guanosine-5--o-(3-thiotriphosphate) and Leukemia

Influx of Ca(2+) represents an important regulatory signal in the process of cell proliferation. However, little is known about how Ca(2+) entry changes during the cell-cycle. Patch-clamp experiments and microfluorimetry show that store-operated Ca(2+) entry was substantially reduced in rat basophilic leukaemia cells cultured for 24h under serum-free conditions. Likewise, retinoic acid treatment blocked Ca(2+) influx activated by store depletion via inositol 1,4,5-trisphosphate. Both procedures are known to arrest cells at the G0/G1 boundary of the cell-cycle and induced a reduction in 5-bromo 2'-deoxyuridine incorporation into DNA. Ca(2+) release from the stores remained unaltered and two types of K(+) currents were not affected in cells after serum starvation. The specific reduction in Ca(2+) entry was not detected when using aphidicolin, 5-fluorouracil or thymidine to synchronise the cell-cycle. These data suggest that store-operated Ca(2+) influx changed during cell-cycle progression which might have important implications for cell growth.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Aphidicolin; Bromodeoxyuridine; Calcium; Cell Cycle; Cell Division; Culture Media, Serum-Free; Enzyme Inhibitors; Fluorouracil; Guanosine 5'-O-(3-Thiotriphosphate); Inositol 1,4,5-Trisphosphate; Leukemia; Patch-Clamp Techniques; Potassium; Potassium Channels; Rats; Time Factors; Tretinoin; Tumor Cells, Cultured

We studied the relationships between the activation of phospholipase D (PLD) by guanine nucleotides and phorbol esters in permeabilized U937 promonocytes and in solubilized extracts prepared from U937 cell membranes. Treatment of permeabilized cells with phorbol myristate acetate (PMA) strongly potentiated GTP gamma S-dependent PLD activity at free Ca2+ < 100 nM. In the absence of GTP gamma S, PMA stimulated only minor PLD activity. This suggested synergistic interaction between regulatory G-proteins and a protein kinase C (PKC) family kinase. The potential role of PKC was evaluated by testing two mechanistically distinct PKC inhibitors, bisindolylmaleimide (BIM) and calphostin. BIM inhibits PKC enzymes via competition with ATP for binding to the catalytic domain, while calphostin competes with PMA or diglyceride for binding to the regulatory domain. The ability of PMA to potentiate the GTP gamma S-dependent PLD was not inhibited by BIM. In contrast, calphostin strongly inhibited the GTP gamma S-dependent PLD activity, both in the presence and absence of PMA as a potentiating agent. Calphostin also produced complete inhibition of a GTP gamma S-dependent PLD activity, present in solubilized membrane extracts, which was assayed using phospholipid vesicles of defined composition. Treatment of reconstituted membrane/cytosol mixtures with calphostin also produced complete inhibition of the GTP gamma S-induced translocation of Rho A from cytosol to membrane. In contrast to its effects on the U937 cell PLD, calphostin did not inhibit the activity of purified PLD from cabbage. These results suggest that the assembly of active RhoA/PLD signaling complexes on membranes involves a phorbol ester/calphostin-binding protein, but is not dependent on PKC-type catalytic activity.

