Cancer is the end result of a multistage, multifactorial process. The assays for chemical transformation of Syrian hamster embryo fibroblasts (HE) and hamster epidermal cells are excellent models in which to study various factors that may be associated with specific stages in transformation, from the first morphologic changes induced by a carcinogen through progression to the neoplastic state. The objective of this proposal is to determine whether altered expression or activation of cellular ras genes plays a role in chemical transformation of hamster cells, and whether such alterations are associated with any specific stage during the progression from morphologic to neoplastic transformation. The c-ras gene which is most frequently activated in neoplastic transformants of HE and hamster epidermal cells initiated with benzo(a)pyrene (B(a)P) will be determined, using DNA transfection into NIH/3T3 cells and restriction enzyme analysis of transforming activity and of hybridization patterns with specific ras probes. The normal hamster homolog of the activated ras gene will be molecularly cloned, and the coding regions containing the codons for amino acids 12 and 61 subcloned and sequenced. The normal gene sequences subcloned into a single-stranded DNA vector will be used as probes for genomic sequencing of the activated genes in the neoplastic transformants and of homologous regions in their preneoplastic progenitors. It will then be determined when, after treatment of primary cells with B(a)P and clonal isolation of morphologically altered HE cell transformants or differentiation-resistant epidermal cell transformants, a mutation in a ras gene can first be detected by a biochemical test for restriction polymorphism or genomic sequencing of the DNA. We will then determine when, in relation to the progression from morphologic to neoplastic transformation, that mutated ras gene is expressed, as measured by its transfectability into 3T3 cells and/or altered production of p21. These experiments can determine if ras gene activation is or is not a primary event in chemical transformation of hamster cells.
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