DNA Damage Response Resulting from Replication Stress Induced by Synchronization of Cells by Inhibitors of DNA Replication: Analysis by Flow Cytometry.
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 G2and 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.
Department of Pathology, Brander Cancer Research Institute, New York Medical College, 40 Sunshine Cottage Road, Valhalla, NY, 10595, USA.
Dorota
D
Halicka
Department of Pathology, Brander Cancer Research Institute, New York Medical College, 40 Sunshine Cottage Road, Valhalla, NY, 10595, USA.
Hong
H
Zhao
Department of Pathology, Brander Cancer Research Institute, New York Medical College, 40 Sunshine Cottage Road, Valhalla, NY, 10595, USA.
Jiangwei
J
Li
Department of Pathology, Brander Cancer Research Institute, New York Medical College, 40 Sunshine Cottage Road, Valhalla, NY, 10595, USA.
Jorge
J
Garcia
Department of Hemato-Oncology and Bone Marrow Transplantation, Medical University, Lublin, Poland.
Monika
M
Podhorecka
Department of Pathology, Brander Cancer Research Institute, New York Medical College, 40 Sunshine Cottage Road, Valhalla, NY, 10595, USA. darzynk@nymc.edu.
Zbigniew
Z
Darzynkiewicz
D016590:Aphidicolin / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D053938:DNA Fragmentation / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics / Q000502:physiology
C468783:H2AX protein, human; D006657:Histones; D016590:Aphidicolin; D064007:Ataxia Telangiectasia Mutated Proteins
ATM phosphorylation; Aphidicolin; Apoptosis; DNA double-strand breaks; DNA fragmentation; DNA repair; G1/S boundary; γH2AX