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Mutations of Human Topoisomerase IIα Affecting Multidrug Resistance and Sensitivity

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Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, and Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101
Cite this: Biochemistry 1999, 38, 33, 10793–10800
Publication Date (Web):July 29, 1999
https://doi.org/10.1021/bi9909804
Copyright © 1999 American Chemical Society
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Abstract

Two mutations, R450Q and P803S, in the coding region of the human topoisomerase IIα gene have been identified in the atypical multidrug resistant (at-MDR) cell line, CEM/VM-1, which exhibits resistance to many structurally diverse topoisomerase II-targeting antitumor drugs such as VM-26, doxorubicin, m-AMSA, and mitoxantrone. The R450Q mutation mapped in the ATP utilization domain, while the P803S mutation mapped in the vicinity of the active site tyrosine of human topoisomerase IIα. However, the roles of these two mutations in conferring multidrug resistance are unclear. To study the roles of these two mutations in conferring multidrug resistance, we have characterized the recombinant human DNA topoisomerase IIα containing either single or double mutations. We show that both R450Q and P803S mutations confer resistance in the absence of ATP. However, in the presence of ATP, the R450Q, but not the P803S, mutation can confer multidrug resistance. The R450Q enzyme was shown to exhibit impaired ATP utilization both for enzyme catalysis and for its ability to form the circular protein clamp. Interestingly, an unrelated mutation, G437E, which is also located in the same domain as the R450Q mutation, exhibited multidrug hypersensitivity in the absence of ATP. However, in the presence of ATP, the G437E enzyme is only minimally hypersensitive to various topoisomerase II drugs. In contrast to the R450Q enzyme, the G437E enzyme exhibited enhanced ATP utilization for enzyme catalysis. In the aggregate, these results support the notion that the multidrug resistance and sensitivity of these mutant enzymes are due to a specific defect in ATP utilization during enzyme catalysis.

 This work was supported by NIH Grants CA39962 and GM27731 (to L.F.L.), CA21765 and CA52814 (to J.L.N.), and GM29006 (to T.-S.H.).

 University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School.

§

 Duke University Medical Center.

 St. Jude Children's Research Hospital.

*

 To whom correspondence should be addressed:  Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Telephone:  (732) 235-4592. Fax:  (732) 235-4073. E-mail:  [email protected]

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