非小细胞肺癌TRF1的表达及核定位信号序列检测
摘要
研究背景与目的肺癌的发病率已成为恶性肿瘤之首，有关发生机理一直是研究的方向与热点。众多的研究表明，端粒是细胞衰老、死亡和癌变的重要标志，许多端粒蛋白参与了端粒结构稳定性的调控，当端粒蛋白不能保护端粒末端时，端粒酶得以将端粒重复序列TTAGGG加到端粒末端，造成细胞不朝衰老、死亡方向运动，而引起肿瘤。端粒重复序列结合因子(Telomeric Repeat Binding Factor 1，TRF1)就是端粒结合蛋白的一种，TRF1由439个氨基酸组成，分子量约60KD，研究结果表明TRF1是端粒延长的一个抑制因子，呈负反馈调节端粒的长度。TRF1与端粒DNA结合后，顺式抑制端粒酶的作用，从而控制端粒的长度。端粒酶是一种由RNA和蛋白质组成的核糖核蛋白体，具有逆转录酶的功能，能够将端粒重复序列TTAGGG加到染色体的末端。大量的恶性肿瘤细胞表达端粒酶，而大部分正常细胞不表达端粒酶。进一步的研究表明，TRF1对端粒酶的表达和活性没有影响，由此推测TRF1通过阻止端粒酶在端粒末端的作用来控制端粒长度。与TRF1密切相关的另一个端粒蛋白是端锚蛋白(Tankyrase 1，TANK1)，TANK1可以调节体内TRF1在端粒的聚集，在端粒酶阳性的细胞，TANK1长期过表达可使端粒长度进行性地延长，缺失PARP区域的TANK1对TRF1的分布和端粒长度的动力学无影响，因而TANK1可能是体内端粒延长的一种正调控因子，通过阻滞TRF1与端粒的结合发挥作用。根据“端粒长度调节的蛋白计数模型”，当TRF1减少至临界数目时，端粒中的TRF1向端粒酶提供的负反馈信号消失，端粒酶复合物激活，且脱落的TRF1为端粒酶与端粒的结合提供了入口，使激活的端粒酶结合到端粒上并催化合成端粒重复序列。随着端粒的延长，其结合的TRF1重新增至临界值，再次抑制端粒酶复合物与端粒的结合，维持了癌细胞端粒长度的稳定性，从而使癌细胞持续生长繁殖。
综上所述，人类端粒的功能需要端粒蛋白的调控，TRF1具有抑制端粒酶复合物与端粒结合的作用，而TANK1能够清除端粒DNA上的TRF1。因此，TRF1和TANK1有可能成为抑制肿瘤的新靶点。基于以上的认识，我们首先对50例非小细胞肺癌(NSCLC)患者进行端粒酶表达的检测，筛选出端粒酶阳性表达的患者，在此基础上，分别检测TRF1在NSCLC患者癌组织及癌旁组织中转录水平、翻译水平表达的差异，并通过免疫组化了解TRF1在癌组织及癌旁组织中的细胞定位，进一步对预测的核定位序列进行检测。暨希望能通过对TRF1在NSCLC中的一系列检测研究，获得端粒结合蛋白在端粒网络调控中的一个切入点。
第一部分TRF1及TANK1mRNA在端粒酶阳性的非小细胞肺癌中的表达
研究目的对端粒酶表达阳性的NSCLC患者进一步检测TRF1、TANK1mRNA表达水平，并探讨其表达水平与临床病理特征的相关性。
研究方法(1)研究对象2004年11月至2005年11月期间，收集NSCLC患者共50例，所有NSCLC病例均选自浙江大学医学院附属第一医院胸外科住院患者，并经病理切片证实；(2)对50例NSCLC患者首先进行免疫组化的端粒酶逆转录酶(TERT)检测，筛选出端粒酶阳性患者；(3)对端粒酶表达阳性的NSCLC患者，采用RT-PCR对TRF1、TANK1mRNA进行检测。
结果(1)NSCLC中TERT免疫组化结果：TERT在细胞浆及细胞核均有表达，50例NSCLC患者的癌组织中TERT阳性表达例数为36例，阳性率为72.0％(36/50)。(2)
端粒酶阳性表达的NSCLC患者的临床资料：36例患者端粒酶表达阳性，其中男性22例，女性14例；腺癌23例，鳞癌13例；(3)TRF1mRNA在癌组织及癌旁组织的表达：癌组织TRF1mRNA为1.101±0．119，相应的癌旁组织为2.015±2.337，经配对t检验，癌旁组织TRF1mRNA的表达水平明显高于癌组织，差异具有显著性(P=0.02)。(4)癌组织TRF1mRNA表达与病理组织学的关系：36例NSCLC患者中，肺腺癌23，肺鳞癌为13例。肺腺癌癌组织TRF1mRNA表达为1.075±0.823，肺鳞癌癌组织TRF1mRNA表达为1.149±0.514，经t检验，差异无显著性(P=0.773)。(5)癌组织TRF1mRNA表达与淋巴结转移的关系：36例NSCLC患者中，淋巴结阳性为19例，淋巴结阴性为17例。淋巴结阳性患者癌组织TRF1mRNA表达为0.893±0.619，淋巴结阴性患者癌组织TRF1mRNA表达为1.334±0.769，差异无显著性(P=0.070)。(6)癌组织TRF1mRNA表达与临床分期的关系：36例NSCLC中，Ⅰ期患者为9例，癌组织TRF1mRNA表达为1.198±0.619；Ⅱ期患者为9例，癌组织TRF1mRNA表达为1.337±0.939，Ⅲ期为16例，Ⅳ期患者为2例，两期合并统计，癌组织TRF1mRNA表达为0.936±0.637，经One-Way ANOVA分析，差异无显著性(P=0.364)。(7)癌组织TRF1mRNA表达与分化程度的关系：36例NSCLC患者中，分化程度高中度的为21例，TRF1mRNA表达为1.309±0.784；分化程度低的为15例，TRF1mRNA表达为0.811±0.510，差异有显著性(P=0.040)。(8)癌组织及癌旁组织中TANK1mRNA表达：TANK1mRNA在癌组织及癌旁组织均有表达，TANK1mRNA在癌组织表达为0.572±0.423，癌旁组织为0.430±0.336，癌组织中TANK1表达水平比癌旁组织明显增加，差异有显著性(P=0.04)。(9)癌组织中TANK1mRNA表达与临床病理特征的关系：经统计学分析，未发现癌组织中TANK1mRNA表达水平与性别、病理、淋巴结转移、分化程度、临床分期有统计学相关性(P＞0.05)。(10)癌组织中TRF1mRNA表达水平与TANK1mRNA表达水平的相关性分析：经Spearman相关性检验，未发现两者在转录水平上有统计相关性(r=0.098，p=0.605)。
结论(1)NSCLC患者中，癌组织中的TRF1mRNA表达水平比癌旁组织中明显下降；(2)NSCLC患者中，癌组织中的TRF1mRNA表达水平与细胞分化程度相关，低分化的NSCLC患者TRF1mRNA表达水平比高中分化者明显降低；(3)NSCLC患者中，癌组织中的TANK1mRNA表达水平比癌旁组织中明显升高。
第二部分TRF1蛋白在非小细胞肺癌中的表达
研究目的检测TRF1蛋白在NSCLC中的表达水平，探讨TRF1蛋白表达水平与临床病理指标之间的关系。
研究方法应用Western blot方法检测NSCLC患者癌组织及癌旁组织中TRF1蛋白表达水平。
