Modulation of Glucocorticoid-Evoked Lymphocyte Apoptosis by BTG1

描述（由申请人提供）：自杀细胞死亡的过程，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、免疫缺陷等中过多，在自身免疫和癌症等疾病中被破坏。化疗药物，如糖皮质激素（GCs），用于T细胞白血病，在细胞周期的G1期阻滞细胞，并通过改变关键生长调节/死亡诱导基因的表达诱导细胞凋亡。本项目的长期目标是了解细胞凋亡的分子途径，重点研究基因调控变化。利用对gc诱导的细胞凋亡敏感和耐药的人T淋巴细胞白血病姐妹细胞系CEM-C-14和CEM-C1-15， PI获得了抗增殖基因Btg1在G1阻滞和细胞凋亡中的转录和蛋白水平上调的初步数据。cAMP通路的激活使耐药细胞对gc诱发的反应敏感，包括BTG1上调、G1阻滞和凋亡。虽然其他几个基因产物也与gc诱发的白血病T细胞凋亡有关，但在本提案中，我们将重点评估BTG1在调节这一过程中的相对贡献、其作用方式以及与gc引发的其他变化的关系。特异性Aim 1a将利用在CEM-C7-14和CEM-C1-15细胞中转染的启动子-荧光素酶报告基因构建体来评估GCs诱导Btg1启动子转录活性的能力，并将使用放线菌素D和环已酰亚胺来确定GCs在两种细胞系中对Btg1 mRNA和Btg1蛋白稳定性的影响是否存在差异。特异性Aim 1b和c将评估GCs改变蛋白酶体介导的BTG1降解或改变其在两种细胞系中核定位的能力。在Specific Aim 2中，我们将在CEM-C7-14细胞中通过siRNA抑制BTG1的表达，以评估G1阻滞和/或凋亡对BTG1表达的依赖性，并了解BTG1与各种生长抑制和凋亡调节剂（如cyclin D3、p27kip1、c-Myc、c-Jun和Bcl-2家族成员）表达的关系。据报道，BTG1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Specific Aim 3中，BTG1结合和调节GR活性的能力将通过共免疫沉淀法和在Jurkat细胞中瞬时共转染GR、BTG1和GR驱动的荧光素酶报告结构物pHHLuc来评估。这些研究将显著促进我们对t细胞凋亡机制和BTG1功能的认识。7. 叙述：细胞凋亡是维持正常健康所必需的一种生理性细胞死亡形式，由多种基因表达或功能改变触发，其中之一是Btg1，一种已被证明可抑制细胞增殖的基因。诸如阿尔茨海默氏症、神经退行性变、自身免疫、免疫缺陷、骨质疏松症、癌症等疾病都可能是细胞凋亡的一个促成因素。这一应用为了解Btg1在t -白血病细胞生长抑制和凋亡中的作用提供了研究。

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.