Topics: Blotting, Western; Cell Membrane; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine 5'-O-(3-Thiotriphosphate); HL-60 Cells; Humans; Indoles; Leukemia; Liposomes; Maleimides; Naphthalenes; Permeability; Phospholipase D; Plants; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The mechanism by which depletion of intracellular Ca2+ stores activates Ca2+ influx is not understood. We recently showed that primaquine, an inhibitor of vesicular transport, blocks the activation of the calcium release-activated calcium current (ICRAC) in rat megakaryocytes (Somasundaram, B., Norman, J. C., and Mahaut-Smith, M. P. (1995) Biochem. J. 309, 725-729). Since it is well established that vesicular transport is temperature-sensitive, we have investigated the effect of temperature on both the activation and maintenance of store-mediated Ca2+ and Mn2+ influx in the human leukemic cell line KU-812 using a combination of whole cell ICRAC recordings and measurements of Mn2+ photoquench of fura-2. Activation of ICRAC was temperature-sensitive, showing a nonlinear reduction when the temperature was lowered from 27 to 17 degrees C with an abrupt change at 21-22 degrees C and complete inhibition at 17 degrees C. Once activated, ICRAC also displayed an abrupt reduction at 21-22 degrees C but was not completely blocked even when the temperature was reduced to 14 degrees C, suggesting that at least one of the temperature-sensitive components is exclusively involved in ICRAC activation. Activation of store-mediated Mn2+ influx also showed similar nonlinear temperature sensitivity and complete inhibition at 19 degrees C. However, in contrast to ICRAC measurements, lowering the temperature following maximal activation of the influx pathway at 37 degrees C did not result in any detectable residual Mn2+ entry below 19 degrees C. We conclude that the mechanism of store-mediated Ca2+ influx involves temperature-dependent steps in both its maintenance and activation, suggesting dependence on a lipid membrane environment.

Topics: Adenosine Triphosphate; Calcium; Calcium Channels; Cytosol; Egtazic Acid; Fluorescent Dyes; Fura-2; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Leukemia; Manganese; Patch-Clamp Techniques; Potassium; Primaquine; Sodium; Temperature; Thapsigargin; Tumor Cells, Cultured

Receptor-induced binding of the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP [gamma S]), to guanine nucleotide-binding regulatory proteins (G proteins) was measured in various permeabilized cells. In myeloid differentiated human leukemia (HL-60) cells, permeabilized with either digitonin, streptolysin O or Staphylococcus aureus alpha-toxin, binding of GTP[gamma S] induced by three distinct chemoattractant receptors was observed. The extent of receptor-stimulated GTP[gamma S] binding (maximally about 2-fold) was independent of the type of permeabilizing agent used. In human erythroleukemia cells permeabilized with digitonin, agonist activation of thrombin and neuropeptide Y receptors increased GTP[gamma S] binding by 1.8- and 1.5-fold, respectively. Finally, in adherently grown human embryonic kidney cells permeabilized with digitonin, activation of the stably expressed human muscarinic m3 receptor increased GTP[gamma S] binding by about 1.6-fold. In digitonin-permeabilized HL-60 cells, a quantitative analysis of formyl peptide receptors and interacting G proteins was performed. About 50,000 formyl peptide receptors per cell were detected. Agonist binding to these receptors was fully sensitive to regulation by guanine nucleotides and pertussis toxin. The number of high-affinity GTP[gamma S] binding sites, most likely representing heterotrimeric G proteins, was calculated to be about 670,000 per cell. Stimulation of formyl peptide receptors led to the activation of about 130,000 of high-affinity GTP[gamma S] binding sites, indicating a ratio of about three activated G proteins per one agonist-activated receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cells, Cultured; Chemotactic Factors; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Leukemia; N-Formylmethionine Leucyl-Phenylalanine; Pertussis Toxin; Receptors, Formyl Peptide; Receptors, Immunologic; Receptors, Peptide; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

Dibutyryl cAMP-differentiated HL-60 human leukemia cells possess receptors for the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), C5a and leukotriene B4 (LTB4). We compared the effects of these chemoattractants in HL-60 membranes and in intact HL-60 cells. fMLP, C5a and LTB4 stimulated GTP hydrolysis and guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) binding in HL-60 membranes with similar effectiveness and in a pertussis toxin (PTX)-sensitive manner. They also stimulated photolabeling of the alpha-subunits of the guanine nucleotide-binding proteins (G-proteins), Gi2 and Gi3 with similar effectiveness. Chloride salts of monovalent cations differentially enhanced and inhibited chemoattractant-induced GTP hydrolyses. C5a was less effective than fMLP in enhancing cholera toxin-catalysed ADP-ribosylation of Gi alpha 2 and Gi alpha 3, and LTB4 was ineffective. fMLP was more effective than C5a and LTB4 in stimulating Ca2+ influx in HL-60 cells. C5a- and LTB4-induced rises in cytosolic Ca2+ concentration ([Ca2+]i) were PTX-sensitive, whereas the effect of fMLP was partially PTX-insensitive. LTB4-induced rises in [Ca2+]i were more sensitive towards homologous desensitization than those induced by C5a, and the effect of fMLP was resistant in this regard. C5a was considerably less effective than fMLP in activating superoxide anion formation and azurophilic granule release, and LTB4 was ineffective. Our data suggest that fMLP, C5a and LTB4 effectively activate the G-proteins, Gi2 and Gi3, in HL-60 cells and that fMLP may additionally activate PTX-insensitive G-proteins. fMLP, C5a and LTB4 are full, partial and incomplete secretagogues, respectively, and these differences may be due to differences in homologous receptor desensitization and qualitative Gi-protein activation.

Topics: Bucladesine; Calcium; Cell Differentiation; Complement C5a; Enzyme Activation; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydrolysis; Leukemia; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Signal Transduction; Tumor Cells, Cultured

Differentiated human leukemia (HL 60) cells contain high numbers of receptors for the chemotactic factors, N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and complement component 5a (C5a), both coupled to pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins). Agonist activation of either receptor stimulated binding of the GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to membrane G proteins and by a similar extent in a non-additive manner. The possible interaction of the two receptors was studied by measuring agonist binding to one receptor in the presence of the other receptor agonist. fMet-Leu-Phe and C5a had no effects on [125I]C5a and fMet-Leu-[3H]Phe receptor binding, respectively, when studied in the absence of regulatory ligands. Similarly, the inhibitory effects of NaCl and GDP on agonist receptor binding were not altered in the presence of the other receptor agonist. In contrast, in the presence of the GTP analogs, GTP[S] and guanosine 5'-[beta,gamma-imino] triphosphate, fMet-Leu-Phe and C5a reduced the binding of [125I]C5a and fMet-Leu-[3H]Phe, respectively, in a concentration-dependent manner. The potencies of the GTP analogs to inhibit binding of [125I]C5a and fMet-Leu-[3H]Phe was increased about 3-fold by fMet-Leu-Phe and C5a, respectively. The data presented suggest that fMet-Leu-Phe and C5a receptors share the same G protein pool in membranes of HL 60 cells and that activation of these G proteins by one of the two receptors decreases the availability of G proteins for the other receptor.

Topics: Cell Membrane; Chemotactic Factors; Chemotaxis, Leukocyte; Complement C5a; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Leukemia; N-Formylmethionine Leucyl-Phenylalanine; Receptors, Immunologic; Tumor Cells, Cultured

Macrophages derived from phorbol ester-induced human leukemic (HL-60) cells exhibit a voltage-activated inward rectifying potassium conductance which was modulated by macrophage colony-stimulating factor (Wieland, S. J., Chou, R. H., and Gong, Q. H. (1990) J. Cell. Physiol. 142, 643-651). Roles of intracellular messengers in this regulatory mechanism were investigated. Intracellular dialysis with inositol 1,3,4,5-tetrakisphosphate (IP4) or inositol 1,4,5-trisphosphate during tight-seal whole cell recording produced a rapid increase in the inward rectifying conductance. Changes in intracellular Ca2+ levels alone did not reproduce the stimulatory effect of these modulators. Intracellular dialysis with guanosine 5'-O-(thiotriphosphate) (GTP gamma S) resulted in profound inhibition of this conductance. These data suggest a novel cellular function for inositol polyphosphates, particularly IP4, and show antagonistic modulation with GTP gamma S on a human macrophage inward rectifier.

Topics: Calcium; Electric Conductivity; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Leukemia; Macrophage Colony-Stimulating Factor; Membrane Potentials; Potassium; Tumor Cells, Cultured