Abstract
DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand breaks (DSBs) repair, and it consists of DNA-PKcs, Ku70, and Ku86. It has been shown that the promoter regions of these three genes have Sp1 binding sites, and the expression levels are correlated with that of Sp1.The purpose of this study is to clarify the contribution of Sp1 to radiosensitivity of cells through the transcriptional regulation of DSBs-repair genes. We investigated whether Sp1 affects the expression level of DNA-PKcs, Ku70, Ku86, and XRCC4. In addition, we examined the DSBs repair activity in Sp1-down-regulated cells. A human transformed kidney cell line, 293T cells were transfected with siRNA vector targeting Sp1 (siSp1) or the control vector (siC). The vector-transfected cells were selected with G418 sulfate. After seven days selection, protein levels of DNA-PKcs, Ku70, Ku86, and XRCC4 were evaluated by western blotting. The DSBs repair kinetics were determined by pulsed-field gel electrophoresis (PFGE) after 80 Gy X-irradiation. The protein level of Sp1 in siSp1-transfected cells was 31% compared with siC-transfected cells. DNA-PKcs, Ku70, and Ku86 were down-regulated to 6%, 3%, and 53% of the control level, respectively. The protein level of XRCC4 was not changed. These results indicate Sp1-dependent transcriptional regulation of DNA-PK. The PFGE study revealed the apparently lower efficiency of DSBs repair in siSp1-transfected cells than siC-transfected cells. This study showed that Sp1 regulates the transcription of DNA-PK and possibly determines the cellular radiosensitivity.
