乳腺良恶性病变组织端粒长度和端粒酶活性检测

目的　比较乳腺良恶性病变端粒长度改变及其在肿瘤发生发展中的意义 ;探讨端粒酶活性与临床病理参数的关系及其在乳腺癌诊断中的价值。方法　Southern印迹杂交检测TRF长度 ,端粒重复扩增分析 (TRAP)方法检测端粒酶活性。结果　乳腺癌组织平均TRF为 (5 2± 2 8)kb ,与正常组织比较明显缩短 (P <0 0 0 1) ,从正常乳腺组织到乳腺良性病变、乳腺原位癌及浸润性癌平均TRF呈递减趋势。 5 8例乳腺癌中 4 9例端粒酶阳性 (84 7% ) ,端粒酶活性与临床病理参数无相关性 ;癌旁组织端粒酶为阴性 ,而 7例乳腺增生症和 6例乳腺纤维腺瘤中分别有 1例端粒酶阳性 ,与乳腺癌比其差异有显著性 (P <0 0 0 1)。结论　端粒长度在肿瘤发生发展过程中渐进性缩短 ,并最终触发端粒酶的激活 ;端粒酶活性检测有望成为乳腺癌诊断的可靠标记物     

6A8α-甘露糖苷酶表达对人鼻咽癌细胞CNE-2L2端粒酶活性和端粒长度的影响

探讨端粒长度与端粒酶活性在人鼻咽癌细胞CNE-2L2恶性行为改变前后的变化，建立研究恶性行为改变与端粒长度与端粒酶活性间关系的细胞模型。与6A8α-甘露糖苷酶表达正常的CNE-2L2细胞(野生型细胞W，转导空载体的细胞M及转导无关DNA片段的细胞S)相比，6A8α-甘露糖苷酶表达低下的细胞(AS)接种裸鼠皮下后的肿瘤性生长受抑。用Telo TAGGG Telomere Length Assay Kit及Telomerase PCR ELISA Kit分别测定端粒长度及端粒酶活性，用RT-PCR方法分析端粒重复序列结合因子(TRF)的转录水平。见AS细胞的端粒明显缩短(6．78Kb，W细胞为8．40Kb，M细胞为8．34kb，S细胞为9．56kb)，但端粒酶活性及端粒重复序列结合因子l和2(TRFl和2)的转录水平未见改变。实验表明，恶性行为降低的CNE-2L2细胞的端粒变短，但与端粒酶活性及TRF1／2无关，提示在CNE-2L2细胞中可能存在着端粒酶及TRF1／2以外的调节端粒长度的机制。这为研究肿瘤细胞恶性行为改变与端粒长度与端粒酶活性间关系提供了一个模型。     

人骨髓间质干细胞中的端粒调控机制

目的：〖HT5"SS〗研究人骨髓来源间充质干细胞(MSCs)端粒长度的调控机制。方法：以贴壁培养法从人骨髓中分离MSCs并用MSCs及造血干细胞相关表面抗体作表型鉴定,用Southern blotting检测MSCs的端粒长度;应用免疫荧光染色技术检测端粒重复序列结合因子1（TRF1）和早幼粒细胞白血病蛋白小体（PML）的定位;以端粒重复序列扩增法（TRAP）和/或Western blotting法检测传代及分化成脂肪细胞的MSCs和经同步化处理被阻断在S期的MSCs的端粒酶表达。结果：与端粒酶阴性ALT细胞株WI-38-2RA细胞相比,MSCs的端粒长度较短并且端粒长度变异度不大;端粒调控相关蛋白TRF1和PML在MSCs中的定位则与端粒酶阳性细胞HeLa细胞相同,两者呈非共定位,而在端粒酶阴性WI-38-2RA细胞中两者呈共定位状态。MSCs中不存在有染色体外端粒重复序列DNA（ECTR DNA）。TRAP法检测传代培养的MSCs端粒酶呈阴性表达,但分化成脂肪的MSCs端粒酶呈阳性表达。Western blotting 检测同步化处理前MSCs端粒酶呈微弱表达,经同步化处理被阻断在S期时,MSCs的端粒酶表达明显增高,并且与S期的细胞比例呈正相关。结论：MSCs中不存在ALT相关的早幼粒细胞白血病蛋白小体(APBs)、染色体外端粒重复序列DNA(ECTR DNA)和端粒长度较长、端粒长度变异度大等ALT机制相关分子特征;非同步化在S期处理的MSCs,端粒酶呈微弱表达,但诱导向脂肪细胞分化或处在S期时,MSCs的端粒酶表达明显增高,并且与S期的细胞比例呈正相关。本研究提示MSCs是通过端粒酶机制而不是端粒延长旁路途径（ALT）机制调控其端粒末端。     

