The role of nitric oxide radicals in removal of hyper-radiosensitivity by priming irradiation

Abstract

In this study, a mechanism in which low-dose hyper-radiosensitivity (HRS) is permanently removed, induced by low-dose-rate (LDR) (0.2–0.3 Gy/h for 1 h) but not by high-dose-rate priming (0.3 Gy at 40 Gy/h) was investigated. One HRS-negative cell line (NHIK 3025) and two HRS-positive cell lines (T-47D, T98G) were used. The effects of different pretreatments on HRS were investigated using the colony assay. Cell-based ELISA was used to measure nitric oxide synthase (NOS) levels, and microarray analysis to compare gene expression in primed and unprimed cells. The data show how permanent removal of HRS, previously found to be induced by LDR priming irradiation, can also be induced by addition of nitric oxide (NO)-donor DEANO combined with either high-dose-rate priming or exposure to prolonged cycling hypoxia followed by reoxygenation, a treatment not involving radiation. The removal of HRS appears not to involve DNA damage induced during priming irradiation as it was also induced by LDR irradiation of cell-conditioned medium without cells present. The permanent removal of HRS in LDR-primed cells was reversed by treatment with inducible nitric oxide synthase (iNOS) inhibitor 1400W. Furthermore, 1400W could also induce HRS in an HRS-negative cell line. The data suggest that LDR irradiation for 1 h, but not 15 min, activates iNOS, and also that sustained iNOS activation is necessary for the permanent removal of HRS by LDR priming. The data indicate that nitric oxide production is involved in the regulatory processes determining cellular responses to low-dose-rate irradiation.

The roles of TGF-β3 and peroxynitrite in removal of hyper-radiosensitivity by priming irradiation

“…We have previously shown that NO is involved in the response to LDR irradiation (Edin et al 2013). Attempts to measure NO levels in LDR-primed cells have been tried extensively in our laboratory but have failed.…”

“…The cells were grown as monolayer cultures in RPMI (Roswell Park Memorial Institute) 1640 medium (JRH Biosciences, Kansas, USA), supplemented with 10% fetal calf serum (Gibco, Paisley, UK), 2 mM L-glutamine (Sigma, St Louis, MO, USA), 200 units l 21 insulin (SIGMA), and 1% penicillin/streptomycin (Gibco) at 37°C in air containing 5% CO 2 (Edin et al 2012(Edin et al , 2013. The cells were grown as monolayer cultures in RPMI (Roswell Park Memorial Institute) 1640 medium (JRH Biosciences, Kansas, USA), supplemented with 10% fetal calf serum (Gibco, Paisley, UK), 2 mM L-glutamine (Sigma, St Louis, MO, USA), 200 units l 21 insulin (SIGMA), and 1% penicillin/streptomycin (Gibco) at 37°C in air containing 5% CO 2 (Edin et al 2012(Edin et al , 2013.…”

“…In order to confirm that TGF-b3 is able to remove HRS, experiments were performed where recombinant TGF-b3 was added to the cell medium of unirradiated cells and even concentrations as low as 0.001 ng/ml TGF-b3 removed the hypersensitive response to doses in the HRS-range in the recipient T98G cells. Only in combination with NO-donor DEANO (Diethylamine NONOate sodium salt hydrate) did HDR priming have the same effect as LDR priming (Edin et al 2013). Interestingly, when the TGF-b3 concentration was increased to 0.01 ng/ml, an increase in surviving fraction was observed also for radiation doses above the acids tyrosine, tryptophan and phenylalanine (van der Vliet et al 1994, Alvarez et al 1996) and a possible explanation of the data in Figure 6 is that peroxynitrite works as a scavenger for selective inhibition of LAP-3 in a way similar to that of NO for LAP-1.…”

“…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(Edin et al , 2013. 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(Edin et al , 2013.…”

Low-Dose-Rate Irradiation for 1 Hour Induces Protection Against Lethal Radiation Doses but Does Not Affect Life Span of DBA/2 Mice

“…The protective effect vanished if the priming irradiation time was reduced to 15 minutes. 26 In the experiments with reporter cells exposed to serum from irradiated mice, the effect was also seen for 3 cGy/h. 24 The effect seems thus to be dose independent as long as the dose rate is within a certain window and the irradiation time is long enough.…”