Heterogeneous methylation of the O6-methylguanine-DNA methyltransferase promoter in immortalized IMR90 cell lines
| Autor: | Sherie R. Howell, Thomas P. Brent, Rebecca P. Danam, Joanna S. Remack |
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| Rok vydání: | 2001 |
| Předmět: |
Cancer Research
Methyltransferase Biology DNA methyltransferase Cell Line O(6)-Methylguanine-DNA Methyltransferase Humans Promoter Regions Genetic Antineoplastic Agents Alkylating Lung neoplasms Gene Cells Cultured DNA Primers Genome Human Reverse Transcriptase Polymerase Chain Reaction Chromosome Mapping Methylation DNA Methylation Fibroblasts Cell cycle Carmustine Molecular biology digestive system diseases Oncology CpG site Cell culture DNA methylation Cancer research CpG Islands |
| Zdroj: | International Journal of Oncology. |
| ISSN: | 1791-2423 1019-6439 |
| DOI: | 10.3892/ijo.18.6.1187 |
| Popis: | Transcriptional silencing of the DNA repair protein, O 6 -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 hot spots' 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 'hot spot' 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. |
| Databáze: | OpenAIRE |
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