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DNA topoisomerase II isozymes involved in anticancer drug action and resistance
D J Fernandes1, J Qiu, C V Catapano
1Department of Experimental Oncology, Hollings Cancer Center, Medical University of South Carolina, Charleston 29425, USA.
Abstract:
DNA topoisomerase II is a major protein of the nuclear matrix. The enzyme appears to have a central role in both DNA organization and replication. The importance of nuclear matrix topoisomerase II alpha as a target for certain anticancer agents was evaluated in CEM human leukemia cells. Studies were done to determine the extent to which the alpha (170 kDa) and beta (180 kDa) isozymes of topoisomerase II form covalent enzyme-DNA complexes in whole cells and in the nuclear matrix and nonmatrix fractions of CEM cells that are either sensitive or resistant to topoisomerase II-active anticancer agents. Topoisomerase II alpha was detected in both the high salt-soluble (nonmatrix) and matrix fractions of nuclei from parental CEM cells. Most of the matrix topoisomerase II alpha was tightly bound to DNA in cells incubated with VM-26. In contrast, topoisomerase II beta was detected only in the high salt-soluble (nonmatrix) fraction of the nucleus. The subnuclear distribution of the alpha and beta topoisomerase II isozymes in CEM/VM-1 cells resistant to topoisomerase-active drugs was similar to that in drug-sensitive CEM cells. However, the amount and activity of topoisomerase II alpha in nuclear matrices of CEM/VM-1 cells were decreased 3- to 6-fold relative to that of CEM cells. The differences observed in the subnuclear distribution and DNA binding pattern of the topoisomerase II isozymes support the hypotheses that each isozyme has a distinct cellular function. Furthermore, these results provide evidence that topoisomerase II alpha is the nuclear matrix target for VM-26, and that depletion of the nuclear matrix isozyme contributes to cellular resistance to this anticancer agent.
Insights
DNA topoisomerase II alpha, a nuclear matrix protein, is a key target for anticancer drugs like VM-26. Its depletion in resistant leukemia cells explains their reduced drug sensitivity.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- DNA topoisomerase II is crucial for DNA organization and replication.
- It exists as alpha and beta isozymes with potentially distinct functions.
- Nuclear matrix localization is important for enzyme activity and drug targeting.
Purpose of the Study:
- To investigate the role of DNA topoisomerase II alpha in nuclear matrix and its significance as an anticancer drug target.
- To compare the distribution and DNA-binding of topoisomerase II alpha and beta isozymes in drug-sensitive and resistant leukemia cells.
- To elucidate the mechanism of resistance to topoisomerase II-active agents.
Main Methods:
- Cellular fractionation to isolate nuclear matrix and nonmatrix components.
- Analysis of topoisomerase II alpha and beta isozyme distribution and DNA complex formation.
- Comparison between drug-sensitive (CEM) and drug-resistant (CEM/VM-1) human leukemia cells.
Main Results:
- Topoisomerase II alpha was found in both matrix and nonmatrix fractions, while beta was only in the nonmatrix fraction.
- VM-26 treatment induced covalent DNA complex formation primarily with matrix-bound topoisomerase II alpha.
- Drug-resistant cells showed a 3- to 6-fold decrease in nuclear matrix topoisomerase II alpha amount and activity.
Conclusions:
- Topoisomerase II alpha and beta isozymes exhibit distinct subnuclear localization and DNA-binding patterns, suggesting different cellular roles.
- Topoisomerase II alpha in the nuclear matrix is the primary target for the anticancer agent VM-26.
- Reduced levels of nuclear matrix-associated topoisomerase II alpha contribute to cellular resistance against VM-26.