Reed Kerry, Hembruff Stacey L, Laberge Monique L, Villeneuve David J, Côté Gilbert B, Parissenti Amadeo M
Department of Biomolecular Sciences, Laurentian University, Sudbury, Ontario, Canada.
Epigenetics. 2008 Sep;3(5):270-80. doi: 10.4161/epi.3.5.6868.
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.
药物转运体与实体瘤和非实体瘤对多种化疗药物的耐药性有关。尽管确切机制尚不清楚，但在耐药肿瘤细胞中经常观察到ABCB1药物转运体的高表达。在选择MCF-7细胞在多西他赛浓度不断增加的环境中存活（MCF-7TXT细胞）的过程中，我们在本研究中观察到在选择剂量9时获得多西他赛耐药性与ABCB1表达增加之间存在时间相关性。随着选择剂量进一步提高，多西他赛耐药程度和ABCB1表达水平均上升。我们还通过亚硫酸氢盐测序实验观察到，获得耐药性（选择剂量10-12）后，ABCB1下游启动子甲基化程度越来越高。在最高选择剂量下，MCF-7TXT细胞中的转录仅归因于上游ABCB1启动子，这表明高甲基化导致了启动子使用的转变。高甲基化还伴随着包含ABCB1基因及其邻近基因ABCB4但不包括DBF-4的7号染色体区域扩增。ABCB1基因的扩增与ABCB1下游启动子的高甲基化（r=0.90）和ABCB1表达增加（r=0.78）均呈正相关。此外，5-氮杂-2'-脱氧胞苷处理诱导的ABCB1下游启动子去甲基化降低了MCF-7TXT细胞中ABCB1 mRNA的表达。综上所述，我们的研究结果表明，乳腺癌细胞获得多西他赛耐药性后ABCB1表达增加可能是多因素的，涉及启动子使用的表观遗传变化和区域染色体扩增。
