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Heterogeneous methylation of the O6-methylguanine-DNA methyltransferase promoter in immortalized IMR90 cell lines

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Transcriptional silencing of the DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT), occurs only in malignant or transformed cell lines, and such MGMT-deficient cells are hypersensitive to chemotherapeutic alkylating agents such as 1, 3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide. Previously we demonstrated in a panel of established cell lines that the lack of gene expression correlated with methylation within the CpG island in the MGMT 5' gene flank. Now, we investigated the relationship between CpG methylation, MGMT suppression and drug-sensitivity in normal, diploid MGMT-expressing IMR90 cells and five immortalized sublines (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 promoter methylation, whereas the MGMT-silenced immortal sublines were more drug-sensitive and promoter-methylated. Surprisingly, the sole MGMT-positive immortal subline, (AA) showed some promoter methylation although it was relatively drug-resistant; and an apparently MGMT-negative subline, (EE) showed unexpectedly low levels of methylation. We determined if these discrepancies were due to heterogeneity (cellular or allelic) and if this reflected transitional states between expressing and silenced phenotypes. Analysis of the methylation status of CpGs by genomic sequencing of cloned single copy DNA confirmed heterogeneity in both these sublines. With increasing cell culture passage, CpG methylation progressively increased with a concomitant trend to a completely MGMT-silenced phenotype in these sublines.
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Document Type: Research Article

Affiliations: Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA

Publication date: June 1, 2001

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  • The International Journal of Oncology provides an international forum for the publication of the latest, cutting-edge research in the broad area of oncology and cancer treatment. The journal accepts original high quality works and reviews on all aspects of oncology research including carcinogenesis, metastasis, epidemiology, chemotherapy and viral oncology. Through fair and efficient peer review, the journal is dedicated to publishing top tier research in the field, offering authors rapid publication as well as high standards of copy-editing and production. The International Journal of Oncology is published on a monthly basis in both print and early online.
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