The isolation and identification of cis-diamminedichloroplatinum (II)-DNA adducts by anion exchange HPLC and inductively coupled plasma mass spectrometry
Morrison JG., Bissett D., Stephens IFD., McKay K., Brown R., Graham MA., Fichtinger-Schepman AM., Kerr DJ.
The adducts produced by thc reaction of cisdiamminedichloroplatinum(II) with DNA have previously been isolated and characterised. These adducts may be measured at the cellular level by immunochemical detection but the accuracy of this assay is dependent on the number of adducts per nucleotide. We have developed a novel assay for cisplatin-DNA adducts, utilising an established method in which platinated DNA is digested to form a mixture of nucleotides and adducts; these are then separated by anion exchange HPLC. The number of cisplatin-DNA adducts is determined by measurement of the platinum content of the HPLC fractions by inductively coupled plasma mass spectrometry. The assay has been validated by cochromatography of purified drug-DNA adducts whose identity has been confirmed by NMR. We describe an application of the assay, namely the measurement of in vitro removal of cisplatin-DNA adducts from calf thymus DNA by cell free extracts derived from tumour cell lines. Adduct removal is dependent on both the amount of extract protein and the duration of the reaction. Almost 70% of adducts are removed from 5 μg of DNA (drug:nucleotide ratio 0.08) by 80 μg of extract. Other potential applications of the assay are discussed.