The ultimate goal of this research project is to determine the genetic and molecular basis of signal transduction mechanisms of the tumor promoting phorbol ester TPA in cultured mouse cells. We are approaching the proposed goals through the use of somatic cell genetics in combination with molecular and biochemical techniques. Specific aims of our investigation are the following: 1. Continued characterization of the TPA-nonresponsive 3T3-L1 variants in terms of (a) A new role of collagen (TIE-5) gene expression in mitogenic response, (b) Expression of the known proto-oncogenes and growth-related genes in TPA-nonresponsive VT-1 cells upon TPA-treatment, (c) Identification of new protein components whose phosphorylation is enhanced upon cell's mitogenic response and cDNA cloning, and (d) Involvement of cytoskeletal components in the translocation of protein kinase C and 80K protein upon TPA stimulation. 2. Comprehensive analysis of the as yet unidentified TIE (TPA Inducible Early) genes in terms of (a) Sequence analysis of the three TIE gene (TIE-4, -10B and -44), (b) Expression patterns of the three TIE genes in 3T3-L1 cells upon stimulation by TPA and growth factors and in various mouse tissues, (c) Functional analysis of the three TIE genes using antisense expression vector, (d) Biochemical characterization and subcellular localization of the three TIE gene products, and (e) Genomic cloning and regulation of TIE gene expression. 3. Alternate approach for isolation of the genes for signal transduction using gene transfer technique for variant cells. 4. Activation of DNA topoisomerase I (Topo I) by protein kinase C-mediated phosphorylation as a significant nuclear event of signal transduction. 5. Chromosomal mapping of growth-related early genes using cell hybrid DNA panel and flow-sorted human chromosomes. 6. Future attempts to isolate more variants affected in mitogenic response. The cell and molecular genetic approaches to the dissection of transmembrane signalling mechanisms are unique, and the information and materials to be generated should prove useful for our further understanding the regulation of cell growth and differentiation and the malignant cell transformation during tumor development.