Epidermal growth factor‐stimulated DNA synthesis requires an influx of extracellular calcium

The dependency of normal cell proliferation on adequate extracellular Ca 2+ levels was further investigated by determining the role of Ca 2+ influx in epidermal growth factor (EGF)‐induced rat liver epithelial (T51B) cell DNA synthesis. Fura‐2‐loaded T51B cells responded with an increase in [Ca 2+ ] i to EGF (5–50 ng/ml) that was blocked by low (25 μM) extracellular Ca 2+ or by pretreatment with 50 μM La 3+ to inhibit plasma membrane Ca 2+ flux. Confluent T51B cells treated for 24 h with EGF (0.1–50 ng/ml) dose‐dependently incorporated [ 3 H]‐thymidine into cell nuclei. Low extracellular Ca 2+ or addition of La 3+ prevented the EGF stimulated rise in labeled nuclei, indicating that a movement of Ca 2+ into the cell was required for DNA synthesis. This was supported by our findings that bradykinin, which induced a rise in [Ca 2+ ] i by opening plasma membrane Ca 2+ channels in T51B cells (but not A23187, thrombin or ATP, which raise [Ca 2+ ] i primary through mobilization of intracellular Ca 2+ stores), potentiated DNA synthesis stimulated by submaximal doses of EGF. Potentiation of the action of EGF by the tumor promoter 12‐0‐tctradecanoyl‐phorbol‐13‐acetatc (TPA), indicates that activation of protein kinase C and an influx of Ca 2+ share a common mechanism for initiating DNA synthesis.