端粒酶活性抑制与肿瘤治疗的研究

端粒酶是一种核糖蛋白,包括人端粒酶RNA、端粒酶相关蛋白和端粒酶催化蛋白亚单位三个主要组成部分,可向染色体末端添加端粒DNA序列.端粒酶在85％～95％的恶性肿瘤组织中有阳性表达,而正常体细胞则一般阴性,是进行肿瘤基因治疗的理想靶点.目前以抑制端粒酶活性为靶点的肿瘤治疗研究较多,其主要策略有:①阻断人端粒酶RNA的模板作用;②抑制端粒酶催化蛋白亚单位;③核苷类似物竞争性抑制反转录过程;④细胞分化诱导剂抑制端粒酶活性;⑤对细胞内调节机制进行调控;⑥其它抑制剂对端粒酶活性的调节.     

端粒相关蛋白TIN2

TIN2(TRF1相互作用核蛋白2)是一重要的端粒相关蛋白。人TIN2蛋白包括N端,TRF1交互作用区(TRF1-Int)和C端3个结构域。它在TRF1复合物、TRF2复合物和Shelterin的功能中扮演关键角色,协同其它端粒蛋白维持端粒长度、结构和功能。TIN2与个体发育、细胞分化和肿瘤发生密切相关。     

端粒/端粒酶在恶性血液病中的研究进展

端粒是真核细胞染色体末端特有的一段DNA序列和几种特异性蛋白质构成的复合物。端粒酶是一种核糖核酶,能以自身RNA为模板,合成端粒DNA,从而维持端粒的长度。端粒/端粒酶的表达调控对肿瘤的发生发展及细胞的衰老、永生化起着重要的作用,深入研究端粒/端粒酶的结构功能及其在恶性血液病细胞和正常造血细胞中的表达和调控,将有助于达到控制和治疗恶性血液病的目的。     

端粒重复因子2与p53的体外结合

目的 通过分析端粒主要结合蛋白端粒重复因子2(TRF2)与p53的体外结合,探讨p53通过端粒途径调节细胞增殖、衰老和凋亡的分子机制。方法 4种不同的p53-谷胱甘肽S转移酶(glutathione S-transferase,GST)融合蛋白和GST经大肠杆菌表达、谷胱甘肽-Sepharose^TM 4B纯化,其中的人重组p53包括野生型(1～393)、C端缺失体p53 N5(2～293)、N端缺失体p53 N5(95～393)、第175位氨基酸突变体(R→H)。将各纯化蛋白和人乳腺癌细胞MCF-7的细胞蛋白进行体外结合反应(pull down),Western blot检测反应物中p53和TRF2的结合。结果 纯化的GST和p53-GST融合蛋白纯度均在90％以上,且相对分子质量与预计的完全一致。TRF2的Western blot显示：野生型p53和p53-R 175H均能沉淀MCF-7中的TRF2,且结合力相似,而单独的GST则无沉淀TRF2的作用。与野生型p53和p53 R175H相比,p53 2C与TRF2的结合力相对增加,p53 N5与TRF2的结合力相对大大减弱。结论 p53和TRF2可以进行直接而特异的体外结合,且其结合部位在p53的C端(293～393)。p53和TRF2的C端依赖性结合可能与端粒动态变化所诱导的细胞活动有关。     

人端粒保护蛋白1(hPOT1)研究进展

随着对端粒、端粒酶研究的深入,一类被称为端粒结合蛋白的核蛋白越来越受到重视。人端粒保护蛋白1(human protection of telomeres 1,hPOT1)及其基因在2001年被鉴定,它是一种单链端粒结合蛋白,有两大功能相关结构域:OB fold(oligonucleotide/oligosaccharide binding fold)和TRF1(TTAGGG repeatbinding factor 1)相互作用蛋白结构域。它与端粒DNA的结合具有高度特异性和对单链端粒DNA游离3′末端结合位点的优先性。hPOT1蛋白可能具有多种重要的功能。目前有关hPOT1蛋白的研究越来越多,现就hPOT1的生物学性质作一综述。     

端粒、端粒酶的结构功能与肿瘤研究新进展

从ＤＮＡ、ＲＮＡ和蛋白质三方面，综合分析以往关于端粒和端粒酶的结构和功能的研究进展，及今年以来多项关于端粒结合蛋白及端粒酶结构蛋白的研究新突破，阐明了端粒长度及端粒酶活性与细胞凋亡、肿瘤发生和进展具有十分密切的关系。另外还注意到，随着“克隆羊”的诞生，以往关于端粒、端粒酶的理论势必面临新的挑战。     

