PURPOSE: The E3 ubiquitin ligase RNF8 regulates the accumulation or removal of a number of proteins at DNA lesions, thereby playing a critically important role in DNA damage response. The present study investigated the possibility of using RNF8 as a new target in the radiation treatment of human non-small cell lung cancer. METHODS AND MATERIALS: We used RNA interference technology to silence the expression of RNF8 in A549 cells, and then detected the radiation response by colony forming assays. DNA repair was monitored by γ-H2AX foci formation after RNF8 depletion. Expression of Ku70 and Rad51 were assessed by immunofluorescent staining and Western blotting. Cell cycle and apoptosis were measured by flow cytometry assays. RESULTS: After lentivirus-mediated siRNA transfection, expression of RNF8 in A549 cells downregulated which led to an increased radiosensitivity and impaired DNA repair. RNF8 knockdown did not affect Ku70 expression, however, Rad51, a key player in homologous recombination (HR) repair, was abrogated at sites of DNA damage. Furthermore, we observed an extended G2/M arrest and an increased induction of apoptosis after ionizing radiation in the absence of RNF8. CONCLUSIONS: RNF8 silencing effectively downregulates Rad51 therefore maybe impairing HR repair, and prolongs the G2/M accumulation as well as cell apoptosis upon radiation, which all suggest an enhanced radiosensitivity on A549 cells.
PURPOSE: The E3 ubiquitin ligase RNF8 regulates the accumulation or removal of a number of proteins at DNA lesions, thereby playing a critically important role in DNA damage response. The present study investigated the possibility of using RNF8 as a new target in the radiation treatment of humannon-small cell lung cancer. METHODS AND MATERIALS: We used RNA interference technology to silence the expression of RNF8 in A549 cells, and then detected the radiation response by colony forming assays. DNA repair was monitored by γ-H2AX foci formation after RNF8 depletion. Expression of Ku70 and Rad51 were assessed by immunofluorescent staining and Western blotting. Cell cycle and apoptosis were measured by flow cytometry assays. RESULTS: After lentivirus-mediated siRNA transfection, expression of RNF8 in A549 cells downregulated which led to an increased radiosensitivity and impaired DNA repair. RNF8 knockdown did not affect Ku70 expression, however, Rad51, a key player in homologous recombination (HR) repair, was abrogated at sites of DNA damage. Furthermore, we observed an extended G2/M arrest and an increased induction of apoptosis after ionizing radiation in the absence of RNF8. CONCLUSIONS:RNF8 silencing effectively downregulates Rad51 therefore maybe impairing HR repair, and prolongs the G2/M accumulation as well as cell apoptosis upon radiation, which all suggest an enhanced radiosensitivity on A549 cells.