DNA binding by epipodophyllotoxins and N-acyl anthracyclines: implications for mechanism of topoisomerase II inhibition.

Previous evidence suggests that epipodophyllotoxins, such as etoposide and teniposide, and the N-acyl anthracycline AD41 inhibit topoisomerase II resealing even though they apparently do not bind to DNA. Using experimental conditions designed to detect limited numbers of DNA binding sites, we now report that both epipodophyllotoxins and the N-acyl anthracyclines AD41 and AD32 bind to DNA. Binding was greater to kinetoplast DNA than to pUC18 plasmid DNA. There was also greater etoposide binding to single-stranded DNA than to double-stranded linear or supercoiled DNA. Based on binding competition experiments, etoposide and teniposide appear to have equal affinity for DNA, in spite of the fact that the latter is more potent as a topoisomerase inhibitor. This suggests that the difference in the drugs relates to protein interaction. There are 3- to 7-fold more binding sites for AD41 than for AD32, depending on the DNA substrate employed, and both drugs, unlike adriamycin, exhibit saturation of binding sites over a concentration range of 0-50 microM when kinetoplast DNA is the substrate. Evidence for DNA intercalation by AD41 is provided by the observation that the drug introduces positive supercoils into covalently closed plasmid DNA. Based on these data, a hypothesis is proposed that would provide a general mechanism whereby intercalating agents and epipodophyllotoxins alter topoisomerase function and presumably exert their antitumor effects.