Mechanism of Inhibition of the ATPase Domain of Human Topoisomerase IIα by 1,4-Benzoquinone, 1,2-Naphthoquinone, 1,4-Naphthoquinone, and 9,10-Phenanthroquinone
| Autor: | Alok Dhawan, Swatantra Kumar Jain, Ravishankar Ramachandran, Julian Laubenthal, Alok K. Pandey, Ashutosh Kumar, Vandna Kukshal, Diana Anderson, Ashish Arora, Deepak Gurbani, Sarita Tripathi |
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| Rok vydání: | 2012 |
| Předmět: |
Models
Molecular ATPase Toxicology medicine.disease_cause Cell Line Inhibitory Concentration 50 chemistry.chemical_compound Antigens Neoplasm Tandem Mass Spectrometry medicine Humans Topoisomerase II Inhibitors Binding site Adenosine Triphosphatases chemistry.chemical_classification biology Quinones Isothermal titration calorimetry Flow Cytometry Immunohistochemistry Molecular biology Naphthoquinone DNA-Binding Proteins Kinetics DNA Topoisomerases Type II Enzyme chemistry Biochemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Premature chromosome condensation biology.protein Electrophoresis Polyacrylamide Gel DNA Genotoxicity Chromatography Liquid |
| Zdroj: | Toxicological Sciences. 126:372-390 |
| ISSN: | 1096-0929 1096-6080 |
| DOI: | 10.1093/toxsci/kfr345 |
| Popis: | The inhibition of human topoisomerase IIα (Hu-TopoIIα), a major enzyme involved in maintaining DNA topology, repair, and chromosome condensation/decondensation results in loss of genomic integrity. In the present study, the inhibition of ATPase domain of Hu-TopoIIα as a possible mechanism of genotoxicity of 1,4-benzoquinone (BQ), hydroquinone (HQ), naphthoquinone (1,2-NQ and 1,4-NQ), and 9,10-phenanthroquinone (9,10-PQ) was investigated. In silico modeling predicted that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ could interact with Ser-148, Ser-149, Asn-150, and Asn-91 residues of the ATPase domain of Hu-TopoIIα. Biochemical inhibition assays with the purified ATPase domain of Hu-TopoIIα revealed that 1,4-BQ is the most potent inhibitor followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ. Ligand-binding studies using isothermal titration calorimetry revealed that 1,4-BQ, HQ, 1,4-NQ, 1,2-NQ, and 9,10-PQ enter into four sequentially binding site models inside the domain. 1,4-BQ exhibited the strongest binding, followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ, as revealed by their average K(d) values. The cellular fate of such inhibition was further evidenced by an increase in the number of Hu-TopoIIα-DNA cleavage complexes in the human lung epithelial cells (BEAS-2B) using trapped in agarose DNA immunostaining (TARDIS) assay, which utilizes antibody specific for Hu-TopoIIα. Furthermore, the increase in γ-H2A.X levels quantitated by flow cytometry and visualized by immunofluorescence microscopy illustrated that accumulation of DNA double-strand breaks inside the cells can be attributed to the inhibition of Hu-TopoIIα. These findings collectively suggest that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ inhibit the ATPase domain and potentially result in Hu-TopoIIα-mediated clastogenic and leukemogenic events. |
| Databáze: | OpenAIRE |
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