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研究院开展端粒长度及端粒酶活性检测项目
点击数:   更新时间:2012-06-18 11:12 作者:超级管理员

研究院开展端粒长度及端粒酶活性检测项目

  端粒-引起人类衰老和罹患癌症等严重疾病的秘密

  项目简介

  在外貌上,脸部的皱纹会泄露我们的年龄,而在身体内,染色体尾端结构也会泄露细胞的年龄,敲响生命的警钟,这些尾端结构被称之为端粒,对承载基因的染色体起着保护作用,会随着我们年龄的增加而老化,因此它们可能用来预测我们的寿命。多项研究表明,那些细胞中有较短端粒的人更容易患像癌症、心脏病和老年痴呆症(阿尔茨海默症)之类的疾病,甚至会较早死亡,小鼠实验则表明,延长端粒能够增加寿命的长度。

  河北燕达医学研究院成功的建立了一套检测端粒长度及端粒酶活性的技术平台。该技术平台将端粒长度检测的“金标准”—Southern blot法与生物信息计算技术相结合,对端粒的长度及端粒酶活性进行分析,预测你的生物学年龄,评估你罹患衰老相关疾病(如心脑血管病、癌症及老年痴呆症等)的风险,并判断你的生活方式,指导你改善不良的生活习惯,延缓端粒的缩短,有助于你活得更健康。

  项目依据

  1、2009 年度诺贝尔生理学或医学奖

  2009年10月5日,瑞典皇家科学院诺贝尔奖委员会宣布将2009 年度诺贝尔生理学或医学奖授予3 位美国科学家伊丽莎白·布莱克本(Elizabeth H.Blackburn)、卡罗尔·格雷德(Carol W. Greider)和杰克·绍斯塔克(Jack W. Szostak),以表彰他们“发现端粒和端粒酶是如何保护染色体的”,被世界医学界誉为找到了“引起人类衰老和罹患癌症等严重疾病的秘密”。

  2009年诺贝尔生理学或医学奖获得者

  卡罗尔▪格雷德(左),伊丽莎白▪布莱克本(中),杰克▪绍斯塔克(右)

  Winners of the 2009 Nobel Prize in Physiology or Medicine

  Carol W.Greider (1eft),Elizabeth H.Blackburn (middle),Jack W.Szostak(right)

  2、端粒—人类“寿命时钟”
 

  端粒是位于人染色体末端的特殊结构,与细胞的衰老和死亡密切相关。当细胞分裂一次,每条染色体的端粒就会逐次变短一些,随着体细胞的不断增殖,端粒会逐渐缩短,当端粒缩短至一定程度,细胞停止分裂,进入静止状态,故有人称端粒为正常细胞的“分裂钟”(mistosis clock),因此,端粒可以揭示人的生物学年龄,以及与年龄有关疾病的发生机制,所以,端粒又被称为“寿命时钟”

  3、端粒酶—人类“寿命天使”

  端粒酶的主要生物学功能是通过其逆转录酶活性复制和延长端粒DNA来稳定染色体端粒DNA的长度,使得端粒的长度和结构得以稳定。细胞随着端粒的变短而衰老,而当端粒酶的活性足以维护端粒的长度时,细胞将会延迟衰老。在正常细胞中,端粒上的端粒酶是个“寿命天使”,如果我们能控制正常细胞的端粒酶,使之具备相当的活性,正常细胞的寿命就可能延长,起到抗衰老作用。

 

  端粒的丢失与保护(The erosion and maintenance of telomere)

  左侧:在没有端粒酶的保护下,随着细胞的分裂,端粒的丢失导致染色体损伤;

  右侧:端粒酶保护端粒,使整个染色体在每一轮细胞分裂中都得到完整的复制

  项目优势

  1、 结果准确可靠

  运用国际承认的“金标准”检测方法,检测结果准确可靠

  2 、无创伤性

  只需抽取2ml外周血(无需空腹)即可进行检测

  3 、通量高、易于自动化操作

  适合大样本量集中检测,自动化程度高,降低人为干预

  4 、严格的质量控制体系

  实验室建立了一套严格的质量控制体系,严格的试剂盒组装加上自动化的实验操作,最大程度上降低了实验的人为误差。

  5、完善的实验室管理、一流的实验室设备

 

  长度及端粒酶活性分析服务简介

  1、服务流程

  2、标本采集要求

  采集外周血3ml于标准真空采血管内(EDTA抗凝),充分混匀,及时送至检测部门,进行下步处理。

  3、服务周期

  仅需10个工作日

  项目背景介绍

  一、什么是端粒和端粒酶

  端粒(Telomere )是染色体末端的特殊结构,能保护染色体末端免于融合和退化,在染色体定位、复制、保护和控制细胞生长及寿命方面具有重要作用,并与细胞凋亡、细胞转化和永生化密切相关。染色体末端的帽子结构—端粒,常被比喻成鞋带头上包裹鞋带、防止鞋带散开的塑料片,它就像这个塑料片保护鞋带一样保护着染色体上的遗传基因不在细胞分裂中丢失。

 

  端粒在染色体末端形成帽子结构

  (The telomeres forms caps at the ends of chromosomes)

  端粒酶(Telomerase)是一种自身携带模板的逆转录酶,由RNA和蛋白质组成,RNA组分中含有一段短的模板序列与端粒DNA的重复序列互补,而其蛋白质组分具有逆转录酶活性,以RNA为模板催化端粒DNA的合成,将其加到端粒的3′端,以维持端粒长度及功能。端粒酶是细胞永生的关键调控因子,它能够减慢端粒的缩短。
 

  端粒酶合成端粒示意图

  ( Telomerase operates at the end of the chromosome)

  二、端粒缩短究竟会对人体产生什么影响呢?

