guanosine-triphosphate and Leukemia--Myeloid--Acute

The 21-kD protein Ras of the low-molecular-weight GTP-binding (LMWG) family plays an important role in transduction of extracellular signals. Ras functions as a 'molecular switch' in transduction of signals from the membrane receptors of many growth factors, cytokines, and other second messengers to the cell nucleus. Numerous studies have shown that in multiple malignant tumors and hematopoietic malignancies, faulty signal transduction via the Ras pathway plays a key role in tumorigenesis. In this work, a non-radioactive assay was used to quantify Ras activity in hematologic malignancies. Ras activation was measured in six different cell lines and 24 patient samples, and sequence analysis of N- and K-ras was performed. The 24 patient samples comprised of seven acute myelogenous leukemia (AML) samples, five acute lymphocytic leukemia (ALL) samples, four myeloproliferative disease (MPD) samples, four lymphoma samples, four juvenile myelomonocytic leukemia (JMML) samples, and WBC from a healthy donor. The purpose of this study was to compare Ras activity determined by percentage of Ras-GTP with the mutational status of the Ras gene in the hematopoietic cells of the patients. Mutation analysis revealed ras mutations in two of the seven AML samples, one in codon 12 and one in codon 61; ras mutations were also found in two of the four JMML samples, and in one of the four lymphoma samples (codon 12). We found a mean Ras activation of 23.1% in cell lines with known constitutively activating ras mutations, which was significantly different from cell lines with ras wildtype sequence (Ras activation of 4.8%). Two of the five activating ras mutations in the patient samples correlated with increased Ras activation. In the other three samples, Ras was probably activated through "upstream" or "downstream" mechanisms.

Topics: DNA Mutational Analysis; Guanosine Diphosphate; Guanosine Triphosphate; Hematologic Neoplasms; Humans; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Juvenile; Lymphoma; Mutation; Myeloproliferative Disorders; Oncogenes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; ras Proteins; Signal Transduction; Tumor Cells, Cultured

Inactivation of the NF1 tumor suppressor causes myeloproliferative diseases. NF1 encodes a GTPase activating protein (GAP) for Ras. Myeloid cells with loss of NF1 have high levels of Ras-GTP, functionally equivalent to the effects of RAS oncogenes. We investigated the effects of the NF1 GAP-related domain (GRD) in proliferation, apoptosis and Ras-GTP levels in Nf1-negative acute myeloid leukemia (AML) cells. In AML cells, with cooperating mutations, the expression of the neurofibromin GRD causes significant reductions of N- and K-Ras-GTP levels, which is not incompatible with AML cell survival, but which is strongly selected against due to suppression of proliferation.

Topics: Animals; Blotting, Western; Cell Proliferation; Down-Regulation; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Leukemia, Myeloid, Acute; Mice; Mice, SCID; Neurofibromin 1; Protein Structure, Tertiary; Proto-Oncogene Proteins p21(ras); Tumor Cells, Cultured

The differentiation capability of guanosine 5'-triphosphate (GTP) was studied using U937 and KG1 cells.. Cell cycle was analyzed by PI staining using flow cytometry. Apoptosis was measured by Annexin-V-FITC/PI double staining using flow cytometry. Differentiation was observed by morphological criteria, Wright-Giemsa staining and expression of cell surface markers CD11b and CD14.. Variable GTP concentrations (25-200 micromol/L) at short treatment times (up to 24 h) showed significant anti-proliferative activities among both cell types. However, longer treatment times (up to 72 h) were required to trigger apoptosis. Cell-cycle analyses of the GTP-treated cells indicated an increase in S-phase population by 48 h followed by the appearance of a sub-G(1) peak after 72 h of treatment. The effects of GTP on U937 and KG1 cells were accompanied with differentiation toward monocyte/macrophage lineage. This was evidenced by a sharp increase in the extent of CD11b and CD14 expression after 24 h of exposure to GTP. The viability of both cell types did not significantly change during the first 24 h. However, at longer treatment times (72-96 h), dramatic decreases in both the extent of CD14 expression and the cell viabilities were observed. Simultaneous measurement of apoptosis and CD14 expression in GTP-treated U937 cells indicated that cells with lower CD14 content underwent more apoptosis.. These finding may pave the way for further pharmaceutical evaluation of GTP as a suitable differentiating agent for acute myeloblastic leukemia (AML) therapy.

Topics: Antineoplastic Agents; Apoptosis; CD11b Antigen; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Guanosine Triphosphate; Humans; Leukemia, Myeloid, Acute; Lipopolysaccharide Receptors; Time; U937 Cells

RAS genes, predominantly N-RAS and K-RAS, have been implicated in the pathogenesis of acute myeloid leukemia (AML), due to activating RAS mutations detectable in approximately 20% of AML patients. In the present study, RAS proteins were detected in their activated, GTP-bound form, in AML patients (n = 10) not expressing mutated forms of H-RAS, K-RAS and N-RAS. Further analysis revealed the simultaneous presence of N-RAS and K-RAS proteins in the GTP-bound state in seven out of 10 AML samples. In four out of 10 samples the levels of RAS-GTP were comparable to an AML cell line (TF-1) with an activating N-RAS mutation (Q61P). The detection of RAS-GTP in AML patients without RAS mutations further supports a functional role of RAS proteins in the pathogenesis of AML and may explain the observed effects of RAS inhibitors in some AML patients in the absence of activating RAS mutations.

