Related Experiment Videos
Mutations of human topoisomerase II alpha affecting multidrug resistance and sensitivity
1Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854, USA.
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.
Insights
Mutations in human topoisomerase II alpha affect drug resistance by altering ATP utilization. These findings clarify the roles of specific mutations in conferring multidrug resistance and sensitivity.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Atypical multidrug resistance (at-MDR) in cancer cells poses a significant challenge to chemotherapy.
- Topoisomerase II-targeting drugs are crucial in cancer treatment, but resistance mechanisms limit their efficacy.
- Specific mutations in the human topoisomerase II alpha gene have been identified in drug-resistant cell lines.
Purpose of the Study:
- To investigate the roles of R450Q and P803S mutations in human topoisomerase II alpha in conferring multidrug resistance.
- To characterize the functional impact of these mutations on enzyme activity, particularly ATP utilization.
- To explore the relationship between ATP utilization defects and drug resistance/sensitivity.
Main Methods:
- Site-directed mutagenesis to create recombinant human topoisomerase II alpha with single or double mutations (R450Q, P803S, G437E).
- Biochemical assays to assess enzyme catalysis, ATP utilization, and protein clamp formation.
- Drug sensitivity testing of cells expressing mutant topoisomerase II alpha enzymes.
Main Results:
- Both R450Q and P803S mutations conferred drug resistance in the absence of ATP.
- In the presence of ATP, only the R450Q mutation conferred multidrug resistance.
- The R450Q mutation impaired ATP utilization for catalysis and clamp formation, while G437E enhanced it.
- The G437E mutation caused multidrug hypersensitivity in the absence of ATP, with minimal effect in the presence of ATP.
Conclusions:
- The study demonstrates that defects in ATP utilization by human topoisomerase II alpha are directly linked to multidrug resistance and sensitivity.
- The R450Q mutation's role in resistance is mediated by impaired ATP binding/utilization.
- These findings provide insights into novel therapeutic strategies targeting topoisomerase II alpha function and drug resistance in cancer.