Cisplatin has been shown to create interstrand DNA cross-links by several methods. These experiments were performed to determine if cisplatin coordinates cross-links between deoxyguanylic and deoxycytidylic acids. After incubation at pH 7.2 with cisplatin at various platinum/base nucleotide ratios, ri, for 3 days at 20°, polydeoxyguanylic acid·polydeoxycytidylic acid [poly(dG)·poly(dC)] was centrifuged in alkaline cesium sulfate for 3 days at 35,000 rpm. Untreated samples resulted in a low-density deoxycytidylic acid peak and a high-density deoxyguanylic acid peak, as identified by the ultraviolet absorption spectrum. The density gradient profiles of cisplatin-treated poly(dG). poly(dC) had an additional hybrid-density peak the area of which increased with the dose (ri) as the areas under the polydeoxycytidylic acid [poly(dC)] and polydeoxyguanylic acid [poly(dG)] peaks diminished. With increasing ri, the densities of both the poly(dG) and hybrid peaks increased while that of the poly(dC) peak remained unchanged. Similarly, the amount of bound platinum in the poly(dG) and hybrid-density peaks increased with ri, while no platinum could be detected in the poly(dC) peak. Therefore, cisplatin appears to react first with the deoxyguanylic acid strand, followed by a second reaction with the deoxycytidylic acid strand to form the interstrand cross-links that are necessary for the formation of the hybrid-density bands. Based upon the intercept of the percent cross-linked poly(dG)·poly(dC) versus ri curve with the abscissa, the minimum amount of cisplatin or the threshold amount of cisplatin required to observe cross-links was ri = 0.017. When multiplied by the molecular weight of the poly(dG)·poly(dC) used, this was equivalent to 1 cross-link per 15 molecules of bound platinum. However, based upon the amount of bound drug, the frequency of cross-links per platinum reaction was calculated to range from one of 50 to 67 reactions. Due to limitations in the sensitivity of the platinum assay and to a progressively lower recovery of the cross-linked material at lower ri values, it was not possible to calculate the cross-linking efficiency based upon the amount of bound drug for ri values equal to or less than 0.025. The monofunctional analogue, [diethylenetriaminechloroplatinum] chloride, did not cause a hybrid peak to form. These studies demonstrate that cisplatin coordinates interstrand cross-links between poly(dG) and poly(dC).
This investigation was supported by National Cancer Institute Grant CA25736.