ROLE OF CA-2+ IN STIMULATION OF DNA-SYNTHESIS BY EPIDERMAL GROWTH-FACTOR AND TUMOR PROMOTERS IN CULTURED RAT HEPATOCYTES

This study examines the effects of extracellular Ca2+ concentrations, [Ca2+]o, and of treatments known to modulate intracellular Ca2+ levels on the extent and timing of DNA synthesis in primary cultures of adult rat hepatocytes. In cultures exposed to insulin and EGF, the extent of DNA synthesis between 40 h and 70 h in culture was independent of [Ca2+]o in the range 25-1,800 muM, although the peak of DNA synthesis occurred 5-10 h earlier with 1.2 mM Ca2+ than with 25 muM Ca2+. Complete removal of extracellular Ca2+ Using EGTA blocked DNA synthesis if Ca2+ was removed on the second day after EGF addition but not if Ca2+ WaS absent only on day 1. Treatment of cultures in 1.2 mM Ca2+-containing media with Ca2+-ionophore A23187 or with thapsigargin, agents expected to raise cytosolic [Ca2+], failed to augment the stimulation of DNA synthesis by EGF. These observations suggest that hepatocytes may have a permissive requirement for [Ca2+]o > 0 at least late in the sequence of events leading from growth factor stimulation to DNA synthesis. However, sustained elevation of cytosolic [Ca2+] does not appear to be important as an early signalling event either in mediating or augmenting EGF action in hepatocytes. The ability of liver tumor promoters alpha-hexachlorocyclohexane or DDT to stimulate DNA synthesis in combination with EGF was independent of [Ca2+]o. By contrast, the skin tumor-promoting phorbol ester, TPA, or liver tumor promoter, phenobarbital, were without effect or inhibitory at low [Ca2+]. but in combination with EGF, stimulated DNA synthesis at [Ca2+]o > 0.4 mM, suggesting that Ca2+ may have some role in mediating or modulating the stimulatory effects of these agents.