sq-23377 and Leukemia--Myeloid--Acute

The relationship between calcium ions and the differentiation of human promyelocytic leukemia HL-60 cells was investigated. Proliferation of HL-60 cells incubated in calcium-free medium was inhibited without cell differentiation. On the other hand, incubation with 100 microM verapamil markedly inhibited cell proliferation and caused slight cell differentiation into monocytes. Both calcium-free medium and 100 microM verapamil enhanced HL-60 cell differentiation after treatment with 1 nM 1 alpha,25-dihydroxyvitamin D3, 1 nM beta-all-trans-retinoic acid, or 0.75% dimethyl sulfoxide. However, no enhancement was obtained by treatment with 1 nM 12-O-tetradecanoylphorbol-13-acetate. The free cytosolic calcium concentration was measured by the intracellularly trappable fluorescent calcium indicator, quin 2. The increase of intracellular calcium induced by 250 nM ionomycin was completely blocked by 100 microM verapamil in calcium-free medium, suggesting that the high concentration of verapamil (100 microM) blocks the intracellular calcium mobilization in HL-60 cells. Therefore, the enhancing effect of calcium deprivation or verapamil of HL-60 cell differentiation seemed to be closely related to the inhibition of intracellular calcium mobilization. This speculation is supported by the finding that 50 microM 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate, an intracellular calcium antagonist, also enhanced HL-60 cell differentiation induced by 1 alpha,25-dihydroxyvitamin D3, beta-all-trans-retinoic acid, or dimethyl sulfoxide.

Topics: Calcitriol; Calcium; Cell Differentiation; Cell Division; Cell Line; Dimethyl Sulfoxide; Ethers; Gallic Acid; Humans; Ionomycin; Leukemia, Myeloid, Acute; Tetradecanoylphorbol Acetate; Tretinoin; Verapamil

The generation of the two inositol trisphosphate (IP3) isomers, 1,4,5-IP3 and 1,3,4-IP3, and its relation to changes in the cytosolic free calcium concentration, [Ca2+]i, in response to the chemotactic peptide fMet-Leu-Phe was studied in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Stimulation by fMet-Leu-Phe within seconds transiently elevates 1,4,5-IP3 to peak values averaging 8-fold basal levels, and leads to a concomitant rise in [Ca2+]i and to degranulation. These responses are followed by a slower and more sustained rise in 1,3,4-IP3. Alterations in [Ca2+]i modulate differentially the generation of the two IP3 isomers. At [Ca2+]i lower than 30 nM, no IP3 is generated upon fMet-Leu-Phe stimulation. Working at normal resting [Ca2+]i, but preventing the fMet-Leu-Phe induced transient rise in [Ca2+]i (by prior depletion of intracellular Ca2+ stores and working in calcium-free medium) the fMet-Leu-Phe stimulation of 1,3,4-IP3 levels is attenuated, whereas the response of 1,4,5-IP3 is not significantly altered. Maintained elevation of [Ca2+]i to micromolar levels with the Ca2+ ionophore ionomycin generates enhanced 1,3,4-IP3 levels in the absence of fMet-Leu-Phe, whereas the fMet-Leu-Phe stimulation of 1,4,5-IP3 generation is markedly inhibited. Pertussis toxin selectively abolishes the fMet-Leu-Phe-induced IP3 production, whereas ionomycin stimulation of 1,3,4-IP3 generation is unaffected. These findings indicate that in intact cells: receptor-triggered phosphatidylinositol bisphosphate phosphodiesterase activation has a minimal Ca2+ requirement, but does not depend on a previous or concomitant rise in [Ca2+]i; Ca2+ elevations above micromolar levels decrease the fMet-Leu-Phe-induced generation of 1,4,5-IP3; and 1,3,4-IP3 generation is not directly linked to receptor activation and appears to result both from increased [Ca2+]i and 1,4,5-IP3 levels.

Topics: Calcium; Cell Line; Chromatography, High Pressure Liquid; Cytosol; Ethers; Exocytosis; Hexosaminidases; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ionomycin; Leukemia, Myeloid, Acute; N-Formylmethionine Leucyl-Phenylalanine; Pertussis Toxin; Receptors, Formyl Peptide; Receptors, Immunologic; 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