylamide gels. The purified rap GAPm was inactive toward the GTP-bound forms of p21ras, rho, G25K, and rac-1 and did not stimulate dissociation of guanine nucleotide from rap.

Topics: Amino Acid Sequence; Antibodies; Cell Line; Cell Membrane; Chromatography; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Durapatite; Electrophoresis, Polyacrylamide Gel; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Hydroxyapatites; Kinetics; Leukemia, Promyelocytic, Acute; Molecular Sequence Data; Molecular Weight; Peptides; Phosphorus Radioisotopes; Proteins; rap GTP-Binding Proteins; ras GTPase-Activating Proteins

The possible role of tyrosine phosphorylation in the activation of granulocytic HL60 cells was examined using vanadate, a phosphotyrosine phosphatase inhibitor. Treatment of permeabilized cells with micromolar concentrations of vanadate resulted in a substantial accumulation of tyrosine-phosphorylated proteins, detected by immunoblotting. At comparable concentrations, vanadate was also found to elicit an NADPH-dependent burst of oxygen utilization. Actin assembly, studied using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, was similarly stimulated by vanadate, though considerably higher concentrations were required to observe this effect. In contrast with these responses, the secretion of lysozyme was not stimulated by vanadate, nor did vanadate affect calcium-induced secretion. Therefore, accumulation of tyrosine-phosphorylated proteins is associated with stimulation of some, but not all, of the responses characteristic of granulocytic cell activation. This indicates that the effects of vanadate are selective and suggests divergence of the signalling pathways leading to the individual effectors.

Topics: Actins; Adenosine Triphosphate; Amanitins; Calcium; Cell Membrane Permeability; Exocytosis; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Immunoblotting; Leukemia, Promyelocytic, Acute; Muramidase; NADP; Neutrophils; Oxygen Consumption; Phosphotyrosine; Polymers; Signal Transduction; Thionucleotides; Tumor Cells, Cultured; Tyrosine; Vanadates

The marine toxin maitotoxin (MTX) and the chemotactic peptide fMet-Leu-Phe (fMLP) induce the formation of inositol phosphates in HL-60 cells differentiated with dibutyryl cyclic AMP. The increase in [3H]inositol(1,4,5)-trisphosphate is rapid but transient after fMLP stimulation, whereas MTX-induced increase in [3H]inositol(1,4,5)-trisphosphate occurs at a slower rate and is sustained over time. In both cases increases in [Ca++]i, measured with fura-2, parallel the formation of inositol trisphosphate. MTX-mediated stimulation of inositol phosphate formation is inhibited in the absence of calcium, whereas the response to fMLP is not. The calcium ionophore ionomycin stimulates the formation of inositol phosphates in differentiated HL-60 cells. The magnitude of the response is smaller than that obtained with MTX. Ionomycin also induces a rapid but sustained increase of [Ca++]i. In undifferentiated HL-60 cells, neither fMLP nor ionomycin induce significant inositol phosphate formation, and the increase in [Ca++]i elicited by ionomycin is transient. In contrast, the effects of MTX on phosphoinositide breakdown and on [Ca++]i in undifferentiated cells are nearly identical to those elicited by MTX in differentiated cells. In the presence of the intracellular calcium chelator BAPTA, fMLP, ionomycin and MTX still stimulate the generation of inositol phosphates. Guanyl nucleotides and calcium stimulate phospholipase C activity in membrane preparations from differentiated HL-60 cells. fMLP stimulates the enzyme only in the presence of GTP. MTX has no effect on membrane phospholipase C activity.

