Objective
hMLH1 protein serves to detect the DNA damage caused by cisplatin (DDP) and destroys the cell. The absence of hMLH1 expression has been correlated with acquired resistance of ovarian cancer cells to platinum. The aim of this study was to determine the possible role of DNA methylation and histone H3 lysine 9 (H3-K9) acetylation on the loss of hMLH1 expression, and to evaluate the reversal effects of 5-Aza-2′-deoxycytidine (5-Aza-dC) and Trichostatin A (TSA) on DDP-resistance in ovarian cancer cell lines.
Methods
Two human ovarian cancer cell lines, COC1 and its DDP-resistant subline, COC1/DDP were cultured. The two cancer cells were treated with 5-Aza-dC or TSA. Using COC1 cells as a control, we used methylation-specific PCR (MSP) to analyze DNA methylation at hMLH1 gene promoter. hMLH1 mRNA and protein expressions were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. Chromatin immunoprecipitation assay (ChIP) was used to test the levels of histone H3-K9 acetylation at hMLH1 gene promoter.
Results
In COC1 cells, there was no DNA methylation at hMLH1 gene promoter, while there were hMLH1 mRNA and protein expression. In COC1/DDP cells, there was DNA hypermethylation at hMLH1 gene promoter, while there was no hMLH1 mRNA or protein expression. The treatment with 5-Aza-dC resulted in DNA demethylation at the promoter region, as well as restoration of hMLH1 expression in COC1/DDP cells. The treatment with TSA had no effects on DNA demethylation or restoration of hMLH1 expression in COC1/DDP cells.
Conclusion
Hypermethylation of DNA at the promoter is related to the silencing of hMLH1 in COC1/DDP ovarian cancer cells. DNA methylation at hMLH1 promoter could play a significant role in determining the sensitivity of ovarian cancer to DDP. The drug resistance mediated by methylation of hMLH1 could be overcome by 5-Aza-dC.
