Effects of DNA intercalating agents on topoisomerase II induced DNA strand cleavage in isolated mammalian cell nuclei

Abstract

Intercalator-induced DNA double-strand breaks (DSB) presumably represent topoisomerase II DNA cleavage sites in mammalian cells. Isolated L1210 cell nuclei were used to determine the saturability of this reaction at high drug concentrations. 4''-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) and 5-iminodaunorubicin (5-ID) both produced DSB in a concentration-dependent manner, and the production of these breaks leveled off above 10 .mu.M. Addition of m-AMSA to 5-ID-treated nuclei did not raise the plateau level. Thus, both drugs seemed to interact similarly on identical targets. The ellipticine derivative 2-methyl-9-hydroxyellipticinium (2-Me-9-OH-E+) had two effects on the production of DSB. Below 10 .mu.M, 2-Me-9-OH-E+ produced DSD as did ellipticine, m-AMSA, or 5-ID. Above 10 .mu.M, 2-Me-9-OH-E+ did not induce DSB and inhibited the DSB induced by m-AMSA, 5-ID, or ellipticine. 2-Me-9-OH-E+ and m-AMSA competed with each other to produce either double-strand break formation (m-AMSA-induced reaction) or double-strand break inhibition (2-Me-9-OH-E+-induced reaction at concentrations greater than 10 .mu.M). Because these results were reproduced in experiments using DNA topoisomerase II isolated from L1210 nuclei, it is likely that the intercalator-induced protein-associated DNA breaks detected by alkaline elution in nuclei represent DNA topoisomerase II-DNA complexes. The intercalators appear to interact reversibly and competitively, but in different ways, with these complexes, m-AMSA, 5-ID, ellipticine, and 2-Me-9-OH-E+, the last two at concentrations below 10 .mu.M, probably stabilize the enzyme-DNA complexes in an open (cleaved) conformation of DNA, whereas high concentrations of 2-Me-9-OH-E+ destabilize the DNA topoisomerase II DNA cleavage intermediates.