OVERCOMING CELLULAR SENESCENCE IN HUMAN EPITHELIAL CELLS

该项目的主要目标是研究人类如何 上皮细胞变得永生。细胞永生化是一个关键事件， 恶性肿瘤的发展，上皮细胞引起的大多数 人类的癌症。有待检验的假设是，废除 视网膜母细胞瘤通路和端粒酶的激活对于 上皮细胞克服衰老。通过使用独特的上皮细胞模型 在实验室开发的系统中，PI建议确定因素 参与衰老过程中Rb通路的诱导，并确定如何 当细胞变得永生时，端粒酶被激活。终端的作用 将检查Rb通路活化和衰老的分化 通过几种方法，包括调节p16 INK 4a的表达， cdk 4/细胞周期蛋白D抑制剂，在上皮细胞中特异性丢失 p16 INK 4a表达在用hTERT（p16 INK 4a的催化组分）永生化后， 端粒酶还将鉴定参与p16 INK 4a上调的基因 通过使用cDNA逆转录病毒文库绕过Rb介导的阻断。因为 p16 INK 4a的表达通常在人类癌症中丢失，PI观察到， 这项工作很可能会大大提高我们对 癌症发生的早期阶段。以前的研究表明，人类 乳头瘤病毒E6激活端粒酶。为了确定这一机制， 激活，将使用差异激活端粒酶的HPV E6突变体 在cDNA表达阵列分析和酵母双杂交筛选中鉴定 参与HPV E6端粒酶激活的因素。了解E6如何 将有助于阐明端粒酶如何被激活 梗概. PI观察到这些研究有可能导致 新的和创新的战略，以预防、诊断和治疗 人类癌症这项计划的总体目标是研究人类如何 上皮细胞变得不朽，这是发展中的关键事件。 恶性肿瘤这一建议的核心假设是， 视网膜母细胞瘤途径和端粒酶活化对于 上皮细胞克服衰老。通过使用独特的上皮细胞模型 系统开发在他的实验室，PI将追求以下具体 目的：（1）确定分化在Rb介导的衰老中的作用， 人类上皮细胞;（2）识别和表征上游基因 参与诱导Rb介导的人上皮细胞衰老;和 (3)以确定HPV 16 E6如何激活上皮细胞中的端粒酶。这些 研究应该有可能导致新的和创新的战略， 用于预防、诊断和治疗人类癌症。

The major goal of the proposed project is to study how human epithelial cells become immortal. Cellular immortalization is a key event in the development of malignancy, and epithelial cells give rise to the majority of human cancers. The hypothesis to be tested is that both the abrogation of the retinoblastoma pathway and activation of telomerase is necessary for epithelial cells to overcome senescence. By using unique epithelial cell model systems developed in the laboratory, the PI proposes to identify factors involved in the induction of the Rb pathway during senescence and determine how telomerase is activated as cells become immortal. The role of terminal differentiation in activation of the Rb pathway and senescence will be examined by several approaches, including regulated expression of p16INK4a, a cdk4/cyclin D inhibitor, in epithelial cells that have specifically lost p16INK4a expression upon immortalization with hTERT, the catalytic component of telomerase. Genes involved in upregulation of p16INK4a will also be identified by using cDNA retroviral libraries to bypass the Rb mediated block. Because expression of p16INK4a is commonly lost in human cancers, the PI observes that this work is likely to lead to a significant increase in our understanding of the early stages of carcinogenesis. Previous studies demonstrated that human pappiloma virus E6 activates telomerase. To determine the mechanism of this activation, HPV E6 mutants that differentially activate telomerase will be used in cDNA expression array analysis and yeast two-hybrid screens to identify factors involved in telomerase activation by HPV E6. Understanding how E6 activates telomerase will be useful in elucidating how telomerase is activated in general. The PI observes that these studies have the potential of leading to new and innovative strategies for the prevention, diagnosis and treatment of human cancer. The overall goal of this proposal is to study how human epithelial cells become immortal, which is a key event in the development of malignancy. The hypothesis central to this proposal is that both abrogation of the retinoblastoma pathway and activation of telomerase are necessary for epithelial cells to overcome senescence. By using unique epithelial cell model systems developed in his laboratory the PI will pursue the following specific aims: (1) to determine the role of differentiation in Rb-mediated senescence of human epithelial cells; (2) to identify and characterize upstream genes involved in induction of Rb-mediated senescence in human epithelial cells; and (3) to determine how HPV 16 E6 activates telomerase in epithelial cells. These studies should have the potential of leading to new and innovative strategies for the prevention, diagnosis and treatment of human cancer.