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 Ca 2+ concentrations, [Ca 2+ ] o , and of treatments known to modulate intracellular Ca 2+ 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 [Ca 2+ ] o in the range 25–1,800 μM, although the peak of DNA synthesis occurred 5–10 h earlier with 1.2 mM Ca 2+ than with 25 μM Ca 2+ . Complete removal of extracellular Ca 2+ using EGTA blocked DNA synthesis if Ca 2+ was removed on the second day after EGF addition but not if Ca 2+ was absent only on day 1. Treatment of cultures in 1.2 mM Ca 2+ ‐containing media with Ca 2+ ‐ionophore A23187 or with thapsigargin, agents expected to raise cytosolic [Ca 2+ ], failed to augment the stimulation of DNA synthesis by EGF. These observations suggest that hepatocytes may have a permissive requirement for [Ca 2+ ] o > 0 at least late in the sequence of events leading from growth factor stimulation to DNA synthesis. However, sustained elevation of cytosolic [Ca 2+ ] 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 α‐hexachlorocyclohexane or DDT to stimulate DNA synthesis in combination with EGF was independent of [Ca 2+ ] o . By contrast, the skin tumor‐promoting phorbol ester, TPA, or liver tumor promoter, phenobarbital, were without effect or inhibitory at low [Ca 2+ ] o but in combination with EGF, stimulated DNA synthesis at [Ca 2+ ] o > 0.4 mM, suggesting that Ca 2+ may have some role in mediating or modulating the stimulatory effects of these agents. © 1993 Wiley‐Liss, Inc.