Topics: Adult; Aged; DNA Mutational Analysis; Female; Gene Expression Regulation, Neoplastic; Genes, ras; Guanosine Triphosphate; HeLa Cells; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mutation; Phosphorylation; Protein Binding; ras Proteins

In IPC-81 cells, the adenylyl-cyclase activation by cholera toxin produces an elevation of cAMP that causes a rapid cytolysis. A resistant clone with deficient cholera toxin-induced cyclase activity (yet sensitive to cAMP) showed a rapid decrease in the amount of membrane-bound Gs alpha (42-47 kDa) detectable soon after ADP-ribosylation of these proteins; pertussis toxin-sensitive G proteins (41 kDa) were not affected. Resistant cells showed a rapid decrease of Gs alpha that is consistent with the finding that cAMP did not accumulate in these cells. Cholera toxin treatment of resistant cells had long-lasting effects (several weeks) on the level of Gs alpha in the cell membrane. The duration of Gs alpha decrease does not correspond to the probable life of catalytically active cholera toxin in the cells, and suggests a regulated process more complex than a proteolytic degradation targeted on ADP-ribosylated molecules.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cell Communication; Cholera Toxin; Drug Resistance; Enzyme Activation; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Immunoblotting; Leukemia, Myeloid, Acute; Membrane Proteins; Pertussis Toxin; Rats; Thionucleotides; Tumor Cells, Cultured; Virulence Factors, Bordetella

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC 286193), a selective inhibitor of the activity of IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, provided in end stage leukemic patients a rapid decrease of IMP dehydrogenase activity and GTP concentration in the blast cells and a subsequent decline in blast cell count. Sixteen consecutive patients with end stage acute nonlymphocytic leukemia or myeloid blast crisis of chronic granulocytic leukemia were treated with tiazofurin. Allopurinol was also given to inhibit xanthine oxidase activity to decrease uric acid excretion and to elevate the serum concentration of hypoxanthine, which should competitively inhibit the activity of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), the salvage enzyme of guanylate synthesis. Assays of IMP dehydrogenase activity and GTP concentration in leukemic cells provided a method to monitor the impact of tiazofurin and allopurinol and to adjust the drug doses. In this group of patients with poor prognosis, five attained a complete hematological remission and one showed a hematological improvement. A marked antileukemic effect was seen in two other patients. All five evaluable patients with myeloid blast crisis of chronic granulocytic leukemia reentered the chronic phase of their disease. Five patients with acute nonlymphocytic leukemia were refractory to tiazofurin and three were unevaluable for hematological effect because of early severe complications. Responses with intermittent 5- to 15-day courses of tiazofurin lasted 3-10 months. Tiazofurin had a clear antiproliferative effect, but the pattern of hematological response indicated that it appeared to induce differentiation of leukemic cells. In spite of toxicity with severe or life-threatening complications in 11 of 16 patients, tiazofurin was better tolerated in most patients than other antileukemic treatment modalities and provided a rational, biochemically targeted, and biochemically monitored chemotherapy which should be of interest in the treatment of leukemias and as a paradigm in enzyme pattern-targeted chemotherapy.

Topics: Antimetabolites, Antineoplastic; Blast Crisis; Blood Cell Count; Bone Marrow; Enzyme Inhibitors; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Ketone Oxidoreductases; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Ribavirin; Ribonucleosides

The cellular phosphotyrosine content of the HL-60 promyelocytic leukemia markedly decreased during the induced granulocytic and monocytic maturation of these cells. This occurs in the face of major increases in tyrosine kinase and protein phosphotyrosine phosphatase activities (D. A. Frank and A. C. Sartorelli, Biochem. Biophys. Res. Commun., 140: 440-447, 1986). In the present work, these two activities were characterized in the particulate fraction of HL-60 cells, since both enzymes are membrane bound. The tyrosine kinase activity utilized ATP as a phosphate donor, although GTP and other nucleotides were competitive with ATP. The enzyme was temperature sensitive, had a pH optimum of 6.5, and required Mg2+ or Mn2+ for activity, with additional stimulation of activity being produced by Zn2+. Agents such as epidermal growth factor and insulin, which stimulate other tyrosine kinase enzymes, were without effect on the tyrosine kinase activity of HL-60 cells. Enzyme activity was stimulated, however, by non-ionic detergents and was inhibited by quercetin. The protein phosphotyrosine phosphatase activity was paralleled by that of p-nitrophenyl phosphatase, was inhibited by VO3-4, Zn2+ and F-, and was maximally active at a pH of 7 to 8. The characteristics of the tyrosine kinase and the protein phosphotyrosine phosphatase activities were distinct from those of other known proteins of these classes. Tyrosine kinase activity was predominantly located on the plasma membrane, while the protein phosphotyrosine phosphatase activity was concentrated on internal membranes. The activities of both enzymes present on the plasma membrane appeared to exist on the cytoplasmic face of this membrane. Further characterization of the activities of these enzyme systems and their contribution to the regulation of tyrosine phosphorylation would appear to be important to an understanding of the control of cellular proliferation and differentiation.

