HeLa细胞端粒酶最适反应条件及活性重建

端粒是真核细胞染色体末端的重复ＤＮＡ序列 ,其生物学功能是防止染色体ＤＮＡ降解、末端融合、非正常重组和染色体的缺失[1] .由于存在“末端复制问题” ,随着老化人体细胞端粒重复序列长度不断缩短 ,但在生殖细胞中由于端粒酶的存在 ,端粒序列并不缩短 .端粒酶是由蛋白质和ＲＮＡ构成的核蛋白 ,是依赖ＲＮＡ的ＤＮＡ聚合酶 ,在ＤＮＡ3’端合成端粒重复序列[2 ] .研究表明 ,在 85 %～ 95 %的人肿瘤细胞中可以检测到端粒酶的活性[3 ,4 ] ,而在正常体细胞中除生殖细胞和造血干细胞等极少数细胞中存在端粒酶活性外 ,均检测不到端粒酶活性 ,这…     

肿瘤治疗研究的新热点──端粒酶活性的抑制

端粒是染色体末端由ＤＮＡ和蛋白质组成的特殊结构,其ＤＮＡ部分由富含Ｇ的简单重复序列构成,在真核细胞其重复单位为５＇ＴＴＡＧＧＧ３＇。端粒对于维持染色体的稳定性有重要作用。由于ＤＮＡ聚合酶只能沿５＇→３＇方向合成ＤＮＡ,随从链的５＇端ＲＮＡ引物去除后无法填补。因此,端粒长度将随着复制次数增多而逐渐缩短。端粒酶的存在解决了这一"末端复制问题"。端粒酶是一种特殊的逆转录酶,它以自身的ＲＮＡ为模板合成端粒重复序列。有许多研究表明,端粒酶在肿瘤发生过程中起重要作用。因此．端粒酶活性的抑制成为当前肿瘤治疗研究…     

端粒酶与血液系统肿瘤

端粒 (ｔｅｌｏｍｅｒｅ)是真核细胞染色体末端的ＤＮＡ 蛋白质复合物。端粒酶 (ｔｅｌｏｍｅｒａｓｅ)是由ＲＮＡ和蛋白质组成的特殊核糖核蛋白复合体 ,具有逆转录酶活性 ,能以自身ＲＮＡ为模板 ( 5′ ＣＵＡＡＣＣＣＵＡＡＣ 3′)合成端粒。在正常人体细胞中端粒酶活性表达受抑 ,而在肿瘤形成时可被激活。因此在某些永生细胞和肿瘤细胞中随着ＤＮＡ的复制 ,其端粒不会缩短。1 .端粒酶在血液系统肿瘤表达端粒酶在人体细胞大多为阴性 ,但在胚系细胞、精子细胞、小肠陷窝细胞、毛囊细胞、上皮细胞、活化淋巴细胞、子宫内膜细胞和大多数肿瘤细…     

多形胶质母细胞瘤细胞系端粒酶活性的测定

端粒缩短见于星形细胞瘤发展过程中,但其长度在胶质母细胞瘤／细胞系相对稳定,提示胶质瘤细胞内存在端粒修复机制的可能性．为证实此点,利用端粒重复片段扩增技术（ＴＲＡＰ）,对８株人／大鼠多形胶质母细胞系的蛋白提取液中端粒酶活性加以测定．结果显示：８例胶质瘤样本的反应液均可见端粒ＰＣＲ扩增片段;用无ＤＮａｓｅ的ＲＮａｓｅ事先处理蛋白提取液,可明显降低或消除ＰＣＲ产物的出现,说明ＴＲＡＰ反应中的ＰＣＲ扩增是在端粒酶的介导下进行而非ＤＮＡ污染或其它端粒修复因子所致．从而不但建立起检测人癌细胞内端粒酶活性的可靠方法,也为针对端粒酶的胶质母细胞瘤生物／药物治疗提供了实验依据．     

