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Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis

Authors

  • Xuan Huang,

    1. Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA,
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  • H. Dorota Halicka,

    1. Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA,
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  • Frank Traganos,

    1. Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA,
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  • Toshiki Tanaka,

    1. Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA,
    2. First Department of Surgery, Yamaguchi University School of Medicine, Yamaguchi, Japan and
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  • Akira Kurose,

    1. Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA,
    2. Department of Pathology, Iwate Medical University, Iwate, Japan
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  • Zbigniew Darzynkiewicz

    Corresponding author
    1. Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA,
      Dr Z. Darzynkiewicz, Brander Cancer Research Institute at NYMC, 19 Bradhurst Avenue, Suite 2400, Hawthorne, NY, 10532, USA. Tel.: 914-347-2801; Fax: 914-347-2804; E-mail: darzynk@nymc.edu
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Dr Z. Darzynkiewicz, Brander Cancer Research Institute at NYMC, 19 Bradhurst Avenue, Suite 2400, Hawthorne, NY, 10532, USA. Tel.: 914-347-2801; Fax: 914-347-2804; E-mail: darzynk@nymc.edu

Abstract

Abstract.  Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxynucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named γH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of γH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio- or chemotherapy may provide an early marker predictive of response to treatment.

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