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-----Oorspronkelijk bericht----- Van: Hilary Blue <[log in to unmask]> Aan: [log in to unmask] <[log in to unmask]> Datum: woensdag 28 juli 1999 15:31 Onderwerp: PORQUETTA'S BACK >> FEMALE HORMONES and their effect on Parkinson's << Estrogen Receptor-Dependent Gene Expression Carmen A. Peralta, Rice University, Class of 1995 Bert O'Malley, M.D., Department of Cell Biology The major focus of Dr. Bert O'Malley's laboratory has been to characterize the mechanism(s) by which steroid receptors are activated and regulate gene expression. Within this family of receptors, which includes proteins such as steroid, thyroid and vitamin D3 receptors, I focused on the analysis of estrogen receptor (ER) activity. Previous data indicate that ER activation is regulated by hormone, antihormones, epidermal growth factor (EGF), and a neurotransmitter (dopamine) in HeLa cells, but the mechanism(s) by which this is accomplished are yet to be explained completely. By using a modified adenovirus, the ER cDNA was introduced into HeLa cells along with a reporter gene including an estrogen response element and the chloramphenicol acetyl transferase gene (CAT). The cells were treated with estrogen, dopamine, EGF and the estrogen antagonists, 4-hydroxytamoxifen and ICI 164, 384 using sterile tissue culture conditions. After an incubation period of 22-23 hours, the cells were harvested and CAT enzyme activity was measured with a standard protocol. The relative activity of the cells was graphed and compared. The study above was performed on the wild type ER as well as several deletion mutants. The wild type estrogen receptor showed about a 50 fold increase in activity in the presence of estradiol (1nM) with respect to the basal. Dopamine and EGF-treated cells also showed increased activity. 4-hydroxytamoxifen appeared to inhibit the action of estradiol, but not dopamine or EGF while ICI appeared to inhibit all activation. Deletion mutants proved to be a great tool for determining regions of the receptor necessary for activation. Gradual deletion of the carboxy terminus reduced activation, and deletion of the last 60 amino acids inhibited all activation. On the other hand, deletion of the first 121 amino acids appeared to increase activation by estradiol. Several experiments are being performed to find possible causes for this increase in activity such as a beta-galactosidase assay to compare the amount of DNA incorporated into cells and a Western blot to determine receptor levels in the cells. Future experiments will elucidate the mechanisms by which dopamine and EGF pathways interact with known receptor activation pathways and what regions of the ER are involved in this interaction. ---------------------------------------------------------------------------- ---- Copyright © 1996 BCM ---------------------------------------------------------------------------- ---- Author: Wendy Cooper URL: http://mbcr.bcm.tmc.edu/Cellbio/ (Modified: June 12, 1996)