Influence of CpG island methylation status in O6-methylguanine-DNA methyltransferase expression of oral cancer cell lines.

It is known that the O6-methylguanine-DNA methyltransferase (MGMT) gene is susceptible to epigenetic regulation associated with an altered frequency of CpG methylation. To investigate whether epigenetic regulation of the MGMT gene might lead to significant reductions in the expression levels of cancer cells, we sought evidence of a link between the methylation status of the MGMT promoter and the expression levels of seven human oral cancer cell lines. We found two frequently methylated regions: the 5' region extending from nt 690 to nt 893 in the promoter, and the more 3' region extending from nt 1060 to nt 1151 in the untranslated first exon. The 3' region was hypermethylated independently of MGMT expression levels in all cell lines. By contrast, in the three MGMT-downregulated cell lines (SAS, Hep2, HO-1-u-1), the levels of MGMT expression were inversely related to the density of 5' region of the methylated CpGs in the MGMT promoter. Our results implied that the transcriptional inactivation of MGMT might require methylation of the 5' region, but not that of the 3' region in oral cancer cell lines. We further explored the role of methylation in MGMT expression by treating cells with 5-Aza-2'-deoxycytidine (5Aza-dC). 5Aza-dC treatment led to the partial or complete cytosine demethylation of two frequently methylated MGMT regions in all cell lines. 5Aza-dC succeeded in upregulating of the MGMT mRNA levels in only 2 of 7 cell lines (HSC3 and HO-1-u-1), and in fact reduced MGMT mRNA in the other 5 cell lines. Furthermore, 5Aza-dC had an inhibitory effect on MGMT protein levels in all cell lines. Our results suggest that MGMT levels may not revert after 5Aza-dC treatment. Based on our findings, the regulation of MGMT expression appears to be more complex than previously thought, although it is at least partially influenced by CpG methylation. Accordingly, care should be taken interpreting the link between MGMT methylation and expression.