BACR2
Chronic but not acute hypoxia exposure impairs replication restart after reoxygenation via replisome disassembly
Isabel Pires1, Zuzana Bencokova1, Manuela Milani2, Lisa Folkes1, Michael Stratford1, Adrian Harris2, Ester Hammond1
1Gray Institute for Radiation Oncology and Biology, University of Oxford, UK, 2University of Oxford, Weatherall Institute of Molecular Medicine, UK
Introduction
Regions of severe hypoxia in tumours are associated with poor prognosis and increased genetic instability. Severe hypoxia (pO2<0.02%) induces an S-phase arrest associated with replication block which correlates with a decrease in DNA repair proficiency. We have further clarified the nature of the replication arrest and investigated whether replication restart after reoxygenation is also impaired.
Method
The nature of the replication arrest was analysed using HPLC with spectrophotometric detection of dNTP levels. Replication restart was analysed using the DNA fiber technique and FACS analysis. Replisome stability was analysed by Western Blotting in vitro and by IHC in vivo.
Results
We determined that exposure to severe levels of hypoxia correlates with falling dNTP levels. DNA fiber analysis demonstrates that replication is blocked at both initiation and elongation. The ability to resume replication after reoxygenation correlates with the duration of hypoxia, with chronic but not acute exposures impairing replication re-start. Replisome disassembly was observed in chronically exposed cells. Hypoxia-mediated repression of MCM6 was also observed in vivo in a spheroid model and xenograft tumours.
Conclusion
Reoxygenation is accompanied by extensive DNA damage in a cellular background in which DNA repair is already compromised. Cells exposed to hypoxia for chronic periods of time are replication incompetent and do not contribute to increased genomic instability. However, acutely exposed regions will contain cells that are able to continue to replicate their DNA and are repair compromised and could, therefore, potentially become genetically unstable and, further on, contribute to tumourigenesis.