Topics: Adenosine Triphosphate; Cations, Divalent; Cell Line; Ethylmaleimide; Guanosine Triphosphate; Humans; Hydrogen-Ion Concentration; Leukemia, Myeloid, Acute; Peptides; Phosphoprotein Phosphatases; Phosphotyrosine; Polymers; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Tyrosine

Tumor necrosis factor (TNF) is a monokine that induces pleiotropic events in both transformed and normal cells. These effects are initiated by the binding of TNF to high affinity cell surface receptors. The post-receptor events and signaling mechanisms induced by TNF, however, have remained unknown. The present studies demonstrate the presence of a single class of high affinity receptors on membranes prepared from HL-60 promyelocytic leukemic cells. The interaction of TNF with these membrane receptors was associated with a 3.8-fold increase in specific binding of the GTP analogue, GTP gamma S. Scatchard analysis of GTP gamma S binding data demonstrated that TNF stimulates GTP binding by increasing the affinity of available sites. The TNF-induced stimulation of GTP binding was also associated with an increase in GTPase activity. Moreover, the increase in GTPase activity induced by TNF was sensitive to pertussis toxin. The results also demonstrate that TNF similarly increased GTP binding and pertussis toxin-sensitive GTPase activity in membranes from mouse L929 fibroblasts, thus indicating that these effects are not limited to hematopoietic cells. Analysis of HL-60 membranes after treatment with pertussis toxin in the presence of [32P]NAD revealed three substrates with relative molecular masses of approximately Mr 41,000, 40,000, and 30,000. In contrast, L929 cell membranes had only two detectable pertussis toxin substrates of approximately Mr 41,000 and 40,000. Although the Mr 41,000 pertussis toxin substrate represents the guanine nucleotide-binding inhibitory protein Gi, the identities of the Mr 40,000 and Mr 30,000 substrates remain unclear. In any event, inhibition of the TNF-induced increase in GTPase activity and ADP-ribosylation of Gi by pertussis toxin suggested that TNF might act by increasing GTPase activity of the Gi protein. However, the results further indicate that TNF has no detectable effect on basal or prostaglandin E2-stimulated cAMP levels in HL-60 cells. Taken together, these findings indicate that a pertussis toxin-sensitive GTP-binding protein other than Gi, and possibly the Mr 40,000 substrate, is involved in the action of TNF. Finally, the demonstration that pertussis toxin inhibited TNF-induced cytotoxicity in L929 cells supports the presence of a GTP-binding protein which couples TNF-induced signaling to a biologic effect.

Topics: Adenosine Diphosphate Ribose; Animals; Cell Line; Cell Membrane; Cyclic AMP; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Kinetics; L Cells; Leukemia, Myeloid, Acute; Mice; Pertussis Toxin; Phosphoric Monoester Hydrolases; Receptors, Cell Surface; Receptors, Tumor Necrosis Factor; Thionucleotides; Tumor Necrosis Factor-alpha; Virulence Factors, Bordetella

NADPH-oxidase-catalyzed superoxide (O2-) formation in membranes of HL-60 leukemic cells was activated by arachidonic acid in the presence of Mg2+ and HL-60 cytosol. The GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] and guanosine 5'-[beta,gamma-imido]triphosphate, being potent activators of guanine-nucleotide-binding proteins (G proteins), stimulated O2- formation up to 3.5-fold. The adenine analogue of GTP[gamma S], adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), which can serve as donor of thiophosphoryl groups in kinase-mediated reactions, stimulated O2- formation up to 2.5-fold, whereas the non-phosphorylating adenosine 5'-[beta,gamma-imido]triphosphate was inactive. The effect of ATP[gamma S] was half-maximal at a concentration of 2 microM, was observed in the absence of added GDP and occurred with a lag period two times longer than the one with GTP[gamma S]. HL-60 membranes exhibited nucleoside-diphosphate kinase activity, catalyzing the thiophosphorylation of GDP to GTP[gamma S] by ATP[gamma S]. GTP[gamma S] formation was half-maximal at a concentration of 3-4 microM ATP[gamma S] and was suppressed by removal of GDP by creatine kinase/creatine phosphate (CK/CP). The stimulatory effect of ATP[gamma S] on O2- formation was abolished by the nucleoside-diphosphate kinase inhibitor UDP. Mg2+ chelation with EDTA and removal of endogenous GDP by CK/CP abolished NADPH oxidase activation by ATP[gamma S] and considerably diminished stimulation by GTP[gamma S]. GTP[gamma S] also served as a thiophosphoryl group donor to GDP, with an even higher efficiency than ATP[gamma S]. Transthiophosphorylation of GDP to GTP[gamma S] was only partially inhibited by CK/CP. Our results suggest that NADPH oxidase is regulated by a G protein, which may be activated either by exchange of bound GDP by guanosine triphosphate or by thiophosphoryl group transfer to endogenous GDP by nucleoside-diphosphate kinase.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Arachidonic Acid; Arachidonic Acids; Cell Line; GTP-Binding Proteins; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; Leukemia, Myeloid, Acute; NADH, NADPH Oxidoreductases; NADPH Oxidases; Phosphotransferases; Superoxides; Thionucleotides

