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ATM Kinase pp 183–196Cite as

ATM Activation and H2AX Phosphorylation Induced by Genotoxic Agents Assessed by Flow- and Laser Scanning Cytometry

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1599))

Abstract

Activation of Ataxia Telangiectasia Mediated protein kinase (ATM) by its phosphorylation on serine 1981 and phosphorylation of histone H2AX on serine 139 (γH2AX) are the key events reporting DNA damage, primarily formation of DNA double strand breaks. These events are detected immunocytochemically in individual cells using phospho-specific Abs. The protocols are presented that describe the methodology of immunofluorescent labeling of cells in conjunction with specific staining of cellular DNA. Flow- and imaging-cytometry, the latter exemplified as laser scanning cytometry, is used to quantify intensity of cellular fluorescence reporting activation of ATM and induction of γH2AX with respect to cellular DNA content, which in turn reports the cell cycle phase. Different protocols are presented for analysis of cells either grown in suspension or attached to surface of culture vessels. Examples of ATM activation and H2AX phosphorylation in response to DNA damage in leukemic HL-60 cells by DNA topoisomerase I inhibitor topotecan, and in lung carcinoma A549 cells by hydrogen peroxide, are presented.

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References

  1. Bakkenist CJ, Kastan MB (2004) Initiating cellular stress responses. Cell 118:9–17

    Article  CAS  PubMed  Google Scholar 

  2. Bonner WM, Redon CE, Dickey JS, Nakamura AJ, Sedelnikova OA, Solier S, Pommier Y (2008) γH2AX and cancer. Nat Rev Cancer 8:957–966

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kastan MB (2008) DNA damage responses: mechanisms and roles in human disease. G.H.A. cloves memorial award lecture. Mol Cancer Res 6:517–524

    Article  CAS  PubMed  Google Scholar 

  4. Nakamura AJ, Rao VA, Pommier Y, Bonner WM (2010) The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks. Cell Cycle 9:389–398

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lord CJ, Ashworth A (2012) The DNA damage response and cancer therapy. Nature 481:287–294

    Article  CAS  PubMed  Google Scholar 

  6. Helt CE, Cliby WA, Keng PC, Bambara RA, O’Reilly MA (2005) Ataxia telangiectasia mutated (ATM) and ATM and Rad3-related protein exhibit selective target specificities in response to different forms of DNA damage. J Biol Chem 280:1186–1192

    Article  CAS  PubMed  Google Scholar 

  7. Shiloh Y (2014) ATM: expanding roles as a chief guardian of genome stability. Exp Cell Res 329:154–161

    Article  CAS  PubMed  Google Scholar 

  8. Lavin MF, Kozlov S, Gatei M, Kijas AW (2015) ATM-dependent phosphorylation of all three members of the MRN complex: from sensor to adaptor. Biomolecules 5:2877–2902

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Paull TT (2015) Mechanisms of ATM activation. Annu Rev Biochem 84:711–738

    Article  CAS  PubMed  Google Scholar 

  10. Siddiqui MS, François M, Fenech MF, Leifert WR (2015) Persistent γH2AX: a promising molecular marker of DNA damage and aging. Mutat Res Rev Mutat Res 766:1–19

    Article  CAS  PubMed  Google Scholar 

  11. Pozarowski P, Holden E, Darzynkiewicz Z (2013) Laser scanning cytometry: Principles and applications. an update. Methods Mol Biol 913:187–212

    Google Scholar 

  12. Olive PL, Durand RE, Banath JP, Johnston PJ (2001) Analysis of DNA damage in individual cells. Methods Cell Biol 64:235–249

    Article  CAS  PubMed  Google Scholar 

  13. Huang X, Okafuji M, Traganos F, Luther E, Holden E, Darzynkiewicz Z (2004) Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by DNA crosslinking agent cisplatin. Cytometry A 58A:99–110

    Article  CAS  Google Scholar 

  14. Tanaka T, Huang X, Halicka HD, Zhao H, Traganos F, Albino AP, Dai W, 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

