Modulation of Glucocorticoid-Evoked Lymphocyte Apoptosis by BTG1

描述（由申请人提供）：一种自杀性细胞死亡过程，称为细胞凋亡，对正常生理至关重要，因为它调节细胞更新并保持组织活力。细胞凋亡在神经退行性疾病、免疫缺陷等中过度，并且在诸如自身免疫和癌症的疾病中被破坏。用于T细胞白血病的化学治疗剂如糖皮质激素（GC）将细胞阻滞在细胞周期的G1期，并通过改变关键生长调节/死亡诱导基因的表达来诱导细胞凋亡。本PI的长期目标是了解细胞凋亡的分子途径，强调基因调控变化。使用一对人T淋巴细胞白血病姐妹细胞系，CEM-C-14和CEM-C1-15，这是敏感和耐药，分别GC诱发的细胞凋亡，PI已经获得了初步的数据相关的上调转录和蛋白水平的抗增殖基因，Btg 1，G1期阻滞和细胞凋亡。cAMP通路的激活使耐药细胞对GC诱发的反应敏感，包括BTG 1上调、G1期阻滞和凋亡。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BTG 1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。具体目标1a将使用转染到CEM-C7-14和CEM-C1-15细胞中的启动子-荧光素酶报告基因构建体评价GC诱导Btg 1启动子转录活性的能力，并将使用放线菌素D和放线菌酮确定GC是否分别对两种细胞系中Btg 1 mRNA和BTG 1蛋白的稳定性产生差异性影响。特定目标1b和c将评估GC改变蛋白酶体介导的BTG 1降解或改变其在两种细胞系中的核定位的能力。在特定目标2中，将通过siRNA抑制CEM-C7-14细胞中的BTG 1表达，以评估G1停滞和/或凋亡对BTG 1表达的依赖性，并了解BTG 1与各种生长抑制和凋亡调节剂如细胞周期蛋白D3、p27 kip 1、c-Myc、c-Jun和Bcl-2家族成员的表达之间的关系。据报道，BTG 1与核受体结合，类似于GC受体（GR），并促进其调节基因转录的能力。在特定目标3中，将使用共免疫沉淀试验以及GR、BTG 1和GR驱动的荧光素酶报告基因构建体pHHLuc在Jurkat细胞中的瞬时共转染，评价BTG 1结合GR并调节GR活性的能力。这些研究将大大推进我们对T细胞凋亡机制和BTG 1功能的理解。7.说明：细胞凋亡是维持正常健康所必需的生理性细胞死亡的一种形式，并且由多个基因的表达或功能改变触发，其中之一是Btg 1，该基因已被证明抑制细胞增殖。诸如阿尔茨海默氏病、神经变性、自身免疫、免疫缺陷、骨质疏松症、癌症等疾病是细胞凋亡可能是促成因素的病症。本申请提出了理解Btg 1在T-白血病细胞的生长抑制和凋亡中的作用的研究。

DESCRIPTION (provided by applicant): A process of suicidal cell death, termed apoptosis, is vital for normal physiology because it regulates cellular turnover and preserves tissue vitality. Apoptosis is excessive in neurodegenerative diseases, immunodeficiency, etc, and is disrupted in diseases such as autoimmunity and cancer. Chemotherapeutic agents such as glucocorticoids (GCs), used in T cell leukemias, arrest cells in G1 phase of the cell cycle and induce apoptosis by altering expression of key growth regulatory/death inducing genes. The long-term goal of this PI is to understand the molecular pathway for apoptosis, emphasizing on gene regulatory changes. Using a pair of human T lymphocytic leukemia sister cell lines, CEM-C-14 and CEM-C1-15, which are sensitive and resistant, respectively to GC-evoked apoptosis, the PI has obtained preliminary data correlating the up regulation of transcript and protein levels of the anti proliferative gene, Btg1, to G1 arrest and apoptosis. Activation of the cAMP pathway sensitizes the resistant cells to GC-evoked responses, including BTG1 up regulation, G1 arrest 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 BTG1 in modulating the process, its mode of action, and its relationship to other changes triggered by GCs. Specific Aim 1a will evaluate the ability of GCs to induce transcriptional activity from the Btg1 promoter using a promoter- luciferase reporter construct transfected in CEM-C7-14 and CEM-C1-15 cells, and will use actinomycin D and cycloheximide to determine whether GCs differentially affect the stability of Btg1 mRNA and BTG1 protein, respectively, in the two cell lines. Specific Aim 1b and c will assess the ability of GCs to alter proteasome-mediated degradation of BTG1, or alter its nuclear localization in the two cell lines. In Specific Aim 2 BTG1 expression will be repressed in CEM-C7-14 cells by siRNA to evaluate the dependence of G1 arrest and/or apoptosis on BTG1 expression, and to understand the relationship between BTG1 and expression of various growth inhibitory and apoptotic modulators such as cyclin D3, p27kip1, c-Myc, c-Jun, and Bcl-2 family members. BTG1 has been reported to bind to nuclear receptors, similar to the GC receptor (GR), and facilitate their ability to regulate gene transcription. In Specific Aim 3, the ability of BTG1 to bind to and modulate the activity of GR will be evaluated using coimmunoprecipitation assays, and transient cotransfections of GR, BTG1 and a GR-driven luciferase reporter construct, pHHLuc in Jurkat cells. These studies will significantly advance our understanding of the mechanisms of T-cell apoptosis, and the function of BTG1. 7. NARRATIVE: Apoptosis is a form of physiological cell death essential for maintaining normal health, and is triggered by altered expression or function of multiple genes, one of them being Btg1, a gene that has been shown to inhibit cell proliferation. Diseases such as Alzheimer's, neurodegeneration, autoimmunity, immunodeficiency, osteoporosis, cancer, etc are conditions where apoptosis may be a contributing factor. This application proposes studies to understand the role of Btg1 in growth inhibition and apoptosis of T-leukemic cells.