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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA002897-30
Application #
3163122
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1977-04-01
Project End
1988-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
30
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Chicago
Department
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
Razandi, M; Pedram, A; Greene, G L et al. (1999) Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha and ERbeta expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307-19
Greene, M E; Blumberg, B; Kwan, K et al. (1997) Isolation and cloning of human peroxisome proliferator activated receptor gamma cDNA. Adv Exp Med Biol 400A:253-60
Hess, R A; Gist, D H; Bunick, D et al. (1997) Estrogen receptor (alpha and beta) expression in the excurrent ducts of the adult male rat reproductive tract. J Androl 18:602-11
Nyholm, H C; Nielsen, A L; Lyndrup, J et al. (1995) Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay. Anticancer Res 15:649-54
Seielstad, D A; Carlson, K E; Katzenellenbogen, J A et al. (1995) Molecular characterization by mass spectrometry of the human estrogen receptor ligand-binding domain expressed in Escherichia coli. Mol Endocrinol 9:647-58
Seielstad, D A; Carlson, K E; Kushner, P J et al. (1995) Analysis of the structural core of the human estrogen receptor ligand binding domain by selective proteolysis/mass spectrometric analysis. Biochemistry 34:12605-15
Landel, C C; Kushner, P J; Greene, G L (1995) Estrogen receptor accessory proteins: effects on receptor-DNA interactions. Environ Health Perspect 103 Suppl 7:23-8
Pertschuk, L P; Schaeffer, H; Feldman, J G et al. (1995) Immunostaining for prostate cancer androgen receptor in paraffin identifies a subset of men with a poor prognosis. Lab Invest 73:302-5
Landel, C C; Kushner, P J; Greene, G L (1994) The interaction of human estrogen receptor with DNA is modulated by receptor-associated proteins. Mol Endocrinol 8:1407-19
Nardulli, A M; Greene, G L; Shapiro, D J (1993) Human estrogen receptor bound to an estrogen response element bends DNA. Mol Endocrinol 7:331-40

Showing the most recent 10 out of 32 publications