title: New distinct compartments in the G2 phase of the cell cycle defined by the levels of γH2AX.
abstract: Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (γH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G1 cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G2. Intriguingly, G2 cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed "G2H" compartment), to cells that there were almost negative and had about 2 foci (termed "G2L" compartment). EdU-labeling of S phase cells revealed that G2H was directly populated from S phase, while G2L was populated from G2H, but in control cells also directly from S phase. The length of G2H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G2H, and enter mitosis after a 2-3 h delay in G2L.
authors: a Group for Molecular Radiation Biology; Department of Radiation Biology; The Norwegian Radium Hospital ; Oslo , Norway.
authors: Idun
authors: I
authors: Dale Rein
authors: b The Intervention Centre, Oslo University Hospital ; Oslo , Norway.|c Faculty of Health Sciences, Oslo and Akershus University College of Applied Sciences ; Oslo , Norway.
authors: Caroline
authors: C
authors: Stokke
authors: d The Francis Crick Institute ; London , UK.
authors: Marwa
authors: M
authors: Jalal
authors: e Group for Lymphoma and Lymphocyte Biology; Department of Cancer Immunology; The Norwegian Radium Hospital ; Oslo , Norway.|f Centre for Cancer Biomedicine, University of Oslo ; Oslo , Norway.
authors: June H
authors: JH
authors: Myklebust
authors: a Group for Molecular Radiation Biology; Department of Radiation Biology; The Norwegian Radium Hospital ; Oslo , Norway.
authors: Sebastian
authors: S
authors: Patzke
authors: a Group for Molecular Radiation Biology; Department of Radiation Biology; The Norwegian Radium Hospital ; Oslo , Norway.
authors: Trond
authors: T
authors: Stokke
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology; D016195:G2 Phase / Q000187:drug effects / Q000502:physiology*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000494:pharmacology
chemical_list: C468783:H2AX protein, human; D006657:Histones; D010750:Phosphoproteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors
keywords: DNA repair; G2 cell cycle phase; PARP inhibition; ionizing radiation; γH2AX