Formation and longevity of idarubicin-induced DNA topoisomerase II cleavable complexes in K562 human leukaemia cells.
Idarubicin (IDA) is an anthracycline used during treatment of acute myelogenous leukaemia (AML) and is clinically important because of its potency and lipophilicity (compared to the related compounds daunorubicin and doxorubicin). These drugs target DNA topoisomerase II (topo II), a nuclear enzyme that regulates DNA topology. Topo II poisoning leads to the trapping of an intermediate in the enzyme's cycle termed the "cleavable complex." This study aims to increase understanding of drug interactions by use of the "TARDIS" (trapped in agarose DNA immunostaining) assay to measure drug-induced topo II cleavable complexes in individual cells treated with anthracyclines. Mammalian cells contain two isoforms of topo II (alpha and beta) and the TARDIS assay enables visualisation of isoform-specific complexes. Drug-treated cells were embedded in agarose, lysed and incubated with anti-topo II antibodies to microscopically detect topo IIalpha or beta complexes. Results for K562 cells (at clinically relevant concentrations) showed that IDA and idarubicinol, its metabolite, formed mainly topo IIalpha cleavable complexes, the level of which decreases at doses > 1 microM for IDA. Our data suggest that this decrease is due to catalytic inhibition by IDA at these doses. Doxorubicin formed low levels of topo IIalpha complexes and negligible topo IIbeta complexes. In cytotoxicity studies, IDA and idarubicinol were equipotent, but both were more potent than daunorubicin and doxorubicin. We showed for the first time that there was a persistent increase in levels of topo IIalpha cleavable complexes after removal of IDA, suggesting that its greater effectiveness may be associated with both the longevity and high levels of these complexes.
Willmore
Elaine
E
School of Biochemistry & Genetics, Medical School, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne NE2 4HH, UK.
Errington
Fiona
F
Tilby
Michael J
MJ
Austin
Caroline A
CA
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D020134:Catalytic Domain / Q000187:drug effects; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D003630:Daunorubicin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D015255:Idarubicin / Q000494:pharmacology*; D020014:K562 Cells; D007938:Leukemia / Q000473:pathology; D059005:Topoisomerase II Inhibitors
D000903:Antibiotics, Antineoplastic; D004791:Enzyme Inhibitors; D059005:Topoisomerase II Inhibitors; D004247:DNA; D004250:DNA Topoisomerases, Type II; D015255:Idarubicin; D003630:Daunorubicin