端粒及端粒酶的研究进展

端粒是真核细胞染色体末端的特有结构,是由端粒结合蛋白和一段重复序列的端粒DNA组成的一个高度精密的复合体,在维持染色体末端稳定性,避免染色体被核酸酶降解等方面起着重要的作用。端粒的长度、结构及组织形式受多种端粒结合因子的调控。由于端粒的重要性,在哺乳动物细胞里,端粒的长度或端粒结构变化与癌症发生及细胞衰老有密切的关系。由于末端复制问题的存在,随着细胞分裂次数的增加,端粒不断缩短,细胞不可避免的走向衰老或凋亡。由于在细胞分裂过程中端粒长度的不断缩短与细胞分裂代数增加具有相关性,即端粒长度反应了细胞的分裂次数,因此有人将端粒形象的比喻为生物时钟。在90%的癌细胞中,端粒酶被重新激活,以此来维持端粒的长度,使细胞走向永生化。简要综述了端粒、端粒酶及端粒酶结合蛋白的最新研究进展。     

端粒与端粒酶研究进展

细胞分裂中染色体因其末端（端粒）的DNA不能完全复制而短缩,使细胞逐渐失去增殖能力而衰老．端粒酶可延长染色体末端DNA,端粒酶的活化使细胞无限增殖．85％左右的恶性肿瘤端粒酶表达阳性,生殖细胞和无限繁殖的细胞系中端粒酶表达也呈阳性．文章综述了端粒的构成和功能、端粒酶在端粒合成中的作用,介绍了端粒酶活性的测定方法、细胞恶变与端粒酶激活的关系,并论及通过抑制端粒酶活性来治疗癌症的可能性．     

端粒和端粒酶在癌症中的研究进展及意义

端粒是位于染色体末端的DNA串联重复序列,对基因组稳定性和完整性起保护作用。端粒的长度与细胞周期密切相关。其长度变化机制分为依赖端粒酶活性和端粒重组两类,氧化应激和铅(Pb)与端粒酶的功能蛋白相结合抑制其活性,致使端粒缩短,硒(Se)与二者具有拮抗作用,延缓衰老。相关数据表明85%肿瘤细胞与端粒酶活性成正相关,以端粒酶活性作为肿瘤治疗靶标称为当代热点之一。主要对肺癌、乳腺癌等恶性肿瘤与端粒的相关性进行了综述,以期为端粒和端粒酶在癌症治疗研究提供参考依据。     

端粒结合蛋白与端粒长度调控

真核细胞端粒DNA序列的丢失与细胞的衰老及凋亡有关.端粒酶的激活可维持端粒长度并使细胞获得无限增殖的能力.端粒结合蛋白则可能通过调节端粒酶或其他相关因子的行为参与对端粒长度的调控.近年有关端粒结合蛋白的研究取得了突破性进展并在此基础上建立了端粒长度调控模型.     

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.     

骨肿瘤端粒长度变化与相关蛋白表达的关系

目的:研究骨肿瘤端粒长度变化与端粒结合蛋白即端粒重复结合因子1(TRF1)和端粒保护因子(POT1),端粒酶催化亚单位(hTERT),肿瘤相关基因P53、c-myc表达的关系,以了解骨肿瘤的分子特征。方法:采用免疫组织化学、端粒定量荧光原位杂交(Telo-FISH)和原位杂交检测了20例骨肉瘤、25例软骨肉瘤、14例骨的纤维结构不良中端粒长度、TRF1、POT1、hTERT、P53、c-myc的表达情况,并进行统计分析。结果:20例骨肉瘤平均长度为0.31,25例软骨肉瘤为0.41,14例骨的纤维结构不良为0.52。统计显示三者间端粒长度有显著差异(P<0.05)。骨肉瘤和软骨肉瘤TRF1、POT1阳性率均显著低于骨纤维结构不良(P<0.05)。而骨肉瘤和软骨肉瘤hTERT基因表达显著高于骨纤维结构不良(P<0.05)。骨肉瘤、软骨肉瘤P53、c-myc阳性率高于骨纤维结构不良(P<0.05)。统计分析骨肿瘤端粒长度变化与端粒结合蛋白TRF1、POT1的表达呈负相关性,与端粒酶hTERT基因表达、与P53蛋白核聚积,以及c-myc癌基因表达呈正相关性。结论:骨肿瘤端粒长度与恶性表型有关、端粒短缩与肿瘤基因突变相关。     

端粒酶活性调节的分子机制

人端粒酶由RNA亚基、hTERT催化亚基和hTEP1调节蛋白等组成。端粒酶对端粒结构的稳定起着重要的作用,而端粒结构和端粒结合蛋白也影响着端粒酶活性。某些化疗药物通过破坏端粒结构下调端粒酶活性。端粒酶的激活需要hTERT基因的从头转录和各个蛋白亚基正确装配为端粒酶全酶。端粒酶活性调节的分子机制包括：（1）TERT基因的表达和转录是决定端粒酶活性的重要环节,受多种因素调控;（2）蛋白激酶Cα和蛋白激酶B磷酸化端粒酶蛋白而激活端粒酶,蛋白磷酸酯酶2A（PP2A）可逆转这一过程,下调端粒酶活性;（3）多种癌基因和抑癌基因及其编码的蛋白质也直接或间接与端粒蛋白、端粒酶蛋白反应,参与端粒酶活性的调控。     

