Analysis of bisdioxopiperazine dexrazoxane binding to human DNA topoisomerase II alpha: decreased binding as a mechanism of drug resistance.
Topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double stranded DNA segment and transporting it through another DNA molecule. Despite the extensive use of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, much remains unknown concerning the interactions between these agents and topoisomerase II. To identify the interaction of the bisdioxopiperazine dexrazoxane (ICRF-187) with topoisomerase II, we developed a rapid gel-filtration assay and characterized the binding of ((3)H)-dexrazoxane to human topoisomerase II alpha. Dexrazoxane binds to human topoisomerase II alpha in the presence of DNA and ATP with an apparent K(d) of 23 microM and a stoichiometry of 1 drug molecule per enzyme dimer. Various N-terminal single amino acid substitutions in human topoisomerase II alpha that were previously shown to confer specific bisdioxopiperazine resistance either totally abolished drug binding or resulted in less efficient binding. The effect of the various mutations on drug binding correlated well with their effect on drug resistance in vivo and in vitro. Interestingly, an altered active site tyrosine mutant of human topoisomerase II alpha, which is incapable of carrying out DNA strand passage, was unable to bind dexrazoxane, which agrees with the drug's proposed mechanism of action late in the topoisomerase II catalytic cycle. The direct correlation between the level of drug binding and dexrazoxane resistance is consistent with a decreased drug binding mechanism of action for these dexrazoxane resistance conferring mutations.
Renodon-Cornière
Axelle
A
Department of Pathology, Laboratory and Finsen Centers, Rigshospitalet 5444, DK-2100 Copenhagen, Denmark.
Jensen
Lars H
LH
Nitiss
John L
JL
Jensen
Peter B
PB
Sehested
Maxwell
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D016831:Crithidia fasciculata / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018105:DNA, Kinetoplast / Q000378:metabolism; D004351:Drug Resistance; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D011929:Razoxane / Q000031:analogs & derivatives / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D059005:Topoisomerase II Inhibitors; D014316:Tritium
D018105:DNA, Kinetoplast; D011994:Recombinant Proteins; D059005:Topoisomerase II Inhibitors; D014316:Tritium; D011929:Razoxane; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II