结果(1)TRF1蛋白在NSCLC癌组织及癌旁组织中的表达：36例NSCLC患者癌组织中TRF1蛋白表达阳性者为30例，癌旁组织表达阳性为33例，为了配对t检验，30例样本进入统计分析。(2)NSCLC患者TRF1在癌组织及癌旁组织的表达：30例NSCLC患者癌组织TRF1蛋白为0.552±0.329，相应的癌旁组织为0.652±0.476，经配对t检验，癌旁组织TRF1的蛋白表达水平明显高于癌组织，且差异具有显著性(P=0.028)。(3)癌组织TRF1蛋白表达水平与病理组织学的关系：30例NSCLC中，肺腺癌19，肺鳞癌为11例。肺腺癌癌组织TRF1蛋白表达为0.602±0.397，肺鳞癌癌组织TRF1蛋白表达为0.466±0.132，经t检验，差异无显著性(P=0.601)。(4)癌组织TRF1蛋白表达与淋巴结转移的关系：30例NSCLC中，淋巴结阳性者为15例，淋巴结阴性者为15例。淋巴结阳性患者癌组织TRF1蛋白表达为0.593±0.309，淋巴结阴性患者癌组织TRF1蛋白表达为0.512±0.354，差异无显著性(P=0.509)。(5)癌组织TRF1蛋白表达与临床分期的关系：30例NSCLC中，Ⅰ期患者为8例，癌组织TRF1蛋白表达为0.481±0.250；Ⅱ期患者为7例，癌组织TRF1蛋白表达为0.797±0.487，Ⅲ期为14例，Ⅳ期患者为1例，两期合并统计，癌组织TRF1表达为0.477±0.229，经One-Way ANOVA分析，差异无显著性(P=0.076)。(6)癌组织TRF1蛋白表达与分化程度的关系：30例NSCLC患者中，分化程度高中度的为16例，TRF1表达为0.481±0.148；分化程度低下的为14例，TRF1表达为0.634±0.451，差异无显著性(P=0.240)。(7) TRF1mRNA表达水平与TRF1蛋白表达水平的相关性检验：将其与相对应的TRF1mRNA的检测标本进行Spearman相关性检验，结果提示NSCLC中TRF1mRNA表达水平与TRF1蛋白表达水平无统计相关性(r=0.099，t=0.603，P＞0.05)。
结论(1)NSCLC患者中，TRF1蛋白在癌组织中的表达水平比癌旁组织明显降低。(2)NSCLC患者中，癌组织的TRF1蛋白表达水平在不同病理分型、淋巴结转移、临床分期、分化程度方面的差异无显著性。
第三部分TRF1蛋白在非小细胞肺癌中的免疫组化定位
研究目的探讨TRF1蛋白在NSCLC中的细胞定位，为深入了解TRF1在NSCLC中的作用提供依据。
研究方法免疫组化方法检测TRF1蛋白在NSCLC中的分布。
结果TRF1蛋白阳性信号为细颗粒状或片状染色，不论癌组织或癌旁组织阳性表达均较弱，为局限性的表达，浅棕黄色染色．主要表达于细胞浆内，少量表达于细胞核内。TRF1蛋白在癌旁组织中的阳性表达率为50.0％，癌组织中为44.44％，差异无显著性(P＞0.05)；癌组织中核浆均阳性表达为22.22％，癌旁组织核浆均阳性表达为33.33％。差异无显著性(P＞0.05)；腺癌、鳞癌之间的TRF1表达阳性率无显著性差异(P＞0.05)。
结论(1)免疫组化结果表明，NSCLC患者中肺癌组织的TRF1的阳性表达率为44.44％，癌旁组织为50.0％；(2)免疫组化结果表明，NSCLC患者中TRF1的表达主要在细胞浆中，部分呈浆、核表达双阳性。
第四部分非小细胞肺癌中TRF1蛋白的核定位信号序列检测
研究目的探讨TRF1蛋白在NSCLC中的细胞核定位信号序列是否存在碱基突变或缺失，为深入了解TRF1蛋白在NSCLC中的作用提供依据。
研究方法PSORTⅡ程序预测TRF1蛋白的核定位信号，PCR扩增相应的序列，并进行测序。
结果将NSCLC患者的肺癌组织、癌旁组织检测结果进行对比，核定位信号序列中未发现有突变或缺失的碱基。
结论对预测的核定位信号序列进行测序，结果表明NSCLC患者中，未发现TRF1蛋白核定位信号序列存在碱基突变或缺失。
Background and Objective Non-small cell lung cancer (NSCLC) is the most common human malignant tumor. The goals and hotpoints of current studies are the molecular mechanisms underlying NSCLC etiology. In recent years many factors associated with telomere control have been found. Telomeres are composed of tandem arrays of a short DNA sequence, 5'(TTAGGG)n3' in vertebrates, and associated proteins. They are essential genetic elements to stabilize the natural ends of linear eukaryotic chromosomes and protect the DNA ends from degradation and fusion. Telomeric DNA-binding proteins have an essential role both in regulation of the length of the telomeric DNA tract and in protection against chromosome end-to-end fusion. Telomeric repeat binding factor 1 (TRF1), one of the important telomeric binding proteins, consists of 438 amino acid and its molecular weight is about 60KD. In human cells, the changes of telomeric length have been implicated in the molecular clock controlling cell senescence and as a step in tumorigenesis. The maintenance of telomeric length is important for cancer cells to keep immortality. TRF1 plays pivotal roles in telomere protection and maintenance in mammalian cells. TRF1 can
