DNA cleavage induced by inhibitors of topoisomerases I (camptothecin) and II (anthracyclines, amsacrine, VP-16, VM-26, and ellipticines) can be produced by purified mouse leukemia L1210 topoisomerases in (32)P-end labeled DNA fragments. Both purification of topoisomerases and preparation of DNA fragments were performed in the laboratory. DNA sequence analysis of cleavage sites induced by different inhibitors were investigated in order to study the molecular interactions between drugs and topoisomerase-DNA complexes. For some drugs (anthracyclines and camptothecins), a model emerged in which the drug seems to bind inside a cavity formed by the base pairs flanking the cleavage site (-l and +1 bases) and the enzyme. Drug effects in chromatin were also investigated. First, we found that SV40 DNA contained a nuclear matrix attachment site (MAR) located in the early transcription region (nucleotides 4071-4377) and that the same region contained the highest density of anthracycline-induced cleavage sites. Secondly, we found that topoisomerase II-mediated DNA cleavage was suppressed in reconstituted nucleosomes, and therefore drug activity depends upon chromatin structure. Finally, a major m-AMSA-induced DNA cleavage site was found in the promoter region of the c-myc gene, suggesting a role of topoisomerase II in transcription regulation and that the induction of cleavage sites in gene promoter regions may be important for the antitumor activity of topoisomerase II inhibitors.