    Article  CAS  Google Scholar 

  15. Zhao H, Traganos F, Darzynkiewicz Z (2010) Kinetics of the UV-induced DNA damage response in relation to cell cycle phase. Correlation with DNA replication. Cytometry A 77A:285–293

    CAS  Google Scholar 

  16. Darzynkiewicz Z, Zhao H, Halicka HD, Rybak P, Dobrucki J, Wlodkowic D (2012) DNA damage signaling assessed in individual cells in relation to the cell cycle phase and induction of apoptosis. Crit Rev Clin Lab Sci 49:199–217

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Olive PL (2011) Retention of γH2AX foci as an indication of lethal DNA damage. Radiother Oncol 101:18–23

    Article  CAS  PubMed  Google Scholar 

  18. Halicka HD, Ozkaynak MF, Levendoglu-Tugal O, Sandoval C, Seiter K, Kajstura M et al (2009) DNA damage response as a biomarker in treatment of leukemias. Cell Cycle 8:1720–1724

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li P, Du CR, Xu WC, Shi ZL, Zhang Q et al (2013) Correlation of dynamic changes in γ-H2AX expression in peripheral blood lymphocytes from head and neck cancer patients with radiation-induced oral mucositis. Radiat Oncol 8:155

    Article  PubMed  PubMed Central  Google Scholar 

  20. Vici P, Di Benedetto A, Ercolani C, Pizzuti L, Di Lauro L, Sergi D et al (2015) Predictive significance of DNA damage and repair biomarkers in triple-negative breast cancer patients treated with neoadjuvant chemotherapy: an exploratory analysis. Oncotarget 6:42773–42780

    PubMed  PubMed Central  Google Scholar 

  21. Kurose A, Tanaka T, Huang X, Halicka HD, Traganos F, Dai W, 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 2005(68A):1–9

    Article  Google Scholar 

  22. Zhao H, Tanaka T, Halicka HD, Traganos F, Zarebski M, Dobrucki J, Darzynkiewicz Z (2007) Cytometric assessment of DNA damage by exogenous and endogenous oxidants reports the aging-related processes. Cytometry A 71A:905–914

    Article  CAS  Google Scholar 

  23. Tanaka T, Halicka HD, Huang X, Traganos F, Darzynkiewicz Z (2006) Constitutive histone H2AX phosphorylation and ATM activation, the reporters of DNA damage by endogenous oxidants. Cell Cycle 5:1940–1945

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gorczyca W, Gong J, Darzynkiewicz Z (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 53:1945–1951

    CAS  PubMed  Google Scholar 

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Acknowledgment

Supported by NIH R01 28 705 and The Robert A. Welke Cancer Research Foundation.

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Authors and Affiliations

  1. Department of Pathology, Brander Cancer Research Institute, New York Medical College, Basic Sciences Building, 15 Dana Road, Valhalla, NY, 10595, USA

    Hong Zhao, H. Dorota Halicka, Jorge Garcia, Jiangwei Li & Zbigniew Darzynkiewicz

Authors
  1. Hong Zhao
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  2. H. Dorota Halicka
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  3. Jorge Garcia
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  4. Jiangwei Li
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  5. Zbigniew Darzynkiewicz
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Corresponding author

Correspondence to Zbigniew Darzynkiewicz .

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Editors and Affiliations

  1. University of Queensland Centre for Clinical Research (UQCCR), University of Queensland, Herston, Queensland, Australia

    Sergei V. Kozlov

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Zhao, H., Halicka, H.D., Garcia, J., Li, J., Darzynkiewicz, Z. (2017). ATM Activation and H2AX Phosphorylation Induced by Genotoxic Agents Assessed by Flow- and Laser Scanning Cytometry. In: Kozlov, S. (eds) ATM Kinase. Methods in Molecular Biology, vol 1599. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6955-5_14

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  • DOI: https://doi.org/10.1007/978-1-4939-6955-5_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6953-1

  • Online ISBN: 978-1-4939-6955-5

  • eBook Packages: Springer Protocols

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