Inactivation of O6-methylguanine-DNA methyltransferase by promoter CpG island hypermethylation in gastric cancers

Abstract

Promoter hypermethylation of CpG islands in tumour suppressor genes can lead to transcriptional inactivation. To investigate the association between methylation and expression at O6-methylguanine-DNA methyltransferase, we performed methylation-specific PCR and immunohistochemistry in 149 gastric carcinomas. Promoter methylation was found in 14.1% of tumours and loss of expression was detected in 11.4% of tumours. To examine correlation between the O6-methylguanine-DNA methyltransferase expression and the clinical data, we investigated O6-methylguanine-DNA methyltransferase expression in 315 consecutive gastric carcinomas. A similar frequency of loss of O6-methylguanine-DNA methyltransferase expression was confirmed in these cases. The loss of O6-methylguanine-DNA methyltransferase expression was significantly associated with pTNM stage (P=0.037), tumour invasion (P=0.02), microsatellite instability (P=0.041) and overall survival (P=0.01). Among 11 gastric cancer cell lines, SNU-620 showed the loss of O6-methylguanine-DNA methyltransferase expression as well as promoter methylation. After treatment with 5-aza-2-deoxycytidine, a demethylating agent, SNU-620 re-expressed O6-methylguanine-DNA methyltransferase mRNA. In summary, we suggest that during gastric carcinogenesis, the loss of O6-methylguanine-DNA methyltransferase expression frequently occurs via the hypermethylation of the CpG islands of the promoter region, and that this is significantly associated with the clinicopathological characteristics.

Figure 1

Methylation-specific PCR result of MGMT in primary gastric carcinomas and the gastric cancer cell lines. (A) In gastric carcinomas, matched normal tissues (N) showed only unmethylated bands but tumours (T) showed both unmethylated and methylated bands. (B) The SNU-620 cell line showed only the methylated allele but the SNU-719 cell line showed both methylated and unmethylated alleles. Methylated product was not detected in other cell lines. U, unmethylated allele; M, methylated allele.

Figure 2

Expression of MGMT in gastric carcinomas. On immunohistochemistry, MGMT protein expressed in the nuclei of normal cells and cancer cells (A). In some cases, nuclear MGMT expression is lost completely (B) or focally (C).

Figure 3

Overall survival of the gastric cancer patients according to MGMT expression. Cases with loss of MGMT expression showed poor prognosis compared to those with normal expression pattern (P=0.01).

Figure 4

Expression of MGMT in gastric cancer cell lines. (A) Protein expression by Western blot analysis. All but SNU-620 cell line express MGMT protein. (B) mRNA expression by RT–PCR. MGMT mRNA was not detected in the SNU-620 cell line. β-actin was used as a control. (C) MGMT mRNA expression after 5-aza-2′-deoxycytidine treatment. The SNU-620 cell line expressed mRNA after 10 uM of 5-aza-2′-deoxycytidine treatment for 10 days. The SNU-1 was used as a positive control.