Immunodetection of human topoisomerase I-DNA covalent complexes

• Published in: Nucleic acids research Vol. 44; no. 6; pp. 2816 - 2826
• Main Authors: Patel, Anand G, Flatten, Karen S, Peterson, Kevin L, Beito, Thomas G, Schneider, Paula A, Perkins, Angela L, Harki, Daniel A, Kaufmann, Scott H
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 07-04-2016
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - isolation & purification; Antineoplastic Agents, Phytogenic - pharmacology; Apoptosis - drug effects; Benzodioxoles - pharmacology; Cell Line, Tumor; Cisplatin - pharmacology; DNA - genetics; DNA - metabolism; DNA Breaks, Double-Stranded; DNA Topoisomerases, Type I - genetics; DNA Topoisomerases, Type I - metabolism; Gene Expression Regulation, Neoplastic; HCT116 Cells; Histones - genetics; Histones - metabolism; Humans; Isoquinolines - pharmacology; K562 Cells; Mice; Molecular Sequence Data; Nucleic Acid Enzymes; Protein Binding - drug effects; Rad51 Recombinase - genetics; Rad51 Recombinase - metabolism; Sequence Alignment; Structure-Activity Relationship; Topoisomerase I Inhibitors - pharmacology; Topotecan - pharmacology
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkw109

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.