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Modulation of human DNA topoisomerase IIalpha function by interaction with 14-3-3epsilon
E U Kurz1, K B Leader, D J Kroll
1Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center and University of Colorado Cancer Center, Denver, Colorado 80262, USA.
Abstract:
Human DNA topoisomerase IIalpha (topo II), a ubiquitous nuclear enzyme, is essential for normal and neoplastic cellular proliferation and survival. Several common anticancer drugs exert their cytotoxic effects through interaction with topo II. In experimental systems, altered topo II expression has been associated with the appearance of drug resistance. This mechanism, however, does not adequately account for clinical cases of resistance to topo II-directed drugs. Modulation by protein-protein interactions represents one mechanism of topo II regulation that has not been extensively defined. Our laboratory has identified 14-3-3epsilon as a topo II-interacting protein. In this study, glutathione S-transferase co-precipitation, affinity column chromatography, and immunoprecipitations confirm the authenticity of these interactions. Three assays evaluate the impact of 14-3-3epsilon on distinct topo II functional properties. Using both a modified alkaline comet assay and a DNA cleavage assay, we demonstrate that 14-3-3epsilon negatively affects the ability of the chemotherapeutic, etoposide, to trap topo II in cleavable complexes with DNA, thereby preventing DNA strand breaks. By electrophoretic mobility shift assay, this appears to be due to reduced DNA binding activity. The association of topo II with 14-3-3 proteins does not extend to all 14-3-3 isoforms. No protein interaction or disruption of topo II function was observed with 14-3-3final sigma.
Insights
The protein 14-3-3epsilon interacts with human DNA topoisomerase IIalpha (topo II), inhibiting its function. This interaction prevents etoposide from forming DNA-damaging complexes, potentially explaining drug resistance.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Human DNA topoisomerase IIalpha (topo II) is crucial for cell proliferation and targeted by anticancer drugs.
- Drug resistance to topo II-targeting therapies is a clinical challenge not fully explained by expression levels.
- Protein-protein interactions are an understudied mechanism for regulating topo II function.
Purpose of the Study:
- To investigate the interaction between 14-3-3epsilon and human topo II.
- To determine the functional consequences of this interaction on topo II activity.
- To explore the role of 14-3-3epsilon in modulating the efficacy of topo II-targeting drugs like etoposide.
Main Methods:
- Glutathione S-transferase co-precipitation, affinity column chromatography, and immunoprecipitations were used to confirm protein interactions.
- Modified alkaline comet assay and DNA cleavage assays were employed to assess topo II function.
- Electrophoretic mobility shift assay (EMSA) was used to evaluate DNA binding activity.
Main Results:
- 14-3-3epsilon physically interacts with human topo II.
- 14-3-3epsilon inhibits etoposide-induced DNA damage by preventing the formation of cleavable complexes.
- This inhibition is associated with reduced DNA binding activity of topo II.
- The interaction is specific to certain 14-3-3 isoforms, with 14-3-3sigma showing no interaction or functional effect.
Conclusions:
- 14-3-3epsilon negatively regulates human topo II activity.
- This interaction may represent a novel mechanism contributing to resistance against etoposide and potentially other topo II-targeting anticancer drugs.
- Targeting the topo II-14-3-3epsilon interaction could offer new therapeutic strategies in cancer treatment.