Treatment of quiescent density-arrested A31 T6 proadipocytes with medium supplemented with either 12-0-tetradecanoylphorbol-I 3-acetate (TPA), insulin, or cholera toxin alone did not stimulate Go/G1 traverse and initiation of DNA synthesis. Combinations of either TPA and cholera toxin or insulin and cholera toxin caused a small stimulation of proliferation. Addition of medium supplemented with TPA and insulin caused a marked stimulation of cell cycle traverse which was significantly potentiated by the coaddition of cholera toxin. The actions of cholera toxin were mimicked by forskolin. Expression of c-fos was regulated in a manner that reflected the results of the mitogenic experiments. TPA caused a marked induction of expression, while only a small increase in transcript levels was seen after treatment with cholera toxin. Addition of a combination of cholera toxin and TPA caused a synergistic induction of c-fos expression. The model system described in this paper allows a detailed analysis of the regulation, by independent second messenger systems, of the transcription of a gene in a mitogenically relevant manner. B 1992 WiIey-Liss, ~nc.
Cellular proliferation is regulated by a family of extracellular mitogens that act by binding to cell surface receptors and, by a process that is largely yet to be characterized, increasing the transcription of a set of growth-regulated genes [Lau and Nathans, 19911. In nontransformed BALB/c-3T3 cells, a combination of mitogens is required for optimal mitogenesis. This was first demonstrated with the observation that neither platelet-derived growth factor (PDGF) nor platelet-poor plasma (PPP) alone are efficient mitogens, although a combination of PDGF and PPP causes a marked stimulation of DNA synthesis in quiescent, density-arrested cells [Pledger et al., 19781. It was later shown that the comitogenic effects of PPP can be replaced with a combination of epidermal growth factor and insulin-like activity [Leof et al., 19821. In NIH 3T3 cells, the addition of medium supplemented with PDGF alone causes maximal mitogenic stimulation. This appears to be due to the fact that PDGF uniquely stimulates multiple intermediate pathways in these cells, an effect mimicked ~ ~~