Heterogeneous methylation of the O-6-methylguanine-DNA methyltransferase promoter in immortalized IMR90 cell lines

Transcriptional silencing of the DNA repair protein, O-6-methylguanine-DNA methyltransferase (MGMT), occurs only in malignant or transformed cell line s, and such MGMT-deficient cells are hypersensitive to chemotherapeutic alk ylating agents such as 1, 3-bis (2-chloroethyl)-1-nitrosourea. (BCNU) and t emozolomide. Previously we demonstrated in a panel of established cell line s that the lack of gene expression correlated with methylation 'hot spots' within the Cpg island in the MGMT 5' gene flank. Now, we investigated the r elationship between CpG methylation, MGMT suppression and drug-sensitivity in normal, diploid MGMT-expressing IMR90 cells and five immortalized sublin es (AA, EE, J, KK and Pool), four of which have silenced MGMT. As expected, the MGMT-expressing parental cells were most drug-resistant and free of pr omoter methylation, whereas the MGMT-silenced immortal sublines were more d rug-sensitive and promoter-methylated. Surprisingly, the sole MGMT-positive immortal subline, (AA) showed some promoter methylation although it was re latively drug-resistant; and an apparently MGMT-negative subline, (EE) show ed unexpectedly low levels of methylation. We determined if these discrepan cies were due to heterogeneity (cellular or allelic) and if this reflected transitional states between expressing and silenced phenotypes. Analysis of : the methylation status of 'hot spot' CpGs by genomic sequencing of cloned single copy DNA confirmed heterogeneity in both these sublines. With incre asing cell culture passage, CpG methylation progressively increased with a concomitant trend to a completely MGMT-silenced phenotype in these sublines .