端粒酶抑制剂在肿瘤治疗中的进展

端粒是真核细胞染色体末端含有ＴＴＡＧＧＧ重复结构的复合体 ,有防止染色体降解丢失 ,端端融合和重组建作用 ,端粒酶是一种逆转录酶 ,以自身ＲＮＡ为模板 ,逆转录维持染色体的稳定。目前肿瘤组织端粒酶检出率约 85 - 90 %,而正常组织或良性肿瘤端粒酶检出率〈5 %,说明端粒酶与恶性肿瘤密切相关。端粒酶抑制剂作为新的抗肿瘤策略正成为肿瘤研究的热点 ,现就端粒酶抑制剂在肿瘤治疗中的研究状况做一简单概述。1、阻断端粒酶ＲＮＡ的模板作用对端粒酶活性的抑制1. 1反义核苷酸、反义肽苷及硫代反义核苷酸对端粒酶活性的抑制端粒酶ＲＮＡ序列中…     

肾癌组织中端粒酶活性的测定与分析

端粒酶 (Ｔｅｌｏｍｅｒａｓｅ)是一种核糖核蛋白 ,依赖酶分子中的ＲＮＡ模板 ,通过逆转录合成染色体末端的端粒。近年来研究证实 ,端粒酶与细胞衰老及细胞分裂过程密切相关 ,尤其在恶性肿瘤组织中活性异常高。因此通过测定肿瘤组织、癌旁细胞及脱落细胞等端粒酶活性 ,可用于癌症的临床诊断、疗效观察、愈后及病理机制的研究。国外已经有对肾癌组织的端粒酶活性进行测定但都为定性 ,国内未见报道。本实验采用ＴＲＡＰ -ＥＬＩＳＡ技术对肾癌组织中端粒酶活性进行定量检测 ,现将结果报告如下。1　材料与方法1 1　样本来源 :组织标本均来自白…     

端粒酶活性的定量分析方法

端粒酶是目前已知最为广谱的肿瘤分子标记物,并可能成为今后肿瘤诊断和预后判断的新指标以及肿瘤治疗的新靶点。对端粒酶活性的测定,在ＴＲＡＰ法的基础上发展建立了各种定量和半定量方法。     

以端粒酶为靶点的药物设计和基因治疗策略

端粒亦称端区,位于真核细胞染色体末端,随细胞分裂而脱失,缩短至一定程度时体细胞死亡,癌细胞因具有合成端粒的端粒酶而获永生。端粒酶亦称端聚酶,为一含ＲＮＡ的核糖核蛋白,在９０％的癌细胞中过表达,成为肿瘤治疗的新靶点。以端粒酶为靶点的药物设计策略包括端粒酶ＲＮＡ和编码蛋白质的基因以及编码端粒结合蛋白基因的操作。另外,染色体转移、诱导分化、细胞周期调节及常规药物筛选亦为重要的策略。其中针对端粒酶ＲＮＡ模     

甘蔗和烟草幼叶原生质体的核酸含量与核酸酶活性及其影响因素

与幼叶组织相比,酶法新鲜分离的甘蔗和烟草幼叶原生质体内的ＲＮＡ、ＤＮＡ及总核酸含量均降低。其原因可能是刚游离的原生质体内酸性和碱性ＲＮＡ酶与ＤＮＡ酶等活性提高所致。甘蔗叶原生质体内的核酸降低量和ＲＮＡ酶与ＤＮＡ酶活性的增加程度均高于烟草。随用作渗透压稳定剂的甘露醇浓度增加,甘蔗和烟草叶原生质体的ＲＮＡ酶和ＤＮＡ酶活性均相应提高。其中以甘蔗叶原生质体的核酸酶活性增加水平较明显。在细胞壁降解产物的作用下,除了甘蔗原生质体内的ＲＮＡ酶活性略被促进外,其ＤＮＡ酶和烟草叶原生质体内的核酸酶均不受影响     

端粒酶缺乏影响高增殖器官的发育

近年来对体外单个细胞和单细胞生物的研究表明，端粒酶缺乏可致细胞衰老、死亡，而端粒酶活性增强是肿瘤细胞增殖过度的原因，但端粒酶对哺乳动物整体的细胞生长和生存的影响未进行研究。有鉴于此，汉城国立大学的Ｌｅｅ等对一种缺乏端粒酶ＲＮＡ的小鼠进行连续传代，研究...     

