title: Amiloride intercalates into DNA and inhibits DNA topoisomerase II.
abstract: Amiloride is capable of inhibiting DNA synthesis in mammalian cells in culture. Recent evidence indicates that the enzyme, DNA topoisomerase II, is probably required for DNA synthesis to occur in situ. In experiments to determine the mechanism of inhibition of DNA synthesis by amiloride, we observed that amiloride inhibited both the catalytic activity of purified DNA topoisomerase II in vitro and DNA topoisomerase II-dependent cell functions in vivo. Many compounds capable of inhibiting DNA topoisomerase II are DNA intercalators. Thus, we performed studies to determine if and how amiloride bound to DNA. Results indicated that amiloride 1) shifted the thermal denaturation profile of DNA, 2) increased the viscosity of linear DNA, and 3) unwound circular DNA, all behavior consistent with a DNA intercalation mechanism. Furthermore, quantitative and qualitative measurements of amiloride fluorescence indicated that amiloride (a) bound reversibly to purified DNA under conditions of physiologic ionic strength, and (b) bound to purified nuclei in a highly cooperative manner. Lastly, amiloride did not promote the cleavage of DNA in the presence of DNA topoisomerase II, indicating that the mechanism by which amiloride inhibited DNA topoisomerase II was not through the stabilization of a "cleavable complex" formed between DNA topoisomerase II, DNA, and amiloride. The ability of amiloride to intercalate with DNA and inhibit topoisomerase II is consistent with the proposed planar, hydrogen-bonded, tricyclic nature of amiloride's most stable conformation. Thus, DNA and DNA topoisomerase II must be considered as new cellular targets of amiloride action.
authors.affiliation: Department of Molecular Biology, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
authors.forename: J M
authors.initials: JM
authors.lastname: Besterman
authors.forename: L P
authors.initials: LP
authors.lastname: Elwell
authors.forename: S G
authors.initials: SG
authors.lastname: Blanchard
authors.forename: M
authors.initials: M
authors.lastname: Cory
mesh_terms: D000584:Amiloride / Q000494:pharmacology*; D002273:Carcinogens / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007364:Intercalating Agents; D009675:Novobiocin / Q000494:pharmacology; D009691:Nucleic Acid Denaturation; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000494:pharmacology; D010957:Plasmids; D059005:Topoisomerase II Inhibitors; D014783:Viscosity
chemical_list: D002273:Carcinogens; D007364:Intercalating Agents; D010703:Phorbol Esters; D059005:Topoisomerase II Inhibitors; D009675:Novobiocin; D015240:Phorbol 12,13-Dibutyrate; D000584:Amiloride; D004247:DNA