Gene mutations in experimental models of carcinogenesis and their effects on responses to chemopreventive agents in the azoxymethane-treated rat model

Abstract

The Ki-ras oncogene and the p53 tumor suppressor gene are mutated in a high percentage of human colon cancers. The mutation status of these two genes were assessed in colon adenocarcinomas obtained from azoxymethane (AOM)- and dimethylhydrazine (DMH)-treated rats, widely used experimental models of human colon carcinogenesis. The status of p53 mutations was examined using polymerase chain reaction (PCR) amplification of these sequences. In so doing, it was discovered that the rat p53 gene is structurally distinct from the human p53 gene, since it is missing one intron between exons 6 and 7. Further analysis using single stranded DNA conformational polymorphism (SSCP) analysis and direct DNA sequencing of the highly conserved regions of rat exons 5-7, highly mutated in human colon cancers, revealed no p53 mutations in any of these regions. Mutations at codon 12 of the Ki-ras gene were also characterized. Mutation frequency was approximately 60% which is close to the frequency of Ki-ras mutations found in human cancers. Since Ki-ras mutations have been shown to occur early in human colon carcinogenesis, while p53 mutations are thought to occur in late stage tumors, these data suggest this model may be a good for early, but not late, events of human colon carcinogenesis. It was also found that the non-steroidal anti-inflammatory drug (NSAID), sulindac sulfone, could reduce Ki-ras mutation frequency in this model. NSAIDs are agents being studied as a possible chemopreventive agent in both human colon cancers and the AOM model. Previous studies have found that the NSAIDs, sulindac and its sulfone metabolite, decrease the frequency of colon tumors in the AOM model. In this study, sulindac and sulindac sulfone specifically induced apoptosis in normal rat embryo cells (RECs) transfected with either an EJ-ras or activated myc and ras but not in non-transfected RECs. These data suggest that sulindac and sulindac sulfone may be reducing tumor frequency and Ki-ras frequency by inducing apoptosis in those cells with specific mutations that give them carcinogenic potential, possibly through non-prostaglandin dependent pathways.