Transformation of Human Endometrial Epithelial Cells

描述（申请人提供）：子宫内膜癌是美国女性中最常见的生殖道癌症，在女性癌症总发病率中排名第四。因此，对我们对这种疾病的理解做出重大贡献的发现，以及为发现新疗法提供新方法的发现，将对妇女癌症产生重大影响。在这项应用中，我们提出了一项培养正常子宫内膜上皮细胞的初步研究，这些细胞在培养中寿命非常有限，并从它们中获得体外寿命和增殖能力大大增加的培养物。在过去，我们做过SV40大T抗原的转移，但结果细胞明显异常，表现得像早期恶性子宫内膜细胞的严重增生。此外，我们使用了一种似乎在子宫内膜癌发展中没有作用的药物。在这项初步研究中，我们试图培养可繁殖的人类子宫内膜上皮细胞，这种细胞经过基因修饰，更接近子宫内膜癌变过程中发生的变化。我们寻求开发具有增强增殖能力的细胞。为了实现这一目标，我们提出以下三个具体目标：1)确定适用于正常上皮子宫内膜细胞原代培养的最佳遗传转移剂和技术。2)测试破坏这些细胞中PTEN功能的最佳方法，重现子宫内膜癌发生的一个公认的早期事件，并确定这种改变是否导致细胞增殖能力增加。3)利用各种基因文库作为“虚拟诱变剂”，发现与子宫内膜癌发展特异性相关的进一步突变。这些研究将评估细胞增殖能力的增加，并将采用新的选择方案，模拟子宫组织中基底膜和基质细胞施加的选择压力。通过识别这一过程中产生的细胞生理变化，我们希望阐明致癌子宫内膜上皮细胞逃避子宫内膜组织内正常运作的细胞增殖稳态控制的机制。

DESCRIPTION (provided by applicant): Endometrial cancer is the most abundant cancer of the genital tract in American women and ranks fourth in overall incidence among cancers in women. Therefore, discoveries that contribute significantly to our understanding of this disease and hat provide new methods with which novel therapies may be discovered, would have a high impact on cancer in women. In this application we propose a pilot study to culture normal endometrial epithelial cells, which have very limited longevity in culture, and derive cultures from them that have a greatly increased longevity and capacity for proliferation in vitro. In the past we did with transfers of SV40 large T antigen but the cells that result are notably abnormal, behaving like severely hyperplastic to early malignant endometrial cells. Furthermore, we used an agent that appears to have no role in endometrial cancer development. In this pilot study we seek to develop propagable human endometrial epithelial cells that have been genetically modified in a manner that better approximates changes occurring during endometrial carcinogenesis. We seek to develop cells with an enhanced capacity for proliferation. To accomplish this we propose the following three specific aims 1) to determine the best genetic transfer agents and techniques applicable to primary cultures of normal epithelial endometrial cells. 2) to test the best way to impair PTEN function in these cells, recreating a recognized early event in endometrial carcinogenesis, and determine whether this change results in cells with increased capacity for cell proliferation. 3) to discover further mutations specifically relevant to development of endometrial cancer using various genetic libraries as "virtual mutagens." These studies will assess increased capacity for cell proliferation and will employ novel selection protocols that will mimic the selection pressures exerted by basement membrane and stromal cells in the uterine tissue. By identifying the alterations in cell physiology produced by this process, we hope to clarify the mechanisms that allow carcinogenic endometrial epithelial cells to escape the homeostatic controls on cell proliferation that normally operate in within endometrial tissue.