Ethanol, Zn²⁺ and insulin interact as progression factors to enhance DNA synthesis synergistically in the presence of Ca²⁺ and other cell cycle initiators in fibroblasts

In serum-starved NIH 3T3 fibroblasts, ethanol (30-80 mM) promoted the effects of insulin and insulin-like growth factor I (IGF-I) on DNA synthesis in a Zn²⁺-dependent manner. Ethanol and Zn²⁺ were most effective when added shortly before or after insulin, indicating that all these agents facilitated cell cycle progression. The synergistic effects of ethanol, Zn²⁺ and insulin (or IGF-I) on DNA synthesis required 1.1-2.3 mM Ca²⁺, which seemed to act as the cell cycle initiator. When serum-starved cells were pretreated for 2 h with other cell cycle initiators such as 10% (v/v) serum, 50 ng/ml platelet-derived growth factor or 2 ng/ml fibroblast growth factor, subsequent co-treatments with 60 mM ethanol, Zn²⁺ and insulin for an 18 h period again synergistically increased DNA synthesis. Of the various signal transducing events examined, ethanol stimulated cellular uptake of ⁴⁵Ca and it enhanced the stimulatory effects of insulin on p70 S6 kinase activity in a Zn²⁺-dependent manner. In contrast, ethanol inhibited insulin-induced activating phosphorylation of p42/p44 mitogen-activated protein kinases; these inhibitory ethanol effects were prevented by Zn²⁺. The results show that, in NIH 3T3 fibroblasts, ethanol can promote cell cycle progression in the presence of a cell cycle initiator as well as Zn²⁺ and insulin (or IGF-I).