Topoisomerase II poisoning by ICRF-193

Antineoplastic bis(dioxopiperazine)s, such as meso-2,3-bis(2,6-dioxopiperaz in-4-yl)butane (ICRF-193), are widely believed to be only catalytic inhibit ors of topoisomerase II. However, topoisomerase inhibitors have little or n o antineoplastic activity unless they are topoisomerase poisons, a special subclass of topoisomerase-targeting drugs that stabilize topoisomerase-DNA strand passing intermediates and thus cause the topoisomerase to become a c ytotoxic DNA-damaging agent. Here we report that ICRF-193 is a very signifi cant topoisomerase II poison. Detection of topoisomerase II Poisoning by IC RF-193 required the use of a chaotropic protein denaturant in the topoisome rase poisoning assays. ICRF-193 caused dose-dependent cross-linking of huma n topoisomerase II beta to DNA and stimulated topoisomerase II beta -mediat ed DNA cleavage at specific sites on P-32-end-labeled DNA. Human topoisomer ase Ha-mediated DNA cleavage was stimulated to a lesser extent by ICRF-193. In vivo experiments with MCF-7 cells also showed the requirement of a chao tropic protein denaturant in the assays and selectivity for the beta -isozy me of human topoisomerase II. Studies with two topoisomerase II beta -negat ive cell model systems confirmed significant topoisomerase II poisoning by ICRF-193 in the wild type cells and were consistent with beta -isozyme sele ctivity. Common use of only the detergent, SDS, in assays may have led to f ailure to detect topoisomerase II poisoning by ICRF-193 in earlier studies.