Phorbol Ester Response and Resistance in Thymoma Cells

描述(由申请人提供)：肿瘤细胞的增殖和存活涉及细胞黏附和有丝分裂信号转导之间的复杂相互作用。EL4是一种小鼠胸腺瘤细胞系，是研究这些事件所涉及的分子步骤的独特模型系统。EL4细胞存在PMA敏感和PMA耐药两种基本表型。这些表型是指细胞在肿瘤促进剂佛波酯(PMA)存在下存活和增殖的能力。对两种细胞的信号转导和蛋白表达的研究表明，PMA耐药细胞不能完全激活ERK丝裂原激活的蛋白激酶级联反应PMA。PMA诱导的PMA敏感细胞生长停滞需要ERK的激活。其他发现是，只有PMA耐药的细胞表达粘着斑激酶(FAK)，而PMA敏感的细胞表达更高水平的PYK2，一种FAK相关的激酶。此外，PMA敏感细胞比耐药细胞表达更高水平的RasGRP，RasGRP是RAS的一种鸟嘌呤核苷酸交换因子。RasGRP是一种佛波酯受体，当与PMA结合时可以诱导RAS的激活。这项拟议的工作测试了从这些先前的观察中产生的两个假设。首先，在EL4细胞中，佛波酯介导的ERK激活是通过调节RAS激活的蛋白质来实现的。第二，FAK的表达增强了EL4胸腺瘤细胞的黏附性、存活率和致瘤性。其具体目的是：1)检测PKC激活对EL4细胞RAS上游蛋白的影响，2)确定RasGRP和PYK2在PMA诱导的ERK激活中的作用，3)确定黏附蛋白在EL4细胞PMA反应中的作用，4)检测FAK和PMA敏感性在EL4细胞体内肿瘤发生中的作用。这项拟议的工作将利用一系列克隆EL4细胞系。这些细胞中蛋白质表达的变化类似于某些肿瘤中的变化。EL4细胞为研究黏附蛋白在信号转导中的作用提供了一个特别新颖的模型。这项工作的长期目标是描述对佛波醇酯和二甘油酯的主要反应途径。所获得的信息将为抑制肿瘤细胞进展提供新的治疗方法。

DESCRIPTION (provided by applicant): Proliferation and survival of tumor cells involves a complex interplay between cell adhesion and mitogenic signal transduction cascades. EL4, a murine thymoma cell line, is a unique model system in which to investigate the molecular steps involved in these events. EL4 cells exist in two basic phenotypes, PMA-sensitive and PMA-resistant. These phenotypes refer to the ability of the cells to survive and proliferate in the presence of phorbol 12-myristate 13-acetate (PMA), a tumor promoter. Characterization of signal transduction and protein expression in the two cell types has revealed that PMA-resistant cells are unable to fully activate the Erk mitogen-activated protein kinase cascade in response to PMA. Erk activation is required for PMA-induced growth arrest in PMA-sensitive cells. Additional findings are that only PMA-resistant cells express focal adhesion kinase (FAK), while PMA-sensitive cells express higher levels of Pyk2, a FAK-related kinase. In addition, PMA-sensitive cells express higher levels of RasGRP, a guanine nucleotide exchange factor for Ras, than do resistant cells. RasGRP is a phorbol ester receptor that induces activation of Ras when bound to PMA. The proposed work tests two hypotheses that have arisen from these previous observations. The first is that, in EL4 cells, phorbol ester-mediated ERK activation occurs via proteins that regulate activation of Ras. The second is that expression of FAK enhances attachment, survival, and tumorigenicity of EL4 thymoma cells. The Specific Aims are: 1) To examine the effects of PKC activation on proteins upstream of Ras in EL4 cells, 2) To determine the roles of RasGRP and Pyk2 in PMA-induced Erk activation, 3) To define the roles of adhesion proteins in PMA response in EL4 cells, and 4) To examine the role of FAK and PMA sensitivity in tumorigenesis of EL4 cells in vivo. The proposed work will utilize a series of clonal EL4 cell lines. The alterations in protein expression that occur in these cells mimic those occurring in some tumors. EL4 cells provide a particularly novel model in which to study the roles of adhesion proteins in signaling. The long-term goal of this work is to delineate major pathways for response to phorbol ester and diglycerides. The information gained will suggest new therapeutic approaches to inhibit tumor cell progression.