Binding of etoposide to topoisomerase II in the absence of DNA: decreased affinity as a mechanism of drug resistance.
Despite the prevalence of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, interactions between these agents and topoisomerase II are not well understood. Therefore, to further define interactions between anticancer drugs and the type II enzyme, a nitrocellulose filter assay was used to characterize the binding of etoposide to yeast topoisomerase II. Results indicate that etoposide binds to the enzyme in the absence of DNA. The apparent Kd value for the interaction was approximately 5 microM drug. Etoposide also bound to ytop2H1012Y, a mutant yeast type II enzyme that is approximately 3-4-fold resistant to etoposide. However, the apparent Kd value for the drug (approximately 16 microM) was approximately 3 times higher than that determined for wild-type topoisomerase II. Although it has been widely speculated that resistance to topoisomerase II-targeted anticancer agents results from a decreased drug-enzyme binding affinity, these data provide the first direct evidence in support of this hypothesis. Finally, the ability of yeast topoisomerase II to bind etoposide was dependent on the presence of the hydroxyl moiety of Tyr783, suggesting specific interactions between etoposide and the active site residue that is involved in DNA scission.
Kingma
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PS
Departments of Biochemistry and Medicine (Hematology/Oncology), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Burden
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DA
Osheroff
N
N
D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005047:Etoposide / Q000378:metabolism* / Q000494:pharmacology; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014443:Tyrosine / Q000378:metabolism
D000970:Antineoplastic Agents; D014443:Tyrosine; D005047:Etoposide; D004247:DNA; D004250:DNA Topoisomerases, Type II