Role of promoter methylation in the induction of ABCB1 gene expression in anthracycline-resistant and paclitaxel-resistant breast tumor cells

4291 A major obstacle in the successful treatment of breast cancer is the acquisition of multidrug resistance (MDR), which in vitro often involves the elevated expression and activity of the ABCB1 drug transporter. While the exact mechanism responsible for increased ABCB1 expression in drug-resistant cancer cells remains unclear, it has been proposed that changes in the methylation status of a CpG island within the ABCB1 promoter may be involved.
 A novel system to study the role of epigenetics in the acquisition of drug resistance within breast cancer cells has been developed within our laboratory. Three panels of drug-resistant MCF-7 cell lines were established by selection in increasing concentrations of various chemotherapy agents. Resistance to doxorubicin, epirubicin and paclitaxel was acquired at specific threshold doses (29.1 nM, 31.5 nM and 3.66 nM respectively) and resistance levels continued to increase with higher selection doses. Gene expression analysis of the drug-resistant cell lines in comparison to MCF-7 co-cultured control cells by quantitative-PCR (Q-PCR) revealed that ABCB1 expression was dramatically higher in epirubicin-resistant (MCF-7 EPI ) and paclitaxel-resistant (MCF-7 TAX-2 ) cell lines at or above the threshold dose [X 2 (4)=11.067,p 2 (4)=11.725,p DOX-2 ) did not express ABCB1 even at the highest selection dose. These observations were confirmed at the protein level at the maximum selection dose using flow cytometry. No gene amplifications at the ABCB1 locus or its neighbor loci ABCB4 and DBF-4 were detected by Q-PCR using genomic DNA from drug-resistant cells lines compared to co-cultured control cells. Bisulfite sequencing data indicated that compared to co-cultured control MCF-7 cells, the ABCB1 promoters in MCF-7 EPI and MCF-7 TAX-2 cells were significantly hypomethylated following the acquisition of drug resistance (threshold dose p DOX-2 cells. Treatment of MCF-7 cells with a demethylating agent (5’aza-2’deoxycytodine) increased ABCB1 expression by approximately 9.2 ± 2.8 fold, indicating that alterations in methylation at the ABCB1 promoter can result in increased gene expression. Thus, changes in ABCB1 promoter methylation appear to accompany both the acquisition of drug resistance and increased ABCB1 expression in MCF-7 cells.