CATALYTIC SUBUNIT OF THE TELOMERASE GENE HEST2

描述(申请人提供)：许多类型的哺乳动物细胞，当被放入培养时，停止增殖并进入一种称为衰老的不生长状态。流行的理论认为，衰老的开始是由染色体末端端粒的双链DNA区域的长度决定的。相反，我们已经证明，衰老不是由ds端粒DNA的总长度控制的，而是由从端粒的dsDNA部分(SsOH)末端伸出的一小段单链DNA的长度控制的。当细胞进入衰老时，SSOH大量丢失。我们将开发各种方法来原位测量ssOH和dsDNA部分的长度。使用这些分析以及更直接的分子测量，我们将测试一个分子模型，在该模型中，细胞遭受的生理应激引起ssOH的丢失，并且这种丢失反过来诱导依赖于p53的DNA损伤反应，从而导致细胞衰老的表型。我们将考察这样的概念，即ssOH的丢失是触发衰老的一种常见机制，以响应各种生理压力，并且这种丢失通常是由细胞遭受的累积氧化损伤引起的。使用原位测量细胞内端粒的ssOH和dsDNA部分将使我们能够确定ssOH的丢失是在单个细胞中以一致的方式发生的，因此是主动激发的，还是以异步、随机的方式发生的。此外，这些测量将使我们能够确定在活组织内的细胞中是否发生了ssOH和相关的P53激活的丢失，从而提供了细胞衰老状态在体内发生的证据，并可能有助于在衰老组织和各种病理状态下细胞的增殖能力的丧失。

DESCRIPTION (provided by applicant): Many types of mammalian cells, when placed into culture, halt proliferation and enter into a non-growing state termed senescence. The prevailing theory says that the onset of senescence is dictated by the length of double strand (ds) DNA regions of the telomeres at the ends of chromosomes. We have demonstrated that, on the contrary, senescence is not controlled by the overall length of the ds telomeric DNA, but instead by the length of a short stretch of single-strand DNA that protrudes from (overhangs) from the ends of the dsDNA portion of a telomere (ssOH). ssOH is largely lost when cells enter into senescence. We shall develop assays to gauge the lengths in situ of the ssOH and dsDNA portions. Using these assays as well as more direct molecular measurements, we will test a molecular model whereby physiologic stress suffered by cells provokes loss of the ssOH, and that this loss, in turn, induces a p53-dependent DNA damage response that results in the senescent cell phenotype. We will examine the notion that loss of the ssOH is a common mechanism for triggering senescence in response to a variety of physiologic stresses, and that this loss, is often provoked by cumulative oxidative damage suffered by cells. Use of in situ measurements of the ssOH and dsDNA portions of the telomeres within cells will allow us to determine whether the loss of ssOH occurs in a concerted fashion in individual cells and is thus actively provoked or in an asynchronous, stochastic fashion. In addition, these measurements will allow us to determine whether loss of ssOH and associated p53 activation occur in cells within living tissues, thereby providing evidence that the state of cell senescence occurs in vivo and may contribute to the loss of proliferative potential of cells in aging tissues and in various pathological states.