Lin Chiao Yun, Liang Yu Chun, Yung Benjamin Yat-Ming
Cancer Biochemistry Laboratory, Department of Pharmacology, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan 333, Taiwan, R.O.C.
Cell Signal. 2006 Nov;18(11):2041-8. doi: 10.1016/j.cellsig.2006.04.001. Epub 2006 Apr 22.
Expression of nucleophosmin/B23 and E2F1 and E2F1-dependent transcription increased in U1 bladder cancer cells upon serum stimulation from quiescence. Nucleophosmin/B23-siRNA treatment abrogated such increase of E2F1-dependent transcriptional activity. In identifying physiologically important factors that may occupy E2F1 promoter and regulate its activity in vivo, we found that the pattern of NF-kappaB, E2F1 and pRB recruitment to E2F1 promoter changed in a strikingly dynamic fashion as cells progressed from quiescence into serum-stimulated growth. E2F1 promoter activity in quiescent cells was associated with recruitment of NF-kappaB. NF-kappaB was replaced largely by E2F1 in concert with gene activation during the early stage (12 h) of serum stimulation. At late stage (24 h) of serum stimulation, pRB was then recruited to the E2F1-promoter complex to counterbalance its activity. Upon siRNA-mediated reduction of intracellular nucleophosmin/B23, E2F1 and pRB were recruited to the promoter with the dissociation of NF-kappaB concomitant with gene inactivation. Based on immunoprecipitation experiments, nucleophosmin/B23 was found to be associated with NF-kappaB in cells grown in serum-supplemented but not in serum-deprived medium. Furthermore, nucleophosmin/B23 could also be co-immunoprecipitated with ppRB at the early stage (12 h) but not at the late stage (24 h) of serum stimulation. The results demonstrate a novel mechanism for transcriptional regulation of E2F1 and identify the functional role of nucleophosmin/B23 in modulating the binding of NF-kappaB, E2F1 and pRB to activate E2F1 promoter.
在血清刺激下，U1膀胱癌细胞从静止状态进入增殖状态时，核磷蛋白/B23、E2F1以及E2F1依赖的转录表达均增加。核磷蛋白/B23-siRNA处理可消除E2F1依赖的转录活性的这种增加。在鉴定可能占据E2F1启动子并在体内调节其活性的生理重要因子时，我们发现，随着细胞从静止状态进入血清刺激生长状态，NF-κB、E2F1和pRB募集至E2F1启动子的模式以显著动态的方式发生变化。静止细胞中的E2F1启动子活性与NF-κB的募集相关。在血清刺激的早期阶段（12小时），随着基因激活，NF-κB在很大程度上被E2F1取代。在血清刺激的后期阶段（24小时），pRB随后被募集至E2F1启动子复合物以平衡其活性。在siRNA介导的细胞内核磷蛋白/B23减少后，E2F1和pRB被募集至启动子，同时NF-κB解离并伴随基因失活。基于免疫沉淀实验，发现核磷蛋白/B23在补充血清的培养基中生长的细胞中与NF-κB相关，但在无血清培养基中生长的细胞中则不然。此外，在血清刺激的早期阶段（12小时），核磷蛋白/B23也可与ppRB进行共免疫沉淀，但在血清刺激的后期阶段（24小时）则不能。这些结果证明了E2F1转录调控的一种新机制，并确定了核磷蛋白/B23在调节NF-κB、E2F1和pRB结合以激活E2F1启动子中的功能作用。
