mIgM and mIgD Receptor Signaling in B Lymphoma Apoptosis

B 淋巴瘤细胞凋亡中的 mIgM 和 mIgD 受体信号转导

• 批准号: 6620894
• 负责人: GREGORY B CAREY
• 金额: $ 10.75万
• 依托单位: AMERICAN NATIONAL RED CROSS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-10 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6620894
• 关键词: B cell lymphoma; B cell receptor; CD8 molecule; antibody receptor; apoptosis; biological signal transduction; chimeric proteins; gene expression; immunocytochemistry; immunoglobulin D; immunoglobulin M; laboratory mouse; neoplastic growth; phosphatidylinositol 3 kinase; phosphomonoesterases; tissue /cell culture

DESCRIPTION (provided by applicant): Our broad aim is to better understand the roles of surface IgM and IgD (slgM and slgD respectively) receptors in B lymphomas and normal cells. Using anti-mu and anti-delta antibodies to crosslink and activate slgM and slgD receptors respectively, we established that anti-mu and not anti-delta induced growth arrest and apoptosis in B lymphoma cells. Although anti-mu, and anti-delta both stimulate c-Myc, only anti-mu, ultimately ablates c-Myc protein expression and mediates a large induction of p27Kip1 protein. We also demonstrated that c-Myc protein expression is controlled through the ITAM and we recently established PI-3K regulation as the mediator of anti-mu, and anti-delta action. Therefore, our first aim is to explore the role of ITAMS and accessory BCR proteins in PI-3K recruitment and signaling. This will be performed by using CD8-Ig-alpha fusion protein constructs which contain ITAM and non-ITAM mutations first using immunohistochemical then molecular methods. We will also directly test our hypothesis that aborted apoptotic signaling via the slgD receptor results from its failure to co-localize negative regulators which could include adapter proteins, kinase(s), phosphatase(s) or even possibly ITIM-containing co-receptors. Our second aim is to directly establish the role(s) of PI-3K pathway proteins in anti-mu driven growth arrest and apoptosis. We will subclone active and dominant negative PI-3K modulators and effector cDNAs into HIV-TAT fusion vectors. These fusion proteins will be transduced into lymphoma cell lines and primary cells to determine their roles and the hierarchy of their influence on slgM or slgD receptor signaling. Our third aim is to further explore and contrast positive and negative signaling in BCR induced apoptosis. We recently observed that anti-mu massively stimulates PTEN protein expression, whereas anti-delta does not. Therefore, I will establish the mechanisms that regulate PTEN protein expression following BCR engagement and determine its role in BCR mediated outcomes. Transduction of dominant negative PTEN should render lymphoma cells resistant to anti-mu mediated growth arrest whereas transduction of active PTEN should mimic or augment the anti-mu (apoptotic) and anti-delta (pro-apoptotic) responses. Our last aim is to compare and contrast selected gene expression with slgM and slgD receptor signaling. Using a novel gene expression technique, RAGE, we recently observed that anti-mu, specifically modulates a number of cDNAs. We will perform kinetic analysis of the expression of their corresponding proteins and determine their role(s) in the anti-mu effect. Together, these studies will delineate proteins and signaling events, modules and programs initiated by the surface IgM or surface IgD receptors to effect survival, growth arrest and/or apoptosis in B-lymphoma cells.

描述(由申请人提供)：我们的广泛目标是更好地理解 B细胞表面免疫球蛋白M和免疫球蛋白D(分别为sLgM和sLgD)受体的作用 淋巴瘤和正常细胞。使用抗MU和抗Delta抗体 分别交联和激活slgM和slgD受体，我们建立了 抗Mu抗体和非抗Delta抗体诱导B细胞生长停滞和细胞凋亡 淋巴瘤细胞。虽然抗Mu和抗Delta都能刺激c-Myc，但 抗Mu，最终抑制c-Myc蛋白的表达，并介导大量 P27Kip1蛋白的诱导表达。我们还证明了c-Myc蛋白 表达通过ITAM控制，我们最近建立了PI-3K 调节作为抗MU和抗三角洲作用的中介。因此，我们的 第一个目的是探索ITAM和辅助BCR蛋白在PI-3K中的作用 招募和发信号。这将通过使用CD8-Ig-α来执行 首先含有ITAM和非ITAM突变的融合蛋白构建体 用免疫组织化学方法，然后用分子方法。我们还将直接测试 我们的假设是通过slgD受体终止了细胞凋亡信号 由于未能对负面监管机构进行共同本地化，这可能包括 适配蛋白、激酶(S)、磷酸酶(S)，甚至可能含有ITIM 共同受体。我们的第二个目标是直接确立Pi-3K的角色(S 抗MU途径蛋白导致生长停滞和细胞凋亡。我们会 亚克隆活性和显性负性PI-3K调节子和效应子cDNA HIV-TAT融合载体。这些融合蛋白将被转导到淋巴瘤细胞系和原代细胞中，以确定它们的作用和 它们对slgM或slgD受体信号的影响等级。我们的第三个 目的是进一步探索和对比BCR中的积极和消极信号 诱导细胞凋亡。我们最近观察到，反MU会大量刺激 PTEN蛋白表达，而抗Delta蛋白不表达。因此，我会 建立BCR后调节PTEN蛋白表达的机制 并确定其在BCR介导的结果中的作用。转导 显性阴性PTEN应使淋巴瘤细胞对抗Mu产生耐药性 介导的生长停滞，而活性PTEN的转导应该模仿或 增强抗MU(凋亡)和抗Delta(促凋亡)反应。我们的 最后的目的是比较和对比选定的基因表达与slgM和 SlgD受体信号转导。使用一种新的基因表达技术RAGE，我们 最近观察到，抗Mu，特异性地调节了一些cDNA。我们 将对其相应的表达进行动力学分析 并确定其在蛋白质中的作用(S)的抗母作用。加在一起，这些 研究将描绘蛋白质和信号事件、模块和程序 由表面IgM或表面IGD受体启动以影响生存， B淋巴瘤细胞的生长停滞和/或凋亡。