527 International Journal of Radiation Biology, July 2014; 90(7): 527–537 © 2014 Informa UK, Ltd. ISSN 0955-3002 print / ISSN 1362-3095 online DOI: 10.3109/09553002.2014.906767 Correspondence: Nina Jeppesen Edin, Department of Physics, Biophysics Group, P.B. 1048 Blindern, N-0316 Oslo, Norway. Tel:  47 2285 5492. Fax:  47 2285 5671. E-mail: n.f.j.edin@fys.uio.no (Received 20 June 2013; revised 5 March 2014; accepted 5 March 2014) The roles of TGF- b3 and peroxynitrite in removal of hyper-radiosensitivity by priming irradiation Nina Jeppesen Edin 1,2 , Joe Alexander Sandvik 1 , Chang Cheng 3 , Linda Bergersen 3 & Erik Olai Pettersen 1 1 Department of Physics, University of Oslo, 2 Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, and 3 The Brain and Muscle Energy Group, Department of Anatomy and Centre for Molecular Biology and Neuroscience, University of Oslo, Norway a more resistant response in the dose range ~ 0.5–1 Gy called ‘increased radioresistance’ (IRR) (Lambin et al. 1993, Marples and Joiner 1993). HRS is, in all probability, the default response of cells to small doses of both high- and low-LET ionizing radiations (Marples et al. 2004). In the following the term HRS-response is used instead of HRS/ IRR-response for simplification. Exposing HRS-proficient cells to a high dose-rate (HDR) priming dose of 0.2–0.3 Gy transiently abolishes the HRS-response to subsequent HDR challenge irradiation (Marples and Joiner 1995, Joiner et al. 1996, Wouters and Skarsgard 1997, Short et al. 2001, Edin et al. 2007). However, previous studies from our laboratory has shown that if the priming dose of 0.3 Gy is given as a protracted irradiation at a low dose-rate of 0.3 Gy/h (LDR), the removal of HRS in T-47D human breast cancer and T98G human glioma cells becomes permanent (Edin et al. 2007). Furthermore, medium conditioned by these LDR-primed cells, when added to unprimed (i.e., control) cells, led to removal of HRS in the recipient cells. at effect was transient, lasting for about 2 weeks. Our conclusion was that the cells exposed to the LDR priming dose probably secrete a signaling fac- tor into the medium which transiently removes HRS in unprimed recipient cells (Edin et al. 2009a). at the effect is transient was taken as an indication that this factor has a limited lifetime and that it is not produced in an active form by the unprimed recipient cells. We then tested the effect of adding medium conditioned by unprimed cells to recipient cells and found that this did not influence HRS in the recipient cells. However, to our surprise it completely removed HRS in the recipient cells if the conditioned medium itself (without cells present) was LDR irradiated (0.3 Gy) before it was added to the recipient cells (Edin et al. 2009b, 2013). us, the possibility was that unprimed cells secrete a factor which is activated by LDR-irradiation itself. Furthermore, it was considered a possibility that the active factor secreted by the primed cells could be the same as the one activated by LDR in cell-conditioned medium. Abstract Purpose: To investigate the mechanisms inducing and maintaining the permanent elimination of low dose hyper-radiosensitivity (HRS) in cells given a dose of 0.3 Gy at low dose-rate (LDR) (0.3 Gy/h). Materials and methods: Two human HRS-positive cell lines (T-47D, T98G) were used. The effects of pretreatments with transforming growth factor beta (TGF-b) neutralizers, TGF-b3 or peroxynitrite scavenger on HRS were investigated using the colony assay. Cyto- plasmic levels of TGF-b3 were measured using post-embedding immunogold electron microscopic analysis. Results: TGF-b3 neutralizer inhibited the removal of HRS by LDR irradiation. Adding 0.001 ng/ml TGF-b3 to cells removed HRS in T98G cells while 0.01 ng/ml additionally induced resistance to higher doses. Cytoplasmic levels of TGF-b3 were higher in LDR-primed cells than in unirradiated cells. The presence of the peroxynitrite scavenger uric acid inhibited the effect of LDR irradiation. Furthermore, the permanent elimination of HRS in LDR-primed cells was reversed by treatment with uric acid. The removal of HRS by medium from hypoxic cells was inhibited by adding TGF-b3 neutralizer to the medium before transfer or by adding hypoxia inducible factor 1 (HIF-1) inhibitor chetomin to the cell medium during hypoxia. Conclusions: TGF-b3 is involved in the regulation of cellular responses to small doses of acute irradiation. TGF-b3 activation seems to be induced by low dose-rate irradiation by a mecha- nism involving inducible nitric oxide (iNOS) and peroxynitrite, or during cycling hypoxia by a mechanism most likely involving HIF-1. The study suggests methods to turn resistance to doses in the HRS-range on (by TGF-b3) or off (by TGF-b3 neutralizer or by peroxynitrite inhibition). Keywords: TGF-β3, low dose hyper-radiosensitivity, low dose-rate irradiation, peroxynitrite Introduction Low dose hyper-radiosensitivity (HRS) is characterized by a high sensitivity per unit dose for doses below ~ 0.5 Gy. For low LET (linear energy transfer) radiation this is followed by Int J Radiat Biol Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 09/22/14 For personal use only.