Phorbol Ester Response and Resistance in Thymoma Cells

胸腺瘤细胞中的佛波酯反应和耐药性

• 批准号: 6726998
• 负责人: Kathryn E Meier
• 金额: $ 23.19万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-16 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6726998
• 关键词: cell adhesion molecules; cell growth regulation; cell line; cell proliferation; densitometry; flow cytometry; focal adhesion kinase; green fluorescent proteins; guanine nucleotide binding protein; immunoprecipitation; laboratory mouse; mitogen activated protein kinase; neoplastic cell; phorbols; phosphorylation; polymerase chain reaction; posttranslational modifications; protein kinase C; protein protein interaction; thymus neoplasms; transfection; tumor promoters; two dimensional gel electrophoresis; western blottings

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.

描述（由申请人提供）：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复杂的相互作用。 EL4 是一种小鼠胸腺瘤细胞系，是一个独特的模型系统，可用于研究这些事件中涉及的分子步骤。 EL4细胞存在两种基本表型：PMA敏感型和PMA抗性型。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。两种细胞类型中信号转导和蛋白表达的表征表明，PMA 抗性细胞无法完全激活 Erk 丝裂原激活蛋白激酶级联反应以响应 PMA。 PMA 诱导的 PMA 敏感细胞生长停滞需要 Erk 激活。其他发现是，只有 PMA 抗性细胞表达粘着斑激酶 (FAK)，而 PMA 敏感细胞表达更高水平的 Pyk2（一种 FAK 相关激酶）。此外，PMA 敏感细胞比耐药细胞表达更高水平的 RasGRP（Ras 的鸟嘌呤核苷酸交换因子）。 RasGRP 是一种佛波酯受体，与 PMA 结合时可诱导 Ras 激活。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 激活的蛋白质发生的。其次，FAK 的表达增强了 EL4 胸腺瘤细胞的附着、存活和致瘤性。具体目标是：1) 检查 EL4 细胞中 PKC 激活对 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) 确定粘附蛋白在 EL4 细胞 PMA 反应中的作用，4) 检查 FAK 和 PMA 敏感性在 EL4 细胞体内肿瘤发生中的作用。拟议的工作将利用一系列克隆 EL4 细胞系。这些细胞中发生的蛋白质表达的变化类似于某些肿瘤中发生的变化。 EL4 细胞提供了一种特别新颖的模型来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。获得的信息将提出抑制肿瘤细胞进展的新治疗方法。