Effects of insulin and the phorbol ester tumor promoter TPA on cell growth and transcript levels of the growth-associated enzyme, ornithine decarboxylase

Insulin and a phorbol ester tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) were examined for their capabilities to affect the growth of rat hepatoma cells; growth was measured by the incorporation of tritiated thymidine into acid-precipitable DNA. In quiescent cells, insulin induced growth, TPA alone had no effect, and TPA in the presence of insulin caused an inhibition of the insulin-stimulated growth (an effect seen only with the tumor-promoting phorbol esters). Through timed addition and removal of TPA it was discovered that TPA was maximally effective in its inhibitory actions if applied with the first four hours (early G1 phase) of the insulin-stimulated cell cycle. It was proposed that a TPA-sensitive event occurred sometime within this early G1 phase. It was questionable whether insulin and TPA could be acting through a common mechanism. In an atten5)t to further qualify the observed effects of insulin and TPA on cell growth, the enzyme ornithine decarboxylase was selected as a marker enzyme. Insulin and TPA both increased messenger RNA (mRNA) levels of ornithine decarboxylase and were previously shown to increase the enzyme's activity as well. The time course of induction by each affector molecule was different, but the timing of enzyme activity increases in both cases closely parallelled the increases in mRNA. With the addition of both insulin and TPA, ornithine decarboxylase transcript levels exhibited an additive increase. These increases might be attributed to increased transcription rates or increased mRNA stability. The latter of these possibilities was partially addressed through experiments using the protein synthesis inhibitor cycloheximide, since the possibility exists that the mRNA may be either degraded or stabilized by a newly synthesized protein. Initial observations appear to indicate that separate post-transcriptional mechanisms are set into motion by insulin and TPA which result in different effects on ornithine decarboxylase mRNA.