negatively regulate telomeric length by inhibiting the interaction between telomerase and telomere. Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA at the end of chromosomes and compensates for the end replication problems. It has been shown that telomerase is activated in a large majority of human cancer tissues, but not in most normal tissues and tissues adjacent to malignant or benign tumors. A similar protein-counting model was proposed for telomeric length homeostasis. The expression of a dominant negative allele of TRF1, which removes endogenous TRF1 from telomeres, leads to telomere elongation. In this system, TRF1 did not affect the activity of telomerase detectable in cell extracts, which suggests that TRF1 does not affect telomerase activity globally in the cell. Instead, TRF1 acts in cis as a negative length regulator at each individual telomere.
Tankyrase(TANK1), originally identitied as a TRF1 binding protein, is a member of the growing family of poly(ADP-ribose) polymerase(PARPs). TANK1 mediated ADP-ribosylation inhibits binding of TRF1 into telomeric repeats in vitro. Ribosylation by TANK1 displaces TRF1 from telomeric DNA. This finding suggested that TANK1 might be a positive regulator of telomeric length in telomerase -expressing cells. Indeed, when TANK1 protein overexpressed, telomere length increased in telomerase-positive tumor cells. According to the protein-counting model, very short telomeres would not bind sufficient amounts of TRF1. Long telomere would recruit a large amount of TRF1 protein, blocking telomerase-mediated telomeric elongation.
Based on the current datas, the emerging view is that long telomeres recruit a larger amount of TRF1 and TANK1 mediated ADP-ribosylation inhibits binding of TRF1 into telomeric repeats. This indicates that TRF1 and TANK1 may serve as a potential target for cancer therapy. On the basis of the results from the studies above, we used immunohistochemical techniques to measure expression of telomerase
reverse transcriptase (TERT) in 50 patients and detect the expression of TRF1 with RT-PCR and Western blot in 36 patients of telomerase positive. The cellular localization of TRF1 in NSCLC was observed by immunohistochemistry and the sequence of cellular nuclear localization signal was measured by DNA sequencing techniques. As the molecular mechanism of the TRF1 in NSCLC has not yet been clarified, this study could help to provide clues to the role of TRF1 in the telomere regulation.
Part I The expression of TRF1 mRNA and TANK1 mRNA in Non-Small Cell Lung Cancer of telomerase positive
Objective To investigate the expression of TRF1 mRNA and TANK1 mRNA in NSCLC of telomerase positive and analyze the relationship between the TRF1 mRNA and TANK1 mRNA levels.
