Ethanol has multiple effects on DNA synthesis in fibroblasts depending on the presence of secreted growth regulators and zinc as well as the level ofprotein kinase C activation

Earlier we showed that in serum-starved (27 h), washed mouse fibroblasts an d other cell lines 40-80 mM concentrations of ethanol (EtOH) potentiate, in a zinc (Zn2+)-dependent manner, the combined stimulatory effects of calciu m (Ca2+) and insulin (Ins) on DNA synthesis. We now report that the promito genic EtOH effects require removal of the used medium at least 6 h prior to treatments with EtOH, Zn2+, and Ins. If serum-starved (21 h) cells were co ntinuously incubated for another 18-h period without replacing the medium, a secreted cellular factor moderately enhanced the mitogenic effect of Ins and simultaneously blocked the potentiating effect of EtOH on DNA synthesis measured during the last hour of treatments. However, the presence of Ca2 (2.8 mM) plus Zn2+ (25 mu M) or 25-300 nM phorbol 12-myristate 13-acetate (PMA) during the serum starvation period partially restored the promitogeni c effect of EtOH. The PMA effect was blocked by the protein kinase C (PKC) inhibitor GF 109203X added for the second (18 h) period. Even at 300 nM, PM A failed to fully downregulate PKC-alpha, the major PKC isoform, over a 28- h period, suggesting that an activated PKC enzyme was involved in the resto ration of EtOH effect. When EtOH (40-80 mM) was added for the entire serum starvation period and the incubations were continued for 18 h without remov ing the medium, EtOH inhibited both the combined actions of Ins and cellula r factor as well as the promoting effect of newly added EtOH on Ins-depende nt DNA synthesis. Coaddition of Zn2+ and PMA with EtOH prevented these inhi bitory effects of EtOH. The results indicate that in mouse fibroblasts EtOH can both enhance and inhibit Ins-dependent DNA synthesis depending on the timing of EtOH treatment as well as the presence of Zn2+, cellular factors, and activators of the PKC system. (C) 2000 Academic Press.