FORMATION OF DNA-ADDUCTS BY THE ANTICANCER DRUG CARBOPLATIN - DIFFERENT NUCLEOTIDE-SEQUENCE PREFERENCES IN-VITRO AND IN CELLS

We have studied the formation of adducts upon carboplatin treatment of isolated DNA and in cells. The major adduct formed in vitro, determined with atomic absorption spectroscopy and enzyme-linked immunosorbent assay, was the intrastrand cross-link cis-Pt(NH3)(2)d(pGpG)(Pt-GG) (58%). cis-Pt-(NH3)(2)d(pApG) (Pt-AG) (11%), cis-Pt(NH3)(2)d(GMP)(2) (G-Pt-G) (9%), and monofunctionally bound platinum (cis-Pt(NH3)(3)dGMP (Pt-G), 22%) were formed in smaller amounts. These relative occurrences of-the adducts, average values found between 1 and 16 h of incubation, are comparable with those after incubation with cisplatin. The formation of carboplatin-DNA adducts was slow, and about 230-fold more carboplatin than cisplatin (molar dose) was required to obtain equal levels of platination after 4 h of incubation. However, less than 20 times more carboplatin was needed to obtain equal levels of cytotoxicity after 1 h of-exposure of CHO cells. The percentages of the carboplatin-DNA adducts after 7-12 h postincubation of the cells (determined with ELISA), Pt-GG (30%), Pt-AG (16%), G-Pt-G (40%), and Pt-G (14%), were different from those of the in vitro data. After 12 h postincubation, the number of interstrand cross-links (determined by alkaline elution) amounted to about 10% of the G-Pt-G adducts and 3-4% of the total amount of adducts. The immunocytochemical detection (with antiserum NKI-A59) of the platinum-DNA modifications showed a pattern similar to that found for the various bifunctional adducts: the initially low levels slowly increased to maximum values within 7-12 h and then slowly decreased. In conclusion, carboplatin forms the same bifunctional adducts as cisplatin. However, in cells no preference for carboplatin binding on pGpG sequences was found.