Methods (1) From 2004 Nov to 2005 Nov, 50 patients with NSCLCs had undergone radical operations in The First Affiliated Hospital,College of Medicine,Zhejiang University. The diagnosis of lung cancer was obtained according to the pathohistologic examination in all cases. (2) The expressions of TERT were detected by immunohistochemistry. (3) The expressions of TRF1 mRNA and TANK1 mRNA in NSCLC with telomerase positive were investigated by RT-PCR.
Results (1) Immunohistochemistry: TERT expression in NSCLC was found at the cell nuclear and cytoplasm. In 50 lung cancers specimens studied with the immunohistochemical method, 36(36/50, 72.0%) were showed telomerase positive. (2) Clinical information of telomerase positive patients: 36 patients with telomerase positive patients included 22 males and 14 females. There were 23 samples of adenocarcinoma and 13 samples of squamous cell carcinoma. (3) Expression of TRF1 mRNA in cancer tissues and paried noncancerous tissues: The mean±s of
TRF1 mRNA in cancer tissues and paired noncancerous tissues was ±0.119 and 2.015±2.337, respectively . It showed that TRF1mRNA levels were higher in paired noncancerous tissues than in cancer tissues (p=0.02). (4) There were no significant differences in TRF1 mRNA expression between adenocarcinoma and squamous cell carcinoma (P=0.773) . (5) The mean± s of TRF1 mRNA in lymph node metastasis and no lymph node metastasis was 0.893±0.619 and 1.334±0.769, respectively. No significant difference was found between in lymph node metastasis and no lymph node metastasis (P=0.070) . (6) One-Way ANOVA was employed to evaluate the TRF1 mRNA expression in clinical stages. There were no significant differences in TRF1 mRNA expression among different clinical stages (P=0.364). (7) Expression of TRF1mRNA in variable grade of differentiation : The mean±s of TRF1mRNA in moderate differentiated and in poorly differentiated was 1.309±0.784 and 0.811±0.510, respectively. The results showed that the expression of TRF1 mRNA in poorly differentiated tumor was significantly down-regulated when compared with the moderated differentiated tumor (P=0.040) . (8) TANK1 mRNA was significantly up-regulated in cancer tissue when compared with the paired noncancerous tissue (P=0.04). (9) No significant difference of TANK1 mRNA level was found among sexes, different clinical stages, pathological subtypes and lymph node metastasis (p>0.05). (10) Spearman test was employed to evaluate the correlation between TRF1 mRNA and TANK1 mRNA. No correlation was observed between TRF1 mRNA and TANK1 mRNA levels (r=0.098, P=0.605).
Conclusions (1) Down-regulation of TRF1 mRNA expression was found in lung cancer tissues. (2) The expression of TRF1 mRNA was significantly associated with grade of tumor differentiation. (3) Up-regulation of TANK1 mRNA was found in lung cancer tissues.
Part II The expression of TRF1 protein in Non-Small Cell Lung Cancer
Objective To detect the expression of TRF1 protein in NSCLC and analyze the relationship between the TRF1 and clinic factors.
Methods The levels of TRF1 in cancer tissues and paired noncancerous tissues were measured by Western blot.
Results (1) Expression of TRF1 in cancer tissues and paried noncancerous tissues: 36 lung cancers samples studied with Western blot method, however, the expression of TRF1 in cancer tissues and paried noncancerous tissues was 30 and 33 samples, respectively. (2) TRF1 expression of cancer tissues and paried noncancerous tissues in 30 cases: The mean±s of TRF1 in cancer tissues and in paired noncancerous tissues was 0.552±0.329 and 0.652±0.476, respectively. The results showed that TRF1 levels were higher in paired noncancerous tissues than in cancer tissues (p=0.028). (3) There were no significant differences in TRF1 expression between adenocarcinoma and squamous cell carcinoma (P=0.601) .(4) The mean±s of TRF1 in the positive lymph node and in the negative lymph node was 0.593±0.309 and 0.512±0.354, respectively . No significant difference was found between in lymph node metastasis and no lymph node metastasis (P=0.509) . (5) One-Way ANOVA was employed to evaluate the relationship between TRF1 expression and clinical stages. There was no significant difference in TRF1 expression levels among clinical stages (P=0.076). (6) Expression of TRF1 in variable grade of differentiation : The mean±s of TRF1 in tumor with moderate differentiation and in tumor with poor differentiation was 0.481±0.148and 0.634±0.451, respectively . The results showed that the expression of TRF1 was no significantly different between moderate differentiated tumor and poorly moderate
differentiated (P=0.24) (10) Spearman test was employed to evaluate the correlation between TRF1 mRNA and TRF1 protein. No correlation was observed between TRF1 mRNA and TRF1 protein levels (r=0. 099, t=0.603, P>0.05).
Conclusions (1)Down-regulation of TRF1 expression was found in lung cancer tissues.(2) No significant difference of TRF1 expression was found among sexes, different clinical stages, pathological subtypes and lymph node metastasis
Part III Cellular localization of TRF1 protein in Non-Small Cell Lung Cancer by immunohistochemistry
Objective To investigate the cellular localization of TRF1 in NSCLC.
Methods Cellular localization of TRF1 in NSCLC was evaluated by immunohistochemistry.
