The level of substrate ornithine can alter polyamine‐dependent DNA synthesis following phorbolester stimulation of cultured hepatoma cells

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Abstract
Although the precise intracellular function(s) of the polyamines remain incompletely defined, a myraid of evidence now shows that the polyamines must accumulate or be maintained at a specific intracellular concentration in order for all mammalian cells to grow or divide. The initial step in polyamine biosynthesis normally involves the decarboxylation of ornithine by the enzyme ornithine decaboxylase (ODCase E.C. 4.1.1.17) to yield putrescine. Increases in the steady-state level of intracellular ornithine have been reported to markedly alter the accumulation of the polyamines following stimulation of Reuber H35 Hepatoma cells with 12-O-tetradecanoylphorbol-β-acetate (TPA) in the presence of serum (Wu and Byus:(Biochem. Biophys. Acta 804:89–99, 1984)) Wu et al.: (Cancer Res. 41:3384–3391, 1981). We wished to determine whether or not incubation of H35 hepatoma cells with exogenous ornithine would result in a stimulation of DNA synthesis following treatment with the mitogens TPA and insulin. For these studies, H35 cells were maintained under serum-free conditions for 2–3 days in order to obtain synchronous cultures suitable for analysis of the level of DNA synthesis. Cultures treated in this manner were highly viable, maintained similar growth rates, and possessed the equivalent levels of intracellular ornithine and polyamines as the serum-containing cultures. Arginine levels, however, were approximately twofold higher following culture under serum-restricted conditions for 3 days. The addition of exogenous ornithine (0.5 mM) was accompanied by a 4–5-fold increase in intracellular steady-state ornithine levels and by a 6–8-fold increase in the presence of TPA and ornithine. In a manner identical to the serum-containing cultures (Wu and Byus (1984)) the addition of TPA and exogenous ornithine to the serum-free cells caused a dose-dependent increase in intracellular putrescine (up to 5-fold) and a concomitant decrease in ODC activity in comparison to stimulation with TPA alone. The addition of TPA led to a 3–5-fold increase in the incorporation of tritiated thymidine into DNA. In the presence of exogenous ornithine, TPA-induced DNA synthesis was further stimulated more than twofold in a dose-dependent manner. Insulin (10−10–10−8 M) proved to be more efficacious as a mitogen in the H35 cells and led to greater stimulation of DNA synthesis than TPA. Insulin alone also resulted in a higher steady-state level of ornithine and putrescine in comparison with TPA alone. However, ornithine addition to the culture medium was not accompanied by any further increase in the insulin-mediated elevation in DNA synthesis. The data is discussed in relation to the selective ability of mitogens to alter the flux through extracellular and intracellular pools of ornithine as required to supply sufficient polyamines to support maximal rates of DNA synthesis. The results furthers support the suggestion that the high levels of intracellular putrescine observed following TPA + ornithine might enhance any of a number of the specific or unique transductive events employed by TPA, in comparison with insulin, which lead to the synthesis of DNA.