Promoter hypermethylation of MGMT is associated with protein loss in gastric carcinoma

Abstract

Key words: DNA methylation, gastric carcinoma, MGMTN-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) have been shown to induce gastric carcinoma at a high frequency in rats and mice when given in their drinking water. 1,2 MNNG and MNU produce O 6 -methylguanine, which mispairs with thymine during replication, resulting in conversion of a guanine-cytosine pair to an adenine-thymine pair if the adduct is not removed. 3,4 The O 6 -methylguanine-DNA methyltransferase, MGMT, is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O 6 position of guanine induced by alkylating agents such as MNNG and MNU. 5 Therefore, inactivation of the MGMT gene can lead to G to A mutation. In fact, MNU has been shown to induce tumors such as thymic lymphomas and lung adenomas MGMT knockout mice. 6 Epigenetic alterations, in addition to multiple genetic abnormalities, are deeply involved in the genesis and progression of human cancers. Methylation status of the CpG island in promoter regions is an important determinant of gene expression. Aberrant hypermethylation of the CpG island is associated with silencing of tumor suppressor genes in various tumors. Promoter hypermethylation and reduced MGMT expression has been found in some primary human carcinomas. [7][8][9] Data have also indicated that wild-type p53 may negatively regulate basal MGMT promoter activity. in vitro and in vivo studies have shown that induction of MGMT in the presence of DNAdamaging agents involves p53. 10,11 Transfection of MGMT promoter together with a p53-expression vector in both p53 knockout and p53 wild-type cells significantly reduced MGMT promoter activity. 10,12 No study, however, has been conducted to clarify the role and mechanism of MGMT alteration in gastric carcinoma.We examined DNA methylation of CpG islands of the MGMT gene by methylation specific PCR in 26 primary gastric carcinoma tissues and 8 gastric carcinoma cell lines and compared the results with levels of MGMT protein expression to know whether transcriptional silencing of MGMT occurs due to promoter hypermethylation. In addition, we investigated p53 mutation status in the same tissues by PCR-SSCP analysis for comparison with the levels of MGMT protein expression. MATERIAL AND METHODS Tissue samplesA total of 26 gastric carcinoma tissue samples from 26 cases were studied. Tumor tissues and their corresponding non-neoplastic mucosae were surgically removed, frozen immediately in liquid nitrogen, and stored at Ϫ80°C until use. We confirmed microscopically that the tumor-tissue specimens consisted mainly of carcinoma tissue and that non-neoplastic mucosa did not exhibit any tumor-cell invasion or show significant inflammatory involvement. Histologic classification and tumor staging were done according to Lauren classification system 13 and the TNM Stage Grouping. 14 Cell linesEight cell lines derived from human gastric carcinoma were used. The TMK-1 cell line was established in our laboratory from poorly-differentiated adenocarcinoma. 15 Five ga...

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

“…In the present study, promoter methylation was detected in 14.1% of gastric carcinomas and a loss of MGMT expression was found in 11.4 -13.3% of tumours. Our result is lower than those of Oue et al (2001) or Park et al (2001) but is similar to those of Esteller et al (2001a). The study by Park et al (2001) showed that there was no significant difference in the protein expression between cases with MGMT methylated and unmethylated tumours.…”

“…The study by Park et al (2001) showed that there was no significant difference in the protein expression between cases with MGMT methylated and unmethylated tumours. But in our results, as well as the study by Oue et al (2001), there were significant differences associated between the loss of MGMT protein expression and promoter methylation (P50.001).…”

Accumulation of DNA methylation is associated with tumor stage in gastric cancer

“…22 In brief, each target sequence was amplified in a 20 L reaction volume containing 10 -20 ng of genomic DNA, 0.2 M 2Јde-oxynucleoside-SЈtriphosphate, 10 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, 2 mM MgCl2, 0.3 M of each primer, and 0.75 units (U) of Ampli Taq Gold (Perkin-Elmer, Norwalk, CT). PCR amplification consisted of 35 cycles of 94°C for 30 seconds, 60°C or 55°C for 30 seconds, and 72°C for 30 seconds, after the initial activation step of 94°C for 10 minutes.…”

“…16,[21][22][23][24][25][26][27] Inactivation of the hMLH1 gene by hypermethylation is associated with microsatellite instability in GC. 21 MGMT is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O 6 position of guanine induced by alkylating agents such as MNNG and MNU.…”

DNA methylation of multiple genes in gastric carcinoma: Association with histological type and CpG island methylator phenotype

“…Although several reports have been published about methylation of various genes in gastric carcinoma, [5][6][7][8][9][10][11][12][13][14][15] in most of these studies, the methylation status was investigated for just a single gene and no reports have described an association between DNA methylation pattern and CIMP status or histological classification. Kang et al 16) studied DNA methylation of multiple genes and reported that EpsteinBarr virus (EBV)-positive gastric carcinoma frequently shows aberrant methylation; however, they did not investigate whether any association exists between DNA hypermethylation and CIMP status or histological classification.…”