Development of Comprehensive Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry Assays to Quantitate Cisplatin-Induced DNA-DNA Cross-Links

ABSTRACT

Cisplatin (CP) is a common anti-tumor drug used to treat many solid tumors. The activity of CP is attributed to the formation of DNA-DNA cross-links, which consist of 1,2-intra-, 1,3-intra-, and interstrand cross-links. To better understand how each intrastrand cross-link contributes to the activity of CP, we have developed comprehensive ultraperformance liquid chromatography-selective ion monitoring (UPLC-SIM) assays to quantify 1,2-GG, 1,2-AG, 1,3-GCG, and 1,3-GTG-intrastrand cross-links. The limit of quantitation for the developed assays ranged from 5 – 50 fmol, or as low as 6 cross-links per 10⁸ nucleotides. To demonstrate the utility of the UPLC-SIM assays, we first performed in vitro cross-link formation kinetics experiments. We confirmed 1,2-GG-intrastrand cross-links were the most abundant intrastrand cross-link and formed at a faster rate compared to 1,2-AG- and 1,3-intrastrand cross-links. Furthermore, we investigated the repair kinetics of intrastrand cross-links in CP-treated wild type and nucleotide excision repair (NER)-deficient U2OS cells. We observed slow repair of both 1,2- and 1,3-intrastrand cross-links in wild type cells, and no evidence of repair in the NER-deficient cells. Taken together, we have demonstrated that our assay is capable of accurately quantifying intrastrand cross-links in CP-treated samples and can be utilized to better understand the activity of CP.