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Cell Cycle Synchronization pp 107–119Cite as

DNA Damage Response Resulting from Replication Stress Induced by Synchronization of Cells by Inhibitors of DNA Replication: Analysis by Flow Cytometry

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1524))

Abstract

Cell synchronization is often achieved by transient inhibition of DNA replication. When cultured in the presence of such inhibitors as hydroxyurea, aphidicolin or excess of thymidine the cells that become arrested at the entrance to S-phase upon release from the block initiate progression through S then G2 and M. However, exposure to these inhibitors at concentrations commonly used to synchronize cells leads to activation of ATR and ATM protein kinases as well as phosphorylation of Ser139 of histone H2AX. This observation of DNA damage signaling implies that synchronization of cells by these inhibitors is inducing replication stress. Thus, a caution should be exercised while interpreting data obtained with use of cells synchronized this way since they do not represent unperturbed cell populations in a natural metabolic state. This chapter critically outlines virtues and vices of most cell synchronization methods. It also presents the protocol describing an assessment of phosphorylation of Ser139 on H2AX and activation of ATM in cells treated with aphidicolin, as a demonstrative of one of several DNA replication inhibitors that are being used for cell synchronization. Phosphorylation of Ser139H2AX and Ser1981ATM in individual cells is detected immunocytochemically with phospho-specific Abs and intensity of immunofluorescence is measured by flow cytometry. Concurrent measurement of cellular DNA content followed by multiparameter analysis allows one to correlate the extent of phosphorylation of these proteins in response to aphidicolin with the cell cycle phase.

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Acknowledgment

Supported by NCI CA RO1 28 704 and 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, 40 Sunshine Cottage Road, Valhalla, NY, 10595, USA

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

  2. Department of Hemato-Oncology and Bone Marrow Transplantation, Medical University, Lublin, Poland

    Monika Podhorecka

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

Correspondence to Zbigniew Darzynkiewicz .

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Editors and Affiliations

  1. Department of Biotechnology and Microbiology, University of Debrecen, Debrecen, Hungary

    Gaspar Banfalvi

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Halicka, D., Zhao, H., Li, J., Garcia, J., Podhorecka, M., Darzynkiewicz, Z. (2017). DNA Damage Response Resulting from Replication Stress Induced by Synchronization of Cells by Inhibitors of DNA Replication: Analysis by Flow Cytometry. In: Banfalvi, G. (eds) Cell Cycle Synchronization. Methods in Molecular Biology, vol 1524. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6603-5_7

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  • DOI: https://doi.org/10.1007/978-1-4939-6603-5_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6602-8

  • Online ISBN: 978-1-4939-6603-5

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