Role of calcium in prolactin-stimulated c-myc gene expression and mitogenesis in Nb2 lymphoma cells.
Receptor-activated transmembrane calcium flux has been implicated as a mediator of the actions of many growth factors and hormones. We examined the effects of PRL, calcium ionophores, and calcium antagonists on 45Ca2+ flux, c-myc gene expression, and DNA synthesis in the PRL-dependent rat Nb2 lymphoma cell line. PRL had no detectable effects on 45Ca2+ uptake or efflux, and the mitogenic effects of PRL could not be reproduced by the calcium ionophore A23187 alone or in combination with the tumor-promoting phorbol ester 12-O-tetra-decanoyl-phorbol-13 acetate (TPA). PRL, but not A23187 or TPA, stimulated c-myc gene expression in quiescent Nb2 cells. Exposure to PRL for brief periods (15 min to 4 h), followed by extensive washing, resulted in a time- and dose-dependent activation of DNA synthesis measured 16 h later. This activation was not blocked by addition of excess anti-PRL antiserum after the wash steps, indicating that the observed stimulation was not due to residual PRL. Despite the marked increase in DNA synthesis, removal of PRL after 4 h prevented mitosis, suggesting that PRL may be required throughout the cell cycle for Nb2 cell proliferation. Although continuous incubation with calcium antagonists resulted in a dose-dependent inhibition of PRL-stimulated DNA synthesis, activation of DNA synthesis by brief exposure to PRL was not inhibited by the presence of EGTA, calcium channel blockers (nifedipine, cobalt chloride), or calmodulin inhibitors (trifluoperazine, N-6-aminohexyl-5-chloronaphthalene sulfonamide). PRL-stimulated c-myc expression was attenuated, but not blocked, by the calcium channel antagonists. However, the putative intracellular calcium antagonist TMB-8 inhibited both c-myc expression and DNA synthesis in a dose-dependent manner (IC50 = 16 microM). Nb2 cells were sensitive to TMB-8 throughout G1 of the cell cycle, but inhibition of DNA synthesis was greatest when TMB-8 was present during the first 3 h of mitogen presentation, indicating a block in the transition from G0 to G1 of the cell cycle. The effect of TMB-8 suggests that release of a small intracellular calcium pool may mediate the early actions of PRL in Nb2 cells.
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002132:Calcium Radioisotopes; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D004533:Egtazic Acid / Q000494:pharmacology; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D008223:Lymphoma / Q000235:genetics*; D008297:Male; D011388:Prolactin / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016271:Proto-Oncogene Proteins c-myc; D011519:Proto-Oncogenes; D051381:Rats; D011919:Rats, Inbred Strains; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002121:Calcium Channel Blockers; D002132:Calcium Radioisotopes; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D000001:Calcimycin; D004533:Egtazic Acid; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D011388:Prolactin; D004247:DNA; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium