Estrogens are the major carcinogen in the environment of most females with exposure to unopposed estrogen increasing the risk of breast and endometrial cancer. Conversely, it has become increasingly apparent that estrogens are essential for the well being of women (and men) throughout life. Progesterone acts to oppose the effects of estrogen on cell proliferation and, consequently, it is used in the treatment of endometrial cancer and it is an essential component of hormone replacement therapy designed to alleviate post-menopausal symptoms in women. It is, therefore, of fundamental importance to understand the mechanism of action of these hormones on cell proliferation. In adult ovariectomized mice, a single injection of estradiol-17beta (E2) results in the stimulation of a wave of DNA synthesis and cell proliferation that is restricted to the uterine epithelium. This proliferation is completely inhibited by pretreatment with progesterone (P4). The uterine epithelium can be isolated with great purity in a state suitable for biochemical analysis. This method together with defined hormonal regimens provides a controllable model in which to study the mechanism of action of these hormones in vivo. In tissue culture cells the cell cycle is regulated by the orderly activation of cyclins and their dependent kinases (Cdk). These include the cyclin D-Cdk4 and cyclin D-Cdk6 complexes acting early in G1 and the cyclin E-Cdk2 complex acting at the G1 to S-phase boundary. Our studies in the uterine epithelium have shown that E2 induces the re-localization of cyclin D1 and Cdk-4 to the nucleus and, results in orderly activation of cyclin-E and cyclin ACdk-2 activities and hyper-phosphorylation of pRb and p107. Progesterone pre- treatment prohibited the cyclin D1/Cdk-4 relocalization to the nucleus with a consequent inhibition of pRb and p107 phosphorylation. In addition, P4 abrogated the E2 induced cyclin E and cyclin A-Cdk2 activities.
The specific aims of this grant are: 1) To determine the mechanism whereby P4 prohibits cyclin D1/Cdk4 nuclear accumulation following E2 treatment; 2) To determine the mechanism of action of P4-inhibition of Cdk-2 activation; 3) identify differentially regulated genes in the uterine epithelium following E2 treatment in the presence and absence of P4; 4) to develop methods to interfere with signaling pathways in the uterine epithelium in vivo. It is expected that by the end of the grant that the mechanisms of cyclin D1/Cdk4 exclusion can be identified and novel proteins associated with this process isolated. Furthermore, novel E2 and P4-regulated genes that play important roles in the control of epithelial cell proliferation should be identified. These studies will define specific mechanisms that may result in the development of therapeutics that would inhibit estrogen's mitogenic effects in tumors as well as in benign proliferative diseases such as endometrial polyps and endometriosis.
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