Primary cilium participates in radiation-induced bystander effects through TGF-β1 signaling

Abstract

Many studies have indicated that tumor growth factor-beta (TGF-β) signaling mediates radiation-induced bystander effects (RIBEs). The primary cilium (PC) coordinates several signaling pathways including TGF-β signaling to regulate diverse cellular processes. But whether the PC participates in TGF-β induced RIBEs remains unclear. The cellular levels of TGF-β1 were detected by western blot analysis and the secretion of TGF-β1 was measured by ELISA kit. The ciliogenesis was altered by CytoD treatment, STIL siRNA transfection, IFT88 siRNA transfection, or KIF3a siRNA transfection, separately, and was detected by western blot analysis and immunofluorescence staining. G₀ /G₁ phase cells were arrested by serum starvation and S phase cells were induced by double thymidine block. The TGF-β1 signaling was interfered by LY2109761, a TGF-β receptor 1 (TβR1) inhibitor, or TGF-β1 neutral antibody. The DNA damages were induced by TGF-β1 or radiated conditional medium (RCM) from irradiated cells and were reflected by p21 expression, 53BP1 foci, and γH2AX foci. Compared with unirradiated control, both A549 and Beas-2B cells expressed and secreted more TGF-β1 after carbon ion beam or X-ray irradiation. RCM collected from irradiated cells or TGF-β1 treatment caused an increase of DNA damage in cocultured unirradiated Beas-2B cells while blockage of TGF-β signaling by TβR1 inhibitor or TGF-β1 neutral antibody alleviates this phenomenon. IFT88 siRNA or KIF3a siRNA impaired PC formation resulted in an aggravated DNA damage in bystander cells, while elevated PC formation by CytoD or STIL siRNA resulted in a decrease of DNA damage. Furthermore, TGF-β1 induced more DNA damages in S phases cells which showed lower PC formation rate and less DNA damages in G₀ /G₁ phase cells which showed higher PC formation rate. This study demonstrates the particular role of primary cilia during RCM induced DNA damages through TGF-β1 signaling restriction and thereby provides a functional link between primary cilia and RIBEs.

Keywords: DNA damages; TGF-β1; ionizing radiation; primary cilium; radiation-induced bystander effects.