Immunodetection of human topoisomerase I-DNA covalent complexes

Anand G. Patel, Karen S. Flatten, Kevin L. Peterson, Thomas G. Beito, Paula A. Schneider, Angela L. Perkins, Daniel A. Harki, Scott H Kaufmann

Research output: Contribution to journalArticle

16 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2816-2826
Number of pages11
JournalNucleic Acids Research
Volume44
Issue number6
DOIs
StatePublished - Feb 24 2016

Fingerprint

Type I DNA Topoisomerase
Topotecan
NSC 724998
Topoisomerase Inhibitors
human TOP1 protein
Investigational Drugs
Camptothecin
Double-Stranded DNA Breaks
Nucleosides
Immunoblotting
Cisplatin
Fluorescent Antibody Technique
Flow Cytometry
Monoclonal Antibodies
Staining and Labeling

ASJC Scopus subject areas

  • Genetics

Cite this

Patel, A. G., Flatten, K. S., Peterson, K. L., Beito, T. G., Schneider, P. A., Perkins, A. L., ... Kaufmann, S. H. (2016). Immunodetection of human topoisomerase I-DNA covalent complexes. Nucleic Acids Research, 44(6), 2816-2826. https://doi.org/10.1093/nar/gkw109

Immunodetection of human topoisomerase I-DNA covalent complexes. / Patel, Anand G.; Flatten, Karen S.; Peterson, Kevin L.; Beito, Thomas G.; Schneider, Paula A.; Perkins, Angela L.; Harki, Daniel A.; Kaufmann, Scott H.

In: Nucleic Acids Research, Vol. 44, No. 6, 24.02.2016, p. 2816-2826.

Research output: Contribution to journalArticle

Patel, AG, Flatten, KS, Peterson, KL, Beito, TG, Schneider, PA, Perkins, AL, Harki, DA & Kaufmann, SH 2016, 'Immunodetection of human topoisomerase I-DNA covalent complexes', Nucleic Acids Research, vol. 44, no. 6, pp. 2816-2826. https://doi.org/10.1093/nar/gkw109
Patel AG, Flatten KS, Peterson KL, Beito TG, Schneider PA, Perkins AL et al. Immunodetection of human topoisomerase I-DNA covalent complexes. Nucleic Acids Research. 2016 Feb 24;44(6):2816-2826. https://doi.org/10.1093/nar/gkw109
Patel, Anand G. ; Flatten, Karen S. ; Peterson, Kevin L. ; Beito, Thomas G. ; Schneider, Paula A. ; Perkins, Angela L. ; Harki, Daniel A. ; Kaufmann, Scott H. / Immunodetection of human topoisomerase I-DNA covalent complexes. In: Nucleic Acids Research. 2016 ; Vol. 44, No. 6. pp. 2816-2826.
@article{2a544e5c363f4ac1b854622f878f033f,
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.",
author = "Patel, {Anand G.} and Flatten, {Karen S.} and Peterson, {Kevin L.} and Beito, {Thomas G.} and Schneider, {Paula A.} and Perkins, {Angela L.} and Harki, {Daniel A.} and Kaufmann, {Scott H}",
year = "2016",
month = "2",
day = "24",
doi = "10.1093/nar/gkw109",
language = "English (US)",
volume = "44",
pages = "2816--2826",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "6",

}

TY - JOUR

T1 - Immunodetection of human topoisomerase I-DNA covalent complexes

AU - Patel, Anand G.

AU - Flatten, Karen S.

AU - Peterson, Kevin L.

AU - Beito, Thomas G.

AU - Schneider, Paula A.

AU - Perkins, Angela L.

AU - Harki, Daniel A.

AU - Kaufmann, Scott H

PY - 2016/2/24

Y1 - 2016/2/24

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84963825596&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84963825596&partnerID=8YFLogxK

U2 - 10.1093/nar/gkw109

DO - 10.1093/nar/gkw109

M3 - Article

C2 - 26917015

AN - SCOPUS:84963825596

VL - 44

SP - 2816

EP - 2826

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 6

ER -