OVERCOMING CELLULAR SENESCENCE IN HUMAN EPITHELIAL CELLS

克服人类上皮细胞的细胞衰老

• 批准号: 6642035
• 负责人: ALOYSIUS John KLINGELHUTZ
• 金额: $ 22.05万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6642035
• 关键词: cell differentiation; cell growth regulation; cell line; cell senescence; cell transformation; cyclins; enzyme activity; enzyme inhibitors; epithelium; human papillomavirus; human tissue; retinoblastoma protein; telomerase; virus infection mechanism; yeast two hybrid system

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.

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，上皮细胞产生了大多数 人类癌症。要检验的假设是，废除 视网膜母细胞瘤途径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 途径的诱导并确定如何 当细胞变得永生时，端粒酶被激活。终端的作用 将检查 Rb 途径激活和衰老的差异 通过多种方法，包括调节 p16INK4a 的表达，a cdk4/cyclin D 抑制剂，在特异性丢失的上皮细胞中 p16INK4a 在 hTERT 永生化后的表达，hTERT 是 端粒酶。参与 p16INK4a 上调的基因也将被鉴定 通过使用 cDNA 逆转录病毒文库绕过 Rb 介导的阻断。因为 p16INK4a 的表达在人类癌症中通常缺失，PI 观察到 这项工作可能会显着增加我们对 癌变的早期阶段。先前的研究表明，人类 乳头瘤病毒 E6 激活端粒酶。为了确定这个机制 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 HPV E6 参与端粒酶激活的因素。了解E6如何 激活端粒酶将有助于阐明端粒酶是如何被激活的 一般来说。 PI 观察到这些研究有可能导致 预防、诊断和治疗的新的和创新的策略 人类癌症。该提案的总体目标是研究人类如何 上皮细胞变得永生，这是发育过程中的关键事件 恶性肿瘤。该提案的核心假设是废除 视网膜母细胞瘤途径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 在其实验室开发的系统中，PI 将追求以下具体目标 目的：(1) 确定分化在 Rb 介导的衰老中的作用 人上皮细胞； (2) 鉴定和表征上游基因 参与诱导人上皮细胞 Rb 介导的衰老；和 (3)确定HPV 16 E6如何激活上皮细胞中的端粒酶。这些 研究应该有可能带来新的和创新的策略 用于预防、诊断和治疗人类癌症。