Initial studies have shown that the structure of the estrogen ligand can dictate the range of genes induced and the concentration limits for the ligand to stimulate an estrogen response in MCF-7 human breast cancer cells. Structural modifications to the 17beta-estradiol (E2) molecule in the A and D rings are influential in discriminating among the range of estrogen responsive genes. The proposed studies are designed to first evaluate the effects of these E2 derivatives on induction of four E2-responsive genes (pS2, progesterone receptor, tissue plasminogen activator, and cathepsin D) and MCF-7 cell growth. Further """"""""mapping"""""""" of the estrogen molecule for other components which may be important in the induction of responsive genes will be carried out with a series of B-and C-ring based derivatives of E2. Studies of the aberrant ER complex formed with E2 analogues will be pursued by documenting structural alterations in the analogue-receptor complex in solution by sucrose density gradient analysis and when bound to the estrogen responsive element (ERE) by gel retardation analysis. It is hypothesized that ER complexed with structurally altered estrogens may bind with different affinities and/or conformations to the consensus ERE and functional EREs that vary from it by one or more nucleotides. Furthermore, the possibility that analogue-ER complexes may be interacting with other regulatory elements known to influence estrogen responsive gene expression will be considered. Investigations concerned with the gene-specific induction exhibited by E2 isomers will be pursued by examining the ability of each estrogen analogue to activate the CAT gene in MCF-7 cells transfected with plasmid constructs in which CAT expression is regulated by different EREs or other regulatory elements. The abnormal activity of specific estrogen analogues will also be examined in experiments which document variances in transcription rate, half-life of mRNAs of gene products, and the effect of estrogen structure on ER dimer formation. Knowledge gained from these studies will contribute to the understanding of gene regulation by estrogens and aid in the design of attenuated estrogens for use in the treatment of hormone dependent neoplasms.
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