Laboratory of DNA Damage Signaling, Chiba Cancer Center Research Institute, Chiba 260-8717, Japan.
Biochem Biophys Res Commun. 2012 Apr 27;421(1):57-63. doi: 10.1016/j.bbrc.2012.03.108. Epub 2012 Mar 29.
Appropriate regulation of DNA damage response is pivotal for maintaining genome stability. p53 as well as E2F-1 plays a critical role during DNA damage response, however, the physiological significance of their interaction has been elusive. In the present study, we found that E2F-1 has an inhibitory effect on p53 during adriamycin (ADR)-mediated DNA damage response. Upon ADR exposure, p53 and E2F-1 were markedly induced at protein and mRNA levels in p53-procifient U2OS and HCT116 cells, and formed a stable complex as examined by co-immunoprecipitation experiments. Of note, chromatin immunoprecipitation (ChIP) experiments revealed that ADR-mediated induction coincides with the efficient recruitment of p53 and E2F-1 onto the promoters of p53-target genes, such as p21(WAF1) and BAX. Subsequent RT-PCR and luciferase reporter assays demonstrated that E2F-1 strongly attenuates p53-dependent transactivation of p53-target genes. Importantly, siRNA-mediated knockdown of E2F-1 stimulated apoptosis in response to ADR, which was associated with an accelerated response of p21(WAF1) and BAX. Collectively, our present findings suggest that E2F-1 participates in p53-mediated DNA damage response and might have a checkpoint function to limit overactive p53.
适当调节 DNA 损伤反应对于维持基因组稳定性至关重要。p53 和 E2F-1 在 DNA 损伤反应中起着关键作用，然而，它们相互作用的生理意义一直难以捉摸。在本研究中，我们发现 E2F-1 在阿霉素（ADR）介导的 DNA 损伤反应中对 p53 具有抑制作用。在 ADR 暴露后，p53 和 E2F-1 在 p53 阳性的 U2OS 和 HCT116 细胞中在蛋白质和 mRNA 水平上均明显诱导，并通过共免疫沉淀实验形成稳定的复合物。值得注意的是，染色质免疫沉淀（ChIP）实验表明，ADR 介导的诱导与 p53 和 E2F-1 有效招募到 p53 靶基因的启动子上一致，如 p21(WAF1)和 BAX。随后的 RT-PCR 和荧光素酶报告基因检测表明，E2F-1 强烈抑制 p53 依赖性 p53 靶基因的转录激活。重要的是，siRNA 介导的 E2F-1 敲低可刺激 ADR 诱导的细胞凋亡，这与 p21(WAF1)和 BAX 的快速反应相关。综上所述，我们的研究结果表明，E2F-1 参与了 p53 介导的 DNA 损伤反应，并且可能具有检查点功能以限制过度活跃的 p53。
