PROGESTERONE, CELL CYCLE AND CANCER

雌激素是大多数女性环境中的主要致癌物，暴露于无对抗性雌激素会增加患乳腺癌和子宫内膜癌的风险。相反，越来越明显的是，雌激素对女性（和男性）一生的健康至关重要。黄体酮的作用是对抗雌激素对细胞增殖的影响，因此，它被用于治疗子宫内膜癌，它是旨在减轻妇女绝经后症状的激素替代疗法的重要组成部分。因此，了解这些激素对细胞增殖的作用机制具有重要意义。在切除卵巢的成年小鼠中，单次注射雌二醇-17 β (E2)可刺激一波DNA合成和细胞增殖，这种刺激仅限于子宫上皮。孕酮预处理可完全抑制这种增殖（P4）。子宫上皮可以在适合生化分析的状态下以很高的纯度分离。这种方法与确定的激素方案一起提供了一个可控的模型来研究这些激素在体内的作用机制。在组织培养细胞中，细胞周期是由细胞周期蛋白及其依赖激酶（Cdk）的有序激活来调节的。其中包括在G1期早期作用的周期蛋白D-Cdk4和周期蛋白D-Cdk6复合物，以及在G1期至s期边界作用的周期蛋白E-Cdk2复合物。我们在子宫上皮的研究表明，E2诱导cyclin D1和Cdk-4重新定位到细胞核，导致cyclin- e和cyclin ACdk-2活性有序激活，pRb和p107超磷酸化。孕酮预处理可抑制细胞周期蛋白D1/Cdk-4向细胞核的再定位，从而抑制pRb和p107的磷酸化。此外，P4可抑制E2诱导的细胞周期蛋白E和细胞周期蛋白A-Cdk2活性。该资助的具体目的是：1)确定E2治疗后P4阻止cyclin D1/Cdk4核积累的机制；2)确定p4抑制Cdk-2活化的作用机制；3)在P4存在和不存在的情况下，鉴定E2治疗后子宫上皮的差异调控基因；4)研究在体内干扰子宫上皮信号通路的方法。预计到本研究结束时，可以确定cyclin D1/Cdk4排斥的机制，并分离出与该过程相关的新蛋白。此外，还应发现在上皮细胞增殖控制中发挥重要作用的E2和p4调控基因。这些研究将确定特定的机制，从而可能导致治疗方法的发展，从而抑制雌激素在肿瘤以及良性增殖性疾病（如子宫内膜息肉和子宫内膜异位症）中的有丝分裂作用。

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.