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.
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