Mutations of human topoisomerase II alpha affecting multidrug resistance and sensitivity

Biochemistry. 1999 Aug 17;38(33):10793-800. doi: 10.1021/bi9909804.

Abstract

Two mutations, R450Q and P803S, in the coding region of the human topoisomerase II alpha 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 II alpha. 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 II alpha 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Adenosine Triphosphate / pharmacology
  • Antigens, Neoplasm
  • Antineoplastic Agents / pharmacology*
  • Arginine / genetics
  • Catalysis
  • DNA Topoisomerases, Type II* / genetics*
  • DNA Topoisomerases, Type II* / isolation & purification
  • DNA Topoisomerases, Type II* / metabolism
  • DNA-Binding Proteins
  • Dose-Response Relationship, Drug
  • Drug Resistance, Multiple / genetics*
  • Drug Resistance, Neoplasm / genetics
  • Enzyme Activation / drug effects
  • Enzyme Activation / genetics
  • Glutamic Acid / genetics
  • Glutamine / genetics
  • Glycine / genetics
  • Humans
  • Isoenzymes / genetics*
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism
  • Mutagenesis, Site-Directed*
  • Proline / genetics
  • Serine / genetics
  • Teniposide / pharmacology
  • Tumor Cells, Cultured / drug effects
  • Tumor Cells, Cultured / enzymology

Substances

  • Antigens, Neoplasm
  • Antineoplastic Agents
  • DNA-Binding Proteins
  • Isoenzymes
  • Glutamine
  • Glutamic Acid
  • Serine
  • Adenosine Triphosphate
  • Arginine
  • Teniposide
  • Proline
  • DNA Topoisomerases, Type II
  • Glycine