Liver tumor promotion: effect of phenobarbital on EGF and protein kinase C signal transduction and transforming growth factor-beta 1 expression.

Phenobarbital (PB) added to the medium of cultured rat hepatocytes alters epidermal growth factor (EGF) dependent mitogenesis in a biphasic manner; PB concentrations less than 1.5 mM are growth stimulatory but higher concentrations significantly inhibit normal hepatocyte proliferation. In contrast, the growth of putative preneoplastic cells is inhibited less by high concentrations of PB. Mechanistic studies designed to test the ability of PB to alter the early events of EGF signal transduction demonstrate that PB neither competes with EGF for binding to the EGF receptor nor alters EGF-induced receptor down-regulation. However, pretreatment with PB (greater than 1 mM) results in a transient inhibition of EGF binding to hepatocytes. The kinetics of this effect are similar to those obtained when hepatocytes are exposed to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a skin tumor promoter and activator of Ca2+/phospholipid-dependent protein kinase C. However, several observations suggest that distinct mechanisms mediate the responses to these two tumor promoters. First, the inhibitory effects of PB and TPA on EGF binding are additive. Also down-regulation of EGF receptors in response to TPA occurs with hepatocytes, A431 epidermal carcinoma cells, HepG2 hepatoma cells, and rat liver epithelial cells, but only hepatocytes are sensitive to PB. Furthermore, translocation of protein kinase C to the membrane occurs in hepatocytes treated with TPA but not in those treated with PB. The chronic treatment of rats with PB further sensitizes hepatocytes to EGF receptor down-regulation by in vitro PB while desensitizing them to EGF receptor down-regulation by TPA. This latter effect is correlated with a decreased ability of TPA to induce translocation of protein kinase C to the membrane. PB significantly increases the intracellular concentration of TGF-beta 1 in periportal hepatocytes but not in putative preneoplastic cells. TGF-beta 1 may therefore have an important function in regulating early stages of cell cycle progression in proliferating hepatocytes.