Cell Synchronization by Inhibitors of DNA Replication Induces Replication Stress and DNA Damage Response: Analysis by Flow Cytometry

  • Zbigniew DarzynkiewiczEmail author
  • H. Dorota Halicka
  • Hong Zhao
  • Monika Podhorecka
Part of the Methods in Molecular Biology book series (MIMB, volume 761)


Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin, or thymidine become arrested at the entrance to S phase and upon release from the block they synchronously progress through S, G2, and M. We recently reported that exposure of cells to these inhibitors at concentrations commonly used to synchronize cell populations led to phosphorylation of histone H2AX on Ser139 (induction of γH2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response signaling pathways and caution about interpreting data obtained with use of cells synchronized such way as representing unperturbed cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual cell immunocytochemically with phospho-specific antibody (Ab) and measured by flow cytometry. Concurrent measurement of cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with cell cycle phase.

Key words

DNA repair DNA double-strand breaks flow cytometry apoptosis DNA fragmentation G1/S boundary 



This project is supported by NCI CA RO1 28 704.


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Zbigniew Darzynkiewicz
    • 1
    Email author
  • H. Dorota Halicka
    • 1
  • Hong Zhao
    • 1
  • Monika Podhorecka
    • 2
  1. 1.Department of PathologyBrander Cancer Research Institute, New York Medical CollegeValhallaUSA
  2. 2.Department of Hemato-Oncology and Bone Marrow TransplantationMedical UniversityLublinPoland

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