Topics: Calcium; Egtazic Acid; Guanosine Triphosphate; Humans; Ionomycin; Leukemia, Promyelocytic, Acute; Marine Toxins; N-Formylmethionine Leucyl-Phenylalanine; Oxocins; Phosphatidylinositols; Tumor Cells, Cultured; Type C Phospholipases

The chronically infected promonocytic clone U1 expresses low-to-undetectable constitutive levels of human immunodeficiency virus (HIV). Virus replication in these cells can be increased up to 25-fold by phorbol esters (phorbol-12-myristate-13-acetate), recombinant cytokines such as tumor necrosis factor-alpha, and cytokine-enriched mononuclear cell supernatants. We have tested specific activators of protein kinases (PK) and PK inhibitors (isoquinolinesulfonamide derivatives), as well as calcium-mobilizing agents, for their effect on constitutive and induced virus expression in U1 cells. Virus expression was measured by reverse transcriptase, Western blot, and nuclear run-on analysis. Activation of PKC by 1-oleyl,2-acetylglycerol, a synthetic analog of the natural ligand 1,2-diacylglycerol, and bryostatin 1 (a recently described specific PKC activator) resulted in a two- to eightfold increase in virus production. In contrast, activators of cyclic-nucleotide-dependent PKs were not effective in inducing virus expression. PK inhibitors were tested for their effect on HIV upregulation by cytokines and other inducing agents. The isoquinolinesulfonamide derivative H7, a potent inhibitor of PKC activation, effectively blocked (70 to 90%) HIV induction by cytokines and phorbol-12-myristate-13-acetate. The derivative HA1004, which is more selective for cyclic-nucleotide-dependent kinases, did not suppress viral induction. In addition, increases in intracellular calcium levels dramatically enhanced HIV production induced by both specific PKC activators and cytokines. These results indicate that activation of PKC is a common pathway involved in the upregulation of HIV expression in chronically infected cells stimulated by cytokines and other inducing agents.

Topics: Biological Factors; Bryostatins; Cell Line; Cell Nucleus; Cytokines; Diglycerides; Enzyme Activation; Guanosine Triphosphate; HIV; Humans; Lactones; Leukemia, Promyelocytic, Acute; Macrolides; Mitogens; Protein Kinase C; Recombinant Proteins; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; Viral Proteins; Virus Replication

P2-purinergic receptor agonists (UTP) and formylated peptide receptor agonists (FMLP) were found to be equally efficacious in eliciting rapid 6-7-fold increases in inositol polyphosphate accumulation in differentiated HL-60 granulocytes. The activation of this response by either agonist was substantially but incompletely inhibited in cells treated with pertussis toxin. Thus, in cells containing only 1-10% of the control level of non-ADP-ribosylated Gi-2/3, UTP induced rapid 2-fold increases in inositol polyphosphate accumulation whereas smaller 50% increases were observed in FMLP-stimulated cells. Washed membranes prepared from control and toxin-treated HL-60 cells were used to characterize this toxin-insensitive activation of phospholipase C further. The agonist-independent stimulation of phospholipase C by either millimolar Ca2+ or the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was only modestly attenuated by toxin treatment. There was a 70-80% decrease in the rate and extent of phospholipase C activity stimulated by GTP per se in the absence of receptor agonists. The rate and extent of FMLP-induced potentiation of GTP-dependent phospholipase C activity were also inhibited by greater than 80% in toxin-treated membranes. Conversely, the potency and efficacy characterizing UTP-induced potentiation of GTP-dependent phospholipase C activity were only modestly attenuated (less than 20% inhibition). The results indicate that P2-purinergic receptors (and perhaps other Ca2(+)-mobilizing receptors) activate both pertussis toxin-sensitive and toxin-insensitive pathways for phospholipase C regulation in phagocytic leukocytes.

Topics: Cell Differentiation; Cell Line; Cell Membrane; Granulocytes; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Inositol Phosphates; Kinetics; Leukemia, Promyelocytic, Acute; Membrane Lipids; N-Formylmethionine Leucyl-Phenylalanine; Pertussis Toxin; Thionucleotides; Type C Phospholipases; Uridine Triphosphate; Virulence Factors, Bordetella

