Role of protein kinase C in modulating epidermal growth factor- and phorbol ester-induced mammary epithelial cell growth in vitro.

Normal mammary epithelial cells isolated from mid-pregnant BALB/c mice were grown within collagen gels and maintained on serum-free media. Chronic treatment with low doses (0.1-0.5 nM) of phorbol 12-myristate 13-acetate (PMA) had no mitogenic action when given alone, but significantly enhanced epidermal growth factor (EGF)-induced growth. In contrast, similar treatment with high doses (10-100 nM) of PMA significantly stimulated mammary epithelial cell growth in the absence of EGF. Furthermore, growth of cells treated with high doses of PMA and EGF was similar to that observed in cells treated with PMA alone. In parallel experiments, treatment with similar doses of 4-alpha-phorbol 12-myristate 13 acetate, a phorbol ester which does not activate PKC, did not significantly alter mammary epithelial cell proliferation when given alone or in combination with EGF. Acute treatment with 10 ng/ml EGF or 20 nM PMA stimulated phospholipid-dependent PKC translocation from the cytosolic to the membrane fraction, and this effect was blocked by prior treatment for 7 days with 20 nM PMA. Western blot analysis showed that chronic treatment with 1-10 nM PMA for 6 days caused only slight decrease in relative PKC alpha levels in the cytosolic and membrane fractions, while similar treatment with 20-100 nM PMA caused a large down-regulation in total cellular phospholipid-dependent PKC alpha levels. Additional studies showed that treatment with 1-2 nM PMA caused an increase, whereas treatment with 5-100 nM PMA caused a dose-related decrease in EGF-dependent EGF-receptor (EGF-R) autophosphorylation, In summary, these findings suggest that submitogenic doses of PMA potentiate EGF-induced cell growth by enhancing EGF-R mitogenic signaling, whereas the mitogenic effects of high doses of PMA alone appear to be mediated through PKC- and EGF-independent mechanisms.