Calcium influx in induced differentiation of murine erythroleukemia cells.
Murine erythroleukemia cells (MELC) have served as a model for examining the regulation of erythroid differentiation. However, the role of Ca2+ in the signal transduction pathways regulating differentiation remains unclear. To begin to address this uncertainty we have characterized the regulation of cytoplasmic Ca2+ and the possible role of calcium channels during induced differentiation in MELC. MELC can be induced to terminal differentiation using the polar/apolar compound hexamethylene bisacetamide (HMBA). We found that HMBA stimulated Ca2+ influx within 3 to 6 minutes and that Ca2+ entry was required but not sufficient for MELC growth and differentiation. Nifedipine (1 to 10 mumol/L), a calcium channel antagonist, blocked HMBA-induced Ca2+ influx and inhibited differentiation by approximately 60%. Depolarization of the MELC membrane did not induce Ca2+ influx and whole-cell patch-clamp recordings failed to detect a voltage-activated Ca2+ current, suggesting that MELC do not express detectable levels of a functional voltage-dependent calcium channel (VDCC). However, a cDNA probe encoding a portion of the alpha 1 subunit of the cardiac VDCC detected an approximately 8-kb mRNA on Northern blots of total MELC RNA. Taken together, these data show that Ca2+ influx is an early event associated with HMBA-induced differentiation in MELC, blockade of this calcium influx inhibits induced differentiation, and a voltage-insensitive dihydropyridine-sensitive calcium channel may be involved in Ca2+ influx in MELC.
Gillo
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Department of Medicine, Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, NY 10029.
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D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000081:Acetamides / Q000494:pharmacology*; D000814:Aniline Compounds; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D005456:Fluorescent Dyes; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D008969:Molecular Sequence Data; D009543:Nifedipine / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014966:Xanthenes
D000081:Acetamides; D000814:Aniline Compounds; D015220:Calcium Channels; D005456:Fluorescent Dyes; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D014966:Xanthenes; C059715:Fluo-3; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D009543:Nifedipine; C014026:hexamethylene bisacetamide; D002118:Calcium