Qi Jun-Juan, Liu Ling, Cao Ji-Xiang, An Guo-Shun, Li Shu-Yan, Li Gang, Jia Hong-Ti, Ni Ju-Hua
Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Xue Yuan Road 38, Beijing, 100191, People's Republic of China.
Mol Cell Biochem. 2015 Jan;399(1-2):179-88. doi: 10.1007/s11010-014-2244-7. Epub 2014 Oct 14.
The p53R2 gene encoding a small subunit of the ribonucleotide reductase has been identified as a p53-inducible gene. Although this gene is discovered as a target for p53 family proteins, the mechanism underlying p53R2 induction by DNA damage in p53-defiencient cells remains to be elucidated. In this study, we demonstrate that transcription factor E2F1 regulates the p53R2 gene expression in p53-deficient cells. We found that p53R2 was a target for E2F1 in DNA damage response (DDR), because ectopic expression of E2F1 in HCT116-p53(-/-) cells resulted in the increase of p53R2 mRNA and protein expression, and silencing E2F1 diminished its basic expression. Combination of luciferase reporter assay with overexpression or knockdown of E2F1 revealed that E2F1 directly activates the p53R2 gene. Chromatin immunoprecipitation (ChIP) assay showed E2F1 directly bound to the site (TTTGGCGG) at position -684 to -677 of the promoter under E2F1 overexpression or adriamycin (ADR) exposure. Moreover, silencing p53R2 could enhance apoptotic cell death in both HCT116-p53(-/-) and HCT116-p53(+/+) compared to ADR exposure, indicating that p53R2 may protect cancer cell from ADR-induced apoptosis. Together, we have identified a new role of E2F1 in the regulation of p53R2 expression in DDR, and silencing p53R2 may sensitize cancer cells to ADR-induced apoptosis. Our data support the notion that p53R2 is a potential target for cancer therapy. The involvement of E2F1-dependent p53R2 activation in DDR will provide further insight into the induction of p53R2 in p53-deficient cells. These data also give us a deeper understanding of E2F1 role in DDR.
编码核糖核苷酸还原酶小亚基的p53R2基因已被鉴定为一种p53诱导基因。尽管该基因是作为p53家族蛋白的一个靶点被发现的，但p53缺陷细胞中DNA损伤诱导p53R2的潜在机制仍有待阐明。在本研究中，我们证明转录因子E2F1在p53缺陷细胞中调节p53R2基因表达。我们发现p53R2是DNA损伤反应（DDR）中E2F1的一个靶点，因为在HCT116-p53(-/-)细胞中异位表达E2F1导致p53R2 mRNA和蛋白表达增加，而沉默E2F1则降低其基础表达。荧光素酶报告基因检测与E2F1过表达或敲低相结合表明E2F1直接激活p53R2基因。染色质免疫沉淀（ChIP）检测显示，在E2F1过表达或阿霉素（ADR）处理下，E2F1直接结合到启动子-684至-677位的位点（TTTGGCGG）。此外，与ADR处理相比，沉默p53R2可增强HCT116-p53(-/-)和HCT116-p53(+/+)细胞中的凋亡性细胞死亡，表明p53R2可能保护癌细胞免受ADR诱导的凋亡。总之，我们确定了E2F1在DDR中调节p53R2表达的新作用，沉默p53R2可能使癌细胞对ADR诱导的凋亡敏感。我们的数据支持p53R2是癌症治疗潜在靶点的观点。E2F1依赖性p53R2激活在DDR中的参与将为深入了解p53缺陷细胞中p53R2的诱导提供进一步的线索。这些数据也让我们对E2F1在DDR中的作用有了更深入的理解。
