Association of K-ras mutations with p16 methylation in human colon cancer

Background & Aims: K-ras mutations are early genetic changes in colon cance r, p16, a tumor-suppressor gene, is inactivated in neoplasms by mutation, d eletion, or methylation. The aims of this study were to determine p16 methy lation status and its possible association with K-ras mutations in human co lon cancer. Methods: DNA isolated from 8 colon cancer cell lines and 41 mic rodissected human colon tissue samples was analyzed. p16 methylation status was determined using two analytical methods. The level of p16 expression w as determined by reverse-transcription polymerase chain reaction and Northe rn Riot, K-ras mutations were determined by DNA sequence analysis. The DNA methyltransferase activity was determined by microassay, Parental and K-ras -transformed IEC-18 cells were used to determine the potential association between K-ras mutations and p16 methylation, Results: Methylated p16 was fo und in 100% of colon cancer cell lines, 55% of colon cancers, 54% of adenom as, and 25% of transitional mucosa but not in normal colonic epithelium, Fo rty-five percent of cancers and 38% of adenomas showed both K-ras mutations and p16 methylation. Of 11 cancers and adenomas With K-ras mutation, 10 sp ecimens showed methylated p16. In contrast, of 13 adenomas and cancers with wild-type K-ras, only 3 specimens showed methylated p16 (P = 0.001), Stabl e transformation of IEC-18 cells with K-ras increased the DNA methyltransfe rase activity, methylated the p16 gene, and suppressed the expression of p1 6, Treatment with a DNA methylation inhibitor (azadeoxycytidine) resulted i n reexpression of p16 in K-ras-transformed IEC-18 cells, suggesting that th e expression of p16 was suppressed by DNA methylation, Conclusions: p16 met hylation occurs frequently in human colonic adenomas and cancers and is clo sely associated with K-ras mutations.