Results (1) The expression of TRF1 protein was evaluated by immunohistochemical staining and it mainly accumulated in the cytoplasm. In most cells, the signal was generally weak. TRF1 immunoreactivity was expressed at variable intensity and distribution. The positive rates of TRF1 were 16 of 36 cases (44.4%) in cancer tissues, while 18 of 36 cases (50.0 % ) in paired noncancerous tissues. There was no significant difference in the positive rates of TRF1 between the cancer tissues and paired noncancerous tissues (P>0.05).The positive rates of both nucleus and cytoplasm in cancer tissues were 22.22%, and 33.33% in paired noncancerous tissues. No significant difference was found between them, and also between adenocarcinoma and squamous cell carcinoma.
Conclusions (1) Immunohistochemistry results showed that the positive staining
percentages of TRF1 were 44.44% in cancer tissues and 50% in paired noncancerous tissues. (2) Immunohistochemistry results indicate that the expression of TRF1 protein mainly accumulated in the cytoplasm.
Part IV DNA sequencing analysis of the nuclear localization signal of TRF1 in Non-Small Cell Lung Cancer
Objective To investigate the base mutation of nuclear localization signal DNA sequence of TRF1 in NSCLC.
Methods The nuclear localization signal (NLS) of TRF1 was predicted by PSORT II, and then the NLS sequence was detected by sequencing.
Results The results showed that there was no difference in the NLS sequence of TRF1 between cancer tissues and paired noncancerous tissues.
Conclusions There was no base mutation in TRF1 NLS sequence of NSCLC.
综上所述，人类端粒的功能需要端粒蛋白的调控，TRF1具有抑制端粒酶复合物与端粒结合的作用，而TANK1能够清除端粒DNA上的TRF1。因此，TRF1和TANK1有可能成为抑制肿瘤的新靶点。基于以上的认识，我们首先对50例非小细胞肺癌(NSCLC)患者进行端粒酶表达的检测，筛选出端粒酶阳性表达的患者，在此基础上，分别检测TRF1在NSCLC患者癌组织及癌旁组织中转录水平、翻译水平表达的差异，并通过免疫组化了解TRF1在癌组织及癌旁组织中的细胞定位，进一步对预测的核定位序列进行检测。暨希望能通过对TRF1在NSCLC中的一系列检测研究，获得端粒结合蛋白在端粒网络调控中的一个切入点。
第一部分TRF1及TANK1mRNA在端粒酶阳性的非小细胞肺癌中的表达
研究目的对端粒酶表达阳性的NSCLC患者进一步检测TRF1、TANK1mRNA表达水平，并探讨其表达水平与临床病理特征的相关性。
研究方法(1)研究对象2004年11月至2005年11月期间，收集NSCLC患者共50例，所有NSCLC病例均选自浙江大学医学院附属第一医院胸外科住院患者，并经病理切片证实；(2)对50例NSCLC患者首先进行免疫组化的端粒酶逆转录酶(TERT)检测，筛选出端粒酶阳性患者；(3)对端粒酶表达阳性的NSCLC患者，采用RT-PCR对TRF1、TANK1mRNA进行检测。
结果(1)NSCLC中TERT免疫组化结果：TERT在细胞浆及细胞核均有表达，50例NSCLC患者的癌组织中TERT阳性表达例数为36例，阳性率为72.0％(36/50)。(2)
端粒酶阳性表达的NSCLC患者的临床资料：36例患者端粒酶表达阳性，其中男性22例，女性14例；腺癌23例，鳞癌13例；(3)TRF1mRNA在癌组织及癌旁组织的表达：癌组织TRF1mRNA为1.101±0．119，相应的癌旁组织为2.015±2.337，经配对t检验，癌旁组织TRF1mRNA的表达水平明显高于癌组织，差异具有显著性(P=0.02)。(4)癌组织TRF1mRNA表达与病理组织学的关系：36例NSCLC患者中，肺腺癌23，肺鳞癌为13例。肺腺癌癌组织TRF1mRNA表达为1.075±0.823，肺鳞癌癌组织TRF1mRNA表达为1.149±0.514，经t检验，差异无显著性(P=0.773)。(5)癌组织TRF1mRNA表达与淋巴结转移的关系：36例NSCLC患者中，淋巴结阳性为19例，淋巴结阴性为17例。淋巴结阳性患者癌组织TRF1mRNA表达为0.893±0.619，淋巴结阴性患者癌组织TRF1mRNA表达为1.334±0.769，差异无显著性(P=0.070)。(6)癌组织TRF1mRNA表达与临床分期的关系：36例NSCLC中，Ⅰ期患者为9例，癌组织TRF1mRNA表达为1.198±0.619；Ⅱ期患者为9例，癌组织TRF1mRNA表达为1.337±0.939，Ⅲ期为16例，Ⅳ期患者为2例，两期合并统计，癌组织TRF1mRNA表达为0.936±0.637，经One-Way ANOVA分析，差异无显著性(P=0.364)。(7)癌组织TRF1mRNA表达与分化程度的关系：36例NSCLC患者中，分化程度高中度的为21例，TRF1mRNA表达为1.309±0.784；分化程度低的为15例，TRF1mRNA表达为0.811±0.510，差异有显著性(P=0.040)。(8)癌组织及癌旁组织中TANK1mRNA表达：TANK1mRNA在癌组织及癌旁组织均有表达，TANK1mRNA在癌组织表达为0.572±0.423，癌旁组织为0.430±0.336，癌组织中TANK1表达水平比癌旁组织明显增加，差异有显著性(P=0.04)。(9)癌组织中TANK1mRNA表达与临床病理特征的关系：经统计学分析，未发现癌组织中TANK1mRNA表达水平与性别、病理、淋巴结转移、分化程度、临床分期有统计学相关性(P＞0.05)。(10)癌组织中TRF1mRNA表达水平与TANK1mRNA表达水平的相关性分析：经Spearman相关性检验，未发现两者在转录水平上有统计相关性(r=0.098，p=0.605)。
