Molecular Control of Cytokine Withdrawal-Induced Apoptosis in Human Lymphocytes

描述(由申请人提供)：感染清除后，反应活跃的淋巴细胞生存所必需的几种细胞因子水平下降，引发淋巴细胞死亡。我们的长期目标是阐明由细胞因子停用引发的人类淋巴细胞凋亡的分子控制。更好地了解调节淋巴细胞这种形式的凋亡的机制，可能会揭示设计治疗自身免疫性疾病和血液系统恶性肿瘤的新疗法的策略。事实上，使用与该项目中建议的方法类似的方法，我们最近发现了一种新的基因缺陷，导致淋巴聚集、自身免疫和癌症综合征。BIM、PUMA和BAD基因敲除小鼠的淋巴细胞对细胞因子撤除诱导的凋亡具有抵抗力。在不同的细胞类型中，有多条途径调节BIM，而在人类淋巴细胞中调节该蛋白的机制尚不清楚。BIM通过与上皮细胞中的动力蛋白轻链结合而失活。叉头盒O(FOXO)家族的转录因子在几个细胞系中转录调节BIM。细胞外信号调节激酶(ERK)/丝裂原活化蛋白激酶(MAPK)通路控制BIM的基础水平，c-Jun氨基末端激酶(JNK)通路在转录和翻译后控制神经元和其他细胞类型的BIM水平。此外，自噬似乎也保护细胞系免受细胞因子停用诱导的细胞死亡。基于这些观察，我们的具体目的是：1)明确S调节细胞因子停用后BIM水平和激活的机制。我们将通过使用siRNA和化学抑制剂，研究FOXO家族、JNK和ERK激酶通路在BIM转录和翻译后调节以及IL-2退出过程中的作用。2)确定PUMA和BAD在人淋巴细胞细胞因子撤除后启动细胞死亡中的相对重要性。我们将使用siRNA来敲除这些蛋白中的每一个，并评估细胞因子停用对细胞凋亡、细胞色素C释放和Bax激活的影响。3)确定原代淋巴细胞中IL-2停用后自噬作为抗凋亡机制的作用。将测量细胞因子停用后自噬的存在，并使用药物治疗来阻断自噬。这些操作对IL-2停用引起的细胞死亡的影响将被量化。我们的长期目标是阐明由细胞因子停用引发的人类淋巴细胞凋亡的分子控制。更好地了解调节淋巴细胞这种形式的凋亡的机制可能会为设计治疗自身免疫性疾病和血液病的新疗法提供策略。

DESCRIPTION (provided by applicant): After clearance of an infection the level of several cytokines essential for the survival of the responding activated lymphocytes falls, triggering lymphocyte death. Our long-term goal is to elucidate the molecular control of apoptosis triggered by cytokine withdrawal in human lymphocytes. A better understanding of the mechanisms regulating this form of apoptosis in lymphocytes may reveal strategies for designing new therapies for autoimmune diseases and hematologic malignancies. In fact, using an approach similar to the proposed in this project we have recently identified a new genetic defect causing a syndrome of lymphoid accumulation, autoimmunity and cancer. Lymphocytes from BIM, PUMA, and BAD knockout mice are resistant to apoptosis induced by cytokine withdrawal. Multiple pathways regulate BIM in different cell types, and the mechanism regulating this protein in human lymphocytes is unknown. BIM is inactivated by binding to dynein light chains in epithelial cells. Transcription factors of the forkhead box o (FOXO) family transcriptionally regulate BIM in several cell lines. The extracellular-signal regulated kinase (ERK)/MAPK pathway can control the baseline levels of BIM, and the c-JUN amino-terminal kinase (JNK) pathway controls the levels of BIM transcriptionally and post-translationally in neurons and other cell types. In addition, autophagy also seems to protect cell lines from cytokine withdrawal-induced cell death. Based on these observations, our specific aims are to: 1) Characterize the mechanism(s) regulating the levels and activation of BIM after cytokine withdrawal. We will investigate the role of the FOXO family, JNK and ERK kinase pathaways on the transcripitional and post- translational regulation of BIM and during IL-2 withdrawal, by the use of siRNA and chemical inhibitors. 2) Determine the relative importance of PUMA and BAD for the initiation of cell death following cytokine withdrawal in human lymphocytes. We will use siRNA to knock down each of these proteins and evaluate the impact on apoptosis, cytochrome C release, and BAX activation induced by cytokine withdrawal. 3) Determine the role of autophagy as an anti-apoptotic mechanism after IL-2 withdrawal in primary lymphocytes. The presence of autophagy after cytokine withdrawal will be measured and drug treatments used to block autophagy. The impact of these manipulations on cell death induced by IL-2 withdrawal will be quantified. Our long-term goal is to elucidate the molecular control of apoptosis triggered by cytokine withdrawal in human lymphocytes. A better understanding of the mechanisms regulating this form of apoptosis in lymphocytes may reveal strategies for designing new therapies for autoimmune diseases and hematologic