The long-term objective of this project is to elucidate the role of specific receptor proteins in the estrogenic regulation of growth and function in reproductive tissues and cancers. To achieve this goal we propose to continue and extend our application of biochemical and immunochemical techniques to the purification, measurement, characterization and localization of estrogen receptor (estrophilin) in responsive tissues. The cytosol form of calf uterine estrophilin will be purified to homogeneity, with the help of steroid affinity chromatography and immunoadsorption to provide material for detailed analysis of amino acid composition and sequence as well as physical, chemical and structural properties of the purified receptor. This work will complement our ongoing effort to purify and characterize estrogen receptor from MCF-7 human breast cancer cells. Our library of specific monoclonal antibodies to calf and human estrophilins will be used 1) to prepare immunoadsorbents for the isolation of steroid-occupied and unoccupied forms and proteolytic fragments of receptor from various sources, 2) to measure receptor in extracts of human breast cancers, other reproductive tissues, and tumor cell cultures by an immunocolorimetric assay, 3) to localize receptor by an immunoperoxidase technique in responsive tissues to determine the inter- and intracellular distribution, at the light and electron microscopic levels, of receptor in the presence and absence of estrogens and antiestrogens, 4) to study the synthesis, cellular interactions and processing of these receptors and 5) to isolate specific receptor-bound chromatin proteins or DNA sequences that are involved in receptor- mediated regulation of transcription. The immunocolorimetric and immunocytochemical assays for estrophilin may have application in the assessment of prognosis and therapy for human breast cancers and other reproductive cancers. An additional goal of this project is to use our library of monoclonal antibodies to aid in the cloning of the cDNA and chromosomal gene that code for MCF-7 human estrogen receptor. This work is being done in collaboration with Professor Pierre Chambon in Strasbourg, France. The availability of such cloned genes will make possible a detailed analysis of factors controlling the expression of steroid receptor genes, and facilitate the production and sequencing of the receptor protein. The achievement of all of these goals is made possible by the development of procedures for purifying calf and human estrogen receptors and by the preparation of specific high-affinity monoclonal antibodies to these proteins.
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