结论(1)NSCLC患者中，癌组织中的TRF1mRNA表达水平比癌旁组织中明显下降；(2)NSCLC患者中，癌组织中的TRF1mRNA表达水平与细胞分化程度相关，低分化的NSCLC患者TRF1mRNA表达水平比高中分化者明显降低；(3)NSCLC患者中，癌组织中的TANK1mRNA表达水平比癌旁组织中明显升高。
第二部分TRF1蛋白在非小细胞肺癌中的表达
研究目的检测TRF1蛋白在NSCLC中的表达水平，探讨TRF1蛋白表达水平与临床病理指标之间的关系。
研究方法应用Western blot方法检测NSCLC患者癌组织及癌旁组织中TRF1蛋白表达水平。
结果(1)TRF1蛋白在NSCLC癌组织及癌旁组织中的表达：36例NSCLC患者癌组织中TRF1蛋白表达阳性者为30例，癌旁组织表达阳性为33例，为了配对t检验，30例样本进入统计分析。(2)NSCLC患者TRF1在癌组织及癌旁组织的表达：30例NSCLC患者癌组织TRF1蛋白为0.552±0.329，相应的癌旁组织为0.652±0.476，经配对t检验，癌旁组织TRF1的蛋白表达水平明显高于癌组织，且差异具有显著性(P=0.028)。(3)癌组织TRF1蛋白表达水平与病理组织学的关系：30例NSCLC中，肺腺癌19，肺鳞癌为11例。肺腺癌癌组织TRF1蛋白表达为0.602±0.397，肺鳞癌癌组织TRF1蛋白表达为0.466±0.132，经t检验，差异无显著性(P=0.601)。(4)癌组织TRF1蛋白表达与淋巴结转移的关系：30例NSCLC中，淋巴结阳性者为15例，淋巴结阴性者为15例。淋巴结阳性患者癌组织TRF1蛋白表达为0.593±0.309，淋巴结阴性患者癌组织TRF1蛋白表达为0.512±0.354，差异无显著性(P=0.509)。(5)癌组织TRF1蛋白表达与临床分期的关系：30例NSCLC中，Ⅰ期患者为8例，癌组织TRF1蛋白表达为0.481±0.250；Ⅱ期患者为7例，癌组织TRF1蛋白表达为0.797±0.487，Ⅲ期为14例，Ⅳ期患者为1例，两期合并统计，癌组织TRF1表达为0.477±0.229，经One-Way ANOVA分析，差异无显著性(P=0.076)。(6)癌组织TRF1蛋白表达与分化程度的关系：30例NSCLC患者中，分化程度高中度的为16例，TRF1表达为0.481±0.148；分化程度低下的为14例，TRF1表达为0.634±0.451，差异无显著性(P=0.240)。(7) TRF1mRNA表达水平与TRF1蛋白表达水平的相关性检验：将其与相对应的TRF1mRNA的检测标本进行Spearman相关性检验，结果提示NSCLC中TRF1mRNA表达水平与TRF1蛋白表达水平无统计相关性(r=0.099，t=0.603，P＞0.05)。
结论(1)NSCLC患者中，TRF1蛋白在癌组织中的表达水平比癌旁组织明显降低。(2)NSCLC患者中，癌组织的TRF1蛋白表达水平在不同病理分型、淋巴结转移、临床分期、分化程度方面的差异无显著性。
第三部分TRF1蛋白在非小细胞肺癌中的免疫组化定位
研究目的探讨TRF1蛋白在NSCLC中的细胞定位，为深入了解TRF1在NSCLC中的作用提供依据。
研究方法免疫组化方法检测TRF1蛋白在NSCLC中的分布。
结果TRF1蛋白阳性信号为细颗粒状或片状染色，不论癌组织或癌旁组织阳性表达均较弱，为局限性的表达，浅棕黄色染色．主要表达于细胞浆内，少量表达于细胞核内。TRF1蛋白在癌旁组织中的阳性表达率为50.0％，癌组织中为44.44％，差异无显著性(P＞0.05)；癌组织中核浆均阳性表达为22.22％，癌旁组织核浆均阳性表达为33.33％。差异无显著性(P＞0.05)；腺癌、鳞癌之间的TRF1表达阳性率无显著性差异(P＞0.05)。
结论(1)免疫组化结果表明，NSCLC患者中肺癌组织的TRF1的阳性表达率为44.44％，癌旁组织为50.0％；(2)免疫组化结果表明，NSCLC患者中TRF1的表达主要在细胞浆中，部分呈浆、核表达双阳性。
第四部分非小细胞肺癌中TRF1蛋白的核定位信号序列检测
研究目的探讨TRF1蛋白在NSCLC中的细胞核定位信号序列是否存在碱基突变或缺失，为深入了解TRF1蛋白在NSCLC中的作用提供依据。
研究方法PSORTⅡ程序预测TRF1蛋白的核定位信号，PCR扩增相应的序列，并进行测序。
结果将NSCLC患者的肺癌组织、癌旁组织检测结果进行对比，核定位信号序列中未发现有突变或缺失的碱基。
结论对预测的核定位信号序列进行测序，结果表明NSCLC患者中，未发现TRF1蛋白核定位信号序列存在碱基突变或缺失。
Background and Objective Non-small cell lung cancer (NSCLC) is the most common human malignant tumor. The goals and hotpoints of current studies are the molecular mechanisms underlying NSCLC etiology. In recent years many factors associated with telomere control have been found. Telomeres are composed of tandem arrays of a short DNA sequence, 5'(TTAGGG)n3' in vertebrates, and associated proteins. They are essential genetic elements to stabilize the natural ends of linear eukaryotic chromosomes and protect the DNA ends from degradation and fusion. Telomeric DNA-binding proteins have an essential role both in regulation of the length of the telomeric DNA tract and in protection against chromosome end-to-end fusion. Telomeric repeat binding factor 1 (TRF1), one of the important telomeric binding proteins, consists of 438 amino acid and its molecular weight is about 60KD. In human cells, the changes of telomeric length have been implicated in the molecular clock controlling cell senescence and as a step in tumorigenesis. The maintenance of telomeric length is important for cancer cells to keep immortality. TRF1 plays pivotal roles in telomere protection and maintenance in mammalian cells. TRF1 can
