Methylation of selected CpGs in the human O-6-methylguanine-DNA methyltransferase promoter region as a marker of gene silencing

O-6-Methylguanine-DNA methyltransferase (MGMT) is a major determinant of su sceptibility to methylating carcinogens and of tumor resistance to anticanc er methylating and chloroethylating drugs. The silencing of MGMT expression that occurs in 20-30% of human tumor lines is tightly linked to methylatio n within the MGMT gene 5' CpG island. Previous studies on a very limited nu mber of cell lines showed that such methylation was uneven, with hot-spots where methylation almost invariably occurred and intervening regions with v ery low incidences of methylation. To ascertain if such hot-spot methylatio n is in fact a ubiquitous hallmark of MGMT-silenced cells, we determined th e methylation status of selected hot-spot CpGs in an extensive panel of MGM T-expressing and -silenced cell lines and xenografts. Using two simple and rapid bisulfite-polymerase chain reaction-based assays, we confirmed that i n MGMT-silenced cells, methylation occurred at these sites whereas it was e ssentially absent in MGMT-expressing cells. Mel. Carcinog. 24:85-89, 1999. (C) 1999 Wiley-Liss, Inc.