Department of Surgery, University of Louisville School of Medicine, and J. Graham Brown Cancer Center, Louisville, KY, USA.
J Cell Mol Med. 2012 Mar;16(3):605-15. doi: 10.1111/j.1582-4934.2011.01338.x.
E2F-1-deleted mutant, 'truncated E2F' (E2Ftr, E2F-1[1-375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, 'downstream regulatory element antagonist modulator' (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3'-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain.
E2F-1 缺失突变体，“截断的 E2F”（E2Ftr，E2F-1[1-375]），缺乏羧基末端转录激活结构域，比野生型 E2F-1（wtE2F-1；全长 E2F-1）更能有效诱导癌细胞凋亡。然而，wtE2F-1 和 E2Ftr 诱导凋亡的机制尚未完全阐明。我们的研究表明，促凋亡 BH3 仅 Bcl-2 家族成员 Harakiri（Hrk）在 wtE2F-1 和 E2Ftr 诱导的黑素瘤细胞凋亡中的分子作用。我们发现，在 wtE2F-1 和 E2Ftr 过表达时，Hrk mRNA 和 Harakiri（HRK）蛋白表达在黑素瘤细胞中高度上调。HRK 的上调不依赖于 E2F-1 转录激活结构域。此外，在癌细胞中，Hrk 基因的上调和 HRK 蛋白的表达不依赖于 p53。用 Hrk siRNA 进行 Hrk 敲低与 wtE2F-1 和 E2Ftr 诱导的凋亡显著减少有关。我们还发现，上游因子“下游调节元件拮抗剂调制器”（DREAM）可能参与 wtE2F-1 和 E2Ftr 过表达引起的 HRK 介导的凋亡。wtE2F-1 和 E2Ftr 过表达后，DREAM 的表达水平增加。Western blot 检测到 DREAM 主要以二聚体形式存在。wtE2F-1 和 E2Ftr 过表达导致 DREAM 同源二聚化，可能导致电迁移阻滞实验显示 DREAM 与 Hrk 基因 3'-非翻译区的结合活性降低。结果表明，wtE2F-1 和 E2Ftr 诱导的凋亡部分由 HRK 介导。HRK 的功能受到 DREAM 的调节。我们的研究结果有助于理解调节 wtE2F-1 和 E2Ftr 诱导凋亡的机制，并为进一步评估 E2Ftr 诱导凋亡如何用于治疗增益提供了思路。
