title: A role of DNA topoisomerases in the active dissociation of DNA minor groove-ligand complexes. A flow cytometric study of inhibitor effects.
abstract: The biological effects of a number of DNA ligands which interact with the minor groove of B-form DNA (e.g. netropsin, distamycin and Hoechst 33258) are thought to arise from the direct disturbance of the processes of DNA replication and transcription. Although ligand binding appears to be an important factor in cytotoxicity, the pathways by which drug molecules can be actively dissociated from nuclear DNA are unknown. Recent evidence suggests that minor groove ligands can distort the manner in which DNA associates with nucleosomal core particles and we have hypothesized that in an intact cell such imposed torsional stress could be subject to the action of cellular topoisomerases. We have used flow cytometry to study the effects of various inhibitors (including topoisomerase-interactive drugs) on the responses of a mutant cell line (HoeR415) to Hoechst 33342, given that the mutant shows resistance to the cytotoxicity of this DNA-specific dye due to an enhanced capacity to dissociate nuclear DNA-dye complexes. Ligand-DNA dissociation in the mutant was found to be energy-dependent but not specifically, affected by the drug-efflux blocker verapamil or by inhibitors of DNA synthesis. The topoisomerase II inhibitors novobiocin, VP16, nalidixic acid and the topoisomerase I-interactive drug camptothecin inhibited ligand-DNA dissociation to various extents with novobiocin being the most effective (100% inhibition at 1 mM). Both novobiocin and camptothecin were without effect on the nuclear loss of a DNA intercalator, adriamycin. We conclude that efficient topoisomerase activity is required for the active dissociation of DNA minor groove-ligand complexes.
authors.affiliation: MRC Clinical Oncology and Radiotherapeutics Unit, MRC Centre, Cambridge, Great Britain.
authors.forename: P J
authors.initials: PJ
authors.lastname: Smith
authors.forename: P G
authors.initials: PG
authors.lastname: Debenham
authors.forename: J V
authors.initials: JV
authors.lastname: Watson
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism*; D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism* / Q000648:ultrastructure; D004264:DNA Topoisomerases, Type I / Q000502:physiology*; D005434:Flow Cytometry; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D009675:Novobiocin / Q000494:pharmacology; D013937:Thymidine Kinase / Q000172:deficiency; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D014965:X-Rays
chemical_list: D001562:Benzimidazoles; D008024:Ligands; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D009675:Novobiocin; D004247:DNA; D013937:Thymidine Kinase; D004264:DNA Topoisomerases, Type I; C017807:bisbenzimide ethoxide trihydrochloride; D002166:Camptothecin