| Objective:Radiation-induced bystander effects(RIBES) refer to the biological changes detected in the unirradiated cells when receiving signals from the irradiated neighboring cells. Along with the acceptance of the definition of RIBES, many researchers believe that it may play a role in radiation-induced cancer and the therapeutic effects and the side effects of radiotherapy. But the detailed molecular mechanisms of RIBES are still unclear. The occurence of bystander effect involves irradiated cells and unirradiated cells. In the present study, we aimed to explore the mechanisms underlying radiation-induced bystander signaling molecules released by irradiated Ha Cat cells and the consequence of bystander effects. First, we investigated weather X-irradiation could induce medium-mediated bystander effects in Ha Cat cells and it’s potential mechanisms.Based on the work, we determined whether X-rays or alpha particles could induced bystander effect in 2D culture model which was consist of Ha Cat and WS1 cells. And we further explored if bystander effect is dependent on LET and its mechanisms. Then we tested the migration ability of unirradiated Ha Cat and WS1 cells which were co-cultured with irradiated Ha Cat cells. Finally, we set up 3D culture model of the skin which could be used to investigate bystander effects in ex vivo.Methods:Conditioned medium transfer and co-culture methods were used to study whether X-rays could induced bystander effects in Ha Cat cells. Induction of MN, 53BP1 foci and cell proliferation were used as endpoints. The level of TGF-β1 in condiationed medium was measured using ELISA. The ROS levels in bystander Ha Cat cells were determined with ROS detection kit.Using a transwell co-culture system, we observed the formation of MN in unirradiated WS1 cells co-cultured with Ha Cat cells irradiated by X-rays or alphaparticles. Then Ha Cat cells were pretreated with SB431542, a potent and selective inhibitor of TGF-β1 receptor kinases, prior to α-irradiation, then co-cultured with unirradiated WS1 cells after irradiation. And we observed if the MN formation in bystander WS1 cells was changed. Using RT-PCR, we measured the changes of mi R-21 expression level in Ha Cat cells after irradiation. Next we overexpressed mi R-21 in Ha Cat cells by transfecting the cells with mi R-21 mimic, then co-cultured the irradiated transfected cells with unirradiated WS1 cells after irradiation. And we observed if the MN formation in bystander WS1 cells was changed. On the other hand, we downregulated mi R-21 expression in Ha Cat cells by transfecting cells with mi R-21 inhibitor, then co-cultured the transfected cells with WS1 cells and observed if there was bystander MN formation in WS1 cells. Using Western Blot we detected the activation/phosphorylation of Smad2 in irradiated Ha Cat cells or the cells were treated with SB431542 or transfected with mi R-21 prior to α-irradiation. Then we detected the activation/phosphorylation of Smad2 in Ha Cat cells which is transfected with mi R-21 inhibitor. Then we measured the changes of mi R-21 expression level in Ha Cat cells were treated with SB431542 prior to α-irradiation. Finally, we measured the levels of ROS in irradiated Ha Cat cells.Using a transwell insert co-culture system, we observed the migration ability of unirrdiated Ha Cat cells and WS1 cells co-cultured with alpha particle irradiated Ha Cat cells.At last, we set up 3D culture model of the skin by using rat tail collagen and Ha Cat cells and WS1 cells.Results:1.Compared with the control cells receiving medium from sham-irradiated cells(SCM), the percentage of cells with MN in bystander Ha Cat cells increased by 28% and58% after cultured with 3h radiation-conditioned medium(3h RCM) for 24 h and 48 h,respectively. The percentage of positive cells with 53BP1 foci in bystander bystander Ha Cat cells increased by 44% after cultured with 3h RCM for 1 h. However, no significant change in cell proliferation was observed in bystander Ha Cat cells after cultured with 3h RCM for 24, 48 and 72 h. The level of TGF-β1 in 3 h RCM was no significant change compared with fresh medium. The ROS levels in bystander Ha Cat cells cultured with 