The hypothesis was tested that the increased IMP dehydrogenase activity in human myelocytic leukemic cells, and along with it guanylate biosynthesis, might be a sensitive target to chemotherapy by tiazofurin. 1. IMP dehydrogenase activity in normal leukocytes was 3.1 +/- 0.5 (means +/- S.E.) nmol/hr/mg protein and in leukemic cells it was elevated 15- to 41-fold. The activity of guanine phosphoribosyltransferase in normal leukocytes was 389 +/- 27 nmol/hr/mg protein and in the leukemic cells it increased 2.8- to 6.8-fold. 2. IMP dehydrogenase was purified 4,900-fold to homogeneity from rat hepatoma 3924A with a yield of 30%. The kinetic properties of the hepatoma enzyme were similar to those of the enzyme in human myelocytic leukemic blast cells because of the similarity of the Km's for IMP (23 microM), NAD (44 and 65 microM); the Ki for TAD was 0.1 microM in both enzymes. 3. There was a selectivity of the in vitro response to tiazofurin in human normal and leukemic leukocytes. When labeled tiazofurin was incubated with leukocytes from normal, healthy volunteers and from leukemic patients, the leukemic leukocytes made 20- to 30-fold more TAD and the GTP content decreased as compared to normal leukocytes. This procedure proved to be a suitable predictive test in a clinical setting because patients with positive tests responded to tiazofurin whereas those with negative ones did not. 4. The National Cancer Institute approved a chemotherapeutic phase I/II trial which concentrates on treatment of refractory acute myelocytic leukemia. Tiazofurin is infused in a 60-minute period with a pump to insure uniform delivery. A novel aspect of the trial was that it was directed primarily by the biochemical impact of tiazofurin on IMP dehydrogenase activity and GTP concentration and the tiazofurin doses were to be adjusted accordingly. Patients received allopurinol as a routine precaution against possible accumulation of uric acid in the kidney. 5. In the first eight patients, there was one complete remission, two entered the chronic phase, two entered into partial remission, one did not respond, and two were not evaluable. In the five patients who responded, there was a rapid, profound decrease in IMP dehydrogenase activity of the blast cells and a gradual decline in GTP concentrations. The blast cell count followed the decrease in the GTP concentration. The white blood cell count was largely preserved. 6. Bone marrow aspirates and peripheral blood samples showed that w

Topics: Adenine; Aged; Allopurinol; Animals; Drug Evaluation; Female; Guanine; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Ketone Oxidoreductases; Leukemia; Leukemia, Myeloid, Acute; Liver Neoplasms, Experimental; Male; Middle Aged; Rats; Ribavirin; Ribonucleosides; Tumor Cells, Cultured

Membranes prepared from DMSO-differentiated HL60 cells labeled with [3H]inositol hydrolyze polyphosphoinositides in a Ca2+-dependent manner, generating inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). Incubation of membranes with GTP or GTP gamma S reduces the concentration of Ca2+ required for activation. This nucleotide effect is potentiated by formyl-Met-Leu-Phe (FMLP). Pertussis toxin inhibits FMLP-induced augmentation, but not the induction of IP2/IP3 formation by GTP or GTP gamma S. These results suggest that differentiated HL60 cells contain a membrane-associated phospholipase C that degrades polyphosphoinositides and that activation of this enzyme is mediated by at least two guanine nucleotide binding proteins, one of which is linked to FMLP receptors and is pertussis toxin sensitive.

