Cisplatin is a platinum-based chemotherapy drug used in the treatment of testicular, ovarian, cervical, lung, and breast cancer. The anti-tumor activity of cisplatin is attributed to the formation of DNA 1,2- and 1,3-intrastrand and interstrand cross-links. Despite its clinical utility, the development of resistance and toxic side effects remain big challenges associated with cisplatin therapy. Potential mechanisms of resistance include changes in drug influx and efflux, cisplatin metabolism/activation, and repair rates of cisplatin-induced DNA damage. All three resistance mechanisms influence the abundance and distribution of DNA-DNA cross-links. Therefore, we hypothesize that the biological response to cisplatin can be accurately predicted by measuring the formation and repair of these cisplatin DNA cross-links in cells or tumors. To address this goal, we have developed an isotope dilution UPLC-SIM assay to quantify cisplatin-induced 1,2-GG, 1,2-AG, 1,3-GCG, and 1,3-GTG intrastrand and interstrand crosslinks. The DNA digestion and monomer enrichment methodology for each analyte were optimized using synthetic DNA oligonucleotides containing specific cross-links and platinated calf thymus DNA. We then monitored the formation and repair of cross-links over time in nucleotide excision repair (NER)-deficient cell lines and isogenic control cell lines and are extending these studies to additional cell lines with mutations in DNA repair genes. Successful application of this assay in cells is expected to be useful in clinical practice to predict the outcome of cisplatin therapy.

Monitoring the formation and repair kinetics of cisplatin-induced DNA-DNA cross-links by comprehensive ultra-performance liquid chromatograph-selective ion monitoring (UPLC-SIM)

ACS Fall 2021

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