  美国心脏协会主办的专业期刊 《动脉硬化、血栓形成和血管生理学学报》 2008年 7月刊报道了一项新近的研究结果,在780例患有稳定型心脏病的患者中,免疫细胞端粒最短的患者 4.4年后死亡率和罹患心力衰竭的危险性是端粒最长患者的两倍,最高风险组患者的端粒长度只有最低风险组患者端粒长度的一半

  三、生活方式影响端粒长短

  高剂量的应激激素、感染、高胰岛素、高血糖,以及吸烟、脂肪含量高的饮食、肥胖、久坐等生活习惯,都与端粒缩短和端粒末端转移酶含量减少有关系。

  如果你吸烟,血液中就会进行氧化反应,与染色体的其它组成部分相比,端粒对氧化应激更敏感,所以它们会不断缩短。如果你承受的压力很大,你的肾上腺就会释放出糖皮质激素,这种激素会杀死免疫系统里的T细胞,于是会有更多的细胞进行分裂以补充死亡的T细胞。但是需要注意的是,细胞增殖越快,端粒缩短的速度也就越快。”

  四、用端粒预测寿命更准确

  研究者认为,与传统的方法相比,通过端粒长度来预测人的寿命也许更加准确。利福尼亚大学的艾培尔博士表示:“检测胆固醇只能说明血脂的情况,检测葡萄糖只能说明血糖的情况,C反应蛋白只能说明炎症的情况,但端粒长度是对多种生物化学平衡的全面反应,是健康状态的一项总指标”。 萨博尔斯基认为:“检查端粒能更好地预测到不良健康状况,与其它指标相比,它的准确度更高,它能反应人体整个系统的耗损程度”。

  五、改善生活方式可增长端粒

  布莱克本、艾培尔还有其他 30名学者在去年发表于《柳叶刀》杂志上的一项先导性研究结果表明:人们可以通过一些自然的方式来提高端粒末端转移酶的活性水平,从而逆转不良生活方式对端粒产生的恶性作用。

  全面改善生活方式,包括低脂饮食、坚持锻炼、通过沉思或是瑜珈减少压力,这样做三个月后端粒末端转移酶活性就可以提高30%。

  六、端粒和端粒酶的临床意义

  1、端粒与细胞衰老

  实验证据表明,端粒、端粒酶与衰老之间存在相关性。通过对不同年龄人群的端粒长度进行检测,研究人员发现细胞中端粒长度与供体年龄呈高度负相关。老年个体的端粒长度较年轻个体短得多,年轻个体细胞中的某些细胞。如淋巴细胞中的端粒酶活性会随年龄的增加而逐渐下降。此外,研究发现干细胞等具有无限分裂能力细胞端粒较长,且具有较高的端粒酶活性,而大多数具有有限增生能力的体细胞的端粒较短,不具有或仅有很低的端粒酶活性。在早老症和Wemer综合征等临床研究的结果也显示,病人的培养细胞中端粒的平均长度明显比正常个体短得多。当端粒缩短到一定程度时,细胞无法维持正常的端粒结构而导致细胞发生不可逆的生长停滞,最终进入细胞衰老状态。

  2、端粒与肿瘤发生

  正常情况下人类体细胞端粒酶的检测结果是阴性的,但在400个肿瘤样品中,85%以上能检测到端粒酶的存在.细胞的端粒酶活性因某些原因被激活,使端粒不断维持在一定的长度,细胞因此逃过死亡而成为无限增殖的细胞—肿瘤细胞。端粒酶活性与恶性肿瘤的这种密切关系,为肿瘤的诊断提供了有效的标志物。端粒酶是正常细胞转变为肿瘤细胞的关键性物质,是抗肿瘤治疗的重要靶点,而且,正常细胞与肿瘤组织中端粒酶的表达、端粒的长度和细胞动力学的差异,使得选择端粒酶作为药物靶标成为相对安全的治疗手段。

  3、端粒长度与心血管疾病

  越来越多的证据表明端粒长度同心脑血管疾病是相关的,较短的端粒不仅同心脑血管疾病及心脑血管疾病危险因素相关,而且端粒缩短的程度同疾病的严重性相关。 一项纳入 203例早发心肌梗死患者(年龄 < 50岁 ) 和 180例对照人群的研究,发现早发心梗患者(50岁之前发生)白细胞端粒长度相当于健康对照组年长11.3岁的端粒长度。值得注意的是,Ogami等人研究发现冠心病患者冠脉内皮细胞端粒长度较年龄配对的非冠心病患者为短,同时在冠心病个体中,发生动脉粥样硬化的冠脉斑块处与非斑块处内皮细胞端粒长度相比表现缩短。这些研究表明端粒缩短导致的内皮细胞功能失调及衰老在冠脉动脉粥样硬化中起到重要作用。端粒的缩短有可能是心血管疾病发生的一个独立因素,白细胞端粒长度可能是心血管疾病意外风险或者预后的预测因子。

