A Practical Site-specific Method for the Detection of Bulky DNA Damages.

Helix-distorting DNA damages block RNA and DNA polymerase, compromising cell function and fate. In human cells, these damages are removed primarily by nucleotide excision repair (NER). Here, we describe damage-sensing PCR (dsPCR), a PCR-based method for the detection of these DNA damages. Exposure to DNA damaging agents results in lower PCR signal in comparison to non-damaged DNA, and repair is measured as the restoration of PCR signal over time. We show that the method successfully detects damages induced by ultraviolet (UV) radiation, by the carcinogenic component of cigarette smoke benzo[a]pyrene diol epoxide (BPDE) and by the chemotherapeutic drug cisplatin. Damage removal measured by dsPCR in a heterochromatic region is less efficient than in a transcribed and accessible region. Furthermore, lower repair is measured in repair-deficient knock-out cells. This straight-forward method could be applied by non-DNA repair experts to study the involvement of their gene-of-interest in repair. Furthermore, this method is fully amenable for high-throughput screening of DNA repair activity.

Genomic Characterization of Cisplatin Response Uncovers Priming of Cisplatin-Induced Genes in a Resistant Cell Line.

Cisplatin is a chemotherapy drug that kills cancer cells by damaging their DNA. In human cells, this damage is repaired primarily by nucleotide excision repair. While cisplatin is generally effective, many cancers exhibit initial or acquired resistance to it. Here, we studied cisplatin resistance in a defined cell line system. We conducted a comprehensive genomic characterization of the cisplatin-sensitive A2780 ovarian cancer cell line compared to A2780cis, its resistant derivative. The resistant cells acquired less damage, but had similar repair kinetics. Genome-wide mapping of nucleotide excision repair showed a shift in the resistant cells from global genome towards transcription-coupled repair. By mapping gene expression changes following cisplatin treatment, we identified 56 upregulated genes that have higher basal expression in the resistant cell line, suggesting they are primed for a cisplatin response. More than half of these genes are novel to cisplatin- or damage-response. Six out of seven primed genes tested were upregulated in response to cisplatin in additional cell lines, making them attractive candidates for future investigation. These novel candidates for cisplatin resistance could prove to be important prognostic markers or targets for tailored combined therapy in the future.