Phorbol Ester Response and Resistance in Thymoma Cells

描述（由申请方提供）：肿瘤细胞的增殖和存活涉及细胞粘附和促有丝分裂信号转导级联之间的复杂相互作用。小鼠胸腺瘤细胞系EL 4是研究这些事件中所涉及的分子步骤的独特模型系统。EL 4细胞存在两种基本表型，PMA敏感和PMA抗性。这些表型是指细胞在佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）（一种肿瘤促进剂）存在下存活和增殖的能力。在两种细胞类型的信号转导和蛋白质表达的表征表明，PMA抗性细胞不能完全激活响应PMA的Erk丝裂原活化蛋白激酶级联。PMA敏感细胞中PMA诱导的生长停滞需要Erk活化。其他发现是，只有PMA耐药细胞表达粘着斑激酶（FAK），而PMA敏感细胞表达更高水平的Pyk 2，一种FAK相关激酶。此外，PMA敏感细胞表达更高水平的RasGRP，一种Ras的鸟嘌呤核苷酸交换因子，比耐药细胞。RasGRP是一种佛波酯受体，当与PMA结合时诱导Ras活化。拟议的工作测试了两个假设，已经从这些以前的观察。首先，在EL 4细胞中，佛波酯介导的ERK激活通过调节Ras激活的蛋白质发生。第二是FAK的表达增强EL 4胸腺瘤细胞的粘附、存活和致瘤性。具体目标是：1）研究PKC激活对EL 4细胞Ras上游蛋白的影响; 2）确定Ras GRP和Pyk 2在PMA诱导的Erk激活中的作用; 3）确定粘附蛋白在EL 4细胞PMA反应中的作用; 4）研究FAK和PMA敏感性在EL 4细胞体内肿瘤发生中的作用。拟议的工作将利用一系列的克隆EL 4细胞系。在这些细胞中发生的蛋白质表达的改变模拟了在一些肿瘤中发生的蛋白质表达的改变。EL 4细胞提供了一个特别新颖的模型，在其中研究粘附蛋白在信号传导中的作用。这项工作的长期目标是描绘佛波酯和甘油二酯反应的主要途径。所获得的信息将提出新的治疗方法来抑制肿瘤细胞的进展。

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.