Combined ataxia telangiectasia mutated and DNA-dependent protein kinase inhibition radiosensitizes Madin-Darby canine kidney cells.

Uncovering radiation toxicity is critical for the adaptation and expansion of advanced radiation therapies and for the development of novel cancer radiotherapy. In the near future, advanced radiotherapies, including heavy ion beam treatment, are expected to be applied in the treatment of dogs, but further basic research on the effects of radiation using canine normal and cancer cells is necessary to actually apply these techniques and achieve high therapeutic efficacy. The radiation sensitivity is varied by the activities of DNA damage response (DDR) and DNA repair. The development of radiosensitizers that target DDR- and DNA repair-kinases, like ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), is progressing and is expected to be introduced into canine radiotherapy. However, there are no cytotoxicity reports on using the combination of radiation and these sensitizers as treatment in canine cells. In this study, we examined the cytotoxic effects of X-rays and/or radiosensitizers on the Madin-Darby canine kidney (MDCK) cell line. Our results show that X-rays suppress MDCK cell colony formation and proliferation in a dose-dependent manner. Additionally, our observations imply that the combination treatment with ATM inhibitor KU-55933 and DNA-PK inhibitor NU7441 significantly increased X-ray cytotoxicity in MDCK cells compared with the drugs alone. Furthermore, our findings further suggest that MDCK cells might be useful in clarifying the cytotoxicity in canine epithelial cells due to radiation and/or radiosensitizers, such as molecule-targeted drugs.

Nuclear factor erythroid 2-related factor 2-antioxidant activation through the action of ataxia telangiectasia-mutated serine/threonine kinase is essential to counteract oxidative stress in bovine mammary epithelial cells.

Nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) is a transcription factor that binds to the antioxidant response element (ARE) in the upstream promoter region of various antioxidant-responsive genes. Hence, at least in nonruminants, the NFE2L2-ARE signaling pathway plays an important role in the cellular antioxidant defense system. Whether oxidative stress in bovine mammary epithelial cells alters NFE2L2 or the NFE2L2-ARE pathway is unclear. Therefore, the objective of this study was to examine the response in NFE2L2- and NFE2L2-ARE-related components in bovine mammary epithelial cells (BMEC) under oxidative stress. An in silico analysis to identify potential phosphorylation sites on NFE2L2 and the protein kinases was performed with Netphos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) and Scansite (http://scansite.mit.edu) software. Isolated BMEC were exposed to H2O2 (600 µM) for 6 h to induce oxidative stress. In silico analysis revealed ataxia telangiectasia-mutated (ATM) serine/threonine kinase as a key kinase responsible for the phosphorylation of NFE2L2. Thus, after the 6 h incubation with H2O2, BMEC were transiently transfected with ATM-small interfering RNA (siRNA) 1, 2, or 3. Compared with the control, transfection with ATM-siRNA3 resulted in proliferation rates that were 60.7 and 36.2% lower with or without H2O2. In addition, production of reactive oxygen species and malondialdehyde increased markedly, but activities of superoxide dismutase, glutathione peroxidase, catalase, and glutathione-S-transferase decreased markedly in transfected cells without or with H2O2 compared with the control. Transfected cells had markedly lower protein and mRNA expression of NFE2L2 without or with H2O2 compared with the control. In addition, fluorescent activity of the ARE in transfected BMEC indicated that NFE2L2-driven transcriptional activation decreased under oxidative stress. Overall, results indicate that ATM is a physiologically relevant NFE2L2 kinase. Furthermore, inhibition of ATM activity can cause marked alterations in oxidative stress leading to cell death as a result of diminished capacity of BMEC to cope with H2O2-induced cytotoxicity. The relevance of this kinase in vivo merits further study.