Abstract
Exposure of cells to inhibitors of DNA topoisomerase I (topo I) or topoisomerase II (topo II) leads to DNA damage that often involves formation of DNA double-strand breaks (DSBs). DNA damage, particularly induction of DSBs, manifests by phosphorylation of histone H2AX on Ser-139 which is mediated by one of the protein kinases of the phosphoinositide kinase family, namely ATM, ATR, and/or DNA-PK. The presence of Ser-139 phosphorylated H2AX (γH2AX) is thus a reporter of DNA damage. This protocol describes quantitative assessment of γH2AX detected immunocytochemically in individual cells combined with quantification of cellular DNA content by cytometry. The bivariate analysis of γH2AX expression versus DNA content allows one to correlate DNA damage with the cell cycle phase or DNA ploidy. The protocol can also be used to assess activation (Ser-1981 phosphorylation) of ATM; this event also revealing DNA damage induced by topo I or topo II inhibitors. Examples where DNA damage was induced by topotecan (topo I) and etoposide (topo II) inhibitors are provided.
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References
Hsiang, Y.H., Lihou, M.G., and Liu, L.F. (1989) Arrest of replication forks by drug stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin. Cancer Res 49, 5077–5082.
D‘Arpa, P., Beardmore, C., and Liu, L.F. (1990) Involvement of nucleic acid synthesis in cell killing mechanisms of topoisomerase poisons. Cancer Res 50, 6916–6924.
Wu, J., and Liu, L.F. (1997) Processing of topoisomerase I cleavable complexes into DNA by transcription. Nucleic Acids Res 25, 4181–4186.
Kapuscinski, J., and Darzynkiewicz, Z. (1986) Relationship between the pharmacological activity of antitumor drugs Ametantrone and Mitoxantrone (Novantrone) and their ability to condense nucleic acids. Proc Natl Acad Sci USA 83, 6302–6306.
Huang, X., Kurose, A., Tanaka, T., Traganos, F., Dai, W., and Darzynkiewicz, Z. (2006) Activation of ATM and histone H2AX phosphorylation induced by mitoxantrone but not by topotecan is prevented by the antioxidant N-acetyl-L-cysteine. Cancer Biol Ther 5, 959–964.
Tanaka, T., Halicka, H.D., Traganos, F., Seiter, K., and Darzynkiewicz, Z. (2007) Induction of ATM activation, histone H2AX phosphorylation and apoptosis by etoposide: relation to the cell cycle phase. Cell Cycle 6, 371–376.
Del Bino, G., Bruno, S., Yi, P.N., and Darzynkiewicz, Z. (1992) Apoptotic cell death triggered by camptothecin or teniposide: the cell cycle specificity and effects of ionizing radiation. Cell Prolif 25, 537–548.
Kurose, A., Tanaka, T., Huang X., Halicka, H.D., Traganos F., Dai, W., and Darzynkiewicz, Z. (2005) Assessment of ATM phosphorylation on Ser-1981 induced by DNA topoisomerase I and II inhibitors in relation to Ser-139-histone H2AX phosphorylation, cell cycle phase and apoptosis. Cytometry A 68A, 1–9.
Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S., and Bonner, W.M. (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273, 5858–5868.
West, M.H., and Bonner, W.M. (1980) Histone 2A, a heteromorphous family of eight protein species. Biochemistry 19, 3238–3245.
Rogakou, E.P., Boon, C., Redon, C., and Bonner, W.M. (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146, 905–916.
Sedelnikova, O.A., Rogakou, E.P., Panuytin, I.G., and Bonner, W. (2002) Quantitative detection of 125IUdr-induced DNA double-strand breaks with γ-H2AX antibody. Radiat Res 158, 486–492
MacPhail, S.H., Banath, J.P., Yu, T.Y., Chu, E.H., Lambur, H., and Olive, P.L. (2003) Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays. Int J Radiat Biol 79, 351–358.
Huang, X, Okafuji, M., Traganos, F., Luther, E., Holden, E., and Darzynkiewicz, Z. (2004) Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by DNA cross-linking agent cisplatin. Cytometry 58A, 99–110.
Huang, X., Halicka, H.D., Traganos, F., Tanaka, T., Kurose, A., and Darzynkiewicz, Z. (2005) Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis. Cell Prolif 38, 223–243.
Tanaka, T., Huang, X., Halicka, H.D., Zhao, H., Traganos, F., Albino, A.P., Dai, W., and Darzynkiewicz, Z. (2007) Cytometry of ATM activation and histone H2AX phosphorylation to estimate extent of DNA damage induced by exogenous agents. Cytometry A 71A, 648–661.
Speit, G., and Hartmann, A. (1999). The comet assay (single cell gel test). Methods Mol Biol 113, 203–212.
Zhao, H., Tanaka, T., Halicka, H.D., Traganos, F., Zarebski, M., Dobrucki, J., and Darzynkiewicz, Z. (2007) Cytometric assessment of DNA damage by exogenous and endogenous oxidants reports the aging-related processes. Cytometry A 71A, 905–914.
Tanaka, T., Halicka, H.D., Huang, X., Traganos, F., and Darzynkiewicz, Z. (2006) Constitutive histone H2AX phosphorylation and ATM activation, the reporters of DNA damage by endogenous oxidants. Cell Cycle 5, 1940–1945.
Kajstura, M., Halicka, H.D., Pryjma, J., and Darzynkiewicz, Z. (2007) Discontinuous fragmentation of nuclear DNA during apoptosis revealed by discrete “sub-G1” peaks on DNA content histograms. Cytometry A 71A, 125–131.
Tanaka, T., Kurose, A., Huang, X., Dai, W., and Darzynkiewicz, Z. (2006) ATM kinase activation and histone H2AX phosphorylation as indicators of DNA damage by DNA topoisomerase I inhibitor topotecan and during apoptosis. Cell Prolif 39, 49–60.
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Supported by NCI grant RO1 28704.
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Darzynkiewicz, Z., Halicka, D.H., Tanaka, T. (2009). Cytometric Assessment of DNA Damage Induced by DNA Topoisomerase Inhibitors. In: Clarke, D. (eds) DNA Topoisomerases. Methods in Molecular Biology™, vol 582. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-340-4_12
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DOI: https://doi.org/10.1007/978-1-60761-340-4_12
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