Modulation of azoxymethane-induced mutational activation of ras protooncogenes by chemopreventive agents in colon carcinogenesis.

Abstract

In our previous study, we demonstrated that azoxymethane (AOM) treatment significantly enhanced the expression of ras p21, the protein product of ras genes, and that the dietary administration of chemopreventive agents such as D,L-alpha-difluoromethylornithine (DFMO), a irreversible inhibitor of ornithine decarboxylase, and piroxicam, a non-steroidal anti-inflammatory drug (NSAID), exerted a significant inhibitory effect on AOM-induced ras p21 expression. In the present study, which is an extension of our earlier investigation, we have determined the effect of DFMO and piroxicam on mutational activation of ras protooncogenes during AOM-induced colon carcinogenesis. Groups of male F344 rats were fed the modified AIN-76A diet containing 0 or 150 p.p.m. piroxicam, or 4000 p.p.m. DFMO and administered s.c. AOM dissolved in normal saline at a dose rate of 15 mg/kg body wt, once weekly, for 4 weeks. Vehicle control groups received s.c. equal volumes of normal saline. Groups of animals were then killed at 0, 4, 16, 24 or 32 weeks after last AOM or saline injection. AOM-induced colon tumors and colonic mucosa from AOM treated as well as saline treated animals were analyzed for point mutations in K- and H-ras protooncogenes by a combination of polymerase chain reaction mediated restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. Our results demonstrate that of the total 65 AOM-induced colon tumors analyzed, 45/50 (90%) obtained from AOM-treated animals fed the control diet, 4/11 (36%) from AOM-treated animals fed piroxicam diet, and 1/4 (25%) from AOM-treated animals fed the DFMO diet, contained single-point mutations occurring specifically at the second nucleotide of codon 12 which were identified exclusively as G to A transitions in case of K-ras, and G to A transitions and also G to T transversions in H-ras. Similar point mutations were identified in colonic mucosa of 21/30 (70%) of AOM-treated animals fed the control diet, 10/30 (33%) of AOM-treated animals fed piroxicam diet, and none of 30 (0%) of AOM-treated animals fed DFMO diet. These results indicate that the administration of piroxicam and DFMO may inhibit the selective amplification of AOM-induced initiated cells carrying mutated ras genes. Dietary DFMO exerted more pronounced inhibition of selective amplification of initiated cells containing AOM-induced mutant ras. Data suggest that determination of ras activation may be a useful marker for chemoprevention of colon cancer.