Growth Regulation of the Normal & Malignant Endometrium

正常的生长调节

• 批准号: 6371197
• 负责人: Leslie Ina Gold
• 金额: $ 27.81万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-10 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6371197
• 关键词: adenocarcinoma; athymic mouse; cell cell interaction; cell growth regulation; clinical research; connective tissue cells; cyclin dependent kinase; endometrium; enzyme activity; enzyme inhibitors; epithelium; estrogens; female; gene targeting; genetically modified animals; hormone regulation /control mechanism; hormone related neoplasm /cancer; human subject; mitogen activated protein kinase; mixed tissue /cell culture; progesterone; transcription factor; transforming growth factors; uterus neoplasms; women's health

DESCRIPTION: (provided by applicant) Our broad long-term objectives are to elucidate mechanisms that cause loss of growth inhibition by TGF- b in endometna adenocarcinoma (ECA) and to define hormone regulation of TGF-b through stromal/epithelial interactions in the endometrium. ECA is induced by estrogenic (E2) agents causing hyperproliferation of uterine epithelial cells (UtE). Progesterone (Pg) is therapeutic due its growth inhibitory effect. TGF-b-mediated growth inhibition is transduced by two cooperating receptors (RI, RII) and the downstream signaling/transcription factors, Smad2/3,that activate genes that block cell cycle progression, such as the cyclin-dependent kinase inhibitor, p27kip1 We have shown that UtE isolated from all grades of ECA escape negative growth control by TGF-b by incurring multiple defects in the TGF-b response pathway including, loss of: TGF-b RH, activated Smad2, and p27kip1. Moreover, in complex hyperplasia (CH), the precursor to ECA, these proteins are already decreased. Thus, disruption of TGF-b action occurs early in endometrial carcinogenesis, providing an opportunity to understand molecular events leading to dysregulated growth. We will use primary cultures of normal, CH, and ECA UtE and co-cultures with stromal cells (UtS), which unlike ECA cell lines, retain many in vivo differentiation characteristics. Specific Aim 1, will determine the molecular mechanisms causing TGF-b receptor downregulation (e.g., transcriptional, translational) and test for defective Smad2/3 signaling using a TGF-b-promoter-responsive reporter assay. We will try to regain TGF-b function by transient transfection of RII cDNA into ECA UtE. Specific Aim 2 will test the hypothesis that loss of p27kip1 in ECA is by degradation via ubiquitin-proteasome pathway, which is E2-dnven directly through a MAPkinase via the Ras/MAPK/ERK1 pathway and that TGF-b normally prevents p27 degradation. Tissue/celilysates, inhibitors of proteasomes and MAPK, and immuno-analytical techniques will be used. Using single cell and co-cultures, we show that UtS from normal but not malignant endometnum mediates Pg-induced growth inhibition of normal UtE. Specific Aim 3 will test the hypotheses that UtS paracrine-mediates Pg-induced growth inhibition of UtE by release of TGF-b in response to Pg. We will use co-cultures of normal and "malignant" UtS and UtE and novel in vivo chimeric tissue recombinants composed of UtS from both, Pg receptor knock-out (PRKO) and wild-type mice and human UtE, that are hormonally manipulated as transplants in nude mice and UtE then analyzed for growth. These studies should elucidate molecular mechanisms of endometrial carcinogenesis, hormonal (dys)regulation of endometrial growth, and identify targets for prevention and therapeutic intervention.

描述：（由申请人提供）我们广泛的长期目标是 阐明导致TGF- B生长抑制作用丧失的机制 子宫内膜腺癌（ECA），并确定TGF-β的激素调节 通过子宫内膜中的基质/上皮相互作用。ECA由以下因素引起： 引起子宫上皮细胞过度增殖的雌激素（E2）剂 （UtE）。前列腺素（Pg）由于其生长抑制作用而具有治疗作用。 TGF-b介导的生长抑制作用是由两种协同受体转导的 (RI，RII）和下游信号传导/转录因子Smad 2/3， 激活阻断细胞周期进程的基因，如细胞周期蛋白依赖性 激酶抑制剂，p27 kip 1我们已经表明，从所有等级的肿瘤中分离的UtE， ECA逃避负生长控制的TGF-β引起的多重缺陷， TGF-b应答途径，包括TGF-b RH、活化的Smad 2和 p27kip1。此外，在复杂增生（CH）中，ECA的前体，这些 蛋白质已经减少。因此，TGF-β作用的破坏发生在早期， 在子宫内膜癌发生过程中， 导致生长失调的事件。我们将使用正常的， CH和ECA UtE以及与基质细胞（UtS）共培养， 系，保留了许多体内分化特征。具体目标1， 将确定导致TGF-β受体下调的分子机制 (e.g.,转录、翻译）和检测缺陷Smad 2/3信号传导 使用TGF-b启动子应答报告分析。我们将尝试重新获得TGF-b 通过将RII cDNA瞬时转染到ECA UtE中来发挥功能。具体目标2 将检验以下假设：ECA中p27 kip 1的丢失是通过以下途径降解 泛素-蛋白酶体途径，它是E2直接通过MAP激酶介导的 通过Ras/MAPK/ERK 1途径，TGF-β通常阻止p27降解。 组织/细胞裂解物、蛋白酶体和MAPK抑制剂以及免疫分析 技术将被使用。使用单细胞和共培养，我们表明，UtS 正常而非恶性子宫内膜介导前列腺素诱导的生长抑制 正常的UtE。具体目标3将检验以下假设： 旁分泌通过释放TGF-β介导Pg诱导的UtE生长抑制 我们将使用正常和“恶性”UtS和UtE的共培养物 以及由来自Pg和Pg的UtS组成的新的体内嵌合组织重组体， 受体敲除（PRKO）和野生型小鼠和人UtE，其在哺乳动物中 在裸鼠和UtE中作为移植物操作，然后分析生长。这些 研究应该阐明子宫内膜癌发生的分子机制， 子宫内膜生长激素（dys）调节，并确定 预防和治疗干预。