3 h RCM increased by 33% compared with untreated control, but were similar to those in unirradiated cells cultured with 3 h SCM. Additionally, afterco-cultured with Ha Cat cells irradiated with 1 Gy of X-rays for 1 h and 48 h, there was a 35% and 54% increase in the 53BP1 foci and MN formation induction in unirradiated bystander Ha Cat cells, respectively.2.I. Using a transwell insert co-culture system, MN formation in unirradiated WS1 cells was chosen as the endpoint for radiation-induced bystander response. We found that after co-cultured with Ha Cat cells irradiated with 0.56 Gy of α-particles for 24 h,there was a 53% increase in the frequency of MN formation in unirradiated bystander WS1 cells compared with the WS1 cells co-cultured with sham irradiated Ha Cat cells.However, no bystander MN formation was observed when using X-rays(1, 2, 5 and 10Gy). This was not due to less damage caused by X-rays. Indeed X-rays(1-10 Gy)caused greater MN formation and cell killing in irradiated Ha Cat cells than 0.56 Gy of alpha particles did.II. Ha Cat cells were pretreated with SB431542 prior to α-irradiation, then co-cultured with unirradiated WS1 cells after irradiation. And we observed no MN formation in bystander WS1 cells.III. Smad2 in Ha Cat cells was phosphorylated immediately after α-irradiation, and the elevated phosphorylation level last for at least 2 hours. But Smad2 was not activated after Ha Cat cells were irradiated with X-rays,evev with higher doses(2-10 Gy).IV. When Ha Cat cells were treated with SB431542 prior to α-irradiation, α-particle-induced phosphorylation of Smad2 was significantly inhibited.V. When Ha Cat cells were irradiated with 0.56 Gy of α-particles, the expression level of mi R-21 decreased about 1.6 and 1.2 folds at 1 and 3 h after irradiation.In contrast, when Ha Cat cells were irradiated with 1 Gy of X-rays, the expression level of mi R-21 decreased about 1.4 folds 1 h after irradiation, but increased 1.7 folds 3 h after irradiation.When WS1 cells were co-cultured with α-irrsdiated Ha Cat cells with high expression of mi R-21, no bystander MN induction was observed. Downregulationg mi R-21 expression in Ha Cat cells alone could induced bystander-like MN formation in WS1 cells after co-culture.VI. Pretreatment of Ha Cat cells with SB431542 prior to α-irradiation rescued the decrease in mi R-21 expression from 1.6 folds to 1.2 folds 1 h after irradiation, and slight increased the mi R-21 expression 3 h after irradiation。VII. When the mi R-21-overexpressed Ha Cat cells were irradiated with α-particles,compared with the parental cells and the cells transfected with the negative control of mi R-21 mimic, the activation of Smad2 was inhibited. Additionally, the phosphorylation of Smad2 was elevated in the Ha Cat cells transfected with mi R-21 inhibitor compared with in the cells transfected with the negative control for mi R-21 inhibitor.VIII. When Ha Cat cells were irradiated with 0.56 Gy of α-particles, the level of ROS was increased by 25% 5 h later. When Ha Cat cells were irradiated with 1 Gy of X-ray, the level of ROS was only increased by 3%.3. Using a transwell insert co-culture system, the ability of cell migration of unirrdiated Ha Cat cells was speed up and the ability of cell migration of unirrdiated WS1 cells was slow down after co-cultured with alpha particle-irradiated Ha Cat cells for 24, 48 and 72 h.4. We set up 3D culture model of the skin.Conclusion:1. X-rays could induce bystander effects in Ha Cat cells.2. Ha Cat cells irradiated with α-particles but not X-rays could elicit bystander MN formation in unirradiated WS1 cells through medium-mediated mechanisms, suggesting that the bystander MN formation in this system is dependent on radiation quality.3. Radiation quality-dependence of bystander effect may be associated with the TGF-β1-Smad2 pathway and mi R-21 in irradiated cells. And they were not independent,they regulated each other.4. The ability of cell migration of unirrdiated Ha Cat cells was speed up and the ability of cell migration of unirrdiated WS1 cells was slow down after co-cultured with alpha particle-irradiated Ha Cat cells. |
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