title: Topoisomerase II poisoning by ICRF-193.
abstract: Antineoplastic bis(dioxopiperazine)s, such as meso-2,3-bis(2,6-dioxopiperazin-4-yl)butane (ICRF-193), are widely believed to be only catalytic inhibitors of topoisomerase II. However, topoisomerase inhibitors have little or no 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 cytotoxic DNA-damaging agent. Here we report that ICRF-193 is a very significant topoisomerase II poison. Detection of topoisomerase II poisoning by ICRF-193 required the use of a chaotropic protein denaturant in the topoisomerase poisoning assays. ICRF-193 caused dose-dependent cross-linking of human topoisomerase IIbeta to DNA and stimulated topoisomerase IIbeta-mediated DNA cleavage at specific sites on (32)P-end-labeled DNA. Human topoisomerase IIalpha-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 chaotropic protein denaturant in the assays and selectivity for the beta-isozyme of human topoisomerase II. Studies with two topoisomerase IIbeta-negative cell model systems confirmed significant topoisomerase II poisoning by ICRF-193 in the wild type cells and were consistent with beta-isozyme selectivity. Common use of only the detergent, SDS, in assays may have led to failure to detect topoisomerase II poisoning by ICRF-193 in earlier studies.
authors.affiliation: Department of Radiology, The Ohio State University College of Medicine, Columbus, Ohio 43210, USA.
authors.forename: K C
authors.initials: KC
authors.lastname: Huang
authors.forename: H
authors.initials: H
authors.lastname: Gao
authors.forename: E F
authors.initials: EF
authors.lastname: Yamasaki
authors.forename: D R
authors.initials: DR
authors.lastname: Grabowski
authors.forename: S
authors.initials: S
authors.lastname: Liu
authors.forename: L L
authors.initials: LL
authors.lastname: Shen
authors.forename: K K
authors.initials: KK
authors.lastname: Chan
authors.forename: R
authors.initials: R
authors.lastname: Ganapathi
authors.forename: R M
authors.initials: RM
authors.lastname: Snapka
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D002467:Cell Nucleus / Q000201:enzymology; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D054659:Diketopiperazines; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D010879:Piperazines / Q000494:pharmacology*; D013379:Substrate Specificity; D013997:Time Factors; D059005:Topoisomerase II Inhibitors; D014407:Tumor Cells, Cultured
chemical_list: D054659:Diketopiperazines; D004791:Enzyme Inhibitors; D010879:Piperazines; D059005:Topoisomerase II Inhibitors; C070899:4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione; D004250:DNA Topoisomerases, Type II