Transformation of Human Endometrial Epithelial Cells

描述（由申请人提供）：子宫内膜癌是美国女性生殖道最常见的癌症，在女性癌症的总发病率中排名第四。因此，这些发现对我们理解这种疾病有重大贡献，并提供了新的方法，可以发现新的疗法，对女性癌症有很大的影响。 在本申请中，我们提出了一个试点研究，培养正常的子宫内膜上皮细胞，这在文化中具有非常有限的寿命，并从他们那里获得文化，有一个大大增加的寿命和体外增殖能力。在过去，我们用SV40大T抗原转移，但结果细胞明显异常，表现为严重增生的早期恶性子宫内膜细胞。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，我们寻求开发可繁殖的人子宫内膜上皮细胞，这些细胞已被基因修饰，以更好地近似子宫内膜癌变过程中发生的变化。 我们寻求开发具有增强的增殖能力的细胞。为了实现这一目标，我们提出了以下三个具体目标：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)以测试在这些细胞中削弱PTEN功能的最佳方式，重现子宫内膜癌发生中公认的早期事件，并确定这种变化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”，进一步发现与子宫内膜癌发生特别相关的突变。“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。通过确定由这一过程产生的细胞生理学改变，我们希望澄清允许致癌子宫内膜上皮细胞逃避通常在子宫内膜组织内运作的细胞增殖的稳态控制的机制。

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.