Host: The Japan Radiation Research Society, Chairman of the 52nd Annual Meeting, Toshiteru Okubo (Radiation Effects Research Foundation)
Transcriptional regulation of DNA double-strand break repair genes by Sp1
DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand breaks (DSBs) repair, and it consists of Ku70, Ku80 and DNA-PKcs. 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 radiation sensitivity of cells through the transcriptional regulation of DSBs-repair genes. We investigated whether Sp1 affects the protein and mRNA levels of Ku70, Ku80, DNA-PKcs, XRCC4, NBS1, MRE11 and MDC1. In addition, we examined the DSBs-repair, DNA-PK activity, cell cycle, and radiation sensitivity in Sp1-dawn-regulated cells. A human transformed kidney cell line 293T was transfected with siRNA vector targeting Sp1 (Sp1-siRNA) or control vector. The vector-transfected cells were selected with G418. After 7 days selection, protein and mRNA levels were evaluated by Western blotting and RT-PCR, respectively. The protein and mRNA levels of Sp1, Ku70, Ku80, DNA-PKcs, XRCC4, NBS1, MRE11 and MDC1 were down regulated by the Sp1-siRNA treatment. The DSBs-repair after 80 Gy irradiation and DNA-PK activity were suppressed by the Sp1-siRNA treatment. The surviving fraction after irradiation was also suppressed by the Sp1-siRNA treatment. However, cell cycle was not affected by the Sp1-siRNA treatment. These results suggest that Sp1 regulates the radiation sensitivity by the transcriptional regulation of DSBs-repair genes.
