Role of E4BP4 in Glucocorticoid-Evoked Lymphocyte Apoptosis

描述（由申请人提供）：细胞凋亡是一种精心策划的生理性细胞死亡途径，通过改变生长调节/死亡诱导基因的表达而激活。本PI的长期目标是了解细胞凋亡的分子途径，重点是基因调控变化。在一对姐妹的人白血病T细胞系CEM-C7-14和CEM-C1-15中，分别对糖皮质激素（GC）诱导的凋亡敏感或难治，我们观察到转录抑制因子E4 BP 4（促凋亡C. Elegans ces-2基因产物与细胞凋亡。虽然其他几种基因产物也涉及GC诱发的白血病T细胞凋亡，在本提案中，我们将重点评估E4 BP 4在调节过程中的相对贡献，其作用模式，以及其与GC引发的其他变化的关系。根据新的初步数据和审评员的意见，对重新提交的具体目标进行了修订。具体目标1将通过报告基因和用激酶和磷酸酶抑制剂处理的细胞的凋亡测定来分析E4 BP 4去磷酸化对其抑制转录和调节凋亡的能力的相关性。在具体目标2中，将通过CEM-C1-15细胞中的异位E4 BP 4表达或CEM-C7-14细胞中siRNA介导的E4 BP 4敲低来评估E4 BP 4调节下游进化保守的凋亡调节剂（Slug/Snail、Bim/Puma和Bcl 2/Bcl-xL）的能力。已经开发了表达异位小鼠E4 BP 4的CE-C1-15衍生克隆，并表现出对GC诱发的细胞凋亡的敏感性。具体目标3将分别使用GRE驱动的荧光素酶报告基因pHH-Luc和c-myc启动子-荧光素酶报告基因HBMLuc研究E4 BP 4调节GR依赖性转录激活和抑制的能力。E4 BP 4和其他已知的T细胞凋亡调节剂（包括细胞周期蛋白D3、p27 kip 1、c-Jun、c-Myc和BTG 1）的表达之间的关系将在被操作以过表达或关闭E4 BP 4表达的CEM细胞中进行测试。 与公共卫生的相关性：异常或不充分的细胞凋亡有助于疾病，如自身免疫，免疫缺陷，神经变性和癌症。这里提出的研究将促进策略的发展，以加强或预防细胞凋亡和管理这些条件。

DESCRIPTION (provided by applicant): Apoptosis is a well-orchestrated pathway of physiological cell death, activated through alterations in the expression of growth regulatory/death-inducing genes. The long-term goal of this PI is to understand the molecular pathway for apoptosis, with emphasis on gene regulatory changes. In a pair of sister human leukemic T cell lines, CEM-C7-14 and CEM-C1-15, that are susceptible or refractory, respectively, to glucocorticoid (GC)-evoked apoptosis, we have observed a correlation between expression and dephosphorylation of the transcriptional repressor E4BP4, a homolog of the pro-apoptotic C. elegans ces-2 gene product, and apoptosis. While several other gene products are also implicated in GC-evoked apoptosis of leukemic T cells, in this proposal we will focus on evaluating the relative contribution of E4BP4 in modulating the process, its mode of action, and its relationship to other changes triggered by GCs. Based on new preliminary data and comments from reviewers, the specific aims of this resubmission have been revised. Specific aim 1 will analyze the relevance of E4BP4 dephosphorylation on its ability to repress transcription and modulate apoptosis through reporter gene and apoptosis assays of cells treated with kinase and phosphatase inhibitors. In specific aim 2, the ability of E4BP4 to regulate downstream evolutionary conserved modulators of apoptosis (Slug/Snail, Bim/Puma, and Bcl2/Bcl-xL) will be evaluated through ectopic E4BP4 expression in CEM-C1-15 cells, or siRNA-mediated E4BP4 knock-down in CEM-C7-14 cells. A CE-C1-15 derived clone expressing ectopic mouse E4BP4 has been developed and exhibits sensitivity to GC-evoked apoptosis. Specific aim 3 will study the ability of E4BP4 to modulate GR-dependent transcriptional activation and repression using the GRE-driven luciferase reporter pHH-Luc, and the c-myc promoter-luciferase reporter HBMLuc, respectively. The relationship between E4BP4 and expression of other known modulators of T cell apoptosis, including cyclin D3, p27kip1, c-Jun, c-Myc and BTG1, will be tested in CEM cells manipulated to either over-express or shut off E4BP4 expression. Relevance to Public Health: Aberrant or inadequate apoptosis contributes to diseases such as autoimmunity, immunodeficiency, neurodegeneration, and cancer. Studies proposed here will promote development of strategies to enforce or prevent apoptosis and manage these conditions.