ROLES OF GROWTH FACTORS IN NEOPLASTIC TRANSFORMATION

这项建议的长期目标是在 刺激细胞的基因和蛋白质机制 在正常生长、发育和异常情况下增殖 导致转化细胞异常生长的调控机制 生长和肿瘤。这项提案现在寻求继续分析 PDGF A链基因的调控，这是一种编码强大生长的基因 高度发育受控的调控因素 在选定的转化细胞系中过表达。按照计划， 鉴定和鉴定5‘侧翼区的调控序列 在PDGF A链基因的第一内含子内，这些内含子在 表达的发育阶段和细胞类型特异性。任何元素 将被鉴定的蛋白质和与之相互作用的蛋白质 这些元素以及在这些元素的作用中起中介作用的元素 将被克隆和鉴定。计划使用任何单元格类型 发现可定向显性负突变的特定增强子 产生内源性PDGF的细胞类型特异性丢失。中的一个函数。 转基因小鼠。计划使用激活剂和抑制剂 确定Wilms‘s肿瘤基因产物中的结构域以进一步 分析Wilms‘s肿瘤基因影响的机制 转录PPDGF A链基因，以识别二聚体蛋白 与WTI或以其他方式相互作用以影响PDGF的转录 A链基因，并寻找+17AA的功能选择性剪接 WT1插入对PDGF A链基因转录的影响。为了寻求 WT1与PDGF A链基因转录的潜在相关性 将WT1的正、负调控域分别转入 间皮瘤细胞同时高表达PDGF A和WT1和 经PDGF A转化的间皮细胞，其表型 这些细胞将表征和转录每个基因的活性 将会受到考验。它计划鉴定和克隆蛋白质以用于 甲基化对血小板衍生生长因子A链基因转录的抑制作用 并利用Southern杂交和基因组测序的方法寻找 特异性血小板衍生生长因子A链基因甲基化的直接证据 在特定的发育时期和肿瘤中。那就是了 计划寻求甲基化对PDGF A转录的作用- 选定的细胞系中的链基因。该项目将使用已建立的 生物化学、分子和细胞生物学技术。它将利用 也是核心项目和项目调查人员之间的互动 推进每个具体目标的项目。希望这一结果 这项研究将确定基因机制，以帮助调节 血小板衍生生长因子A链基因的表达及其调控效应 在肿瘤细胞中，并确定修饰 血小板衍生生长因子A链对癌细胞生长的作用和贡献。

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.