Proto-oncogenes are cruical cellular targets for chemical carcinogens, and introduction of oncogenes activated by these agents into cells mimics many of their effects. A compelling and logical approach to analyzing how chemical carcinogens alter gene expression in carcingoenesis is to study the effects of activated oncogenes on the expression of other genes. Because activated ras genes are among the most potent known oncogenes and are known to be activated by chemical carcinogens in vivo, we plan a comprehensive study of the role of mutated and normal Ha-c-ras gene expression in the expression of cellular genes. These include genes associated with liver cancer in vivo and """"""""nuclear"""""""" oncogenes which may mediate the actions of ras. In cultured rat liver epithelial (RLE) cells, RNA levels of 2 genes which presage liver cancer in vivo (gamma glutamyl-transferase (gamma GT) and glutathione-S-transferase-P (GST-P)) are increased as much as 80 fold in response to transcription from a regulatable metallothionein mutant ras fusion gene (MT ras T24). In contast, alpha tubulin (alpha TUB) RNA and cytochrome P-450c (CP-450c; an enzyme known to fall during cracingoenesis in vivo) RNA levels decline or are unchanged in RLE cells carrying MT ras T24. We will study the mechanism of these effects by analyzing nuclear transcription (run on) and message stability; investigating expression of gamma GT/CAT (chloramphenicol acetyltransferase) and GST-P/CAT constructions in transient expression assays in cultured RLE cells; identifying, purifying and cloning ras-cloning ras-induced trans-acting factors by gel retardation and footprinting assays and a new application of Lambda gt11 cloning; and using in vitro (cell free) transcription to charcterize gaamma GT and GST-P transcription. We will evaluate the consquences of these elevated RNA levels by measuring changes in gamma GT and GST-P protein levels. In previous experiments, we have shown that in Rat 1 fibroblasts carrying MT ras T24 (MR4 and MR5 cells) graded expression of MT ras T24 results in graded phenotypic alterations and progressively increasing maximal cell densities. In preliminary experiments we have found that induction of MT ras T24 expression in MR-4 cells increases c-myc expression at least 3- fold within 1 hr and induces the onset of DNA synthesis at equivalent 10-12 hr. (C-fos and c-myb expresson have not yet been measured.) These changes will be studied in detail in both Rat 1 and RLE cells and their derivatives (see above).
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