HypoxiaCyclic hypoxia does not alter RAD51 expression or PARP inhibitor cell kill in tumor cells
Section snippets
Cell culture
The H1299 human lung carcinoma cell line was grown in 10% fetal calf serum (FCS) and 20 mmol/L HEPES and RPMI 1640. The ME180 cervical carcinoma cell line was grown in 10% FCS and α-Modified Eagle’s Medium (α-MEM).
Hypoxia treatments, PARP inhibitors and cell cycle assays
Logarithmically growing cells were exposed to 1% O2, 0.2% O2 (chronic hypoxia) or 0% O2 (anoxia) with 5% CO2 and balanced N2 using a HypOxystation H35 and H85 (HypOxygen). To achieve cyclic hypoxia, logarithmically growing cells were exposed to 0% O2 for 30 min and 7% O2 for 30 min
Results
We have previously shown that chronic hypoxia decreases the protein expression of HR proteins (e.g., RAD51, RAD54, BRCA1 and BRCA2) in H1299 and a series of prostate cancer cell lines [6], [8]. To examine the effect of PARP inhibition in hypoxic cells at different oxygen concentrations, we treated H1299 and ME180 cells with 21%, 1% O2 or 0.2% O2 (chronic hypoxia) for 72 h or 0% O2 (anoxia) for 16 h. Anoxic (0% O2) gassing decreases the clonogenic survival of H1299 cells by >97% after 72 h [6].
Discussion
In this study, we did not observe PARP inhibitor toxicity when cells were exposed to cyclic hypoxia (acute anoxia (30 min to 3 h) followed by reoxygenation) for a duration of 72 h. We have previously shown that chronic hypoxia (0.2% O2 for 72 h) leads to a DNA repair deficient phenotype with increased sensitivity to mitomycin C, cisplatin, ionizing radiation or PARP inhibition [5], [6]. This was observed under hypoxic conditions that do not affect cell cycle distribution, proliferation or cell
Conflict of interest
None.
Acknowledgments
AZD2281 was generously provided by AstraZeneca. This work is supported by Grants from the Terry Fox Foundation Program Project Grant and also, in part, by the Ontario Ministry of Health and Long-Term Care. The views expressed do not necessarily reflect those of the Ontario Ministry of Health and Long Term Care. R.G. Bristow is a Canadian Cancer Society Research Scientist.
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These authors contributed equally to the manuscript.