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: 6894254
• 负责人: GREGORY B CAREY
• 金额: $ 16.17万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 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/6894254
• 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）：我们的总体目标是更好地了解 表面IgM和IgD（分别为slgM和slgD）受体在B中的作用 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而非抗δ诱导了B细胞生长停滞和凋亡 淋巴瘤细胞虽然抗mu和抗delta都能刺激c-Myc，但只有 抗mu，最终消除c-Myc蛋白表达，并介导大的 p27 Kip 1蛋白诱导。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-3 K 调节作为抗μ和抗δ作用的介质。所以我们的 第一个目的是探索ITAMS和辅助BCR蛋白在PI-3 K中的作用， 招募和信号。这将通过使用CD 8-Ig-α进行 首先含有ITAM和非ITAM突变的融合蛋白构建体 用免疫组织化学和分子生物学方法。我们还将直接测试 我们假设通过slgD受体中止凋亡信号传导， 从它未能共同本地化的负面监管机构，其中可能包括 衔接蛋白、激酶、磷酸酶或甚至可能含有ITIM的 共受体我们的第二个目标是直接确定PI-3 K的作用 抗mu驱动的生长停滞和凋亡中的途径蛋白。我们将 将活性和显性负性PI-3 K调节剂和效应物cDNA亚克隆到 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， 它们对slgM或slgD受体信号传导的影响的层次。我们的第三 目的是进一步探索和对比BCR中的阳性和阴性信号传导 诱导凋亡。我们最近观察到抗μ抗体极大地刺激了 PTEN蛋白表达，而抗δ蛋白不表达。所以我会 建立BCR后PTEN蛋白表达的调控机制 参与并确定其在BCR介导的结果中的作用。转导 显性阴性的PTEN会使淋巴瘤细胞对抗mu 介导的生长停滞，而活性PTEN的转导应该模拟或 增强抗μ（凋亡）和抗δ（促凋亡）应答。我们 最后一个目的是比较和对比选择的基因表达与slgM， slgD受体信号传导。利用一种新的基因表达技术， 最近观察到抗μ特异性地调节许多cDNA。我们 将对其相应的表达进行动力学分析， 蛋白质，并确定其在抗μ作用中的作用。所有这些 研究将描述蛋白质和信号事件，模块和程序 由表面IgM或表面IgD受体引发以影响存活， B淋巴瘤细胞中的生长停滞和/或凋亡。