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 approximately 90% of the total Pt in the DNA, Pt was linked to the N7 atoms of the nucleobases guanine and adenine. The two 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 two guanines as well as from intrastrand cross-links on two guanines separated by one or more bases. For analytical purposes we developed an improved method to determine cis-DDP adducts. Routinely, 40-micrograms 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.

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)2d(pGpG)(Pt-GG) (58%). cis-Pt-(NH3)2d(pApG) (Pt-AG) (11%), cis-Pt(NH3)2d(GMP)2 (G-Pt-G) (9%), and monofunctionally bound platinum (cis-Pt(NH3)3dGMP (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.(ABSTRACT TRUNCATED AT 250 WORDS)

A method has been developed to determine the adducts formed upon interaction of cis- and trans-diamminedichloroplatinum(II) (cis- and trans-DDP) with DNA. After 5 h at 50 degrees C in the dark, the amount of cis-DDP bound to salmon sperm DNA was larger than the amount of the trans-isomer. After enzymatic degradation with deoxyribonucleases to nucleotides and Pt-containing (oligo)nucleotides, the various products were separated by DEAE chromatography and analyzed for Pt by flameless AAS. Indications were obtained for the presence of nucleotides containing monofunctionally bound Pt and of adducts originating from interstrand DNA crosslinks. DEAE chromatography of digests of cis-DDP-treated DNA yielded a product with overall charge -1, which was identified with NMR and CD as cis-[Pt(NH3)2-d(pGpG)], the oligonucleotide derived from intrastrand crosslinks between two adjacent guanines. Another major peak contained Pt-oligonucleotides with overall charge -2, which could be derived from intrastrand crosslinks between two guanines at sites with pGpXpG (X=T,C,A or G) base sequences.

Cell killing and mutation induction by cis-and trans-Pt(NH3)2Cl2 in Escherichia coli were examined by studying forward mutagenesis in the lad gene in cells with different repair capacities. Survival experiments showed that repair-proficient cells were slightly more sensitive for the cis isomer than for the trans isomer, whereas repair-deficient RecA and UvrB cells were extremely sensitive only for the cis compound. cis-Pt(NH3)2CI2 induced mutagenesis in both wild-type cells and RecA cells but not in UvrB cells; whereas no detectable mutagenesis was induced by treatment with the trans compound. Examination of the nature of the mutations induced by cis-Pt(NH3)WCI2, by. using the Lad system, revealed that base-pair substitutions leading to nonsense mutants are only induced in wild-type cells, suggesting that the intact products of both the uvrB and the recA gene are necessary for the repair responsible for this type of mutagenesis. Investigation ofthe nonsense mutants reveals that 70% ofthese mutations result from GC --TA or GC --AT substitutions at sites where the guanine is part of a GAG or GCG sequence. These results are discussed in relation to existing theories on the interaction between Pt compounds and DNA. A model for Pt-DNA adducts, leading to base-pair.substitutions, is proposed.About a decade ago, Rosenberg et aL (1) reported that cisPt(NH3)2CI2 shows antitumor activity against sarcoma 180 and leukemia L1210, whereas trans-Pt(NH3)2C12 is ineffective.Since then it has been shown that several other cis Pt(II) and cis Pt(IV) compounds exhibit antitumor activity (2, 3). In mammalian, as well as in bacterial cells, DNA is the preferential target for Pt compounds. For cis-Pt(NH3)2CI2 this interaction results in lesions that selectively block DNA replication (4, 5). In this respect, cis-Pt compounds behave similar to other drugs such as alkylating and radiomimetic agents.In vitro, cis-Pt(NH3)2C12 binds to bases in DNA, and the order of binding affinity has been shown to be guanine>ad-enine>cytosine>>thymine, with a strong preference for the N-7 position of guanine (6). Monofunctional binding to a single base is unlikely to be the principal lesion through which cisPt(NH3)2CI2 exerts its antitumor activity because, at equitoxic doses, more of the inactive trans compound is bound to DNA (7). Therefore, specific bifunctional binding of cis-Pt(NH3)2CI2 to DNA is thought to be responsible for its antitumor activity.For the bifunctional mode of action, several models have been proposed such as intrabase chelation at the 0-6 and N-7 positions of guanine (6,8), interstrand crosslinking between the N-7 positions of guanines in opposite strands (3,7,9), and intrastrand crosslinks between two, presumably adjacent, guanines in the same strand (10-12).In bacteria (13-17), as well as in eukaryotic cells (18)(19)(20), a correlation between mutagenicity and antitumor activity ofseveral Pt compounds has been found, suggesting that lesions leading to mutation events can also be responsible for antitumor activity. Several...

The interaction of the anti-cancer drug cis-diamminedichloroplatinum(II) (cisPt) with model membranes was studied, with emphasis on the cisPt and phospholipid species involved. Binding studies using large unilamellar vesicles have revealed that: (i) Interaction involved negatively charged phospholipids only, and (ii) Interaction with negatively charged phospholipids was observed only in buffers with low Cl- concentration, indicating that aquated, positively charged cisPt is involved. Binding to all negatively charged phospholipids tested was highest at pH 6.0. At pH 7.4 a high and specific binding was observed with phosphatidic acid and phosphatidylserine. The consequences of cisPt binding on the organization of lipids was investigated with differential scanning calorimetry studies. These studies have indicated a higher ordering of dispersions of negatively charged phospholipids in the presence of divalent cationic cisPt. Summarizing, the interaction of positively charged cisPt species with negatively charged phospholipids is significant and should be considered in in vivo experiments.