title: Immunodetection of human topoisomerase I-DNA covalent complexes.
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.
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Anand G
authors.initials: AG
authors.lastname: Patel
authors.affiliation: Division of Oncology Research, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Karen S
authors.initials: KS
authors.lastname: Flatten
authors.affiliation: Division of Oncology Research, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Peterson
authors.affiliation: Department of Immunology, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Beito
authors.affiliation: Division of Oncology Research, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Paula A
authors.initials: PA
authors.lastname: Schneider
authors.affiliation: Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Angela L
authors.initials: AL
authors.lastname: Perkins
authors.affiliation: Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Harki
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA Division of Oncology Research, Mayo Clinic, Rochester, MN 55905, USA Kaufmann.Scott@Mayo.edu.
authors.forename: Scott H
authors.initials: SH
authors.lastname: Kaufmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D052117:Benzodioxoles / Q000494:pharmacology; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D020014:K562 Cells; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology*; D019772:Topotecan / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D000972:Antineoplastic Agents, Phytogenic; D052117:Benzodioxoles; C468783:H2AX protein, human; D006657:Histones; D007546:Isoquinolines; C553898:NSC 724998; D059004:Topoisomerase I Inhibitors; D019772:Topotecan; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; D002945:Cisplatin