MiR-138对MCF-7细胞端粒酶活性及端粒稳定性的调控作用

 目的 研究MiR-138通过HTERT作为下游靶基因,对人乳腺癌MCF-7细胞端粒酶活性的调控作用及端粒稳定性的影响。方法 在人乳腺癌MCF-7细胞中瞬时转染MiR-138 模拟物,用MTT法检测细胞增殖活性,并于转染后48h,用实时定量RT-PCR检测端粒酶催化亚单位HTERT表达、TRAP Assay检测端粒酶活性,同时对细胞进行53BP1 抗体免疫荧光染色及端粒的FISH染色。结果 转染后48h,MiR-138模拟物处理的MCF-7细胞HTERT表达水平比对照细胞降低2.18倍(2-△△Ct),端粒酶活性比对照细胞降低2.69倍,53BP1聚集形成的凝集点（Foci）,部分与端粒位点重合,比率达到20.62%±1.55% 。结论 MiR-138以HTERT作为下游靶分子,调控MCF-7细胞端粒酶活性,影响细胞端粒稳定性。     

Telomerase abrogation dramatically accelerates TRF2-induced epithelial carcinogenesis

TRF2 is a telomere-binding protein with roles in telomere protection and telomere-length regulation. The fact that TRF2 is up-regulated in some human tumors suggests a role of TRF2 in cancer. Mice that overexpress TRF2 in the skin, K5TRF2 mice, show critically short telomeres and are susceptible to UV-induced carcinogenesis as a result of deregulated XPF/ERCC1 activity, a nuclease involved in UV damage repair. Here we demonstrate that, when in combination with telomerase deficiency, TRF2 acts as a very potent oncogene in vivo. In particular, we show that telomerase deficiency dramatically accelerates TRF2-induced epithelial carcinogenesis in K5TRF2/Terc-/- mice, coinciding with increased chromosomal instability and DNA damage. Telomere recombination is also increased in these mice, suggesting that TRF2 favors the activation of alternative telomere maintenance mechanisms. Together, these results demonstrate that TRF2 increased expression is a potent oncogenic event that along with telomerase deficiency accelerates carcinogenesis, coincidental with a derepression of telomere recombination. These results are of particular relevance given that TRF2 is up-regulated in some human cancers. Furthermore, these data suggest that telomerase inhibition might not be effective to cease the growth of TRF2-overexpressing tumors.     

TRF1 is a critical trans-acting factor required for de novo telomere formation in human cells

The duplex telomere repeat (TTAGGG)(n) is an essential cis-acting element of the mammalian telomere, and an exogenous telomere repeat can induce chromosome breakage and de novo telomere formation at the site of a break (telomere seeding). Telomere seeding requires the telomere repeat (TTAGGG)(n) more stringently than does an in vitro telomerase assay, suggesting that it reflects the activity of a critical trans-acting element of the functional telomere, in addition to telomerase. Furthermore, telomere seeding is induced at a frequency fluctuating widely among human cell lines, suggesting variation in the activity of this hypothetical factor among cells. In this study, we investigated the cellular factor(s) required for telomere formation using the frequency of telomere seeding as an index and identified TRF1, one of the telomere repeat binding proteins, as an essential trans-acting factor. The exogenous telomere repeat induces telomere formation at a frequency determined by the availability of TRF1, even in telomerase-negative cells. Our study shows clearly that TRF1 has a novel physiological significance distinct from its role as a regulator of telomere length in the endogenous chromosome. The possible role of TRF1 in cell aging and immortalization is discussed.     

Critically short telomeres in acute myeloid leukemia with loss or gain of parts of chromosomes

Telomeres, nucleoprotein complexes at chromosome ends, protect chromosomes against end-to-end fusion. Previous in vitro studies in human fibroblast models indicated that telomere dysfunction results in chromosome instability. Loss of telomere function can result either from critical shortening of telomeric DNA or from loss of distinct telomere-capping proteins. It is less clear whether telomere dysfunction has an important role in human cancer development in vivo. Acute myeloid leukemia (AML) is a good model to study mechanisms that generate chromosome instability in human cancer development because distinct groups of AML are characterized either by aberrations that theoretically could result from telomere dysfunction (terminal deletions, gains/losses of chromosome parts, nonreciprocal translocations), or aberrations that are unlikely to result from telomere dysfunction (e.g., reciprocal translocations or inversions). Here we demonstrate that AML with multiple chromosome aberrations that theoretically could result from telomere dysfunction is invariably characterized by critically short telomeres. Short telomeres in this group are not associated with low telomerase activity or decreased expression of essential telomeric capping proteins TRF2 and POT1. In contrast, telomerase activity levels are significantly higher in AML with short telomeres. Notably, short telomeres in the presence of high telomerase may relate to significantly higher expression of TRF1, a negative regulator of telomere length. Our observations suggest that, consistent with previous in vitro fibroblast models, age-related critical telomere shortening may have a role in generating chromosome instability in human AML development.     

