Association of K-ras mutations with p16 methylation in human colon cancer.
OBJECTIVE
K-ras mutations are early genetic changes in colon cancer. p16, a tumor-suppressor gene, is inactivated in neoplasms by mutation, deletion, or methylation. The aims of this study were to determine p16 methylation status and its possible association with K-ras mutations in human colon cancer.


METHODS
DNA isolated from 8 colon cancer cell lines and 41 microdissected human colon tissue samples was analyzed. p16 methylation status was determined using two analytical methods. The level of p16 expression was determined by reverse-transcription polymerase chain reaction and Northern blot. 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 found in 100% of colon cancer cell lines, 55% of colon cancers, 54% of adenomas, and 25% of transitional mucosa but not in normal colonic epithelium. Forty-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 specimens showed methylated p16. In contrast, of 13 adenomas and cancers with wild-type K-ras, only 3 specimens showed methylated p16 (P = 0.001). Stable transformation of IEC-18 cells with K-ras increased the DNA methyltransferase activity, methylated the p16 gene, and suppressed the expression of p16. Treatment with a DNA methylation inhibitor (azadeoxycytidine) resulted in reexpression of p16 in K-ras-transformed IEC-18 cells, suggesting that the expression of p16 was suppressed by DNA methylation.


CONCLUSIONS
p16 methylation occurs frequently in human colonic adenomas and cancers and is closely associated with K-ras mutations.
Guan
R J
RJ
Division of Gastroenterology, Brigham & Women's Hospital, Boston, USA. rguan@mbcrr.harvard.edu
Fu
Y
Y
Holt
P R
PR
Pardee
A B
AB
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000235:genetics*; D019175:DNA Methylation / Q000187:drug effects; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D004252:DNA Mutational Analysis; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019942:Genes, p16 / Q000235:genetics*; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D009154:Mutation; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine