Telomere Length Dynamics in Telomerase-Positive Immortal Human Cell Populations

It has been proposed that the progressive shortening of telomeres in somatic cells eventually results in senescence. Previous experiments have demonstrated that many immortal cell lines have acquired telomerase activity leading to stabilization of telomere length. Telomere dynamics and telomerase activity were examined in the telomerase-positive immortal cell lines HeLa and 293 and subclones derived from them. A mass culture of HeLa cells had a stable mean telomere length over 60 population doublings (PD)in vitro.Subclones of this culture, however, had a range of mean telomere lengths indicating that telomeric heterogeneity exists within a population with a stable mean telomere length. Some of the subclones lacked detectable telomerase activity soon after isolation but regained it by PD 18, suggesting that at least some of the variation in telomere length can be attributed to variations in telomerase activity levels. 293 subclones also varied in telomere length and telomerase activity. Some telomerase-positive 293 subclones contained long telomeres that gradually shortened, demonstrating that factors other than telomerase also act to modulate telomere length. Fluctuations in telomere length in telomerase-positive immortalized cells may contribute to chromosomal instability and clonal evolution.     

亚硒酸钠对肝细胞L-02端粒酶活性和端粒长度的作用

通过研究硒对端粒酶活性和端粒长度的作用 ,探讨硒抗衰老的生物学机制。实验以人肝细胞株L 0 2为研究对象 ,分别补充 0 .5和 2 .5 μmol L亚硒酸钠 ,采用端粒重复序列扩增 焦磷酸根酶联发光法、逆转录聚合酶链式反应法及流式荧光原位杂交法 ,分别检测细胞的端粒酶活性、人端粒酶逆转录酶催化亚基基因 (hTERT)的表达及端粒长度的变化。结果表明 :常规培养的肝细胞株L 0 2的端粒酶活性和hTERT基因表达水平均较低。补充 0 .5和2 .5 μmol L亚硒酸钠三周后细胞生长状况良好、端粒酶活性和hTERT基因表达水平显著性增高 ,且呈一定的剂量 效应关系。细胞补充亚硒酸钠四周后端粒长度显著增长。说明营养浓度的亚硒酸钠可通过提高端粒酶活性和增长端粒长度来减缓L 0 2肝细胞衰老、延长细胞寿命。     

端粒及端粒酶的研究进展

端粒是染色体末端独特的蛋白质-DNA结构,在保护染色体的完整性和维持细胞的复制能力方面起着重要的作用.端粒酶则是由RNA和蛋白质亚基组成的、能够延长端粒的一种特殊反转录酶.端粒长度和端粒酶活性的变化与细胞衰老和癌变密切相关.端粒结合蛋白可能通过调节端粒酶的活性来调节端粒长度,进而控制细胞的衰老、永生化和癌变.研制端粒酶的专一性抑制剂在肿瘤治疗方面有着广阔的前景.     

端粒酶在细胞永生化和癌变中的作用

重点讨论了端粒酶在肿瘤细胞和永生化细胞中的作用和功能,以及它与细胞衰老和永生化的关系.多数真核细胞的端粒酶能将单一重复序列加到端粒DNA的3′末端.端粒酶主要由模板RNA和端粒酶蛋白催化亚基组成,后者以前者为模板起逆转录酶的作用.端粒酶活性存在于肿瘤细胞中,而在良性肿瘤、体细胞中未发现端粒酶.     

Loss of Human TGS1 Hypermethylase Promotes Increased Telomerase RNA and Telomere Elongation

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Polypeptide Components of Telomere Nucleoprotein Complex

Chromosome telomeres of humans and many model organisms contain a structure called a t-loop, which is maintained by TERF, TINF2, Pot1, and other proteins. Increase in TERF1 concentration prevents telomere elongation by telomerase. Decrease in TERF2 concentration (preventing t-loop formation) is accompanied by blockade of proliferation and appearance of other signs of cellular senescence in experiments. Natural regulation of TERF1 involves tankyrase, ATM protein kinase, and fluctuations of the protein level across a cell cycle. The telomere nucleoprotein complex also interacts with various polypeptide macromolecules (e.g., Sir2, PinX1, Rap1, Ku, Rad50/Mre11/Nbs1) responsible for heterochromatin formation, modulation of telomerase activity, DNA repair, and signaling to other cell compartments about telomere state. Study of structure and functioning of telomere nucleoprotein complex may contribute to elucidation of poorly understood mechanisms of aging and processes of tumor transformation of cells.     

Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination

Progressive telomere shortening from cell division (replicative aging) provides a barrier for human tumor progression. This program is not conserved in laboratory mice, which have longer telomeres and constitutive telomerase. Wild species that do/do not use replicative aging have been reported, but the evolution of different phenotypes and a conceptual framework for understanding their uses of telomeres is lacking. We examined telomeres/telomerase in cultured cells from > 60 mammalian species to place different uses of telomeres in a broad mammalian context. Phylogeny‐based statistical analysis reconstructed ancestral states. Our analysis suggested that the ancestral mammalian phenotype included short telomeres (< 20 kb, as we now see in humans) and repressed telomerase. We argue that the repressed telomerase was a response to a higher mutation load brought on by the evolution of homeothermy. With telomerase repressed, we then see the evolution of replicative aging. Telomere length inversely correlated with lifespan, while telomerase expression co‐evolved with body size. Multiple independent times smaller, shorter‐lived species changed to having longer telomeres and expressing telomerase. Trade‐offs involving reducing the energetic/cellular costs of specific oxidative protection mechanisms (needed to protect < 20 kb telomeres in the absence of telomerase) could explain this abandonment of replicative aging. These observations provide a conceptual framework for understanding different uses of telomeres in mammals, support a role for human‐like telomeres in allowing longer lifespans to evolve, demonstrate the need to include telomere length in the analysis of comparative studies of oxidative protection in the biology of aging, and identify which mammals can be used as appropriate model organisms for the study of the role of telomeres in human cancer and aging.     

端粒及端粒酶活性的调控机制

广义的端粒由帽子、双链的串联重复序列的DNA核心部分及亚端粒构成,其结合蛋白是一个复合体,由TRF1、TRF2、TIN2、Pot1、TPP1、RAP1 6个亚单位组成;另外,还结合组蛋白的特定成分H3K9三甲基聚合体和H4K20三甲基聚合体。端粒酶主要由hTerc、hTert、dyskerin构成。端粒的功能主要受端粒酶的活性调控;而端粒酶活性主要受hTert及hTerc的转录水平和转录后的剪切、hTert的翻译等因素的调控。端粒与端粒酶结构和功能的异常与细胞衰老及肿瘤的发生、发展关系密切。     

The N Terminus of the OB Domain of Telomere Protein TPP1 Is Critical for Telomerase Action

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Telomere Biology and Cellular Aging in Nonhuman Primate Cells

To determine how cellular aging is conserved among primates, we analyzed the replicative potential and telomere shortening in skin fibroblasts of anthropoids and prosimians. The average telomere length of the New World primates Ateles geoffroyi (spider monkey) and Saimiri sciureus (squirrel monkey) and the Old World primates Macaca mulatta (rhesus monkey), Pongo pygmaeus (orangutan), and Pan paniscus (pigmy chimpanzee) ranged from 4 to 16 kb. We found that telomere shortening limits the replicative capacity of anthropoid fibroblasts and that the expression of human telomerase produced telomere elongation and the extension of their in vitro life span. In contrast the prosimian Lemur catta (ring-tailed lemur) had both long and short telomeres and telomere shortening did not provide an absolute barrier to immortalization. Following a transient growth arrest a subset of cells showing a reduced number of chromosomes overgrew the cultures without activation of telomerase. Here we show that the presence of continuous TTAGGG repeats at telomeres and rigorous control of replicative aging by telomere shortening appear to be conserved among anthropoid primates but is less effective in prosimian lemurs.     

