Exposure of animals to phenobarbital (PB) results in pleotrophic effects on the liver which range from microsomal enzyme induction, hepatocyte hypertrophy and hyperplasia to the promotion of hepatocellular carcinomas. However, the mechanism(s) of promotion with PB at the cellular and molecular level remain to be elucidated. We have shown with an in vivo transplantation technique tht PB is a growth stimulating agent for adult parenchymal hepatocytes. However, PB is not directly mitogenic, but rather increases hepatocyte proliferation by either enhancing the cells responsiveness to endogenous growth stimuli and/or inducing hepatocytes to produce their own growth factor(s). In contrast to these results, we found that PB decreased EGF stimulation of hepatocyte proliferation in culture. Thus, PB increases the clonability of hepatocytes when assayed in vitro. The reason for this dichotomy is unclear, but it demonstrates the importance of further understanding the mechanism of growth control in both normal and early neoplastic hepatocytes. The effect of PB exposure on the clonogenicity of normal and early neoplastic hepatocytes will be compared. We will investigate the relationship between PB and the growth factors TGF-B and EGF in modulating normal and early neoplastic cell growth. The EGF signal for cellular proliferation is entirely dependent upon the influx of Ca+2 from external sources. Thus, receptor operated Ca+2 channels are of pivotal importance in the transduction of the initial EGF signal for DNA synthesis. By utilizing structurally similar (PB and phenytoin) and dissimilar (flunarizine) calcium entry blockers and the Ca+2 ionophore A23187, we will investigate the importance of blocking the cellular influx of Ca+2 in promoting liver tumors. The effect of PB on the expressionof cellular oncogenes associated with proliferation (c-myc, c-H-ras, c-K-ras and c-erbB) will also be determined for the normal and early neoplastic hepatocytes. The results of these investigations will be compared with the biological responses observed with growth factors so that role of oncogene expression in tumor promotion can be more easily discerned. Valid comparisons between the multiple proposed experiments with early neoplastic hepatocytes are possible because we have isolated and propagated early neoplastic cell lines with an intrahepatic transplantation technique. In summary, we will utilize hepatocyte transplantation, primary culture and molecular biological techniques to investigate at the organismal, cellular and molecular level the mechanism(s) of hepatocellular tumor formation with PB.
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