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(PP290) DNA double strand break repair and hypoxia-mediated genetic instability.
Chan, Norman*,1, Meng, Alice1, Jalali, Farid1, Bindra, Ranjit2, Glazer, Peter2, Powell, Simon3, Bristow, Rob1, 1 Department of Medical Biophysics, Toronto, Ontario, Canada2 Department of Therapeutic Radiology, New Haven, CT, USA3 Department of Radiation Oncology, St. Louis, MO, USA
ABSTRACT- Hypoxic tumour cells have increased rates of mutagenesis and protection from apoptosis leading to malignant progression. Inappropriate repair of DNA double strand breaks (dsb) can also drive genetic instability. Few data are available pertaining to the relative level, fidelity, and potential functional cellular consequences pertaining to homologous (HR) versus non-homologous (NHEJ) recombination in hypoxic cells. We therefore hypothesized that hypoxia mediates a down-regulation of HR and NHEJ leading to faulty resolution of DNA dsbs and increased genomic instability. To study the contribution of the NHEJ and HR pathways under hypoxia, we are conducting both neutral and alkaline comet assays, and also plasmid-based HR assays, following IR and MMC treatments in H1299 lung carcinoma cells. This cell line contains a stably-integrated HR reporter construct (DR-GFP) to track fidelity of HR. The cells were treated for up to 72 hours with 0.2% and 21% O2 gas prior to treatment and tracked for repair up to72 hours after treatment. In this cell line, 48 or 72 hours of 0.2% hypoxia does not induce a change in cell cycle distribution. Comet assays preformed on asynchronous cells show the expected 2 to 3 fold decrease in dsb induction under hypoxia, but the rate of rejoining of breaks may be delayed under hypoxic conditions. Experiments using the DR-GFP construct show a 2 to 5 fold decrease in HR under hypoxia. These cellular data are consistent with decreased Rad51, BRCA2 and Ku70 mRNA and protein levels in hypoxic cells. Data pertaining to the use of synchronized cultures to address S-phase bias and data pertaining to cytogenetic changes and clonogenic and metastatic capacity for hypoxic H1299 cells will be presented. Altered DNA repair under hypoxia may explain aggressive emergence of aggressive tumour clones and be the basis for novel therapeutics (Supported by Terry Fox-NCIC Program).
Key words: Hypoxia, Repair
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