Reoxygenation mediated DNA damage results in a Chk2 dependent G2 arrest

Abstract

795

Solid tumors pose multiple challenges for the patient and clinician alike. Current non-experimental therapies include surgery, radiotherapy and chemotherapy. None of these options, with their varying degrees of efficacy are ideal. Tumor vasculature is irregular and as a result regions become either permanently or transiently hypoxic. During periods of extreme hypoxia (oxygen concentrations of 0.02% or below) cells arrest in both the G1 and S-phases of the cell cycle. However as blood vessels re-open, cells can be subjected to an influx of oxygen and can resume cycling. We have shown previously that after re-oxygenation cells remain arrested in the G1 phase of the cell cycle before entering S phase. This G1 arrest was shown to be dependent on both p21 and p27. Here we show that after re-oxygenation, cells in the G2 phase of the cell cycle also undergo an arrest before cycling normally. Recently it was demonstrated that re-oxygenation leads to a significant level of DNA-damage. This finding led us to hypothesize that the ATM kinase might respond to the re-oxygenation induced DNA-damage and subsequently initiate a G2 arrest. This PI3-kinase is responsible for initiating the checkpoint response to damage caused by ionizing radiation, UV light and ROS. One of ATM’s principle effector molecules is Chk2 which becomes phosphorylated by ATM and then via its action on cdc25 can induce a G2 arrest. Work done to date has shown that Chk 2 was phosphorylated in response to re-oxygenation. Chk 2 has also been demonstrated to phosphorylate p53 on residue serine 20 possibly indicating that p53 might have a role to play in the G2 arrest. However we have shown that p53 null cells exhibit re-oxygenation induced G2 arrest with similar kinetics to wild-type cells. In contrast, when Chk 2 protein levels were reduced with specific siRNAs the re-oxygenation induced G2 arrest was inhibited. This supports our hypothesis that ATM phosphorylates Chk2 in response to re-oxygenation induced DNA-damage and that Chk 2 then induces a G2 arrest. Significantly we found that Chk2 null cells, generated by either siRNA or genetic knockout, showed decreased viability after hypoxia/re-oxygenation treatment. These data suggest that the Chk2 mediated G2 arrest allows cells sufficient time to repair DNA-damage induced by re-oxygenation. Without this checkpoint cells with mutations that allow them to escape apoptosis may be selected for, therefore leading to a more aggressive and harder to treat tumor.