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

The effect of PMA (phorbol 12-myristate, 13-acetate) on PPI-pde (polyphosphoinositide phosphodiesterase) activity in the promyelocytic cell-line HL60 was examined. HL60 cells were pretreated with PMA in a time- and concentration-dependent manner and PPI-pde activity was monitored both in streptolysin O-permeabilized cells and in membranes. PPI-pde activity was stimulated by either GTP gamma S (guanosine 5'-[gamma-thio]triphosphate), fluoride or Ca2+. Both the Ca2(+)-stimulated and the G protein-mediated PPI-pde activity in permeabilized HL60 cells is maximally inhibited (70-90%) after 60 min pretreatment of intact cells with 10nM PMA. PPI-pde activity can also be observed in membranes prepared from HL60 cells although this activity represents only 10% of the total activity seen in permeabilized cells. In membranes, where PPI-pde activity can also be stimulated by either via the G-protein or directly by Ca2+, PMA pretreatment was also inhibitory regardless of the mode of activation. We suggest that both the membrane-bound PPI-pde activity and that present in the permeabilized cells are targets for protein phosphorylation by protein kinase C leading to inhibition of the catalytic function.

Topics: Calcium; Cell Membrane; Cell Membrane Permeability; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases; Protein Kinase C; Tetradecanoylphorbol Acetate; Thionucleotides; Tumor Cells, Cultured

A major peak of tyrosine protein kinase activity was partially purified from a Triton X100 extract of HL-60. This preparation submitted to high pressure gel filtration was eluted at a volume corresponding to a mass of 35/40 kD. This activity was insensitive to EGF and insulin. Autoradiographs of the preparations incubated with [gamma P32]-ATP and separated by electrophoresis do not give any evidence that autophosphorylation occurs for that particular tyrosine protein kinase. Furthermore, we failed to immunoprecipitate the enzyme with a specific antiphosphotyrosine antibody and anti v-src antibody. All the data presented herein suggest that this enzyme has not been previously purified.

Topics: Adenosine Triphosphate; Autoradiography; Binding, Competitive; Chromatography, High Pressure Liquid; Electrophoresis, Agar Gel; Epidermal Growth Factor; Guanosine Triphosphate; Humans; Immunosorbent Techniques; Insulin; Leukemia, Promyelocytic, Acute; Molecular Weight; Octoxynol; Phosphorylation; Polyethylene Glycols; Protein-Tyrosine Kinases; Substrate Specificity; Tumor Cells, Cultured

The novel tetrahydrofolate, 5,10-dideazatetrahydrofolic acid (DDATHF), was designed as an inhibitor of folate metabolism at a site other than dihydrofolate reductase. DDATHF has been shown to inhibit glycinamide ribonucleotide transformylase, a folate-requiring enzyme that catalyzes the first of two one-carbon transfer reactions in the de novo purine nucleotide biosynthetic pathway. Incubation of HL-60 promyelocytic leukemia cells with 5 x 10(-8) to 10(-5) M DDATHF resulted in a marked inhibition of growth after 48 h, with a complete cessation of cellular replication by day 4. Cell cycle analyses of DDATHF-treated HL-60 cells demonstrated an initial block in early S phase by day 3 followed by an accumulation of cells in the G1 and G2 + M phases of the cell cycle. Inhibition of growth was accompanied by a concentration-dependent increase in the percentage of mature myeloid cells that expressed nitroblue tetrazolium positivity, and a small increase in nonspecific esterase activity. Induction of differentiation and inhibition of growth by DDATHF were completely prevented by hypoxanthine and 5(4)-amino-4(5)-imidazole carboxamide, suggesting that depletion of intracellular purine nucleotide pools has an important role in the biological effects of this inhibitor. This possibility was confirmed by the finding that DDATHF caused a pronounced reduction in intracellular GTP and ATP levels within 2 h, with maximum decreases being observed by 24 h, a time interval which preceded the inhibition of cellular proliferation by this agent. Pyrimidine nucleoside triphosphate levels were markedly increased under these conditions. The findings indicate the importance of purine nucleotides to both the inhibition of growth and the induction of differentiation of HL-60 leukemia cells by DDATHF.

Topics: Acyltransferases; Adenosine Triphosphate; Cell Differentiation; Cell Division; Flow Cytometry; Folic Acid Antagonists; Guanosine Triphosphate; Humans; Hydroxymethyl and Formyl Transferases; Interphase; Leukemia, Promyelocytic, Acute; Phosphoribosylglycinamide Formyltransferase; Purine Nucleotides; Tetrahydrofolates; Tumor Cells, Cultured