The stimulation by the tumour promoters 12-O-tetradecanoylphorbol-13-acetate and phenobarbital of the growth of primary neonatal rat hepatocytes.

A single exposure to a wide range of concentrations (10(-15)-10(-4) mol/l) of the tumour promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phenobarbital (PB) significantly stimulated the flow into DNA synthesis and mitosis of neonatal rat hepatocytes in 4-day-old primary cultures kept in high-calcium (1.8 mmol/l) Eagle's FBS-MEM. Maximal effects were observed in the dose range 10(-12)-10(-6) mol/l. Moreover, both xenobiotics retained their full mitogenic effectiveness when given to hepatocytes incubated in calcium-deficient (0.01 mmol/l) FBS-MEM, thereby evoking a neoplastic phenotype in otherwise normal (i.e., non-initiated) cells. Proliferation kinetic studies showed that TPA and PB acted according to the actual cell cycle setting of the cells: they committed a fraction of the quiescent (GO) hepatocytes to grow, and enabled hepatocytes previously poised at the G1/S and G2/M boundaries to start cycling again, but exerted no influence on liver cells already engaged by themselves in active cycling. These diverse activities of TPA and PB were independent of serum (growth) factors; they were also fully elicited in hepatocytes grown in synthetic media (Eagle's MEM or HiWoBa2000), and were not changed by the addition of a mitogenically effective dose (10(-10) mol/l) of epidermal growth factor/urogastrone (EGF) with or without FBS. Conversely, the addition of the specific plasmalemmal calcium chelator, EGTA, or displacer, La3+, or of the anticalmodulin drugs, W-13 and calmidazolium (or R24571: an agent hardly entering cells) fully inhibited the mitogenic activities of tumour promoters in quiescent and intra-cycle-blocked hepatocytes, while having no effect on untreated or xenobiotic-treated liver cells already cycling by themselves. Such inhibitory effects were in all instances independent of the actual extracellular calcium concentration and took place according to time-related double kinetics. Hence, xenobiotic-activated processes taking place at the plasmalemmal level and involving the activation of calcicalmodulin-dependent enzymes were of critical importance for both the G0/G1 and G1/S transitions in primary hepatocytes.