  4、端粒长度与脑血管疾病

  体外及体内均有明确证据表明脑血管疾病危险因素同端粒长度相关,氧化应激减少端粒长度;同此相一致的是吸烟加速端粒缩短,并且呈剂量依赖性;横断面研究显示:端粒长度的缩短同糖尿病、不断增加的体重和胰岛素抵抗相关;纵向研究显示:端粒短的非高血压个体易于发生高血压,端粒短的高血压个体易于发生动脉粥样硬化。除了这些体格和理化的危险因素外,心理和环境方面的脑血管危险因素也同端粒的缩短相关,包括心理压力、慢性应激、抑郁和社会地位的下降等。总之,脑血管疾病危险因素可能促进或者伴有端粒长度的缩短。Annette等对年龄>65岁的老年人群进行10年随访,年龄低于73岁人群中,端粒每缩短1kb,脑卒中的危险性增加3.22倍。从而初步证实端粒的缩短亦可能是脑血管疾病的预测因子。Martin等人试图进一步研究端粒的缩短与脑梗死预后的关系,其对195例脑卒中患者进行前瞻性研究,他们发现较长的端粒长度者对应较低的死亡和认知下降的风险,而短于正常端粒长度的患者,死亡、痴呆和认知下降的风险较高。患者端粒长度每减低一个碱基对,其死亡风险提高将近一倍。据此认为端粒长度可预测卒中后的死亡率、痴呆发生和认知能力的下降。

  展望

  端粒和端粒酶的发现揭示了细胞复制过程中染色体末端的保护机制,揭示了正常细胞有限分裂能力和癌细胞可以无限增殖的秘密,使人们对于细胞衰老和增殖过程有了更加深刻的认识。更重要的是,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,这一发现将衰老、肿瘤、干细胞等生命科学重大领域联系在一起。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。

  RESEARCH

  Telomeres in cancer and ageing

  Luis E. Donate and Maria A. Blasco

  Telomeres and Telomerase Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernandez Almagro, 3, 28029 Madrid, Spain

  Telomeres protect the chromosome ends from unscheduled DNA repair and degradation. Telomeres are heterochromatic domains composed of repetitive DNA (TTAGGG repeats) bound to an array of specialized proteins. The length of telomere repeats and the integrity of telomere-binding proteins are both important for telomere protection. Furthermore, telomere length and integrity are regulated by a number of epigenetic modifications, thus pointing to higher order control of telomere function. In this regard, we have recently discovered that telomeres are transcribed generating long, non-coding RNAs, which remain associated with the telomeric chromatin and are likely to have important roles in telomere regulation. In the past, we showed that telomere length and the catalytic component of telomerase, Tert, are critical determinants for the mobilization of stem cells. These effects of telomerase and telomere length on stem cell behaviour anticipate the premature ageing and cancer phenotypes of telomerase mutant mice. Recently, we have demonstrated the anti-ageing activity of telomerase by forcing telomerase expression in mice with augmented cancer resistance. Shelterin is the major protein complex bound to mammalian telomeres; however, its potential relevance for cancer and ageing remained unaddressed to date. To this end, we have generated mice conditionally deleted for the shelterin proteins TRF1, TPP1 and Rap1. The study of these mice demonstrates that telomere dysfunction, even if telomeres are of a normal length, is sufficient to produce premature tissue degeneration, acquisition of chromosomal aberrations and initiation of neoplastic lesions. These new mouse models, together with the telomerase-deficient mouse model, are valuable tools for understanding human pathologies produced by telomere dysfunction.

  Keywords: telomeres; telomerase; cancer; ageing; shelterins; stem cells

  1. ROLE OF TELOMERASE IN ADULT STEM CELLS

  (a) Telomeres

  Telomeres are ribonucleoprotein complexes at the ends of chromosomes that are essential for chromosome protection and genomic stability. Telomeres consist of tandem repeats of a DNA sequence rich in G bases (TTAGGG in all vertebrates) bound by a six-protein complex known as shelterin. Shelterin encompasses the Pot1-TPP1 heterodimer, the telomere-binding proteins TRF1 and TRF2, and the interacting factors Rap1 and Tin2 [1].

  Telomeric chromatin is also enriched in epigenetic marks that are characteristic of constitutive heterochromatin, such as histone tri-methylation and DNA hypermethylation, which act as negative regulators of telomere length and telomere recombination [2].

  Telomere shortening below a certain threshold length and/or alterations in the functionality of the telomere-binding proteins can result in loss of telomeric protection, leading to end-to-end chromosome fusions, cell cycle arrest and/or apoptosis. Telomeres also perform other functions, which include the transcriptional silencing of genes located close to the telomeres (this phenomenon is termed subtelomeric silencing), as well as ensuring correct chromosome segregation during mitosis.

