ROLES OF GROWTH FACTORS IN NEOPLASTIC TRANSFORMATION

本提案的长期目标是在以下两个层面上理解 基因和蛋白质的机制，细胞被刺激， 在正常的生长和发育中以及在异常的 导致转化细胞异常生长的调节机制 生长和肿瘤。 这项建议现在试图继续分析 调节PDGF A链基因，该基因编码有效的生长因子， 高度发育调节的调节因子， 在选择的转化细胞系中过表达。 计划在 鉴定和表征5 ′侧翼区域内调节序列 在PDGF A链基因的第一个内含子内， 发育阶段和表达的细胞类型特异性。 任何元件 将被鉴定，并与之相互作用的蛋白质 这些元素和介导这些元素的功能 将被克隆和鉴定。 计划使用任何细胞类型 特异性增强子被鉴定为指导显性负突变， 产生内源性PDGF的细胞类型特异性损失。 中的函数 转基因小鼠 计划使用激活和阻遏物 在肾母细胞瘤基因产物中鉴定的结构域， 分析肾母细胞瘤基因影响 PPDGF A链基因的转录，以鉴定二聚化的蛋白质 与WTI相互作用以影响PDGF的转录 A链基因，并寻求+17AA可变剪接的功能 在PDGF A链基因转录时插入WT 1。 寻求 WT1对PDGF A链基因转录的潜在相关性，我们 将WT1的正调控域和负调控域连接到 表达高水平PDGF A和WT 1的间皮瘤细胞， 用PDGF A转化的间皮细胞。 的表型 这些细胞将被表征并且每个基因的转录活性 会得到考验 计划对蛋白质进行表征和克隆， 甲基化对PDGF A链基因转录的抑制活性 并使用Southern印迹和基因组测序的方法来寻找 PDGF A链基因甲基化的直接证据， 组织和在选定的发展时期和肿瘤中。 然后 计划寻找甲基化对PDGF A转录的作用， 链基因在选定的细胞系。 该项目将利用现有的 生物化学、分子和细胞生物学技术。 它将利用 以及核心项目和项目研究人员之间的互动 项目推进每一个具体目标。 希望结果 这项研究的一部分将确定用于调节 PDGF A链基因的表达及其调控的后果 在肿瘤细胞中，并确定用于修饰肿瘤细胞的位点和策略， PDGF A链对癌细胞生长的功能和贡献。

The long-term goals of this proposal are to understand both at the level of the gene and the protein mechanisms by which cells are stimulated to proliferate in normal growth and in development and in the abnormal regulatory mechanisms that lead to aberrant growth in transformed cell growth and tumors. This proposal now seeks to continue the analysis of the regulation of the PDGF A-chain gene, a gene that encodes a potent growth regulatory factor that is highly developmentally regulated and overexpressed in selected transformed cell lines. It is planned to identify and characterize regulatory sequences within 5' flanking region and within the first intron of the PDGF A-chain gene that are important in developmental stage and cell type specificity of expression. Any elements that are identified will be characterized and proteins that interact with these elements and that mediate in trans the function of these elements will be cloned and characterized. It is planned to use any cell type specific enhancers identified to direct dominant negative mutations to generate cell type specific loss of endogenous PDGF. A function in the transgenic mouse. It is planned to use the activation and repressor domains identified in the product of the Wilms' tumor gene to further analyze the mechanisms by which the Wilms' tumor gene influences transcription of the PPDGF A-chain gene, to identify proteins that dimerize with or otherwise interact with WTI to influence transcription of the PDGF A-chain gene, and to seek the function of the +17AA alternatively spliced insert of WT1 on transcription of the PDGF A-chain gene. To seek the potential relevance of WT1 on transcription of the PDGF A-chain gene, we will transfect positive and negative regulatory domains of WT1 into mesothelioma cells that express high levels of both PDGF A and WT1 and mesothelial cells that are transformed with PDGF A. The phenotypes of these cells will be characterized and transcriptional activity of each gene will be tested. It is planned to characterize and clone proteins for the repressor activity of methylation on transcription of the PDGF A-chain gene and to use methods of Southern blotting and genomic sequencing to seek direct evidence for methylation of the PDGF A chain gene in specific tissues and in selected times of development and in tumors. It is then planned to seek the role of methylation on transcription of the PDGF A- chain gene in selected cell lines. The Project will use established techniques of biochemistry and molecular and cell biology. It will utilize also the Core Projects and interactions among investigators of the Program Project to advance each of the Specific Aims. It is hoped that the results of this research will identify genetic mechanisms that serve to regulate expression of the PDGF A-chain gene and the consequences of its regulation in tumor cells and to identify sites and strategies for modifying the function and contributions of the PDGF A-chain to cancer cell growth.