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Immunodetection of human topoisomerase I-DNA covalent complexes
Anand G Patel1, Karen S Flatten2, Kevin L Peterson2
1Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
Abstract:
A number of established and investigational anticancer drugs slow the religation step of DNA topoisomerase I (topo I). These agents induce cytotoxicity by stabilizing topo I-DNA covalent complexes, which in turn interact with advancing replication forks or transcription complexes to generate lethal lesions. Despite the importance of topo I-DNA covalent complexes, it has been difficult to detect these lesions within intact cells and tumors. Here, we report development of a monoclonal antibody that specifically recognizes covalent topo I-DNA complexes, but not free topo I or DNA, by immunoblotting, immunofluorescence or flow cytometry. Utilizing this antibody, we demonstrate readily detectable topo I-DNA covalent complexes after treatment with camptothecins, indenoisoquinolines and cisplatin but not nucleoside analogues. Topotecan-induced topo I-DNA complexes peak at 15-30 min after drug addition and then decrease, whereas indotecan-induced complexes persist for at least 4 h. Interestingly, simultaneous staining for covalent topo I-DNA complexes, phospho-H2AX and Rad51 suggests that topotecan-induced DNA double-strand breaks occur at sites distinct from stabilized topo I-DNA covalent complexes. These studies not only provide new insight into the action of topo I-directed agents, but also illustrate a strategy that can be applied to study additional topoisomerases and their inhibitors in vitro and in vivo.
Insights
Researchers developed a novel antibody to detect DNA topoisomerase I (topo I) covalent complexes in cells. This tool reveals how anticancer drugs like camptothecins and cisplatin stabilize these complexes, aiding cancer therapy research.
Area of Science:
- Molecular Biology
- Cancer Research
- Drug Development
Background:
- Anticancer drugs targeting DNA topoisomerase I (topo I) stabilize topo I-DNA covalent complexes, leading to cell death.
- Detecting these crucial topo I-DNA covalent complexes within intact cells and tumors has been a significant challenge.
Purpose of the Study:
- To develop a specific monoclonal antibody for detecting covalent topo I-DNA complexes in situ.
- To investigate the dynamics and cellular localization of topo I-DNA complexes induced by various anticancer agents.
Main Methods:
- Development and validation of a monoclonal antibody recognizing covalent topo I-DNA complexes.
- Application of the antibody in immunoblotting, immunofluorescence, and flow cytometry.
- Simultaneous staining for DNA damage markers (phospho-H2AX, Rad51) to assess lesion sites.
Main Results:
- The novel antibody specifically detects covalent topo I-DNA complexes, not free topo I or DNA.
- Detectable complexes were observed after treatment with camptothecins, indenoisoquinolines, and cisplatin.
- Topotecan-induced complexes showed transient kinetics, while indotecan-induced complexes were persistent; DNA double-strand breaks occurred at distinct sites.
Conclusions:
- The developed antibody provides a powerful tool for studying topo I-directed anticancer agents in vitro and in vivo.
- The findings offer new insights into the mechanism of action of topoisomerase I inhibitors and their associated DNA damage.
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