Topics: Calcium; Cell Line; Dimethyl Sulfoxide; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Leukemia, Myeloid, Acute; N-Formylmethionine Leucyl-Phenylalanine; Pertussis Toxin; Phosphatidylinositols; Thionucleotides; Type C Phospholipases; Virulence Factors, Bordetella

A GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was partially purified from human leukemic (HL-60) cells that had been differentiated into neutrophil type. The partially purified protein, referred to as GHL, predominantly consisted of at least two polypeptides with molecular masses of 40,000 daltons (alpha) and 36,000 or 35,000 daltons (beta). The structure was similar to Gi or Go previously purified from rat brain as an alpha beta gamma-heterotrimeric IAP substrate (Katada, T., Oinuma, M., and Ui, M. (1986) J. Biol. Chem. 261, 8182-8191), although the existence of the gamma of GHL was unclear. The 40,000-dalton polypeptide contained the site for IAP-catalyzed ADP-ribosylation and the binding site for guanine nucleotide with a high affinity. The 36,000- and 35,000-dalton polypeptides were cross-reacted with the affinity-purified antibody raised against the beta of brain Gi and Go. Limited proteolysis with trypsin and immunoblot analyses with the use of the affinity-purified antibodies raised against the alpha of brain Gi or Go indicated that the alpha of GHL was different from the alpha of Gi or Go. Kinetics of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding to GHL was also quite different from that to brain Gi or Go. Incubation of GHL with GTP gamma S resulted in a resolution into GTP gamma S-bound alpha and beta(gamma) thus purified had abilities to inhibit a membrane-bound adenylate cyclase activity and to associate with the alpha of brain IAP substrate in a fashion similar to the beta gamma of brain IAP substrates, suggesting that there were no significant differences in the biological activities between the beta(gamma) of GHL and those of Gi or Go. Physiological roles of the new GTP-binding protein, GHL, purified from the neutrophil-like cells in receptor-mediated signal transduction are discussed.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Blood Platelets; Cell Differentiation; Cell Line; Cell Membrane; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Leukemia, Myeloid, Acute; Macromolecular Substances; Molecular Weight; Peptide Fragments; Pertussis Toxin; Substrate Specificity; Thionucleotides; Trypsin; Virulence Factors, Bordetella

In membranes of myeloid differentiated HL 60 cells, the chemotactic peptide FMLP stimulates phospholipase C via a pertussis toxin-sensitive G protein. FMLP markedly stimulates the cholera toxin-dependent ADP-ribosylation of a 40 kDa protein in these membranes. This effect of FMLP is inhibited by GTP and GTP[S], and is almost completely abolished in membranes of pertussis toxin-pretreated HL 60 cells. Treatment of HL 60 membranes with cholera toxin and NAD markedly inhibits FMLP-stimulated high affinity GTPase. These results suggest that a 40 kDa G protein sensitive to both pertussis and cholera toxin functionally interacts with the formyl peptide receptor of HL 60 cells and, thus, very likely is the G protein that stimulates phospholipase C in this system.

Topics: Adenosine Diphosphate Ribose; Cell Differentiation; Cholera Toxin; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Leukemia, Myeloid, Acute; Male; N-Formylmethionine Leucyl-Phenylalanine; Neoplasm Proteins; Pertussis Toxin; Receptors, Formyl Peptide; Receptors, Immunologic; Thionucleotides; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

We have isolated the major GTP-binding proteins from myeloid HL-60 cell plasma membranes. Two pertussis toxin substrates with similar apparent molecular masses of 40 and 41 kDa, respectively, are contained in these preparations, with both proteins being ADP-ribosylated to a similar extent. Partial chymotryptic proteolysis of fractions containing the [32P]ADP-ribosylated 40-kDa GTP-binding protein alpha subunit demonstrated production of 32P-labeled peptides of 28 and 16 kDa which were not observed after partial proteolysis of fractions containing solely the 41-kDa protein. Similarly, mild acid hydrolysis produced an additional 28-kDa fragment only from fractions containing the 40-kDa protein. The results presented here indicate the presence of two distinct pertussis toxin substrates in myeloid cells. The 41-kDa pertussis toxin substrate is likely to represent the alpha subunit of the inhibitory GTP-binding regulatory protein of adenylate cyclase, whereas the 40-kDa substrate may represent the alpha subunit of the GTP-binding protein which is coupled to chemoattractant receptors. In addition to the pertussis toxin substrates, an additional major peak of guanosine 5'-(3-O-thio)triphosphate-binding activity closely corresponded to the appearance of a 23-kDa protein.

Topics: Adenylate Cyclase Toxin; Cell Line; Electrophoresis, Polyacrylamide Gel; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Leukemia, Myeloid, Acute; Molecular Weight; Pertussis Toxin; Receptors, Formyl Peptide; Receptors, Immunologic; Thionucleotides; Virulence Factors, Bordetella