  (b) Telomerase

  Shortening of telomeres is associated with each round of cell division owing to the inability of conventional DNA polymerases to replicate the ends of linear chromosomes, the so-called ‘end replication problem’. Telomerase is a cellular enzyme capable of compensating this progressive telomere attrition through de novo addition ofTTAGGG repeats to the chromosome ends [3]. Telomerase encompasses a catalytic subunit with reverse transcriptase activity (Tert), an RNA component (Terc) that acts as a template for DNA synthesis and the protein dyskerin (Dkc1), which binds and stabilizes Terc.

  Robust telomerase expression is a feature of pluripotent stem cells and early stages of embryonic development, although telomerase activity is also present in adult stem cell compartments [4]. Telomerase activity in adult tissues, however, is not sufficient to prevent telomere shortening associated with ageing.

  Mutations in the different components of telomerase (Tert, Terc and Dkc1), as well as in some shelterins (Tin2), have been linked to rare human genetic diseases, such as dyskeratosis congenita, aplastic anaemia and idiopathic pulmonary fibrosis [5–8]. These diseases are associated with the presence of short/dysfunctional telomeres and they all exhibit a characteristic failure in the regenerative capacity of tissues (such as the bone marrow) and severe skin hyperpigmentation.

  Figure 1. Progressive decrease in median and maximum lifespans along successive generations of telomerase-null mice. Cohorts of successive generations of telomerase-deficient mice (wild-type, black dashed line; first generation G1 Terc2/2, blue dashed line; second generation G2 Terc2/2, green dashed line; third generation G3 Terc2/2,red dashed line) were followed up for a period of 32 months. The figure shows a Kaplan–Meier representation of the survival of the following groups of mice: Tercþ/þ, n ¼ 68; G1 Terc2/2, n ¼ 17; G2 Terc2/2, n ¼ 31; G3 Terc2/2, n ¼ 22. Telomerase-deficient mice have a shorter lifespan than the telomerase-proficient mice, which is further shortened with increasing mouse generations.

 

  Figure 2. A stem cell-based model for the role of telomeres in cancer and ageing. The longest telomeres mark the stem cell compartments (niches). In young or adult organisms, stem cells (blue rounded cells) repopulate tissues as needed: they exit from the niche, proliferate and differentiate (square orange cells). During this process, stem cells undergo telomere shortening, which is partially counterbalanced by the action of telomerase. In old organisms, stem cell telomeres are too short. Critically short telomeres are recognized as DNA damage, activating a p53-mediated DNA damage signalling response that impairs stem cell mobilization and, as a consequence, the tissue regeneration is suboptimal leading ultimately to organ failure. A decreased stem cell mobilization reduces the probability of accumulating abnormal cells in tissues, providing a mechanism for cancer protection. If the stem cells express aberrantly high levels of telomerase (by acquisition of tumorigenic, telomerase-reactivating mutations), stem cell mobilization is more efficient than normal. Under these higher mobilization conditions, tissue fitness would be maintained for a longer time, increasing lifespan and also the probabilities of initiating a tumour.

  2. THE TELOMERASE-DEFICIENT MOUSE MODEL

  Generation of telomerase-deficient mice (Terc knock-out mice) allowed the first demonstration that telomerase is required for telomere maintenance in the context of mammalian organisms, as well as its importance for cancer and ageing. Thus, telomerase-deficient cells exhibit an accelerated telomere shortening that eventually leads to loss of telomere protection and end-to-end chromosome fusions [9–12]. Complete survival curves of increasing generations of telomerase-null mice (G1–G3) [13] indicated that telomere shortening along successive mouse generations [12,14–15] was paralleled by a progressive decrease in both median and maximum longevity (figure 1). Telomerase and telomere maintenance are considered, therefore, to be rate limiting for mouse longevity.

  Additionally, telomerase-deficient mice developed premature ageing pathologies with an onset that is anticipated with increasing mouse generations (similarly to the human diseases linked to telomere dysfunction), in agreement with inheritance of shorter telomeres after each generation. Finally, telomerase-deficient mice show increased resistance to cancer, validating telomerase as a promising target for anti-tumour therapies [16].

  (a) Telomerase in adult stem cells

  As telomerase expression is restricted to stem cell compartments in the context of the adult organism, it is of interest to determine the impact of telomere shortening on stem cell ageing. Stem cell functionality, measured as the ability of epidermal stem cells to mobilize and regenerate the skin and the hair, is dramatically impaired in telomerase-deficient mice with critically short telomeres (as is the case of G3 telomerase-null mice), a defect that is rescued by either telomerase re-introduction and elongation of short telomeres, or by p53 suppression [17,18]. In this regard, all stem cell defects associated with the presence of critically short telomeres, such as the length of the hair bulb and the thickness of the interfollicular epidermis, are fully rescued in mice doubly deficient for telomerase and p53 when compared with single telomerase-deficient mice. These results identify p53 as an important checkpoint for stem cell and tissue fitness, in a manner that only those stem cells with sufficiently long telomeres will be allowed to regenerate tissues [19].