Telomerase inhibitor PinX1 provides a link between TRF1 and telomerase to prevent telomere elongation

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.     

Control of human telomere length by TRF1 and TRF2

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.     

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。     

Human protection of telomeres 1 (POT1) is a negative regulator of telomerase activity in vitro

The telomeric single-strand DNA binding protein protection of telomeres 1 (POT1) protects telomeres from rapid degradation in Schizosaccharomyces pombe and has been implicated in positive and negative telomere length regulation in humans. Human POT1 appears to interact with telomeres both through direct binding to the 3' overhanging G-strand DNA and through interaction with the TRF1 duplex telomere DNA binding complex. The influence of POT1 on telomerase activity has not been studied at the molecular level. We show here that POT1 negatively effects telomerase activity in vitro. We find that the DNA binding activity of POT1 is required for telomerase inhibition. Furthermore, POT1 is incapable of inhibiting telomeric repeat addition to substrate primers that are defective for POT1 binding, suggesting that in vivo, POT1 likely affects substrate access to telomerase.     

Cell-Cycle-Dependent Regulation of Telomere Binding Proteins: Roles of Polo-Like Kinase

The telomere is a functional complex at chromosomal termini consisting of repetitiveDNA and associated proteins, and protects the ends against degradation and fusion.Telomeric repeat binding factors TRF1 and TRF2 bind directly to double-strandedtelomeric DNA. Although structurally related, TRF1 and TRF2 contribute to telomeremaintenance in distinct ways: TRF1 negatively regulates telomerase-dependenttelomere lengthening, whereas TRF2 plays an important role in protecting chromosomalends. It is not known how the proteinaceous complex manages DNA metabolism suchas DNA replication, which requires the recruitment of numerous trans-acting factors.We have found that Xenopus TRF1 (xTRF1) specifically associates with mitoticchromatin and dissociates from interphase replicating chromatin. In contrast, XenopusTRF2 (xTRF2) binds to telomeric DNA throughout the cell cycle. Interestingly,telomerase activity is associated with the interphase chromatin, but not with the mitoticchromatin. These results support a model in which telomeres form a semi-openconfiguration that allows access of telomerase and replication machineries, yet protectsthe chromosomal ends in S phase. Interestingly, M phase specific telomere binding ofxTRF1 requires Polo-like kinase, a key regulator of mitosis. We discuss the relevance ofour studies and recent findings of other groups to indicate the possible role of Polo-likekinase in telomere regulation.     

Changes in Telomerase Activity and Telomere Length during Human T Lymphocyte Senescence

It has been proposed that telomeres shorten with every cell cycle because the normal mechanism of DNA replication cannot replicate the end sequences of the lagging DNA strand. Telomerase, a ribonucleoprotein enzyme that synthesizes telomeric DNA repeats at the DNA 3′ ends of eukaryotic chromosomes, can compensate for such shortening, by extending the template of the lagging strand. Telomerase activity has been identified in human germline cells and in neoplastic immortal somatic cells, but not in most normal somatic cells, which senesce after a certain number of cell divisions. We and others have found that telomerase activity is present in normal human lymphocytes and is upregulated when the cells are activated. But, unlike the immortal cell lines, presence of telomerase activity is not sufficient to make T cells immortal and telomeres from these cells shorten continuously duringin vitroculture. After senescence, telomerase activity, as detected by the TRAP technique, was downregulated. A cytotoxic T lymphocyte (CTL) cell line that was established in the laboratory has very short terminal restriction fragments (TRFs). Telomerase activity in this cell line is induced during activation and this activity is tightly correlated with cell proliferation. The level of telomerase activity in activated peripheral blood T cells, the CTL cell line, and two leukemia cell lines does not correlate with the average TRF length, suggesting that other factors besides telomerase activity are involved in the regulation of telomere length.     

Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2

We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2. To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced. We demonstrate that their targeting leads to telomere shortening. This indicates that these proteins act in cis to repress telomere elongation. Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting and telomerase inhibition have additive effects. The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase. In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.     

