Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

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Gamma-H2AX foci detection is the standard method to quantify DNA double-strand break (DSB) induction and repair. In this study, we investigated the induction and decay of γ-H2AX foci of different tumor cell lines and fibroblasts with known mutations in DNA damage repair genes, including ATM, LigIV, DNA-PKcs, Rad51 and Rad54. A radiation dose of 2.4 Gy was used for either an acute single high-dose-rate (sHDR) exposure or a pulsed dose-rate (pDR) exposure over 24 h. The number of γ-H2AX foci was determined at 30 min and 24 h after sHDR irradiation and directly after pDR irradiation. In a similar manner, γ-H2AX foci were also examined in lymphocytes of patients with differences in normal tissue toxicity after a total radiation dose of 1 Gy. In an initial count of the number of foci 30 min after sHDR irradiation, repair-proficient cell types could not be distinguished from repair-deficient cell types. However at 24 h postirradiation, while we observed a large decrease in foci numbers in NHEJ-proficient cells, the amount of γ-H2AX foci in cell types with mutated NHEJ repair remained at high levels. Except for IRS-1SF cells, HR-deficient cell types eventually did show a moderate decrease in foci number over time, albeit to a lesser extent than their corresponding parentals or repair-proficient control cells. In addition, analysis of γ-H2AX foci after sHDR exposure of patients with different sensitivity status clearly showed individual differences in radiation response. Radiosensitive patients could be distinguished from the more radioresistant patients with γ-H2AX foci decay ratios (initial number of foci divided by residual number of foci). Significantly higher decay ratios were observed in patients without toxicities, indicating more proficient repair compared to patients with radiation-induced side effects. After pDR irradiation, no consistent correlation could be found between foci number and radiosensitivity. In conclusion, γ-H2AX formation is a rapid and sensitive cellular response to DNA DSBs. Decay ratios after sHDR exposure elucidated large differences in γ-H2AX foci kinetics between the repair-proficient or -deficient cell types and patients. This assay may be useful for measuring cellular radiosensitivity and could serve as a clinically useful test for predicting radiosensitivity ex vivo before treatment.