Sensing Of DNA-dsbs Under Continual Hypoxia

Main Category: Cancer / Oncology
Also Included In: Genetics;  Blood / Hematology;  Radiology / Nuclear Medicine
Article Date: 22 Sep 2008 - 4:00 PDT

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Intracellular hypoxia has been linked to genetic instability, tumour progression and poor prognosis. Little is known about the effect of hypoxia on DNA-dsb sensing and repair. We tested whether the sensing of DNA-dsbs and chromatin biology is decreased in cells irradiated under continual hypoxic or anoxic conditions. To test this hypothesis, we used normal diploid fibroblast strains, GM05757, synchronised in G0-G1 to preclude DNA replication as a source of DNA repair foci.

The cells were pre-treated with 0.2% O2 (hypoxia), 0.0% O2 (anoxia) or 21% O2 (normoxia) for 16 hours and then irradiated and kept under hypoxic/ anoxic/ normoxic conditions for another 24 hours.

The biomarkers gammaH2AX, MRE11, 53BP1, ATMser1981 and the MRN complexes were tracked using quantitative immunofluorescent microscopy to score intranuclear foci. We observed an expected 2 to 3 fold decrease in initial number of gammaH2AX ATMser1981 and 53BP1 foci at 30 minutes following 2 or 5 Gy post-IR under hypoxia and anoxia consistent with the OER. However, we observed an increase in the relative ATM-dependent residual foci at times between 4 and 24 hours post-IR under continual hypoxia. These were residual DNA-dsbs based on neutral COMET assays and were not associated with increased cell kill following clongenic assays, suggesting misrepair or repair can occur in such cells when releases into S and G2Mphases. We conclude that hypoxic cells have an altered ability during NHEJ to sense and repair DNA-dsbs and could serve as a potential factor in genetic instability and tumour progression.

Supported by a Terry Fox Hypoxia Project Program Grant and CCS Career Award to RGB.

Bristow Robert et al. Princess Margaret Hospital and University of Toronto, Toronto, Canada

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