The quantitative detection of various Pt-DNA-adducts in Chinese hamster ovary cells treated with cisplatin: application of immunochemical techniques

Abstract

With polyclonal antibodies raised against cis-Pt(NH3)2Guo-GMP, small quantities of specific Pt-adducts could be detected in DNA from Chinese hamster ovary (CHO) cells treated with the antitumor agent cisplatin, after the DNA had been digested with nucleases and the degradation products separated by anion-exchange chromatography (FPLC). Directly after treatment with 83 microM cisplatin, resulting in 97 X 10(-6) platinum atoms bound per nucleotide, 35.9 +/- 4.7% of the platinum was recovered as cis-Pt(NH3)2d(pGpG), derived from intrastrand cross-links on two neighboring guanines, 3.1 +/- 1.6% as cis-Pt(NH3)2d(GMP)2, the degradation product of interstrand cross-links on two guanines (0.07%, according to separate studies) and of intrastrand cross-links on two guanines separated by one or more bases. The immunochemical method was not sensitive enough for the detection of monofunctionally bound platinum on guanine residues. The amount of these adducts, present in the digests as Pt(NH3)3dGMP, could be established with atomic absorption spectroscopy (AAS) (38.5% of the total Pt-content of the DNA). After a post-treatment incubation of the cells for 24 h, the total amount of platinum decreased to 59 X 10(-6) atoms per nucleotide, indicating the removal of adducts. In the digests, cis-Pt(NH3)2d(pGpG) accounted for 46.4 +/- 6.8% of the total Pt-content, cis-Pt(NH3)2d(GMP)2 for 3.0 +/- 0.9% (0.34% derived from DNA interstrand cross-links). The amounts of monofunctional adducts had decreased to such an extent that the exact quantities (below 15%) could not be determined. According to AAS-assays, at the elution position of cis-Pt(NH3)2d(pApG) a significant amount of Pt-product was present, both at t = 0 and 24 h, but the signals did not allow quantitative evaluation (however, below 48% and 28%, respectively). The possible role of the individual lesions in the DNA in the biological effects of this platinum compound in CHO cells is discussed.