Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
PLoS One. 2010 Jun 8;5(6):e11002. doi: 10.1371/journal.pone.0011002.
Acquired drug resistance represents a frequent obstacle which hampers efficient chemotherapy of cancers. The contribution of aberrant DNA methylation to the development of drug resistant tumor cells has gained increasing attention over the past decades. Hence, the objective of the presented study was to characterize DNA methylation changes which arise from treatment of tumor cells with the chemotherapeutic drug doxorubicin. DNA methylation levels from CpG islands (CGIs) linked to twenty-eight genes, whose expression levels had previously been shown to contribute to resistance against DNA double strand break inducing drugs or tumor progression in different cancer types were analyzed. High-definition DNA methylation profiles which consisted of methylation levels from 800 CpG sites mapping to CGIs around the transcription start sites of the selected genes were determined. In order to investigate the influence of CGI methylation on the expression of associated genes, their mRNA levels were investigated via qRT-PCR. It was shown that the employed method is suitable for providing highly accurate methylation profiles, comparable to those obtained via clone sequencing, the gold standard for high-definition DNA methylation studies. In breast carcinoma cells with acquired resistance against the double strand break inducing drug doxorubicin, changes in methylation of specific cytosines from CGIs linked to thirteen genes were detected. Moreover, similarities between methylation profiles obtained from breast and ovarian carcinoma cell lines with acquired doxorubicin resistance were found. The expression levels of a subset of analyzed genes were shown to be linked to the methylation levels of the analyzed CGIs. Our results provide detailed DNA methylation information from two separate model systems for acquired doxorubicin resistance and suggest the occurrence of similar methylation changes in both systems upon exposure to the drug.
获得性耐药性是阻碍癌症有效化疗的常见障碍。在过去几十年中，异常 DNA 甲基化对耐药肿瘤细胞发展的贡献引起了越来越多的关注。因此，本研究的目的是描述肿瘤细胞用化疗药物阿霉素治疗后出现的 DNA 甲基化变化。分析了与 28 个基因相关的 CpG 岛 (CGI) 的 DNA 甲基化水平，这些基因的表达水平先前被证明有助于对 DNA 双链断裂诱导药物的耐药性或不同癌症类型的肿瘤进展。高清晰度 DNA 甲基化谱由 800 个 CpG 位点的甲基化水平组成，这些位点映射到所选基因转录起始位点周围的 CGI 上。为了研究 CGI 甲基化对相关基因表达的影响，通过 qRT-PCR 研究了它们的 mRNA 水平。结果表明，所采用的方法适用于提供高度准确的甲基化谱，与克隆测序相当，克隆测序是高清晰度 DNA 甲基化研究的金标准。在对双链断裂诱导药物阿霉素产生获得性耐药的乳腺癌细胞中，检测到与 13 个基因相关的 CGI 中特定胞嘧啶的甲基化变化。此外，还发现了来自具有获得性阿霉素耐药性的乳腺癌和卵巢癌细胞系的甲基化谱之间的相似性。分析基因的子集表达水平与分析的 CGI 的甲基化水平相关。我们的研究结果提供了来自两个独立的获得性阿霉素耐药模型系统的详细 DNA 甲基化信息，并表明在暴露于药物时两个系统中都发生了类似的甲基化变化。
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