New distinct compartments in the G2 phase of the cell cycle defined by the levels of γH2AX

Cell Cycle
Idun Dale ReinTrond Stokke

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 ...Continue Reading

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Citations

Jan 17, 2020·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Idun D ReinTrond Stokke
Mar 20, 2021·Scientific Reports·Morten P OksvoldThea Kristin Våtsveen

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Methods Mentioned

BETA
flow cytometry
X-ray

Software Mentioned

Excel
Visual Basic
H2AXCellCycleSupfile
Sigmaplot

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