Functional interaction between poly(ADP-Ribose) polymerase 2 (PARP-2) and TRF2: PARP activity negatively regulates TRF2

The DNA damage-dependent poly(ADP-ribose) polymerase-2 (PARP-2) is, together with PARP-1, an active player of the base excision repair process, thus defining its key role in genome surveillance and protection. Telomeres are specialized DNA-protein structures that protect chromosome ends from being recognized and processed as DNA strand breaks. In mammals, telomere protection depends on the T(2)AG(3) repeat binding protein TRF2, which has been shown to remodel telomeres into large duplex loops (t-loops). In this work we show that PARP-2 physically binds to TRF2 with high affinity. The association of both proteins requires the N-terminal domain of PARP-2 and the myb domain of TRF2. Both partners colocalize at promyelocytic leukemia bodies in immortalized telomerase-negative cells. In addition, our data show that PARP activity regulates the DNA binding activity of TRF2 via both a covalent heteromodification of the dimerization domain of TRF2 and a noncovalent binding of poly(ADP-ribose) to the myb domain of TRF2. PARP-2(-/-) primary cells show normal telomere length as well as normal telomerase activity compared to wild-type cells but display a spontaneously increased frequency of chromosome and chromatid breaks and of ends lacking detectable T(2)AG(3) repeats. Altogether, these results suggest a functional role of PARP-2 activity in the maintenance of telomere integrity.     

Alterations of DNA and chromatin structures at telomeres and genetic instability in mouse cells defective in DNA polymerase alpha

Telomere length is controlled by a homeostatic mechanism that involves telomerase, telomere-associated proteins, and conventional replication machinery. Specifically, the coordinated actions of the lagging strand synthesis and telomerase have been argued. Although DNA polymerase alpha, an enzyme important for the lagging strand synthesis, has been indicated to function in telomere metabolism in yeasts and ciliates, it has not been characterized in higher eukaryotes. Here, we investigated the impact of compromised polymerase alpha activity on telomeres, using tsFT20 mouse mutant cells harboring a temperature-sensitive polymerase alpha mutant allele. When polymerase alpha was temperature-inducibly inactivated, we observed sequential events that included an initial extension of the G-tail followed by a marked increase in the overall telomere length occurring in telomerase-independent and -dependent manners, respectively. These alterations of telomeric DNA were accompanied by alterations of telomeric chromatin structures as revealed by quantitative chromatin immunoprecipitation and immunofluorescence analyses of TRF1 and POT1. Unexpectedly, polymerase alpha inhibition resulted in a significantly high incidence of Robertsonian chromosome fusions without noticeable increases in other types of chromosomal aberrations. These results indicate that although DNA polymerase alpha is essential for genome-wide DNA replication, hypomorphic activity leads to a rather specific spectrum of chromosomal abnormality.     

Role of the TRF2 Telomeric Protein in Cancer and Aging

Telomeres are the special heterochromatin that forms the ends of chromosomes, consisting of TTAGGG repeats and associated proteins. Telomeres protect the ends from degradation and recombination, and are essential for chromosomal stability. Both a minimal length of telomere repeats and the telomere-binding proteins are required for telomere protection. Telomerase is a DNA polymerase that specifically elongates telomeres, in this way regulating telomere length and function. A minimal telomere length is required to maintain tissue homeostasis. On one hand, critically short telomeres trigger loss of cell viability and premature death in mice deficient for telomerase activity. Furthermore, altered functioning of telomerase and telomere-interacting proteins is present in some human premature ageing syndromes and cancer. A new mouse model with critically short telomeres has been generated by over-expressing the TRF2 telomere-binding protein, K5-TRF2 mice. These mice show short telomeres in the presence of telomerase activity, leading to premature aging and increased cancer. Short telomeres in TRF2 mice can be rescued in the absence of the XPF nuclease, indicating that this enzyme rapidly degrades telomeres in the presence of increased TRF2 expression. K5-TRF2 mice represent a new tool to understand the consequences of critical telomere shortening a telomerase-proficient genetic background, more closely resembling human cancer and aging pathologies.