Abstract
The comet assay is a simple gel electrophoresis method for visualizing and quantifying DNA damage. The comet assay is sensitive and reproducible and can be used to detect single-strand DNA breaks, double-strand DNA breaks, protein-associated DNA strand breaks and DNA crosslinks. The comet assay uses fluorescent DNA-binding dyes to detect both damaged DNA that resides in the tail region and undamaged DNA that is retained in the head region following gel electrophoresis. This assay is a single cell-based assay and thus is highly adaptable for measuring DNA damage in clinical samples. Furthermore, unlike other assays the detection of DNA damage is not dependent on the random incorporation of radiolabeled nucleotides. Again this can be problematic with clinical samples as proliferation rates are often slow and culturing of primary patient specimens for 48 h required to randomly label DNA is often not possible. In this chapter we will outline the comet assay for the detection of DNA damage induced by topoisomerase II inhibitors, cross-linking agents and gamma radiation.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kohn, K.W. 1991. Principles and practice of DNA filter elution. Pharmacol Ther 49:55–77.
Ostling, O., and Johanson, K.J. 1984. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123:291–298.
Tewey, K.M., Rowe, T.C., Yang, L., Halligan, B.D., and Liu, L.F. 1984. Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science 226:466–468.
Hazlehurst, L.A., Krapcho, A.P., and Hacker, M.P. 1995. Comparison of aza-anthracenedione-induced DNA damage and cytotoxicity in experimental tumor cells. Biochem Pharmacol 50:1087–1094.
Hazlehurst, L.A., Valkov, N., Wisner, L., Storey, J.A., Boulware, D., Sullivan, D.M., and Dalton, W.S. 2001. Reduction in drug-induced DNA double-strand breaks associated with beta1 integrin-mediated adhesion correlates with drug resistance in U937 cells. Blood 98:1897–1903.
Olive, P.L., Banath, J.P., and Durand, R.E. 1990. Detection of etoposide resistance by measuring DNA damage in individual Chinese hamster cells. J Natl Cancer Inst 82:779–783.
Pommier, Y., Schwartz, R.E., Zwelling, L.A., Kerrigan, D., Mattern, M.R., Charcosset, J.Y., Jacquemin-Sablon, A., and Kohn, K.W. 1986. Reduced formation of protein-associated DNA strand breaks in Chinese hamster cells resistant to topoisomerase II inhibitors. Cancer Res 46:611–616.
Fox, M.E., and Smith, P.J. 1990. Long-term inhibition of DNA synthesis and the persistence of trapped topoisomerase II complexes in determining the toxicity of the antitumor DNA intercalators mAMSA and mitoxantrone. Cancer Res 50:5813–5818.
Emmons, M., Boulware, D., Sullivan, D.M., and Hazlehurst, L.A. 2006. Topoisomerase II beta levels are a determinant of melphalan-induced DNA crosslinks and sensitivity to cell death. Biochem Pharmacol 72:11–18.
Hazlehurst, L.A., Enkemann, S.A., Beam, C.A., Argilagos, R.F., Painter, J., Shain, K.H., Saporta, S., Boulware, D., Moscinski, L., Alsina, M., et al. 2003. Genotypic and phenotypic comparisons of de novo and acquired melphalan resistance in an isogenic multiple myeloma cell line model. Cancer Res 63:7900–7906.
O'Connor, P.M., and Kohn, K.W. 1990. Comparative pharmacokinetics of DNA lesion formation and removal following treatment of L1210 cells with nitrogen mustards. Cancer Commun 2:387–394.
Olive, P.L., Banath, J.P., and Durand, R.E. 1990. Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. Radiat Res 122:86–94.
Kent, C.R., Eady, J.J., Ross, G.M., and Steel, G.G. 1995. The comet moment as a measure of DNA damage in the comet assay. Int J Radiat Biol 67:655–660.
Bradley, M.O., Erickson, L.C., and Kohn, K.W. 1978. Non-enzymatic DNA strand breaks induced in mammalian cells by fluorescent light. Biochim Biophys Acta 520:11–20.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hazlehurst, L.A. (2009). Detection of DNA Damage Induced by Topoisomerase II Inhibitors, Gamma Radiation and Crosslinking Agents Using the Comet Assay. In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 523. Humana Press. https://doi.org/10.1007/978-1-59745-190-1_12
Download citation
DOI: https://doi.org/10.1007/978-1-59745-190-1_12
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-873-7
Online ISBN: 978-1-59745-190-1
eBook Packages: Springer Protocols