原发性胃癌组织中的端粒及端粒酶表达

目的观察端粒（ＴＲＦ）及端粒酶在胃癌形成及发展中的作用。方法采用ＤＮＡ琼脂糖凝胶杂交技术及端粒重复扩增ＰＣＲ方法检测１７例早期胃癌、８９例进展期胃癌组织及邻近正常组织中的平均ＴＲＦ长度及端粒酶活性。结果早期、进展期胃癌的ＴＲＦ长度及端粒酶活性表达均显著短于或高于邻近胃组织（Ｐ＜０．０５～０．０１），且端粒酶活性的表达主要表现在ＴＲＦ缩短或延长的胃癌组织中，而ＴＲＦ缩短与胃癌的分化程度相一致。结论端粒及端粒酶的异常状态可能与胃癌的发生发展密切相关，端粒酶活性及端粒缩短可以作为胃癌的诊断标志     

Lack of correlation between telomere length and telomerase activity and expression in leukemic cells

The expression of three components of telomerase complex (hTR, hTERT, TP1) along with telomerase activity and telomere length in leukemic cells was investigated. Cells were isolated from peripheral blood and/or bone marrow of children with acute lymphoblastic (ALL) and non-lymphoblastic (ANLL) leukemia. Expression of three components of telomerase as well as telomerase activity was found in all leukemic cells. Chemiluminescent detection of terminal restriction fragments (TRF) from DNA isolated from ALL cells showed variable patterns expressing considerable heterogeneity of telomere length. The ALL cells appeared to have both long and short telomere lengths, in contrast to normal peripheral lymphocytes, which produced limited pattern of TRF. The ANLL cells produced predominantly short telomere pattern despite high telomerase activity and expression. It can be concluded that high telomerase activity and expression in leukemic cells is not always correlated with long telomeres (TRF pattern).     

Telomere‐associated proteins: cross‐talk between telomere maintenance and telomere‐lengthening mechanisms

Telomeres, the ends of eukaryotic chromosomes, have been the subject of intense investigation over the last decade. As telomere dysfunction has been associated with ageing and developing cancer, understanding the exact mechanisms regulating telomere structure and function is essential for the prevention and treatment of human cancers and age‐related diseases. The mechanisms by which cells maintain telomere lengthening involve either telomerase or the alternative lengthening of the telomere pathway, although specific mechanisms of the latter and the relationship between the two are as yet unknown. Many cellular factors directly (TRF1/TRF2) and indirectly (shelterin‐complex, PinX, Apollo and tankyrase) interact with telomeres, and their interplay influences telomere structure and function. One challenge comes from the observation that many DNA damage response proteins are stably associated with telomeres and contribute to several other aspects of telomere function. This review focuses on the different components involved in telomere maintenance and their role in telomere length homeostasis. Special attention is paid to understanding how these telomere‐associated factors, and mainly those involved in double‐strand break repair, perform their activities at the telomere ends. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Post-translational modifications of TRF1 and TRF2 and their roles in telomere maintenance

Telomeres, heterochromatic structures, found at the ends of linear eukaryotic chromosomes, function to protect natural chromosome ends from nucleolytic attack. Human telomeric DNA is bound by a telomere-specific six-subunit protein complex, termed shelterin/telosome. The shelterin subunits TRF1 and TRF2 bind in a sequence-specific manner to double-stranded telomeric DNA, providing a vital platform for recruitment of additional shelterin proteins as well as non-shelterin factors crucial for the maintenance of telomere length and structure. Both TRF1 and TRF2 are engaged in multiple roles at telomeres including telomere protection, telomere replication, sister telomere resolution and telomere length maintenance. Regulation of TRF1 and TRF2 in these various processes is controlled by post-translational modifications, at times in a cell-cycle-dependent manner, affecting key functions such as DNA binding, dimerization, localization, degradation and interactions with other proteins. Here we review the post-translational modifications of TRF1 and TRF2 and discuss the mechanisms by which these modifications contribute to the function of these two proteins.