Zagurovskaya M, Shareef M M, Das A, Reeves A, Gupta S, Sudol M, Bedford M T, Prichard J, Mohiuddin M, Ahmed M M
Weis Center for Research, Geisinger Clinic, Danville, PA, USA.
Oncogene. 2009 Feb 26;28(8):1121-31. doi: 10.1038/onc.2008.461. Epub 2009 Jan 12.
In this study, we investigated the functional role of early growth response-1 (Egr1 gene) in the regulation of radiation-induced clonogenic inhibition and apoptosis in p53 wild-type and mutant prostate cancer cells 22Rv1 and DU145, respectively. 22Rv1 cells were more sensitive to irradiation compared with DU145 cells, and the sensitivity was enhanced by overexpression of EGR-1 in both cells. Dominant-negative EGR-1 mutant (dnEGR-1) or repressor of EGR-1, NGFIA binding protein 1 (NAB1), increased radioresistance of these cells. Significant activation of caspases 3 and 9 and Bcl2-associated X (Bax) with increased poly(ADP-ribose) polymerase (PARP) cleavage and cytochrome c release was observed in radiation-exposed EGR-1 overexpressing cells. Gel shift analysis and chloramphenicol acetyl transferase (CAT) reporter assays indicate that EGR-1 transactivates the promoter of the Bax gene. Interaction of EGR-1 and Yes kinase-associated protein 1 (YAP-1) through the WW domain of YAP-1 enhances the transcriptional activity of EGR-1 on the Bax promoter as shown by chromatin immunoprecipitation and reporter assays. Irradiation of PC3 cell xenografts that were treated with adenoviral EGR-1 showed significant regression in tumor volume. These findings establish the radiation-induced pro-apoptotic action of EGR-1, in a p53-independent manner, by directly transactivating Bax, and prove that alters the B-cell CLL/lymphoma 2 (Bcl-2)/Bax ratio as one of the mechanisms resulting in significant activation of caspases, leading to cell death through the novel interaction of EGR-1 with YAP-1.
在本研究中，我们分别研究了早期生长反应因子1（Egr1基因）在调节p53野生型和突变型前列腺癌细胞22Rv1和DU145辐射诱导的克隆形成抑制和凋亡中的功能作用。与DU145细胞相比，22Rv1细胞对辐射更敏感，并且在两种细胞中过表达EGR-1均可增强这种敏感性。显性负性EGR-1突变体（dnEGR-1）或EGR-1的抑制因子神经生长因子诱导蛋白A结合蛋白1（NAB1）可增加这些细胞的辐射抗性。在辐射暴露的EGR-1过表达细胞中，观察到半胱天冬酶3和9以及Bcl2相关X蛋白（Bax）显著激活，同时聚（ADP-核糖）聚合酶（PARP）裂解增加和细胞色素c释放。凝胶迁移分析和氯霉素乙酰转移酶（CAT）报告基因检测表明，EGR-1可反式激活Bax基因的启动子。染色质免疫沉淀和报告基因检测显示，EGR-1通过与Yes激酶相关蛋白1（YAP-1）的WW结构域相互作用，增强了EGR-1对Bax启动子的转录活性。用腺病毒EGR-1处理的PC3细胞异种移植瘤经照射后，肿瘤体积显著缩小。这些发现确立了EGR-1以p53非依赖的方式通过直接反式激活Bax产生辐射诱导的促凋亡作用，并证明其改变B细胞淋巴瘤/白血病-2（Bcl-2）/Bax比值是导致半胱天冬酶显著激活的机制之一，通过EGR-1与YAP-1的新型相互作用导致细胞死亡。
