Adducts of the antitumor drug cis-diamminedichloroplatinum(II) with DNA: formation, identification, and quantitation

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Abstract
Salmon sperm DNA, treated with the antitumor agent cis-diamminedichloroplatinum(II) (cis-DDP), was enzymatically degraded to (oligo)nucleotides. Four Pt-containing products were identified by 1H NMR after preparative chromatography on a diethylaminoethyl-Sephacel column at pH 8.8. In all identified adducts, comprising .apprx. 90% of the total Pt in the DNA, Pt was linked to the N7 atoms of the nucleobases guanine and adenine. The 2 major adducts were cis-Pt(NH3)2d(pGpG) and cis-Pt(NH3)2d(pApG), both derived from intrastrand cross-links of cis-DDP on neighboring nucleobases. Only the d(pApG) but not the d(pGpA) adduct could be detected. Two minor adducts were Pt(NH3)3dGMP, resulting from monofunctionally bound cis-DDP to guanine, and cis-Pt(NH3)2d(GMP)2, originating from interstrand cross-links on 2 guanines as well as from intrastrand cross-links on 2 guanines separated by 1 or more bases. For analytical purposes an improved method was developed to determine cis-DDP adducts. Routinely, 40-.mu.g samples of enzymatically degraded cis-DDP-treated DNA are now analyzed by separation of the mononucleotides and Pt-containing (oligo)nucleotides on the anion-exchange column Mono Q (FPLC) at pH 8.8 (completed within 14 min) and subsequent determination of the Pt content in the collected fractions by atomic absorption spectroscopy. The method was used to optimize the digestion conditions for cis-DDP-treated DNA. In kinetic studies on the formation of the various adducts, a clear preference of the Pt compound to react with guanines occurring in the base sequence d(pGpG) was established.