HEPATOCARCINOGENESIS IN VITRO USING ACTIVATED FOS GENES

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If we are ever to understand the molecular pathology of neoplasia, a detailed analysis of the molecular changes brought about by activated oncogenes must be undertaken within the context of well-characterized model systems. Expression of activated oncogenes usually results in complex phenotypic changes including anchorage-independent growth and tumorigenicity, and it is likely that some of these changes involve a reprogramming of cellular gene expression by oncogene products. Recent studies suggest that several oncogene pathways may converge in the nucleus through the transcriptional activation of specific cellular genes. It is therefore logical and compelling to analyze directly, at the molecular level, the effects of a well-characterized nuclear oncogene (i.e., fos) on the expression of cellular genes whose activity changes consistently after hepatocarcinogenesis. Newborn-rat liver epithelial (RLE) cells provide a useful in vitro model system in which to study molecular mechanisms associated with hepatocarcinogenesis, since transformation with either chemical carcinogens or an activated ras gene results in the activation of gamma-glutamyl transpeptidase (gamma GT) and glutathione-S-transferase-P (GSTP), two markers of liver carcinogenesis in vivo. I will use a metal-regulatable, metallothionein-c-fos (MTcfos) fusion gene to transfect RLE cells in culture, to identify any "transformed" (i.e., anchorage-independent) clones by growth in soft agarose, and to investigate the correlation between fos-transformation and the activation of gamma GT and GSTP; preliminary results indicate that expression of gamma GT but not GSTP is activated after transformation of RLE cells by low levels of MTcfos. Utilizing the metal-inducibility of the constructs, I will establish the minimum levels of MTcfos mRNA (northern blots) and protein (western blots) necessary for induction of anchorage independent growth, gamma GT expression, and GSTP expression. During the second phase of these studies, I will analyze in detail the mechanisms by which the c-fos gene product activates expression of the gamma GT gene. My efforts will emphasize transcriptional effects of c-fos, since recent reports suggest that the c-fos product may bind to transcriptional control sites on DNA. The following approaches will be used: i) studies in isolated nuclei to verify transcriptional effects of c-fos; ii) transfection with 5'- deletion mutants of gamma GT fused to the CAT (chloramphenicol acetyltransferase) gene to identify fos-dependent regions of the gamma GT gene; and iii) cell-free transcription of the genomic gamma GT gene to identify any cellular factors whose activity is required for fos- dependent activation.