PROGESTERONE, CELL CYCLE AND CANCER

雌激素是大多数女性环境中的主要致癌物质，暴露于未经对抗的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)一生的福祉至关重要。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，它是旨在缓解女性绝经后症状的激素替代疗法的基本组成部分。因此，了解这些激素对细胞增殖的作用机制是非常重要的。在成年去卵巢小鼠中，单次注射雌二醇-17β(E2)可刺激一波仅限于子宫上皮的DNA合成和细胞增殖。孕酮(P4)可完全抑制这种增殖。在适合生化分析的状态下，可以高纯度地分离子宫上皮。这种方法与确定的激素方案一起提供了一个可控的模型，在其中研究这些激素在体内的作用机制。在组织培养细胞中，细胞周期受细胞周期蛋白及其依赖蛋白激酶(CDK)有序激活的调控。其中包括作用于G1早期的细胞周期蛋白D-CDK4和细胞周期蛋白D-CDK6复合体，以及作用于G1-S相界的细胞周期蛋白E-CDK2复合体。我们在子宫上皮细胞中的研究表明，E2诱导细胞周期蛋白D1和CDK-4重新定位于细胞核，导致细胞周期蛋白E和细胞周期蛋白ACdk-2活性的有序激活，以及pRb和p107的过度磷酸化。孕酮处理抑制了细胞周期蛋白D1/CDK-4在细胞核的重新定位，从而抑制了pRb和p107的磷酸化。此外，P4还抑制了E_2诱导的细胞周期蛋白E和细胞周期蛋白A-CDK2活性。这笔赠款的具体目的是：1)确定P4抑制E2治疗后细胞周期蛋白D1/CDK4核聚集的机制；2)确定P4的作用机制--抑制CDK-2的激活；3)确定在有和没有P4的情况下，E2治疗后子宫上皮中的差异调控基因；4)开发在体内干扰子宫上皮信号通路的方法。预计到拨款结束时，可以确定细胞周期蛋白D1/CDK4的排斥机制，并分离与这一过程相关的新蛋白。此外，应确定在控制上皮细胞增殖中发挥重要作用的新的E2和P4调节基因。这些研究将确定特定的机制，这些机制可能导致开发治疗方法，抑制雌激素在肿瘤以及良性增生性疾病(如子宫内膜息肉和子宫内膜异位症)中的促有丝分裂作用。

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.