Using the [3H]inositol-labeled plasma membranes isolated from the differentiated human leukemic (HL-60) cells, the mode of inhibitory action of the Ca2+/phospholipid-dependent enzyme protein kinase C in the chemotactic peptide, fMet-Leu-Phe (fMLP)-induced, phospholipase C-mediated hydrolysis of phosphoinositides was investigated. In this cell-free membrane system, fMLP in the presence of GTP plus Ca2+, GTP in the presence of Ca2+, or Ca2+ alone could induce the formation of inositol bis- and trisphosphate (IP2 and IP3, respectively). When the intact cells were pre-treated with 12-O-tetradecanoylphorbol-13-acetate, the fMLP- and GTP-induced formation of IP2 and IP3 was markedly reduced but the Ca2+-induced reactions were not reduced in the isolated membranes. This result suggests that protein kinase C impairs the coupling of the GTP-binding protein to the phospholipase C. In another experiment, preincubation of the isolated membranes with pure rat brain protein kinase C inhibited the fMLP-induced formation of IP2, but did not inhibit the GTP- or Ca2+-induced reaction. Under the same conditions, protein kinase C did not inhibit the fMLP-, GTP-, or Ca2+-induced formation of IP3. This result suggests that protein kinase C impairs additionally the coupling of the fMLP receptor to the GTP-binding protein leading to the formation of IP2. The reason for the failure of protein kinase C to inhibit the fMLP-induced formation of IP3 in the cell-free membrane system is unknown, but several possible mechanisms are discussed.

Topics: 2,3-Diphosphoglycerate; Calcium; Cell Differentiation; Cell Line; Cell Membrane; Diphosphoglyceric Acids; Guanosine Triphosphate; Humans; Inositol Phosphates; Kinetics; Leukemia, Myeloid, Acute; Membrane Lipids; N-Formylmethionine Leucyl-Phenylalanine; Protein Kinase C; Sugar Phosphates; Tetradecanoylphorbol Acetate

A patient with refractory acute myeloid leukemia was treated with tiazofurin, an agent that causes inhibition of tumor cell proliferation by depressing GTP concentrations in the malignant cells. The initial dose of 1100 mg/m2 was ineffective clinically and biochemically. Dose escalations to 1650, 2200, and finally 3300 mg/m2 resulted in a marked decrease in the absolute number of blasts without causing bone marrow hypoplasia or marked neutropenia. The decrease in the peripheral blast cell count was observed subsequent to a decline in GTP concentrations in the leukemic cells to less than 30% of the pretreatment value. Consecutive bone marrow examinations showed a remarkable shift from myeloblasts to more mature myeloid elements, suggesting an in vivo differentiative action of tiazofurin. Although a total dose of 23,650 mg/m2 was administered over a 13-day period, only very mild side effects were noted. The absence of complications reported by others in Phase I trials with tiazofurin may be related to our slow administration of the drug by pump over a 1-h period in this trial. Tiazofurin appears to be a promising agent in the treatment of leukemia because of its selective action on leukemic cells and the availability of a rapid in vitro method capable of predicting sensitivity of leukemic cells to the agent and monitoring its activity during treatment by measuring thiazole-4-carboxamide adenine dinucleotide and GTP concentrations. These observations are being tested in a larger group of leukemic patients.

Topics: Antineoplastic Agents; Bone Marrow; Guanosine Triphosphate; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Ribavirin; Ribonucleosides

The synthetic "C" nucleoside, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide), its selenium analogue selenazofurin, and the related inhibitor of inosine 5'-phosphate (IMP) dehydrogenase, mycophenolic acid, are effective inducers of the terminal differentiation of HL-60 promyelocytic leukemia cells. The inhibition of cellular replication and the induced maturation produced by these agents appears to be a consequence of the inhibition of IMP dehydrogenase, since growth inhibition is partially reversed and differentiation is completely prevented by the simultaneous exposure of cells treated with inhibitors of IMP dehydrogenase to exogenous guanosine, which serves to circumvent the effects of the blockage of IMP dehydrogenase. The exposure of HL-60 leukemia cells to inhibitors of IMP dehydrogenase caused a marked reduction in the incorporation of [3H]mannose into both cellular glycoproteins and their lipid-linked oligosaccharide precursors; these effects are presumably due to the pronounced decrease in intracellular levels of guanosine triphosphate produced by blockage of IMP dehydrogenase. Maximum effects on glycoprotein biosynthesis occurred within 8 h of exposure to the inhibitors of IMP dehydrogenase. The simultaneous incubation of cells with guanosine and these inducers of differentiation partially prevented the reduction in [3H]mannose incorporation into glycoproteins, supporting a relationship between glycoprotein biosynthesis and guanosine triphosphate formation in the induction of differentiation by inhibitors of IMP dehydrogenase.

Topics: Cell Cycle; Cell Differentiation; Cell Line; Dolichol Monophosphate Mannose; Glycoproteins; Guanosine; Guanosine Triphosphate; Hematopoiesis; Humans; IMP Dehydrogenase; Ketone Oxidoreductases; Leukemia, Myeloid, Acute; Mycophenolic Acid; Organoselenium Compounds; Phagocytosis; Ribavirin; Ribonucleosides; Selenium

