MOLECULAR MARKERS AS INTERMEDIATE END-POINTS IN CHEMOPREVENTION OF COLON-CANCER - MODULATION OF RAS ACTIVATION BY SULINDAC AND PHENYLHEXYLISOTHIOCYANATE DURING COLON CARCINOGENESIS

Recent evidence suggests that activation of ras proto-oncogenes and in activation of suppressor genes induce malignant phenotype in colonic c ells. Thus the identification of clonal population of cells expressing activated ras may lead to a valuable intermediate biomarker to detect premalignant lesions amenable to chemoprevention. Previously, we demo nstrated that sulindac inhibited the carcinogen-induced colon tumor de velopment whereas phenylhexylisothiocyanate (PHITC) promoted the tumor outcome. The present study was conducted to investigate the effect of sulindac and PHITC on azoxymethane (AOM)induced activation of ras pro to-oncogenes in order to explore the plausibility of using ras as an i ntermediate biomarker in chemoprevention of colon cancer. Male F344 ra ts were fed the AIN-76A diet containing 0, 320 ppm sulindac or 640 ppm PHITC and administered s.c. AOM dissolved in normal saline at a dose rate of 15 mg/kg body wt/week for 2 weeks. Vehicle control groups rece ived s.c. equal volume of normal saline. Animals were sacrificed 52 we eks after AOM or saline treatment and their colonic mucosa and tumors were analyzed for mutations in codon 12 and 13 of K-ras and the expres sion of ras p21. As an alternative non-invasive approach, we developed a simple and sensitive one-step mutant-enriched PCR method to detect these genetic lesions in stools collected at 16, and again at 24 weeks after AOM treatment. AOM-induced G to A transitions were observed at the second nucleotide of 12th codon of K-ras substituting amino acid a sp with wild-type gly. Sulindac not only suppressed the selective ampl ification of initiated cells possessing AOM-induced mutated K-ras codo n 12, but significantly inhibited the AOM-induced expression of total and mutant ras-p21. PHITC did not exert any inhibitory effect on AOM-i nduced ras activation. Results indicated a strong correlation between ras activation and tumor outcome. Data suggest that ras activation may be a useful intermediate molecular marker in chemoprevention of colon cancer.