Background
The most common method for defining the action of potential estrogens is the uterotrophic response and the cornification of the vaginal epithelium in immature female rodents. This bioassay is subject to large variability characteristic of in vivo studies. It is also time consuming, expensive, and only examines the activity in a single tissue, whereas response to estrogens and antiestrogens varies in various tissues and cells. A test that would measure the agonist and antagonist activity of estrogen analogs quickly and economically from different estrogen sensitive tissues would facilitate the identification of such compounds and allow the development of novel estrogen agonists and antagonists.

Description
The investigator and coworkers have developed a series of robust, efficient tests to determine the agonist and antagonist activity of estrogen analogs rapidly and inexpensively in cultured cells from various estrogen responsive tissues. Specifically, they have developed two approaches:

(1). The first involves an in-depth comparison of the biological and biochemical properties of promising compounds to three well characterized antiestrogens, Tamoxifen (or 4-hydroxy tamoxifen), ICI 164,384, and Keoxifene (Raloxifene). The candidate and reference compounds are examined for their ability to bind to mouse uterine estrogen receptors and to stimulate proliferation of cultured cells derived from breast, endometrium, liver and bone. The goal is to understand the mechanism of action of candidate compounds in order to develop more effective antiestrogens. The effects of these compounds on the synthesis of estrogen sensitive proteins are also tested. In addition, the ability of these compounds to bind to antiestrogen binding sites (AEBs) and to facilitate binding of the estrogen receptor to a consensus estrogen responsive element (ERE) are determined. The cell lines tested include: MCF-7 human breast cancer cells, Ishikawa human endometrial cancer cells, HepG2 human hepatoma cells and in ROS 17/2.8 rat osteoblast-like osteosarcoma cells stably transfected with a mouse estrogen receptor cDNA (ROS.SMER cells). The estrogen-sensitive proteins to be examined include transforming growth factor (TGF), EGF/TGF receptor, TGFb, cathepsin D, estrogen receptor, sex hormone binding globulin (SHBG), alkaline phosphatase and chloramphenicol acetyl transferase (CAT) activity of an ERE-CAT construct in a transient transfection system. In each case, the ability of the antiestrogen alone to stimulate the expression of the protein as well as to inhibit estrogen-induced expression is determined.

(2) The second approach involves examining the compounds for their potential estrogenic and antiestrogenic activity using a reduced series of tests. All compounds are initially examined for their ability to bind to a partially purified mouse uterine estrogen receptor, and then for their effect on proliferation on the four cell lines described above. The ability to induce the expression of a characteristic protein in each of the four cell lines is also tested. These include the expression of cathepsin D in MCF-7 cells, alkaline phosphatase in Ishikawa and ROS.SMER cells and either SHBG or AT-III in HepG2 cells. The ability of the antiestrogen to effect proliferation and specific protein expression will be determined alone and in the presence of estrogen. A subset of these compounds can be selected for an additional study in which their effects on proliferation and characteristic protein synthesis will be examined after metabolic conversion (i.e., after incubation with a rat liver homogenate).

Applications
These tests are available to commercial parties on a contract basis.

For further information please contact:
New York University
Industrial Liaison/Technology Transfer
650 First Avenue, New York, N.Y. 10016
Tel: (212)263-8178 Fax: (212)263-8189