The NAD analog N'-methylnicotinamide adenine dinucleotide (N'AD) is formed in intact human promyelocytic leukemia HL-60 and in rat pituitary tumor GH3 cells during treatment of the cultured cells with the nicotinamide derivative N'-methylnicotinamide (N'CH3NAm). N'AD formation is associated with the induced maturation of HL-60 cells and increased hormone production by GH3 cells during treatment with the nicotinamide derivative. N'AD is detected by HPLC analysis of cytoplasmic extracts as a peak which elutes near NAD. Four facts indicate that this compound is N'AD. First, a compound which elutes with identical time retention is produced by transglycosylation during reaction of NAD with pig brain NAD glycohydrolase in the presence of excess N'CH3NAm. Second, the putative N'AD is degraded by prolonged digestion with the NAD glycohydrolase to ADP-ribose. Third, N'AD formation is prevented by addition of nicotinamide along with N'CH3NAm to compete with binding of N'CH3NAm to the NAD glycohydrolase. Fourth, radioactive precursor labeling demonstrates that it contains adenosine, but it is not labeled by radioactive nicotinamide. The biological relevance of N'AD formation was evaluated. The appearance of N'AD precedes development of HL-60 maturation, and NAD levels increase, not decrease, as observed in other cell types, during treatment with N'CH3NAm. Therefore, we propose that N'AD, not the pyridine base itself, is the active species in inducing maturation. The results provide support of a role for NAD metabolism, probably ADP-ribosylation, in the regulation of HL-60 maturation and in hormone production by pituitary cells.

Topics: Adenosine Triphosphate; Animals; Cell Line; Guanosine Triphosphate; Humans; Leukemia, Myeloid, Acute; NAD; NAD+ Nucleosidase; Niacinamide; Nucleotidyltransferases; Pituitary Neoplasms; Poly(ADP-ribose) Polymerases; Rats; Substrate Specificity; Time Factors

fMet-Leu-Phe (fMLP) stimulated the formation of inositol bis- and trisphosphate in the [3H]inositol-labeled plasma membranes from the human leukemic (HL-60) cells differentiated to neutrophil-like cells by dibutyryl cyclic AMP. The stimulatory effect of fMLP was completely dependent on the simultaneous presence of GTP and Ca2+. The fMLP-stimulated formation of the phosphorylated inositols was markedly reduced by the prior ADP-ribosylation of the membranes with pertussis toxin. This toxin ADP-ribosylated a Mr approximately 40,000 protein, presumably the alpha subunit of Gi and/or Go, in the membranes. Reconstitution of the membranes ADP-ribosylated by pertussis toxin with Gi or Go purified from rat brain restored the fMLP-stimulated formation of the phosphorylated inositols. The efficiency of the rat brain Gi and Go in this capacity was roughly equal. The rat brain Gi or Go ADP-ribosylated beforehand by pertussis toxin was inactive in this reconstitution. These results indicate that both rat brain Gi and Go have the potency to couple functionally the fMLP receptor to the phospholipase C-mediated polyphosphoinositide hydrolysis and suggest that Gi or Go may be involved in the mechanism of signal transduction from the fMLP receptor to this reaction in the differentiated HL-60 cells.

Topics: Adenosine Diphosphate Ribose; Calcium; Cell Differentiation; Cell Line; Cell Membrane; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Leukemia, Myeloid, Acute; N-Formylmethionine Leucyl-Phenylalanine; Nucleoside Diphosphate Sugars; Pertussis Toxin; Sugar Phosphates; Virulence Factors, Bordetella

The mechanism of neutrophil activation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) has been studied by pretreatment of human neutrophils with pertussis toxin. Upon stimulation with FMLP, the cytosolic-free calcium concentration, [Ca2+]i, is increased both by stimulation of calcium influx and mobilization of cellular calcium. We have measured [Ca2+]i as well as the generation of the phospholipid breakdown product inositol trisphosphate (IP3), which is thought to mediate Ca2+ mobilization. As the phosphoinositide pool in human neutrophils is difficult to prelabel with [3H]myoinositol, experiments were also carried out in the cultured human promyelocytic leukemia cell line HL-60 after differentiation with dimethylsulfoxide. Pertussis toxin pretreatment of both cell types inhibited FMLP stimulated membrane depolarization, exocytosis, and superoxide production in a dose-dependent manner. This toxin effect was selective for the receptor agonist, since stimulation of these parameters by two substances bypassing the transduction mechanism, the calcium ionophore ionomycin and the phorbolester phorbol myristate acetate, were unaffected. Rises in [Ca2+]i, as well as generation of IP3 in response to FMLP, were inhibited in parallel; for the inhibition of functional responses, slightly lower toxin concentrations were required. The attentuation of the [Ca2+]i rise was more marked in the absence of extracellular calcium, i.e., when the rise is due only to calcium mobilization. The results provide evidence that phospholipase C stimulation by FMLP resulting in IP3 generation is involved in the signal transduction mechanism. Coupling of FMLP receptor occupancy to phospholipase C activation is sensitive to pertussis toxin, suggesting the involvement of a GTP binding protein (N protein), which has been shown to be a pertussis toxin substrate. The parallel changes in [Ca2+]i and IP3 further support the hypothesis that IP3 is the calcium-mobilizing mediator in FMLP-activated cells.

