High-definition DNA methylation profiles from breast and ovarian carcinoma cell lines with differing doxorubicin resistance

Abstract

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.

Figure 1. Cell viability after 72 hours of doxorubicin treatment.

A: Viability assay of MCF-7_wt and MCF-7_ADR. Dashed line indicates 50 percent viability of untreated control. B: Viability assay of OVCAR-5, OVCAR-4 and NCI/ADR-RES. C: Summary of the determined IC₅₀ values from each cell line as well as resistance relative to MCF-7_wt (RF).

Figure 2. Methylation profiling – Reproducibility and validation.

A: Reproducibility, illustrated via two replicate hybridizations, of fully methylated and fully unmethylated control fragment pools. B: Methylation profiles determined from CGIs around the TSS of the genes DNAJC15, ESR1 and GSTP1. Shown are hybridizations of fully methylated and unmethylated control fragments as well as fragments amplified from the cell lines MCF-7_wt and MCF-7_ADR. C: Validation of methylation profiles via clone sequencing of the PCR fragments used for microarray hybridizations shown in Figure 2B.

Figure 3. Hyper- and hypo-methylation events occur in distinct CGI sub-regions with increased doxorubicin resistance.

A: A sub-region of a CGI associated with the gene DNAJC15 becomes hyper-methylated and the gene's mRNA levels decrease with increased resistance. B: A sub-region of a CGI associated with the gene ABCB1 becomes hypo-methylated and the gene's mRNA levels increase with increased resistance. Methylation profiles from CGIs linked to all analyzed twenty-eight genes are shown in Table S2 and expression levels of a subset of those genes in Table S3.

Figure 4. Expression levels from a subset of genes.

Levels of mRNA from indicated genes in the cell lines MCF-7_wt, MCF-7_ADR, OVCAR-5, OVCAR-4 and NCI/ADR-RES in logarithmic scale relative to MCF-7_wt. Error bars indicate standard deviation between triplicates. Values are summarized in Table S3.