Hypermethylation of the ABCB1 downstream gene promoter accompanies ABCB1 gene amplification and increased expression in docetaxel-resistant MCF-7 breast tumor cells

Abstract

Drug transporters have been implicated in resistance of solid and non-solid tumors to a variety of chemotherapeutic agents. Higher expression of the ABCB1 drug transporter is often observed in drug-resistant tumor cells, although the precise mechanism remains unclear. During selection of MCF-7 cells for survival in increasing concentrations of docetaxel (MCF-7TXT cells), we observed in this study a temporal correlation between the acquisition of docetaxel resistance at selection dose 9 and the increased expression of ABCB1. Both the magnitude of docetaxel resistance and the level of ABCB1 expression then rose as the selection dose was further elevated. We also observed through bisulfite sequencing experiments that the ABCB1 downstream promoter became increasingly methylated following the acquisition of drug resistance (selection doses 10-12). Transcription was solely attributed to the upstream ABCB1 promoter within MCF-7TXT cells at the highest selection dose suggesting that hypermethylation caused a shift in promoter usage. The hypermethylation was also accompanied by regional amplification of chromosome 7 containing the ABCB1 gene and its neighbor ABCB4 but not DBF-4. The amplification of the ABCB1 gene correlated positively both with the hypermethylation of the ABCB1 downstream promoter (r=0.90) and the increased expression of ABCB1 (r=0.78). Moreover demethylation of the ABCB1 downstream promoter induced by 5-aza-2A'deoxycytidine treatment decreased the expression of ABCB1 mRNA in MCF-7TXT cells. Taken together, our findings suggest that the increased expression of ABCB1 upon acquisition of docetaxel resistance in breast tumor cells can be multifactorial, involving both epigenetic changes in promoter usage and regional chromosome amplification.

The temporal relationship between ABCB1 promoter hypomethylation, ABCB1 expression and acquisition of drug resistance

“…16 A standard curve for ABCB1 was generated using 2, 1, 0.5, 0.25 and 0.125 ng of genomic DNA. The mean quantity of ABCB1 gene was normalized to the S28 gene.…”

“…16 The gene copy number was determined using a Metasystem-equipped microscope with Metafer4/Metacyte software v3.3.101. A minimum of 105 cells were analyzed for each cell line.…”

“…Transcript levels were then determined by quantitative reverse transcription PCR (Q-PCR) as previously described. 13 Bisulfite sequencing PCR The methylation status of CpG sites within the ABCB1 downstream promoter was determined by bisulfite sequencing PCR (BSP), as previously described, 16 with genomic DNA extracted from MCF-7 CC cells at selection doses 1, 8 and 12. ABCB1 promoter methylation was also examined in MCF-7 DOX2 cells at selection doses 8 and 12, and in both MCF-7 EPI and MCF-7 TAX-2 cells at selection doses 8, 9 and 12.…”

“…The methylation status of the upstream promoter was not ABCB1 promoter hypomethylation and drug resistance K Reed et al investigated due to the paucity of CpG islands in this region. 16 Sequencing of positive clones was performed by Mobix DNA sequencing service (McMaster University, Hamilton, ON, Canada). Changes in methylation at individual CpG sites were calculated by subtracting the average percentage methylation for the lower dose from the higher dose at each site.…”

“…16 Mean quantities were determined for triplicate wells and normalized to S28 expression. Fold changes within 12 clones of MCF-7 CC and MCF-7 TAX-2 cells were calculated relative to the normalized mean quantity of their respective populations.…”