BOA19
A radiosensitivity screen of a siRNA library of DNA repair genes identifies DNA polymerase theta as a tumour specific radiosensitiser
Geoff Higgins1, Remko Prevo1, Yin-Fai Lee1, Michio Yoshimura1, Stephen Taylor2, Eric Bernhard1, Gillies McKenna1
1Gray Institute for Radiation Oncology and Biology, Oxford, UK, 2Computational Biology Research Group, Oxford University, Oxford, UK
Background
Tumours differ significantly in their sensitivity to ionizing radiation. Patients with radioresistant tumours have a worse prognosis than those with radiosensitive disease. The molecular determinants for this are largely unknown. We have screened a 200 gene siRNA library of genes involved in DNA repair to identify genes whose knockdown causes tumour radiosensitisation.
Method
An siRNA library consisting of 200 pools of four individual siRNAs (Dharmacon) was used to transfect human HNSCC cells (SQ20B) in 96-well plates. Plates were subjected to 4Gy irradiation 48 hours after transfection. The amount of double strand DNA breaks remaining at 24h after irradiation was used as a surrogate marker for cell death and was measured by quantifying nuclear gamma-H2AX foci using automated microscopy and image analysis. siRNAs causing a significant increase in post-radiation gamma-H2AX foci compared with non-targeting control siRNA were considered hits. Colony forming assays on several tumour cell lines and normal cells were then used to confirm actual radiosensitisation by these siRNAs. Assays using individual siRNAs from the original positive siRNA pools were used to rule out off-target effects.
Results
The screen identified several genes associated with increased gamma-H2AX foci following irradiation. These included genes such as Lig IV and BRCA1 & 2, which are known to alter radiosensitivity as well as novel genes such as PolQ (DNA polymerase theta). Colony forming assays confirmed that PolQ knockdown is associated with increased radiosensitivity. The sensitisation enhancement ratios at 10% surviving fraction were 1.28, 1.26 and 1.19 for T24, HeLa and PSN1 tumour cells respectively. Normal human fibroblasts were not radiosensitised by PolQ knockdown.
Conclusion
The siRNA screen identified PolQ knockdown as causing increased gamma-H2AX foci post-radiation. Colony forming assays confirmed PolQ as a potential target for tumour specific radiosensitisation.