端粒酶RNA的反义cDNA对乳腺癌细胞端区长度的影响

应用反义核酸技术,将端粒酶ＲＮＡ的ｃＤＮＡ反向插入整合型腺病毒载体ｐＡｄＥ１ＣＭＶＩＴＲＥＸｎｅｏ,与ｐＢ－ＨＧ１１Ｘ共转染２９３细胞反获得反义重组病毒。将此反义重组病毒感染乳腺癌细胞ＭＣＦ－７后,使细胞端区长度缩短。     

人端粒酶逆转录酶核酶抑制端粒酶活性

为有效切割端粒酶逆转录酶mRNA以降低端粒酶活性 ,从而使肿瘤细胞生长变慢 ,凋亡增加。设计并合成了针对端粒酶逆转录酶mRNA的锤头状核酶基因 ,构建了该核酶基因的体外转录和真核表达质粒。检测了该核酶对端粒酶逆转录酶mRNA的体外切割效力。并将该核酶基因转染至肿瘤细胞中 ,检测其对肿瘤细胞端粒酶活性和生物学性状的影响。结果表明 ,该核酶在体外和细胞内均能有效切割端粒酶逆转录酶mRNA ;在细胞内能明显抑制端粒酶活性 ,使细胞生长变慢 ,倍增时间延长。因而 ,该核酶可望成为有效的端粒酶抑制剂 ,在抑制肿瘤生长中发挥作用     

Small-Molecule PAPD5 Inhibitors Restore Telomerase Activity in Patient Stem Cells

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高等植物端粒和端粒酶的研究进展

端粒是构成真核生物线状染色体末端重要的DNA-蛋白质复合结构,DNA由简单的串联重复序列组成。它的合成由一个特殊的具有反转录活性的核糖核蛋白-端粒酶完成。端粒对染色体、整个生物基因组,甚至对细胞的稳定都具有重要意义。本文就植物端粒、端粒酶、端粒结合蛋白,以及端粒变化、端粒酶在植物生长发育中的调节作一概述。     

反义hTR抑制人胰腺癌P3细胞系端粒酶活性和增殖的研究

设计针对端粒酶RNA模板序列并经硫代磷酸修饰的正义、反义和随机序列寡核苷酸,以正常人成纤维细胞作对照,观察其对人胰腺癌细胞端粒酶活性和细胞生长增殖的影响作用以及对正常细胞是否有毒副作用。结果表明：针对hTR模板区的反义寡核苷酸能降低胰腺癌P3细胞端粒酶活性,抑制细胞生长,促进细胞周期改变并诱导细胞发生凋亡,而且对正常细胞没有明显的毒副作用。因此我们认为利用反义技术封闭hTR基因很可能成为治疗肿瘤的安全、有效手段之一。 Abstract:This paper is to investigate PS-ODN's (antisense-PS-ODN of hTR,sense-PS-ODN of hTR and random sequence) effects on telomerase activity and proliferation of P3 pancreatic cancer cells,and to find a novel method for gene therapy of pancreatic cancer.The results indicate that the anti-hTR complementary to the template region of hTR is sufficient to inhibit P3 cell telomerase activity and cell proliferation in vitro,and as a result,they can lead to a profound induction of programmed cell death.Telomerase represents an interesting and promising anticancer drug target and antitelomerase technology may have potential significance in tumor therapy.     

端粒与端粒酶的研究——解读2009年诺贝尔生理学或医学奖

三位美国科学家(Elizabeth H. Blackburn, Carol W. Greider 和Jack W. Szostak)因发现“端粒和端粒酶是如何保护染色体的”获得了2009年的诺贝尔生理学或医学奖．端粒是染色体末端的特殊结构,对染色体有保护作用,而端粒酶能合成端粒,使得端粒的长度和结构得以稳定．研究发现,端粒长度和端粒酶活性与细胞的寿命以及很多疾病发生直接相关．随着研究的不断深入,实现合理控制端粒的长度和端粒酶活性成为可能,这将有助于攻克医学领域“癌症、特定遗传病和衰老”三个重要领域的难题,有望研究开发出潜在的新疗法．     

Stabilization of Reversed Replication Forks by Telomerase Drives Telomere Catastrophe

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