The role of 5-methylcytosine in the expression of O(6)-methylguanine DNA-methyltransferase

Regulation of the gene which encodes the human DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is of clinical interest and provides a gene model for the study of CpG methylation's role in transcriptional regulation. Expression of MGMT is important to the maintenance of genomic integrity because it repairs O6-alkylguanine DNA lesions, which, if left unrepaired, can lead to mutation. In the clinical setting of cancer chemotherapy, however, MGMT provides cancer cells with resistance to the nitrosoureas, a class of cancer chemotherapeutics whose cytotoxic activity is via formation of O6-alkylguanine adducts, and includes N,N'-bis(2-chloroethyl)-N'-nitrosourea (BCNU) and N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU). Expression of MGMT is regulated at the level of transcription in a cell-cycle-independent manner. It has been found that up to 25% of cultured cell lines do not express MGMT activity due to a lack of MGMT transcription. This dissertation describes the use of the human multiple myeloma cell line 8226/S and its MGMT negative (MGMT- variant 8226/V to study the role of CpG methylation in repression of MGMT transcription. Bisulfite sequencing showed that 5-methylation of cytosines within the CpG island of the 8226/V MGMT promoter correlated with loss of MGMT expression. Furthermore, the increase in CpG methylation is associated with the formation of nuclease-resistant chromatin structure around the MGMT transcription start site as determined by chromatin accessibility assays. Additional evidence is presented which shows that CpG methylation increased in the 8226/V genome as a result of selection with verapamil, and that the expression of genes which increase the clonigenic capacity of 8226/V were also affected by the selection. Southern analysis of cytosine methylation across the mdr1, c-myc, H19, K-ras, and thymidine kinase genes is presented, along with development of a sensitive control for the technique which utilizes the mitochondrial genome. This work has established the 8226/S and 8226/V cell lines as a model to study the mechanisms and effects of cytosine methylation. The results presented here support the importance of 5-methylcytosine as an epigenetic mechanism of MGMT gene regulation, and suggests epigenotoxicity as a cause for the increased cancer risk seen in patients receiving verapamil and other calcium channel blockers