  (b) Role of telomeres in cancer and ageing: a stem cell-based model

  We have proposed a stem cell-based model for the role of telomeres in cancer and ageing, which is summarized in figure 2. Adult stem cells reside at specific compartments within tissues, the so-called niches, which are enriched in cells with the longest telomeres [20]. In young or adult organisms with sufficient telomere reserve, adult stem cells efficiently repopulate tissues and repair lesions as needed. In old organisms, however, stem cell telomeres may be too short [20], and this could impair the mobilization of stem cells and the ability to repair tissues efficiently. When telomeres have shortened down to a critical length they are recognized as DNA damage, activating a p53-mediated DNA damage signalling response that prevents the mobilization of the stem cells out of their niches. Decreased stem cell mobilization reduces the probability of accumulating abnormal cells in tissues, thus providing a mechanism for cancer protection. However, the ultimate consequence of impaired mobilization of the stem cells will be organ failure owing to tissue degeneration.

  By using mouse models over-expressing telomerase, we and others showed that elevated TERT expression increases stem cell mobilization. Under these conditions of higher mobilization, the fitness of the tissues would be maintained for longer times, therefore increasing the lifespan. The probabilities of initiating a tumour, however, are also higher, especially so if telomerase reactivation occurs in a context of mutations in the tumour suppressors [21].

  3. LONGEVITY EXTENSION BY TELOMERASE

  As discussed in §2, the study of telomerase-deficient mice suggests that telomerase is rate limiting for mouse longevity. We next wondered whether telomerase over-expression can extend longevity and, if so, to what extent. We previously observed that while telomerase deficiency results in a lower incidence of cancer [16], constitutive telomerase over-expression results in a slightly increased incidence of cancer (figure 3)[22]. To counterbalance this undesired effect of telomerase over-expression, we overexpressed telomerase in the context of cancer-resistant mice having increased levels of the tumour suppressors p53, p16 and p19ARF (SUPER-M mice; figure 3). In these mice, the effects of telomerase on cancer and ageing will be dissociated, therefore allowing assessment of the role of telomerase on the ageing and fitness of mice.

  (a) SUPER-M mice

  Characterization of the SUPER-M mice showed a cancer appearance that was significantly delayed: while in wild-type mice, cancer was detectable at 110 weeks of age (and earlier in those mice over-expressing telomerase), in the SUPER-M mice tumours did not appear until 145 weeks of age [23].

  The age for the onset of degenerative lesions is also delayed in SUPER-M mice. In these mice, the symptoms of ageing are also attenuated: for instance, levels of subcutaneous fat are very similar in young and older SUPER-M mice, while the thickness of the subcutaneous fat layer in older wild-type mice is seven times less than that of young wild-type mice. Also, the SUPER-M mice exhibit reduced ageing of the skin; the skin and hair coat of elderly SUPER-M mice are better than of elderly wild-type mice.

  Organism ageing of SUPER-M mice is also less: thus neuromuscular fitness is improved in old mice. All the SUPER-M mice successfully passed the neuromuscular coordination test, while not all the wild-type mice were successful in this test, and over half the aged mice failed. SUPER-M mice also had improved glucose tolerance, three times better than that of wild-type mice. SUPER-M mice have longer telomeres than wild-type mice, and this difference in telomere length is much greater in elderly mice. Also, aged SUPER-M mice showed less damage in their telomeric DNA than the aged wild-type mice.

  Given the above-described anti-ageing activity of telomerase in the SUPER-M mice, we addressed whether the tranagenic Tert (TgTert) allele had an effect on the lifespan of the cancer-resistant Sp53 and Sp53/Sp16/SArf mice. We obtained survival curves covering the entire lifespan of the Sp53/TgTert and the Sp53/Sp16/SArf/TgTert mice (SUPER-M), compared with Sp53 and Sp53/Sp16/SArf controls; all these mice have the same genetic background composition. We used Sp53 mice as a reference for normal longevity in our mouse cohorts because they have been previously shown to have the same longevity as wild-type mice in two independent studies [24,25]. Analysis of the survival curves indicated a significant extension of median lifespan of 9 per cent and 26 per cent by TgTert expression in the context of cancer-resistant Sp53 and Sp53/Sp16/SArf mice, respectively (figure 4a). To further dissociate the effects of TgTert expression on cancer and ageing, we considered separately the lifespan of the cancer-free mice (i.e. those mice that died without malignant tumours; figure 4b). The lifespans of this subgroup are determined by ageing and not by cancer, and we observed an even more evident impact of TgTert expression, resulting in a median lifespan extension of 18 per cent and 38 per cent in the Sp53/TgTert and Sp53/Sp16/SArf/TgTert (SUPER-M) mice, respectively, compared with the Sp53 and Sp53/Sp16/SArf controls (figure 4b). Furthermore, combined TgTert and Sp53/Sp16/SArf transgenes resulted in a 40.2 per cent extension of the median lifespan when compared with single Sp53 mice (our reference for normal longevity), which was further increased to 50 per cent when considering cancer-free mice (figure 4b). We also studied the group of longest lived mice of each genotype to estimate whether TgTert expression had an effect on maximum longevity. The percentage of mice that reached the extremely old age of 3 years was significantly larger for the SUPER-M mice than for their Sp53/Sp16/SArf controls (42% versus 8%), and this extreme survival is further increased when considering cancer-free mice (up to 50%). The mean age of the upper longevity quartile was significantly higher in SUPER-M mice than in their Sp53/Sp16/SArf controls (163 weeks versus 146 weeks). These observations indicate that TgTert expression changes the longevity curve of mice, significantly extending the median lifespan and significantly increasing the percentage of mice that reach extremely old ages. We also wondered whether the observed effects of telomerase on longevity involve its telomere-maintenance activity. To this end, we examined the longevity curve of TgTert mice lacking the RNA component of telomerase (Terc), which is essential for telomerase catalytic activity. We observed that TgTert does not have an effect on the longevity curve of Terc-deficient mice across different generations up to the fourth generation (G2–G4). These results indicate that the anti-ageing activity of Tert requires telomerase catalytic activity, which strongly implicates telomere maintenance as the main mechanism underlying the longevity enhancing effect of telomerase expression.

