Tamoxifen, an estrogen receptor (ER) ligand, is the most commonly used drug for the treatment and prevention of breast cancer. Other ER-modulating drugs include pure ER antagonists, such as ICI 182,780, and selective ER modulators (SERMs) such as raloxifene, idoxifene, clomiphene and GW 7604. These drugs have traditionally been thought to act through interaction with the estrogen receptor, and therefore, the secondary ER-independent mechanisms and alternative effects of these drugs are just being discovered. The primary aim of this grant is to examine the effects of antiestrogenic drugs on the most important cell type involved in angiogenesis, the vascular endothelial cell. Three approaches will examine these effects. The first approach will utilize animal models (corneal pocket assay, dorsal skin-fold windows) to quantify angiogenesis in several tumor types treated with various estrogen receptor modulating drugs. The second approach will isolate and identify a newly described tamoxifen binding site in endothelial cells. Drug interactions with this site appear to be related to endothelial cell toxicity and apoptosis. The third approach will examine cellular events in endothelial cells that are affected by estrogen receptor modulation including migration, calcium signaling, nitric oxide production, and protein transcriptional regulation. All proposed approaches will use a number of mechanistically different estrogen receptor modulators, including tamoxifen, 4-OH tamoxifen, raloxifene, idoxifene, ICI 182,780, GW 7604, and clomiphene. All approaches will employ both estrogen receptor positive (ER+) and estrogen receptor negative (ER-) cancer cell lines and a variety of endothelial cell lines. In addition, this proposal investigates the time frame of action for each observed effect. By examining the time course of actions of each mechanistically different drug, the difference between genomic and non-genomic actions can be ascertained. By identifying and characterizing a new tamoxifen binding site, and further defining ER-independent actions of antiestrogens on endothelial cells that are mediated by this binding site, novel therapeutics can be developed which prevent tamoxifen resistance, reduce thromboembolic complications, and are more effective in inhibiting tumor angiogenesis. The results of this project will have profound implications, especially as clinicians begin to use the newer estrogen receptor modulating drugs for the prevention and treatment of breast cancer.