CATALYTIC SUBUNIT OF THE TELOMERASE GENE HEST2

描述（由申请人提供）：许多类型的哺乳动物细胞，当置于培养中时，停止增殖并进入称为衰老的非生长状态。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。我们已经证明，相反，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）的末端伸出（悬垂）的短单链DNA的长度控制的。当细胞进入衰老时，ssOH大量丢失。我们将开发测定ssOH和dsDNA部分原位长度的方法。利用这些分析以及更直接的分子测量，我们将测试一种分子模型，即细胞遭受的生理应激会引起ssOH的丢失，而这种丢失反过来又会诱导p53依赖的DNA损伤反应，从而导致衰老细胞表型。我们将研究ssOH的丢失是触发衰老的一种常见机制，以应对各种生理压力，而这种丢失通常是由细胞遭受的累积氧化损伤引起的。使用细胞内端粒的ssOH和dsDNA部分的原位测量将使我们能够确定ssOH的损失是在单个细胞中以协调的方式发生，因此是主动引起的，还是以异步、随机的方式发生。此外，这些测量将使我们能够确定ssOH的丧失和相关的p53激活是否发生在活组织内的细胞中，从而提供证据，证明细胞衰老状态发生在体内，并可能导致衰老组织和各种病理状态下细胞增殖潜力的丧失。

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.