甲磺酸甲酯对酵母DNA损伤及端粒酶活性的影响

观察甲磺酸甲酯 (MMS)对酿酒酵母S2 88C细胞染色体DNA的损伤及端粒酶活性的调节。结果表明 ,甲磺酸甲酯引起酵母细胞DNA损伤 ,随着MMS浓度的增加及作用时间的延长 ,DNA损伤程度加重 ,同时明显提高酵母细胞端粒酶活性。当用 0 .4mmol/LMMS作用 72h后 ,端粒酶活性最高 (是对照组的1.4 7倍 ) ,在作用 96h及 12 0h后端粒酶活性逐渐下降 ,但均高于对照组。甲磺酸甲酯对酿酒酵母S2 88C细胞端粒酶活性的上调作用可能与其DNA损伤有关 ,断裂DNA的损伤后修复可能是端粒酶介导的。     

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。     

端粒生物学与肿瘤治疗

端粒的生物学功能主要是保护染色体末端,避免核酸酶对染色体末端的降解,防止染色体之间发生融合和重排。大多数人类肿瘤细胞通常通过端粒酶活性的重新激活来延长端粒,从而稳定染色体端粒DNA的长度。端粒酶是由端粒酶逆转录酶和端粒酶RNA模板组成的具有特殊逆转录活性的核糖核蛋白复合物。抑制端粒酶阳性细胞中的端粒酶活性会导致细胞凋亡或衰老。目前有多种以端粒和端粒酶为靶点来进行肿瘤治疗的策略。     

Deletion of the major peroxiredoxin Tsa1 alters telomere length homeostasis

Reactive oxygen species (ROS) are proposed to play a major role in telomere length alterations during aging. The mechanisms by which ROS disrupt telomeres remain unclear. In Saccharomyces cerevisiae, telomere DNA consists of TG_(1–3) repeats, which are maintained primarily by telomerase. Telomere length maintenance can be modulated by the expression level of telomerase subunits and telomerase activity. Additionally, telomerase‐mediated telomere repeat addition is negatively modulated by the levels of telomere‐bound Rap1‐Rif1‐Rif2 protein complex. Using a yeast strain defective in the major peroxiredoxin Tsa1 that is involved in ROS neutralization, we have investigated the effect of defective ROS detoxification on telomere DNA, telomerase, telomere‐binding proteins, and telomere length. Surprisingly, the tsa1 mutant does not show significant increase in steady‐state levels of oxidative DNA lesions at telomeres. The tsa1 mutant displays abnormal telomere lengthening, and reduction in oxidative exposure alleviates this phenotype. The telomere lengthening in the tsa1 cells was abolished by disruption of Est2, subtelomeric DNA, Rap1 C‐terminus, or Rif2, but not by Rif1 deletion. Although telomerase expression and activity are not altered, telomere‐bound Est2 is increased, while telomere‐bound Rap1 is reduced in the tsa1 mutant. We propose that defective ROS scavenging can interfere with pathways that are critical in controlling telomere length homeostasis.     

Telomerase RNA template mutations reveal sequence-specific requirements for the activation and repression of telomerase action at telomeres

Telomeric DNA is maintained within a length range characteristic of an organism or cell type. Significant deviations outside this range are associated with altered telomere function. The yeast telomere-binding protein Rap1p negatively regulates telomere length. Telomere elongation is responsive to both the number of Rap1p molecules bound to a telomere and the Rap1p-centered DNA-protein complex at the extreme telomeric end. Previously, we showed that a specific trinucleotide substitution in the Saccharomyces cerevisiae telomerase gene (TLC1) RNA template abolished the enzymatic activity of telomerase, causing the same cell senescence and telomere shortening phenotypes as a complete tlc1 deletion. Here we analyze effects of six single- and double-base changes within these same three positions. All six mutant telomerases had in vitro enzymatic activity levels similar to the wild-type levels. The base changes predicted from the mutations all disrupted Rap1p binding in vitro to the corresponding duplex DNAs. However, they caused two classes of effects on telomere homeostasis: (i) rapid, RAD52-independent telomere lengthening and poor length regulation, whose severity correlated with the decrease in in vitro Rap1p binding affinity (this is consistent with loss of negative regulation of telomerase action at these telomeres; and (ii) telomere shortening that, depending on the template mutation, either established a new short telomere set length with normal cell growth or was progressive and led to cellular senescence. Hence, disrupting Rap1p binding at the telomeric terminus is not sufficient to deregulate telomere elongation. This provides further evidence that both positive and negative cis-acting regulators of telomerase act at telomeres.     

