Inhibition of the BER Factor APE1 Disrupts Repair of Double-Strand DNA Damage in Cells Treated with Low Dose-Rate, but Not High Dose-Rate XRadiation

Introduction: Radiotherapy is utilised in the treatment of many cancers, but its efficacy is limited by normal tissue toxicity and new radiotherapy techniques are thus urgently sought. The AP endonuclease APE1 is involved in repair of single strand DNA damage through the break excision repair (BER) pathway and altered levels of APE1 have been found in some cancers. In this study, we investigated the effects of APE1 inhibition, using the APE1-specific inhibitor CRT0044876 (CRT), in tumour cells following exposure to either high dose-rate (HDR) or low dose-rate (LDR) X-irradiation. Materials and Methods: Treatment efficacy was assessed by clonogenic assay followed by isobologram analysis to assess potential synergy. Cell cycle distribution was assessed by propidium iodide staining followed by flow cytometry. Induction of DNA damage and repair was assessed by single cell gel electrophoresis and by H2A.X phosphorylation. Results: In isobologram analysis of clonogenic assays, combinations of CRT and both HDR and LDR X-irradiation resulted in supra-additive levels of cytotoxicity. Cell cycle analysis showed that, while CRT had no effect on cell cycle distribution, HDR or LDR X-irradiation, and CRT-HDR or CRT-LDR combination treatment induced significant G2/M arrest. However, CRT-HDR combinations induced significantly less G2/M accumulation than HDR alone. Analysis of DNA damage indicated that treatment with HDR or LDR X-irradiation and CRT-HDR and CRT-LDR combinations induced significant double-strand DNA damage. Cells treated with CRT-HDR exhibited a significant reduction in γH2A.X foci 24 h after treatment compared to 1 h, suggesting induction of DNA repair mechanisms. However, in cells treated with CRT-LDR, there was no significant difference between H2A.X phosphorylation at 24 h compared to 1 h, suggesting disruption of dsDNA repair pathways. Conclusions: Pharmacological inhibition of APE1 enhances the cytotoxicity of high dose-rate and low dose-rate X-irradiation by different mechanisms.