Balasubramanian Sivaprakasam, Scharadin Tiffany M, Han Bingshe, Xu Wen, Eckert Richard L
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA.
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA; Department of Dermatology, University of Maryland School of Medicine, Baltimore, MD, USA; Department of Obstetrics and Gynecology, University of Maryland School of Medicine, Baltimore, MD, USA; Department of Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA.
Cell Signal. 2015 Jul;27(7):1336-44. doi: 10.1016/j.cellsig.2015.03.021. Epub 2015 Apr 2.
The Bmi-1 Polycomb group (PcG) protein is an important epigenetic regulator of chromatin status. Elevated Bmi-1 expression is observed in skin cancer and contributes to cancer cell survival. (-) Epigallocatechin-3-gallate (EGCG), an important green tea-derived cancer prevention agent, reduces Bmi-1 level resulting in reduced skin cancer cell survival. This is associated with increased p21(Cip1) and p27(Kip1) expression, reduced cyclin, and cyclin dependent kinase expression, and increased cleavage of apoptotic markers. These EGCG-dependent changes are attenuated by vector-mediated maintenance of Bmi-1 expression. In the present study, we identify Bmi-1 functional domains that are required for this response. Bmi-1 expression reverses the EGCG-dependent reduction in SCC-13 cell survival, but Bmi-1 mutants lacking the helix-turn-helix-turn-helix-turn (Bmi-1ΔHT) or ring finger (Bmi-1ΔRF) domains do not reverse the EGCG impact. The reduction in Ring1B ubiquitin ligase activity, observed in the presence of mutant Bmi-1, is associated with reduced ability of these mutants to interact with and activate Ring1B ubiquitin ligase, the major ligase responsible for the ubiquitination of histone H2A during chromatin condensation. This results in less chromatin condensation leading to increased tumor suppressor gene expression and reduced cell survival; thereby making the cells more susceptible to the anti-survival action of EGCG. We further show that these mutants act in a dominant-negative manner to inhibit the action of endogenous Bmi-1. Our results suggest that the HT and RF domains are required for Bmi-1 ability to maintain skin cancer cell survival in response to cancer preventive agents.
Bmi-1多梳蛋白家族（PcG）蛋白是染色质状态的重要表观遗传调节因子。在皮肤癌中观察到Bmi-1表达升高，且其有助于癌细胞存活。（-）表没食子儿茶素-3-没食子酸酯（EGCG）是一种重要的源自绿茶的癌症预防剂，它可降低Bmi-1水平，从而导致皮肤癌细胞存活率降低。这与p21（Cip1）和p27（Kip1）表达增加、细胞周期蛋白及细胞周期蛋白依赖性激酶表达减少以及凋亡标志物的切割增加有关。这些依赖于EGCG的变化会因载体介导的Bmi-1表达维持而减弱。在本研究中，我们确定了这种反应所需的Bmi-1功能结构域。Bmi-1表达可逆转EGCG依赖性的SCC-13细胞存活率降低，但缺乏螺旋-转角-螺旋-转角-螺旋（Bmi-1ΔHT）或指环结构域（Bmi-1ΔRF）的Bmi-1突变体则不能逆转EGCG的影响。在存在突变型Bmi-1的情况下观察到的Ring1B泛素连接酶活性降低，与这些突变体与Ring1B泛素连接酶相互作用并激活该酶的能力降低有关，Ring1B泛素连接酶是染色质浓缩过程中负责组蛋白H2A泛素化的主要连接酶。这导致染色质浓缩减少，从而导致肿瘤抑制基因表达增加和细胞存活率降低；进而使细胞更容易受到EGCG抗存活作用的影响。我们进一步表明，这些突变体以显性负性方式发挥作用，抑制内源性Bmi-1的作用。我们的结果表明，HT和RF结构域是Bmi-1响应癌症预防剂维持皮肤癌细胞存活能力所必需的。