Deletion of Ogg1 DNA glycosylase results in telomere base damage and length alteration in yeast

Telomeres consist of short guanine‐rich repeats. Guanine can be oxidized to 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) and 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine (FapyG). 8‐oxoguanine DNA glycosylase (Ogg1) repairs these oxidative guanine lesions through the base excision repair (BER) pathway. Here we show that in Saccharomyces cerevisiae ablation of Ogg1p leads to an increase in oxidized guanine level in telomeric DNA. The ogg1 deletion (ogg1Δ) strain shows telomere lengthening that is dependent on telomerase and/or Rad52p‐mediated homologous recombination. 8‐oxoG in telomeric repeats attenuates the binding of the telomere binding protein, Rap1p, to telomeric DNA in vitro. Moreover, the amount of telomere‐bound Rap1p and Rif2p is reduced in ogg1Δ strain. These results suggest that oxidized guanines may perturb telomere length equilibrium by attenuating telomere protein complex to function in telomeres, which in turn impedes their regulation of pathways engaged in telomere length maintenance. We propose that Ogg1p is critical in maintaining telomere length homoeostasis through telomere guanine damage repair, and that interfering with telomere length homoeostasis may be one of the mechanism(s) by which oxidative DNA damage inflicts the genome.     

Yeast Est2p affects telomere length by influencing association of Rap1p with telomeric chromatin

In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.     

S. cerevisiae Tel1p and Mre11p are required for normal levels of Est1p and Est2p telomere association

In diverse organisms, the Mre11 complex and phosphoinositide 3-kinase-related kinases (PIKKs), such as Tel1p and Mec1p from S. cerevisiae, are key mediators of DNA repair and DNA damage checkpoints that also function at telomeres. Here, we use chromatin immunoprecipitation (ChIP) to determine if Mre11p, Tel1p, or Mec1p affects telomere maintenance by promoting recruitment of telomerase subunits to S. cerevisiae telomeres. We find that recruitment of Est2p, the catalytic subunit of telomerase, and Est1p, a telomerase accessory protein, was severely reduced in mre11Delta and tel1Delta cells. In contrast, the levels of Est2p and Est1p binding in late S/G2 phase, the period in the cell cycle when yeast telomerase lengthens telomeres, were indistinguishable in wild-type (WT) and mec1Delta cells. These data argue that Mre11p and Tel1p affect telomere length by promoting telomerase recruitment to telomeres, whereas Mec1p has only a minor role in telomerase recruitment in a TEL1 cell.     

Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules

We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable. However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA. The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes. In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear. These results define a novel subcellular localization behavior for hRif1 during the cell cycle.     

The Rap1p-telomere complex does not determine the replicative capacity of telomerase-deficient yeast

Telomeres are nucleoprotein structures that cap the ends of chromosomes and thereby protect their stability and integrity. In the presence of telomerase, the enzyme that synthesizes telomeric repeats, telomere length is controlled primarily by Rap1p, the budding yeast telomeric DNA binding protein which, through its C-terminal domain, nucleates a protein complex that limits telomere lengthening. In the absence of telomerase, telomeres shorten with every cell division, and eventually, cells enter replicative senescence. We have set out to identify the telomeric property that determines the replicative capacity of telomerase-deficient budding yeast. We show that in cells deficient for both telomerase and homologous recombination, replicative capacity is dependent on telomere length but not on the binding of Rap1p to the telomeric repeats. Strikingly, inhibition of Rap1p binding or truncation of the C-terminal tail of Rap1p in Kluyveromyces lactis and deletion of the Rap1p-recruited complex in Saccharomyces cerevisiae lead to a dramatic increase in replicative capacity. The study of the role of telomere binding proteins and telomere length on replicative capacity in yeast may have significant implications for our understanding of cellular senescence in higher organisms.     

Counting of Rif1p and Rif2p on Saccharomyces cerevisiae telomeres regulates telomere length

Telomere length is negatively regulated by proteins of the telomeric DNA-protein complex. Rap1p in Saccharomyces cerevisiae binds the telomeric TG(1-3) repeat DNA, and the Rap1p C terminus interacts with Rif1p and Rif2p. We investigated how these three proteins negatively regulate telomere length. We show that direct tethering of each Rif protein to a telomere shortens that telomere proportionally to the number of tethered molecules, similar to previously reported counting of Rap1p. Surprisingly, Rif proteins could also regulate telomere length even when the Rap1p C terminus was absent, and tethered Rap1p counting was completely dependent on the Rif proteins. Thus, Rap1p counting is in fact Rif protein counting. In genetic settings that cause telomeres to be abnormally long, tethering even a single Rif2p molecule was sufficient for maximal effectiveness in preventing the telomere overelongation. We show that a heterologous protein oligomerization domain, the mammalian PDZ domain, when fused to Rap1p can confer telomere length control. We propose that a nucleation and spreading mechanism is involved in forming the higher-order telomere structure that regulates telomere length.