Overexpression of TOB1 confers radioprotection to bronchial epithelial cells through the MAPK/ERK pathway

Abstract

The aim of this study was to investigate the effects and mechanisms of antiproliferative transducer of erbB2, 1 (TOB1) on the radiosensitivity of the normal human bronchial epithelial cell line HBE. After exposure to different doses of irradiation or a certain dose for different time intervals, the expression of TOB1 mRNA and protein in HBE cells was determined by semi-quantitative RT-PCR and western blot analysis. Liposome-induced recombinant plasmid transfection and G418 selection were performed to establish a stably transfected TOB1-overexpressing HBE cell line. A clonogenic assay was used to determine the radiosensitivity of the HBE cells with different TOB1 expression statuses. The cell cycle distribution was detected by flow cytometry. The ionizing radiation (IR)-induced γ-H2AX foci formation was detected by immunofluorescence assay. The related mechanism was explored by western blot analysis. TOB1 expression in the HBE cells was not induced by IR, neither dose-dependently nor time-dependently. Compared to the parental or 'mock' transfected HBE cells, the radiosensitivity of HBE cells overexpressing TOB1 was significantly decreased (P<0.05). Exogenous TOB1 prevented HBE cells from apoptosis after IR, in contrast to the control cells (P<0.05), and significantly decreased the IR-induced γ-H2AX foci formation. After IR, the expression of DNA damage repair proteins such as XRCC1, MRE11, FEN1 and ATM was increased in the TOB1‑overexpressing HBE cells when compared with the expression levels in the control cells. HBE/TOB1 cells presented a much higher phosphorylated ERK1/2 and phosphorylated p53 when compared with the levels in the control cell lines when receiving 6 Gy of X-rays. Notably, the increased expression of phosphorylated p53 in HBE/TOB1 cells after IR was sufficiently blocked by U0126, a specific inhibitor of MEK1/2. Different from its functions in several lung cancer cell lines, TOB1 demonstrated a radioprotective function in the immortalized normal human bronchial epithelial cell line HBE via the MAPK/ERK signaling pathway.