Abstract
Hypoxic cells have been linked to genetic instability and tumor progression. However, little is known about the exact relationship between DNA repair and genetic instability in hypoxic cells. We therefore tested whether the sensing and repair of DNA double-strand breaks (DNA-dsbs) is altered in irradiated cells kept under continual oxic, hypoxic or anoxic conditions. Synchronized G0-G1 human fibroblasts were irradiated (0-10 Gy) after initial gassing with 0% O(2) (anoxia), 0.2% O(2) (hypoxia) or 21% O(2) (oxia) for 16 hours. The response of phosphorylated histone H2AX (γ-H2AX), phosphorylated ataxia telangiectasia mutated [ATM(Ser1981)], and the p53 binding protein 1 (53BP1) was quantified by intranuclear DNA repair foci and western blotting. At 24 hours following DNA damage, residual γ-H2AX, ATM(Ser1981) and 53BP1 foci were observed in hypoxic cells. This increase in residual DNA-dsbs under hypoxic conditions was confirmed using neutral comet assays. Clonogenic survival was also reduced in chronically hypoxic cells, which is consistent with the observation of elevated G1-associated residual DNA-dsbs. We also observed an increase in the frequency of chromosomal aberrations in chronically hypoxic cells. We conclude that DNA repair under continued hypoxia leads to decreased repair of G1-associated DNA-dsbs, resulting in increased chromosomal instability. Our findings suggest that aberrant DNA-dsb repair under hypoxia is a potential factor in hypoxia-mediated genetic instability.
Highlights
Human cells have evolved specific pathways to repair DNA double-strand breaks (DNA-dsbs) and enact cell cycle checkpoints following DNA damage to ensure genetic stability
The exogenous DNA damage created by ionizing radiation can be studied without the bias from endogenous DNA damage created during DNA replication in the S phase of the cell cycle (Al Rashid et al, 2005)
The induction of c-H2AX, or other DNA damage sensor foci, which occur as a result of replicative breaks or chromatin compaction in the S and G2 phases of the cell cycle, respectively, can be avoided (Liu et al, 2008)
Summary
Human cells have evolved specific pathways to repair DNA double-strand breaks (DNA-dsbs) and enact cell cycle checkpoints following DNA damage to ensure genetic stability These pathways could be oxygen sensitive because endogenous and exogenous DNA damage can be differentially processed and repaired in oxic versus hypoxic cells (Bindra et al, 2007; Bristow and Hill, 2008). Anoxia has been shown to activate the ATM and ATR (ataxia telangiectasia and Rad3-related) kinases, resulting in S and G2 cell cycle checkpoint arrests (Freiberg et al, 2006; Gibson et al, 2005; Hammond et al, 2003) This activation is in the absence of DNA damage and might be dependent on the oxygen level (Bencokova et al, 2009; Chan et al, 2008)
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