  4. TELOMERE REJUVENATION DURING THE GENERATION OF INDUCED PLURIPOTENT STEM CELLS

  To obtain induced pluripotent stem (iPS) cells from adult differentiated cells is a key goal in the development of customized cellular therapies. This type of cell would represent an unlimited source of cells capable of generating all kind of tissues, with the added advantage of avoiding rejection in cell transplantation therapies, as they originate from the same individual. The first approach to nuclear reprogramming was based on nuclear transplantation from somatic cells to enucleated oocytes [26]. Recently, iPS cells have been generated from differentiated cells by the introduction of four transcription factors, Oct4, Sox2, Klf4 and c-Myc [27–28]. However, c-Myc is dispensable in the generation of iPS cells.

  As previously stated, telomeres shorten with age, thus contributing to organism ageing by limiting the proliferative capacity of adult stem cells. Although it was known that telomerase activity was augmented in both human and murine iPS cells, it remained unknown whether telomeres re-elongated during nuclear reprogramming and whether the telomeric chromatin would acquire the same characteristics as those of embryonic stem cells (figure 5).

  Reprogramming of telomeric chromatin can take place in several possible scenarios: telomerase might not be needed for telomere elongation, telomerase might cooperate with recombination-based mechanisms or, finally, telomere elongation during nuclear reprogramming might completely depend on telomerase activity. We demonstrated that in iPS cells, the telomeres greatly elongate with respect to the telomeres of the parental differentiated cells [29]. This elongation occurs independently of the presence or absence of c-Myc. Furthermore, the efficiency of elongation of telomeres during the generation of iPS cells was the same whether the starting cells were derived from young or elderly individuals. These results, therefore, indicate that telomeres efficiently rejuvenate during nuclear reprogramming of differentiated cells. The elongation of telomeres continues post-reprogramming until they reach the length of the telomeres of the embryonic stem cells. During the reprogramming of telomerase-null cells the telomeres do not elongate. This clearly demonstrates that telomere elongation during nuclear reprogramming of differentiated cells is exclusively mediated by telomerase activity [29].

  We also demonstrated that the telomeres of iPS cells acquire the epigenetic marks of the telomeres of embryonic stem cells, among them a low density of trimethylated histones H3K9 and H4K20, and that in the iPS cells there is a loss of telomeric silencing and an increase in the abundance of TERRA (telomeric transcripts) levels [29].

  We observed that the reprogramming efficiency of cells derived from increasing generations of telomerase-deficient mice is drastically reduced, and this defect is annulled after telomerase reintroduction [29]. We also observed that the generation of iPS cells needs a minimum telomere length. In fact, when we employed cells derived from the G3 generation of telomerase-deficient mice, reprogramming is impaired, indicating the existence of a minimum required telomeric length for iPS cells to be obtained [29].

  5. p53 IS A KEY FACTOR LIMITING REPROGRAMMING OF SUBOPTIMAL CELLS

  The fact that cells with short telomeres are not subject to nuclear reprogramming very probably indicates that some ‘reprogramming barriers’ abort the reprogramming of suboptimal parental cells bearing uncapped or dysfunctional telomeres. To explain this observation, we hypothesise low reprogramming rates resulting from the presence of DNA damage in the starting cells. We have demonstrated that the tumour suppressor p53 is a key factor that limits the reprogramming of suboptimal cells, those bearing different kinds of DNA damage such as short telomeres, having deficiencies in their DNA repair systems (ATM- and 53BP1-deficient cells) or exogenously inflicted DNA damage (irradiated cells) [30].

  Figure 3. Lifespan extension by telomerase. To address whether telomerase over-expression had an effect on mouse longevity, we first took into account that the absence of telomerase (the Terc knock-out mice) results in premature ageing and also in a lesser incidence of cancer. Second, the constitutive over-expression of telomerase in the epithelia, while attenuating the ageing phenotypes, also increased the incidence of cancer in the epithelia. We generated SUPER-M mice, a mouse model in which the constitutive over-expression of telomerase takes place in a genetic background of increased resistance to cancer achieved by means of over-expression of the tumour suppressors p53, p16 and p19ARF. In the SUPER-M mice, the effects of telomerase on cancer and ageing will be dissociated.

  Reprogramming in the face of pre-existing but tolerated DNA damage is aborted by the activation of the p53-dependent DNA damage response (DDR) and apoptosis. p53 suppression allows an efficient reprogramming of cells harbouring persistent DNA damage and chromosomal aberrations. We have observed that during reprogramming, the cells increase their intolerance to the different types of DNA damage and that p53 is critical in avoiding the generation of iPS cells from suboptimal parental cells [30]. Finally, given that certain reprogramming factors promote in vivo tumori genesis, it is tempting to propose that the DDR observed in cultures of p53-deficient cells might be equivalent to the oncogene-induced DDR that takes place during malignant transformation. For the two scenarios nuclear reprogramming and malignant transformation, p53 is critical in controlling the dissemination of damaged cells.

