Hypermethylation of CpG island in O6-methylguanine-DNA methyltransferase gene was associated with K-ras G to A mutation in colorectal tumor

Abstract

Aim: To investigate the functions of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) gene in colorectal tumorigenesis and progression.

Methods: The promoter hypermethylation of MGMT gene was detected in 27 sporadic colorectal adenomas, 62 sporadic colorectal carcinomas and 20 normal colorectal mucosa tissues by methylation-specific PCR. At the same time, the expression of MGMT protein was carried out in the same samples using immunohistochemistry. Mutant-allele-specific amplification was used to detect K-ras G to A point mutation in codon 12.

Results: None of the normal colorectal mucosa tissues showed methylated bands. Promoter hypermethylation was detected in 40.7% (11 of 27) of adenomas and 43.5% (27 of 62) of carcinomas. MGMT proteins were expressed in nucleus and cytoplasm of normal colorectal mucosa tissues. Loss of MGMT expression was found in 22.2% (6 of 27) of adenomas and 45.2% (28 of 62) of carcinomas. The difference between them was significant (P = 0.041). In the 6 adenomas and 28 carcinomas losing MGMT expression, 5 and 24 cases presented methylation, respectively (P = 0.027, P<0.001). Thirteen of the 19 colorectal tumors with K-ras G to A point mutation in codon 12 had methylated MGMT (P = 0.011). The frequencies of K-ras G to A point mutation were 35.3% (12 of 34) and 12.7% (7 of 55) in tumors losing MGMT expression and with normal expression, respectively.

Conclusion: Promoter hypermethylation and loss of expression of MGMT gene were common events in colorectal tumorigenesis, and loss of expression of MGMT occurs more frequently in carcinomas than in adenomas in sporadic patients. Hypermethylation of the CpG island of MGMT gene was associated with loss of MGMT expression and K-ras G to A point mutation in colorectal tumor. The frequency of K-ras G to A point mutation was increased in tumors losing MGMT expression. It suggests that epigenetic inactivation of MGMT plays an important role in colorectal neoplasia.

Figure 1

Methylation-specific PCR result of MGMT in colorectal tumors and normal colorectal mucosa. Colorectal carcinomas (45 °C) showed both unmethylated (U) and methylated (M) bands, but normal tissues (N) showed only unmethylated bands.

Figure 2

MGMT expressed in the nuclei of normal colorectal mucosa cells.

Figure 3

Loss of MGMT expression in colorectal carcinomas.

Figure 4

MASA-PCR products for detection of K-ras point mutations in colorectal tumors. Set 1 and set 2 showed the first and second G to A mutation in K-ras codon 12 (GGT), respectively.