Astronauts face considerable dosages of space rays during long-term space missions. because the response is absent in RelA knock-down cells completely. NF-Bs role within the mobile radiation response depends upon rays quality. 0.05, Figure 4A). The reactions from the parental cell range as well as the shRNA control cell range were not considerably different in no case based on a = 2 level of 0.05. Carbon ion induced d2EGFP expression was completely abolished (Figure 4B). Open in a separate window Figure 4 Effect of RelA knock-down on NF-B activation by X-rays and TNF- (A), and by carbon ions (B). HEK-pNF-B-d2EGFP/Neo L2 cells, cells stably transfected with the shRNA control vector (HEK shRNA control) or the RelA shRNA plasmid (HEK shRNA RelA) were seeded in petri dishes, grown for two days, and exposed to X-rays (200 kV, LET ~0.3C3 keV/m), incubated with 10 ng/mL TNF- (A) or irradiated with 13C-ions (75 MeV/n, LET 34 keV/m). 18 h after exposure, cells were harvested by trypsination, fixed with 3.5% formaldehyde and the percentage of d2EGFP(+) cells was determined by flow cytometry. 2.4. Growth of RelA Knock-Down Cells In order to determine whether RelA knock-down affects basic cellular functions such as growth, cell numbers were counted during a growth period of 10 days. HEK hSPRY1 shRNA RelA cells showed a prolonged lag phase compared to HEK-pNF-B-d2EGFP/Neo clone L2 cells (Figure 5). Once proliferation starts, both cell lines grow with the same velocity. Open in a separate window Figure 5 Growth kinetics of HEK shRNA RelA cells compared to the original cell line. 104 cells/cm2 HEK-pNF-B-d2EGFP/Neo L2 cells and cells stably transfected with the RelA shRNA plasmid (HEK shRNA RelA) were seeded in petri dishes. On a daily base, cells were harvested by trypsination and counted in a counting chamber. The graph shows means and standard errors of three independent experiments. 2.5. Survival of RelA Knock-Down Cells After X-ray and after Heavy Ion Exposure The survival curves after exposure of HEK-pNF-B-d2EGFP/Neo clone L2 cells and HEK shRNA RelA cells were of curvilinear shape (Figure 6). The curve of the RelA knock-down cells is significantly steeper, indicating a higher radiosensitivity. The D0 indicating the dose necessary to reduce survival of HEK cells to 37% is 1.12 Gy for the parental cell line compared to 0.82 Gy for the RelA knock-down cells (Table 3). Open in a separate window Physique 6 Clonogenic survival of HEK cells with RelA knock-down compared to the parental cells after X-irradiation (200 kV). HEK-pNF-B-d2EGFP/Neo L2 and HEK shRNA RelA cells were irradiated, incubated and colonies were fixed after 14 to 21 days (means SE of 7C13 impartial experiments with six replicates each). Table 3 Parameters of the survival curves *. = 2= 2 level of 0.05 was considered as significant. Comparison of two regression lines for HEK-pNF-B-d2EGFP/Neo L2 and HEK shRNA RelA cells is based on the hypothesis (2yx)1 (2yx)2 for D0; P, probability. High-LET radiation exposure of HEK cells results in purely exponential survival curves (Physique 7). Based on energy dose, heavy ions with an LET of 55 keV/m are most efficient in cell killing (Physique 7A), while radiation qualities with an LET above or below this range are less efficient in cell killing (Physique 7A,B). The D0 first decreases to 0.47 Gy for silicon ions, then increases with increasing LET to 0.72 Gy for argon ions (Table 3). Open in a separate window Physique 7 Clonogenic survival of HEK cells with RelA knock-down compared to the parental cells after exposure to heavy ions of diffent LET (A), linear energy transfer (LET) 100 keV/m, (B) LET 100 keV/m). HEK-pNF-B-d2EGFP/Neo L2 and HEK shRNA RelA cells were irradiated, incubated and colonies were fixed after 14C21 days (means SE of 1C2 impartial experiments with each six replicates). 2.6. Induction RGH-5526 of NF-B Target Gene Expression by Exposure to Different Radiation RGH-5526 Qualities As NF-B was weakly activated by X-rays and activated by heavy ions to a higher extent, dependent on LET, but only for X-rays, a reduction of survival in case of RelA downregulation was observed, the expression of 88 NF-B target genes was profiled 6 h after RGH-5526 exposure to X-rays and heavy ions (Table 4). Table 4 Relative gene expression in HEK-pNF-B-d2EGFP/Neo L2 (HEK wt) and HEK.