The ability to respond to environmental signals is absolutely essential for proper development and cell survival. Extracellular signals in the form of growth factors affect many aspects of cell development including proliferation, differentiation and survival. The coordination of proliferation and differentiation affects not only embryonic development, but tissue replenishment and repair in the adult. The decision to divide or differentiate is influenced by numerous growth factors acting both individually and in concert to either stimulate or inhibit proliferation. One way in which growth factors act is through changes in gene expression. The elucidation of the molecular components of the pathways through which growth factors alter patterns of gene expression will provide potential targets for the augmentation or inhibition of these signalling pathways. The ability to manipulate these pathways has widespread applications in tissue maintenance and repair as well as in chemotherapeutic rationales. Moreover, the identification of the mediators of signal transduction pathways from the cell surface to the nucleus will provide a better understanding of the potential interactions between growth factors and insight into some of the cellular mechanisms of growth control. Much of our knowledge regarding signal transduction has resulted from the characterization of two predominant pathways active in fibroblasts, one including tyrosine kinase receptors and the other G-protein coupled receptors which activate phospholipases. Yet, elevations in cAMP are mitogenic in a variety of endocrine and other cell types. Moreover, activating mutations in the stimulatory G protein of adenylate cyclase have been isolated from a variety of tumors in which cAMP induces proliferation. We wish to elucidate the molecules which function in cAMP and ras mediated mitogenic pathways since activating mutations in both pathways have been found in human tumors. Thyroid follicular cells provide an ideal model system for this analysis since their growth can be stimulated by multiple signalling pathways including a cAMP mediated pathway stimulated by thyrotropin (TSH). Moreover, activating mutations in Gs and in all three cellular ras genes have been identified in thyroid adenomas, follicular and papillary carcinomas. Using needle microinjection and other molecular approaches, we will perform a molecular dissection of mitogenic pathways which include the ras protooncogene and cAMP as a second messenger. Microinjection allows for the introduction of putative signalling molecules or inhibitors directly into living cells in high concentrations. Unlike other techniques, biological effects in response to injected molecules can be measured in very short times. Since activating mutations in Gs and ras are found in a variety of human tumors, the identification of the molecular components of cAMP and ras mediated proliferation should contribute significantly to our understanding of growth control and transformation.
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