VEGF MRNA STABILIZATION BY HYPOXIA AND TUMOR MUTATIONS

缺氧和肿瘤突变对 VEGF mRNA 的稳定性

• 批准号: 2430790
• 负责人: MARK P KAMPS
• 金额: $ 13.85万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2430790
• 关键词: RNA binding protein; RNase protection assay; angiogenesis factor; biological signal transduction; breast neoplasms; gene expression; genetic models; genetic regulation; genetic regulatory element; genetic transcription; hypoxia; in situ hybridization; messenger RNA; molecular cloning; nucleic acid sequence; plasminogen activator; point mutation; protein engineering; tissue /cell culture; transfection; transposon /insertion element; vascular endothelial growth factors

The objective of this proposal is to determine how the transcript for vascular endothelial growth factor (VEGF) is transiently stabilized by hypoxia in normal cells and becomes constitutively stabilized in a subset of tumor cells that highly overexpress VEGF. VEGF is a potent angiogenic peptide produced by solid tumors by at least two different mechanisms. First, concentrated zones of VEGF expression appear in hypoxic regions of solid tumors and are implicated in mediating hypoxia-induced angiogenesis. and growth. We find that upregulation of VEGF by hypoxia, TPA, and cyclohexamide is accompanied by a 3- to 6-fold stabilization of the VEGF transcript (from t 1/2 of 26-32 minutes to t 1/2 of 103-158 min), and that VEGF transcripts in human tumor cell lines that overexpress VEGF are constitutively stabilized 3- to 6-fold (1\2 of 113 to 181 min.) and exhibit little or no additional stabilization by hypoxia, TPA, or cycloheximide. This suggests that a normal signal transduction pathway involving an oxygen sensor regulates the stability of VEGF mRNA, and that mutations in this regulatory system cause persistent stabilization in some tumors. Our specific aims are designed to identify the primary sequences in VEGF mRNA that mediate basal instability and hypoxia-inducible stabilization, and to determine the molecular basis for constitutive stabilization of VEGF transcripts in breast carcinomas and other VEGF overexpressing tumors. Aim 1: Clone full-length, normal, 4.2 kb human and chicken VEGF transcripts from hypoxia-induced HBL 100 breast epithelial cells and chicken embryo fibroblasts. Evolutionarily conserved sequences represent potential elements that regulate message stability, and provide a framework for initiating mutagenic studies (Aim 3). Aim 2: Determine whether tumor cell lines exhibiting constitutive VEGF mRNA stabilization have mutations in their message stabilization pathways or in their VEGF transcripts. A slightly altered version of the normal human VEGF transcript, which can be distinguished from the endogenous transcript using RNase protection, will be expressed in tumor cell lines exhibiting constitutive VEGF message stabilization Basal and hypoxia- or TPA-induced stability will be measured for both the endogenous and exogenous VEGF mRNA's, using RNAse protection. Lines that confer the stabilized, stimulus-unresponsive half-life on the exogenous transcript would contain stabilization pathway mutations. Those that process the exogenous VEGF mRNA mRNA with a normal half-life would contain VEGF transcript mutations. Aim 3: Identify VEGF RNA sequence elements responsible for basal instability and for hypoxia- or TPA-induced message-stabilization. Determine whether these elements mediate constitutive stabilization in tumor cells. Regulatory RNA elements will be mapped using a panel of NIH3T3 clones expressing deleted forms the normal human VEGF mRNA. Inherent stability and hypoxia- or TPA-induced stability will be measured. Elements will be tested for transferable function in rabbit beta-globin fusion transcripts. Altered function of individual elements in tumor cell lines will be examined in the context of chimeric transcripts. Aim 4: Begin to identify, purify, clone, sequence, and characterize an RNA-binding protein(s) that mediates basal instability and hypoxia-induced upregulation of VEGF in normal cells, and constitutive upregulation in some solid-tumors. These proteins represent potential targets for anti-angiogenic therapy. Their identification and purification will be based on their binding to 32P-labeled RNA probes in nondenaturing gels and their lack of binding to mutant versions of specific RNA elements that were demonstrated to abrogate function in vivo (in Aim 3).

该提案的目的是确定成绩单的 血管内皮生长因子（VEGF）瞬时稳定 正常细胞中的缺氧，并在子集中构成稳定 高表达VEGF的肿瘤细胞。 VEGF是一种有效的血管生成 实体瘤产生的肽至少由两种不同的机制。 首先，VEGF表达的集中区域出现在低氧区域 实体瘤，与介导缺氧诱导的血管生成有关。 和增长。 我们发现缺氧，TPA和 环己酰胺伴随着VEGF的3至6倍稳定 成绩单（从26-32分钟的T 1/2到T 1/2的103-158分钟）， 过表达VEGF的人类肿瘤细胞系中的VEGF转录本为 组成性稳定3至6倍（113至181分钟）并展示 缺氧，TPA或环己酰亚胺几乎没有或没有额外的稳定。 这表明正常信号转导途径涉及氧气 传感器调节VEGF mRNA的稳定性，并在此中进行突变 调节系统会导致某些肿瘤的持续稳定。 我们的 特定目的旨在识别VEGF mRNA中的主要序列 介导基底不稳定性和缺氧诱导的稳定性，并 确定VEGF组成型稳定的分子基础 乳腺癌和其他VEGF过表达肿瘤的转录本。 目的 1：克隆全长，正常，4.2 kb人类和鸡肉VEGF成绩单 从缺氧引起的HBL 100乳腺上皮细胞和鸡胚胎 成纤维细胞。 进化保守的序列代表潜力 调节消息稳定性的元素，并为 启动诱变研究（AIM 3）。 目标2：确定肿瘤细胞是否 表现出构成VEGF mRNA稳定的线在 他们的消息稳定途径或在VEGF成绩单中。 一个 正常人VEGF成绩单的略有变化版本可以是 使用RNase保护区与内源成绩单区分开，将 在表现出构成VEGF消息的肿瘤细胞系中表达 将测量稳定基础和缺氧或TPA诱导的稳定性 对于内源性和外源性VEGF mRNA，都使用RNase保护。 赋予稳定的，刺激的半衰期的线条 外源转录物将包含稳定途径突变。 那些 该过程以正常半衰期的外源性VEGF mRNA mRNA mRNA将 包含VEGF转录物突变。 AIM 3：识别VEGF RNA序列 负责基础不稳定性和缺氧或TPA诱导的要素 消息稳定。 确定这些元素是否介导 肿瘤细胞中的组成稳定。 调节RNA元素将是 使用表达删除的NIH3T3克隆面板映射的绘制形式正常 人VEGF mRNA。 固有的稳定性和缺氧或TPA诱导的稳定性 将测量。 元素将在 兔β-珠蛋白融合转录本。 个体的功能改变 肿瘤细胞系中的元素将在嵌合的背景下检查 成绩单。 目标4：开始识别，净化，克隆，序列和 表征介导基础不稳定性和的RNA结合蛋白 缺氧引起的正常细胞中VEGF的上调，并构成 在某些固体中上调。 这些蛋白质代表潜力 抗血管生成疗法的靶标。 他们的识别和净化 将基于它们与非肾化的32p标记RNA探针的结合 凝胶及其与特定RNA元素的突变版本缺乏结合 证明可以在体内消除功能（在AIM 3中）。