Topics: Calcium; Cell Line; Cytoplasmic Granules; Ethers; Guanosine Triphosphate; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Intracellular Fluid; Ionomycin; Leukemia, Myeloid, Acute; Lymphocyte Activation; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pertussis Toxin; Tetradecanoylphorbol Acetate; Type C Phospholipases; Virulence Factors, Bordetella

An RNA-directed DNA polymerase was isolated from the peripheral blood leukocytes of a patient with acute myelomonocytic leukemia by successive purification of a particulate cytoplasmic fraction with endogenous, ribonuclease-sensitive DNA polymerase activity. Like RNA-directed DNA polymerase from mammalian type-C virus, the human leukemic cell enzyme efficiently utilized (A)(n).(dT)(12-18) and (C)(n).(dG)(12-18) and had an approximate molecular weight of 70,000. Further, the leukemic cell enzyme was strongly inhibited by antisera to RNA-directed DNA polymerase of primate type-C virus in a fashion similar to that noted with an extensively purified RNA-directed DNA polymerase from a person with acute myelogenous leukemia [Todaro, G.J. & Gallo, R.C. (1973), Nature 244, 206]. By these biochemical and immunological results the leukemic cell enzyme could be differentiated from all other known cellular DNA polymerases but could not be distinguished from RNA-directed DNA polymerase of primate type-C virus. We interpret these data, combined with observations published elsewhere, to indicate that human acute myelogenous leukemia cells contain components related to primate type-C virus. The parameters used in this study may provide the specificity and sensitivity required for determining the presence or absence and (if present) the relatedness of RNA-directed DNA polymerase in other cases and types of human leukemia.

Topics: Aged; Animals; Centrifugation, Density Gradient; Chromatography, Gel; Cross Reactions; Epitopes; Guanosine Triphosphate; Haplorhini; Hominidae; Humans; Leukemia, Myeloid, Acute; Male; Molecular Weight; Retroviridae; RNA-Directed DNA Polymerase; Species Specificity; Templates, Genetic; Thymine Nucleotides

Topics: Animals; Avian Leukosis Virus; Avian Myeloblastosis Virus; Cell-Free System; Chickens; Guanosine Triphosphate; Kinetics; Leukemia, Experimental; Leukemia, Myeloid, Acute; Liver; Magnesium; Phenylalanine; Poly U; Potassium; Protein Biosynthesis; Ribosomes

Terminal deoxyribonucleotidyl transferase (EC 2.7.7.31; nucleoside triphosphate:DNA nucleotidylexotransferase) is usually found only in thymus, but has been reported in leukemic cells from children with acute lymphoblastic leukemia. In an unusual adult patient with acute myelomonocytic leukemia, terminal transferase was found at a level of 16 units per 10(8) bone marrow cells and 14 units per 10(8) circulating leukocytes (1 unit = 1 nmol of nucleotide per hr). This activity is comparable to that found in normal thymus. Assays of transferase in marrow and peripheral leukocytes from patients with typical acute and chronic myelogenous leukemias gave average values of 0.5 and 0.3 unit per 10(8) cells, respectively. Transferase activity is also found in normal bone marrow at about 0.07 unit per 10(8) cells. Terminal deoxyribonucleotidyl transferase in all samples of human marrow and peripheral blood had reaction characteristics, sedimentation, and chromatographic properties similar to the homogeneous enzyme from calf thymus.

Topics: Aged; Bone Marrow; Bone Marrow Cells; DNA Nucleotidyltransferases; Female; Guanosine Triphosphate; Humans; Isoelectric Focusing; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukocytes; Tritium

Topics: Animals; Cell-Free System; Chickens; Escherichia coli; Guanosine Triphosphate; Kinetics; Leukemia, Experimental; Leukemia, Myeloid, Acute; Liver; Liver Neoplasms; Phenylalanine; Protein Biosynthesis; Ribosomes; RNA, Messenger; RNA, Transfer; Tritium

Cells from a patient with childhood acute lymphoblastic leukemia contain an apparent DNA polymerase activity that was not found in any other cells except thymus cells. The enzyme has the properties of terminal transferase, an enzyme known to be found in thymocytes. The cells also contain the three major DNA polymerases found in growing cells. The results suggest that these tumor cells arose from a block in the differentiation of thymocytes. Terminal transferase may be a marker for the origin of leukemic cells.

Topics: Adenosine Triphosphate; Avian Leukosis Virus; Cell Line; Child; DNA Nucleotidyltransferases; Guanine Nucleotides; Guanosine Triphosphate; HeLa Cells; Humans; Infectious Mononucleosis; Leukemia, Lymphoid; Leukemia, Myeloid, Acute; Lymphocytes; Lymphoma, Non-Hodgkin; Male; Middle Aged; Moloney murine leukemia virus; Oligonucleotides; Polynucleotides; Templates, Genetic; Thymine Nucleotides; Thymus Gland; Tritium