A36
Characterization of the H2AX-mediated response to severe hypoxia
Zuzana Bencokova, Isabel M. Pires, Ester M. Hammond
Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, UK
Background
Low oxygen tensions or hypoxia is a common feature of all solid tumours. Hypoxic tumours show increased rates of malignant transformation, metastatic progression, resistance to therapy and consequently have a poorer prognosis. Phosphatidylinositol-3 kinase–like (PIKK) family members, ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR) has been shown to drive the DNA damage response pathways and are also activated by severe hypoxia. This response includes the phosphorylation and relocalisation of H2AX. Phoshorylated H2AX is commonly used as a marker of double strand breaks and yet these have not been detected in hypoxic samples using alternate methods such as the comet assay. Our aim is to characterise the H2AX response to hypoxia.
Method
U2OS osteosarcoma cells were subjected to different levels of hypoxia and were compared with the DNA damaging agent Neocarzinostatin (NCS). In particular we have focused on staining hypoxic cells for both H2AX and other proteins known to co-localise to double strand breaks such as 53BP1 and MDC1.
Results
In response to NCS treatment we and others have found that H2AX, MDC1 and 53BP1 all form nuclear foci. In contrast, during exposure to hypoxia we were unable to detect 53BP1 in foci but did observe co-localising H2AX and MDC1 foci.
Conclusion
The DNA damage response is initiated by hypoxia in the absence of double strand breaks and this response is distinct from the response to classical DNA damaging agents.
