ROLE OF PROTEIN-KINASE-C IN MODULATING EPIDERMAL GROWTH FACTOR-INDUCED 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. C hronic treatment with low doses (0.1-5 nM) of phorbol 12-myristate 13- acetate (PMA) had no mitogenic action when given alone, but significan tly enhanced epidermal growth factor (EGF)-induced growth. In contrast , similar treatment with high doses (10-100 nM) of PMA significantly s timulated mammary epithelial cell growth in the absence of EGF. Furthe rmore, growth of cells treated with high doses of PMA and EGF was simi lar to that observed in cells treated with PMA alone. In parallel expe riments, treatment with similar doses of 4-alpha-phorbol 12-myristate 13-acetate, a phorbol ester which does not activate PKC, did not signi ficantly 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 cyto solic to the membrane fraction, and this effect was blocked by prior t reatment for 7 days with 20 nM PMA. Western blot analysis showed that chronic treatment with 1-10 nM PMA for 6 days caused only a slight dec rease in relative PRC alpha levels in the cytosolic and membrane fract ions, while similar treatment with 20-100 nM PMA caused a large down-r egulation in total cellular phospholipid-dependent PKC alpha levels. A dditional studies showed that treatment with 1-2 nM PMA caused an incr ease, whereas treatment with 5-100 nM PMA caused a dose-related decrea se in EGF-dependent EGF-receptor (EGF-R) autophosphorylation. In summa ry, these findings suggest that submitogenic doses of PMA potentiate E GF-induced cell growth by enhancing EGF-R mitogenic signaling, whereas the mitogenic effects of high doses of PMA alone appear to be mediate d through PKC- and EGF-independent mechanisms. (C) 1996 Academic Press , Inc.