negatively regulate telomeric length by inhibiting the interaction between telomerase and telomere. Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA at the end of chromosomes and compensates for the end replication problems. It has been shown that telomerase is activated in a large majority of human cancer tissues, but not in most normal tissues and tissues adjacent to malignant or benign tumors. A similar protein-counting model was proposed for telomeric length homeostasis. The expression of a dominant negative allele of TRF1, which removes endogenous TRF1 from telomeres, leads to telomere elongation. In this system, TRF1 did not affect the activity of telomerase detectable in cell extracts, which suggests that TRF1 does not affect telomerase activity globally in the cell. Instead, TRF1 acts in cis as a negative length regulator at each individual telomere.
Tankyrase(TANK1), originally identitied as a TRF1 binding protein, is a member of the growing family of poly(ADP-ribose) polymerase(PARPs). TANK1 mediated ADP-ribosylation inhibits binding of TRF1 into telomeric repeats in vitro. Ribosylation by TANK1 displaces TRF1 from telomeric DNA. This finding suggested that TANK1 might be a positive regulator of telomeric length in telomerase -expressing cells. Indeed, when TANK1 protein overexpressed, telomere length increased in telomerase-positive tumor cells. According to the protein-counting model, very short telomeres would not bind sufficient amounts of TRF1. Long telomere would recruit a large amount of TRF1 protein, blocking telomerase-mediated telomeric elongation.
Based on the current datas, the emerging view is that long telomeres recruit a larger amount of TRF1 and TANK1 mediated ADP-ribosylation inhibits binding of TRF1 into telomeric repeats. This indicates that TRF1 and TANK1 may serve as a potential target for cancer therapy. On the basis of the results from the studies above, we used immunohistochemical techniques to measure expression of telomerase
reverse transcriptase (TERT) in 50 patients and detect the expression of TRF1 with RT-PCR and Western blot in 36 patients of telomerase positive. The cellular localization of TRF1 in NSCLC was observed by immunohistochemistry and the sequence of cellular nuclear localization signal was measured by DNA sequencing techniques. As the molecular mechanism of the TRF1 in NSCLC has not yet been clarified, this study could help to provide clues to the role of TRF1 in the telomere regulation.
Part I The expression of TRF1 mRNA and TANK1 mRNA in Non-Small Cell Lung Cancer of telomerase positive
Objective To investigate the expression of TRF1 mRNA and TANK1 mRNA in NSCLC of telomerase positive and analyze the relationship between the TRF1 mRNA and TANK1 mRNA levels.
Methods (1) From 2004 Nov to 2005 Nov, 50 patients with NSCLCs had undergone radical operations in The First Affiliated Hospital,College of Medicine,Zhejiang University. The diagnosis of lung cancer was obtained according to the pathohistologic examination in all cases. (2) The expressions of TERT were detected by immunohistochemistry. (3) The expressions of TRF1 mRNA and TANK1 mRNA in NSCLC with telomerase positive were investigated by RT-PCR.