  Our results highlight the importance of telomere biology in the generation and functionality of iPS cells, and have important implications for the clinical translation of iPS cell technologies, particularly in patients afflicted with the so-called ‘telopathies’.

  6. THE IMPORTANCE FOR CANCER AND AGEING OF CANCELLING THE DNA DAMAGE RESPONSE AT CHROMOSOME ENDS

  (a) The shelterin complex

  The TTAGGG repeats at the end of mammalian chromosomes associate to a six-protein complex termed shelterin. Shelterin enables cells to distinguish their natural chromosome ends from breaks in the DNA strand by cancelling a DDR at telomeres, as well as regulating telomere length. Telomeres contain additionally a number of proteins that are not part of the shelterin complex, but also have non-telomere-related functions.

  The specificity of shelterin for telomeric DNA is through direct recognition of the TTAGGG sequence by three of its components. In particular, TRF1 and TRF2 bind to the double stranded region of telomeric DNA, while Pot1 binds to the TTAGGG repeats of the G-overhang. TRF1 and TRF2 recruit the other four components of shelterin: Tin2 (a TRF1 and TRF2 interacting factor), Rap1, TPP1 and Pot1. These last two proteins form a heterodimer. Shelterin can form a stable complex in the absence of telomeric DNA.

  Some mutations in Tin2 as well as genetic variants of TRF1 have been described linked to the rare human diseases dyskeratosis congenita and aplastic anaemia. These conditions [31–33] have as hallmarks epithelial abnormalities, such as skin hyperpigmentation, nail dystrophy and oral leukoplakia.

  7. MOUSE MODELS FOR CONDITIONALLY DELETED SHELTERIN PROTEINS

  (a) TRF1△/△mice

  The conventional deletion of the shelterin protein TRF1 in mice is lethal embryonically very early during the blastocyst stage, and for this reason the characterization of the role of TRF1 in differentiated cells has remained impractical.

  To study the role of TRF1 in telomere biology and disease in the context of a mammalian organism we generated cells and mice in which TRF1 was conditionally deleted specifically in epithelial (TRF1△/△K5-Cre) mice [34]. TRF1 deletion in mouse embryonic fibroblasts (MEFs) did not result in changes in telomere length; on the contrary, it led to a rapid induction of p53/RB-dependent cellular senescence concomitant with the accumulation of abundant damage foci at telomeric DNA. This persistent DNA damage activated phosphorylation of the ATM/ATR kinases and their downstream effectors, the kinases CHK1 and CHK2, resulting in cell cycle arrest. Cells deficient in TRF1 showed abundant end-to-end telomere fusions involving both chromosomes and sister chromatids. We also observed abundant multi-telomeric signals, which are indicative of a high degree of chromosomal fragility that arises from problems in the replication of telomeric DNA [34,35]. Our results demonstrate that TRF1 exerts a protective function against the DDR at telomeres, and that it facilitates the replication of telomeric DNA.

  (b) Consequences of TRF1 deletion in adult stem cells

  In accordance with severe telomere dysfunction, TRF1△/△K5-Cre mice die perinatally and show reduced skin thickness and reduced skin stratification, as well as severe skin hyperpigmentation. Newborn mice also have focal dysplasia in the epithelia of the palate, the non-glandular stomach, oesophagus, tongue and skin. These pathologies are accompanied by activation of a persistent DDR at telomeres, which induces activation of the p53/p21 and p16 pathways resulting in an in vivo cell cycle arrest. The latter produces dramatic alterations in the properties of the epithelial stem cells. Thus, morphological development of the hair follicles and the sebaceous glands is completely impaired [34].

  (c) Effect of p53 suppression in TRF1-deficient mice

  Suppression of p53 in TRF1△/△K5-Cre mice rescues perinatal survival (p532/2/TRF1△/△ K5-Cre mice reach four months of age) and the functionality of epidermal stem cells, as these mice grow hair normally and do not exhibit skin hyperpigmentation [34].

  Longer lived p532/2/TRF1△/△K5-Cre mice, however, develop epithelial abnormalities similar to those of human diseases linked to mutations in the components of shelterin and/or telomerase, such as nail atrophy and oral leukoplakia. Furthermore, lack of p53 in p532/2/TRF1△/△K5-Cre mice leads to the development of spontaneous squamous carcinomas, showing that TRF1 acts as a tumour suppressor by preventing telomere-related genomic instability.

  Our results demonstrate that dysfunction of a single telomeric protein is sufficient to produce severe telomeric damage and loss of telomere capping in the absence of telomere shortening, leading to premature tissue ageing, acquisition of chromosomal aberrations and the development of neoplasic lesions.

  The trf1△/△ mouse model is of relevance since it constitutes the first mouse model of ageing induced by telomere dysfunction in the absence of critical telomere shortening. This model shows that telomere uncapping and increased telomere fragility do impact on cancer and ageing in the absence of telomere shortening. It also suggests a new class of ‘telopathies’ induced by telomere dysfunction in the presence of normal-length telomeres.

  (d) Tpp1△/△ mice

  The role of TPP1 in telomere regulation in vivo and in mouse development and disease has been poorly characterized to date owing to lack of mouse models with complete TPP1 suppression.

  We have recently generated Tpp1-deficient MEFs as well as mice with targeted Tpp1 deletion to the stratified epithelia [36]. Both MEFs and mice deleted for Tpp1 show induction of telomere damage foci and cell cycle arrest, demonstrating that TPP1 protects telomeres from eliciting a DDR. Similarly to TRF1 deficiency, Tpp1-null mice die perinatally and show severe skin hyperpigmentation and defective hair Review. Telomeres, cancer and ageing L. E. Donate & M. A. Blasco 81 Phil. Trans. R. Soc. B (2011) on November 9, 2011 rstb.royalsocietypublishing.org Downloaded from follicle morphogenesis. These phenotypes are rescued by p53 suppression, indicating that p53 is a main effector of proliferative defects associated with Tpp1 deletion.

  Unexpectedly, Tpp1 deletion results in decreased Tert binding to telomeres and accelerated telomere shortening both in MEFs and mice. Tpp1-null cells failed to elongate their telomeres when reprogrammed into pluripotent stem cells, a process that is dependent on telomerase activity [29,30], thus indicating that TPP1 is essential for telomere elongation in vivo. These results suggest a telomere-capping model where TPP1 not only prevents the induction of a DDR at telomeres by preventing fusions and telomere breakage but also is required for telomere elongation by telomerase.

  (e) Rap1△/△ mice

  Rap1 is a component of the shelterin complex at mammalian telomeres, the in vivo role of which in telomere biology has remained largely unknown to date.

  We have recently generated cells and mice deleted for Rap1 [37]; mice with Rap1 deletion in stratified epithelia are viable but had shorter telomeres and developed skin hyperpigmentation at adulthood. We showed that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. We have found that Rap1 binds to both telomeres and to extratelomeric sites. The extratelomeric Rap1-binding sites were enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells.

  Figure 4. Increased longevity of Sp53/Sp16/SArf/TgTert (SUPER-M) mice. (a) Overall survival. Kaplan–Meier representation of the survival curves of the indicated mouse cohorts. Only mice that reached at least up to 50 weeks of age were included. The increase in the median lifespan is indicated. n ¼ number of mice per genotype. Statistical significance was assessed using the log-rank test. (b) Cancer-free survival. Kaplan–Meier representation of the survival curves of mice of the indicated genotype living for more than 50 weeks excluding those mice that presented malignant tumours at the time of their death (cancer-free survival). The increase in the median lifespan is indicated. n ¼ number of mice included in the analysis. Statistical significance was assessed using the log-rank test. (a) pink trace, Sp53 (n ¼ 68); green trace, Sp53/TgTert (p ¼ 0.05; n ¼ 56); red trace, Sp53/Sp16/SArf (n ¼ 39); green trace, Sp53/Sp16/SArf/TgTert (p ¼ 0.05; n ¼ 27)(SUPER-M); pink trace, Sp53 (n ¼ 68); green trace, Sp53/Sp16/SArf/TgTert (p , 0.001; n ¼ 27)(SUPER-M). (b) pink trace, Sp53 (n ¼ 44); green trace, Sp53/TgTert (p ¼ 0.47; n ¼ 35); pink trace, Sp53/Sp16/SArf (n ¼ 33); green trace, Sp53/Sp16/SArf/TgTert (p ¼ 0.001; n ¼ 22)(SUPER-M); pink trace, Sp53 (n ¼ 44); green trace, Sp53/Sp16/SArf/TgTert (p ¼ 0.001; n ¼ 22) (SUPER-M).

  Figure 5. Telomeres and longevity. The mouse embryonic stem cells have very long telomeres which shorten during embryogenesis. Telomere shortening continues during adult life, and is proposed to be a major cause of organism ageing through impairing the regenerative capacity of tissues. Nuclear reprogramming of differentiated cells derived from both young and old mice into induced pluripotent stem (iPS) cells is accompanied by telomerase-dependent telomere elongation. Telomere function is highly dependent on functional shelterin components. Suppression of some shelterins (such as TRF1 and TPP1) results in rapid onset of degenerative pathologies in newborn mice.

  8. TELOMERES AND LONGEVITY

  The embryonic stem cells of mice have long telomeres, which shorten during embryogenesis (figure 5). Telomere shortening continues throughout the adult life of the mice to such a extent that it has been proposed to be a major cause of mouse ageing since telomere shortening produces defects in the regenerative capacity of tissues.

  Nuclear reprogramming of differentiated cells derived from both young and old mice results in the generation of iPS cells. Telomeres rejuvenate in a telomerase-dependent manner during nuclear reprogramming into iPS cells.

  Telomere function is highly dependent on functional shelterin components. The suppression of some shelterin proteins (such as TRF1 and TPP1) results in sudden development of degenerative pathologies. Thus, telomere uncapping, even in the presence of normal length telomeres, is capable of inducing age-related pathologies in young mice.

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