Results (1) Immunohistochemistry: TERT expression in NSCLC was found at the cell nuclear and cytoplasm. In 50 lung cancers specimens studied with the immunohistochemical method, 36(36/50, 72.0%) were showed telomerase positive. (2) Clinical information of telomerase positive patients: 36 patients with telomerase positive patients included 22 males and 14 females. There were 23 samples of adenocarcinoma and 13 samples of squamous cell carcinoma. (3) Expression of TRF1 mRNA in cancer tissues and paried noncancerous tissues: The mean±s of
TRF1 mRNA in cancer tissues and paired noncancerous tissues was ±0.119 and 2.015±2.337, respectively . It showed that TRF1mRNA levels were higher in paired noncancerous tissues than in cancer tissues (p=0.02). (4) There were no significant differences in TRF1 mRNA expression between adenocarcinoma and squamous cell carcinoma (P=0.773) . (5) The mean± s of TRF1 mRNA in lymph node metastasis and no lymph node metastasis was 0.893±0.619 and 1.334±0.769, respectively. No significant difference was found between in lymph node metastasis and no lymph node metastasis (P=0.070) . (6) One-Way ANOVA was employed to evaluate the TRF1 mRNA expression in clinical stages. There were no significant differences in TRF1 mRNA expression among different clinical stages (P=0.364). (7) Expression of TRF1mRNA in variable grade of differentiation : The mean±s of TRF1mRNA in moderate differentiated and in poorly differentiated was 1.309±0.784 and 0.811±0.510, respectively. The results showed that the expression of TRF1 mRNA in poorly differentiated tumor was significantly down-regulated when compared with the moderated differentiated tumor (P=0.040) . (8) TANK1 mRNA was significantly up-regulated in cancer tissue when compared with the paired noncancerous tissue (P=0.04). (9) No significant difference of TANK1 mRNA level was found among sexes, different clinical stages, pathological subtypes and lymph node metastasis (p>0.05). (10) Spearman test was employed to evaluate the correlation between TRF1 mRNA and TANK1 mRNA. No correlation was observed between TRF1 mRNA and TANK1 mRNA levels (r=0.098, P=0.605).
Conclusions (1) Down-regulation of TRF1 mRNA expression was found in lung cancer tissues. (2) The expression of TRF1 mRNA was significantly associated with grade of tumor differentiation. (3) Up-regulation of TANK1 mRNA was found in lung cancer tissues.
Part II The expression of TRF1 protein in Non-Small Cell Lung Cancer
Objective To detect the expression of TRF1 protein in NSCLC and analyze the relationship between the TRF1 and clinic factors.
Methods The levels of TRF1 in cancer tissues and paired noncancerous tissues were measured by Western blot.
Results (1) Expression of TRF1 in cancer tissues and paried noncancerous tissues: 36 lung cancers samples studied with Western blot method, however, the expression of TRF1 in cancer tissues and paried noncancerous tissues was 30 and 33 samples, respectively. (2) TRF1 expression of cancer tissues and paried noncancerous tissues in 30 cases: The mean±s of TRF1 in cancer tissues and in paired noncancerous tissues was 0.552±0.329 and 0.652±0.476, respectively. The results showed that TRF1 levels were higher in paired noncancerous tissues than in cancer tissues (p=0.028). (3) There were no significant differences in TRF1 expression between adenocarcinoma and squamous cell carcinoma (P=0.601) .(4) The mean±s of TRF1 in the positive lymph node and in the negative lymph node was 0.593±0.309 and 0.512±0.354, respectively . No significant difference was found between in lymph node metastasis and no lymph node metastasis (P=0.509) . (5) One-Way ANOVA was employed to evaluate the relationship between TRF1 expression and clinical stages. There was no significant difference in TRF1 expression levels among clinical stages (P=0.076). (6) Expression of TRF1 in variable grade of differentiation : The mean±s of TRF1 in tumor with moderate differentiation and in tumor with poor differentiation was 0.481±0.148and 0.634±0.451, respectively . The results showed that the expression of TRF1 was no significantly different between moderate differentiated tumor and poorly moderate
differentiated (P=0.24) (10) Spearman test was employed to evaluate the correlation between TRF1 mRNA and TRF1 protein. No correlation was observed between TRF1 mRNA and TRF1 protein levels (r=0. 099, t=0.603, P>0.05).
Conclusions (1)Down-regulation of TRF1 expression was found in lung cancer tissues.(2) No significant difference of TRF1 expression was found among sexes, different clinical stages, pathological subtypes and lymph node metastasis
Part III Cellular localization of TRF1 protein in Non-Small Cell Lung Cancer by immunohistochemistry
Objective To investigate the cellular localization of TRF1 in NSCLC.
Methods Cellular localization of TRF1 in NSCLC was evaluated by immunohistochemistry.
Results (1) The expression of TRF1 protein was evaluated by immunohistochemical staining and it mainly accumulated in the cytoplasm. In most cells, the signal was generally weak. TRF1 immunoreactivity was expressed at variable intensity and distribution. The positive rates of TRF1 were 16 of 36 cases (44.4%) in cancer tissues, while 18 of 36 cases (50.0 % ) in paired noncancerous tissues. There was no significant difference in the positive rates of TRF1 between the cancer tissues and paired noncancerous tissues (P>0.05).The positive rates of both nucleus and cytoplasm in cancer tissues were 22.22%, and 33.33% in paired noncancerous tissues. No significant difference was found between them, and also between adenocarcinoma and squamous cell carcinoma.
Conclusions (1) Immunohistochemistry results showed that the positive staining
percentages of TRF1 were 44.44% in cancer tissues and 50% in paired noncancerous tissues. (2) Immunohistochemistry results indicate that the expression of TRF1 protein mainly accumulated in the cytoplasm.
Part IV DNA sequencing analysis of the nuclear localization signal of TRF1 in Non-Small Cell Lung Cancer
Objective To investigate the base mutation of nuclear localization signal DNA sequence of TRF1 in NSCLC.
Methods The nuclear localization signal (NLS) of TRF1 was predicted by PSORT II, and then the NLS sequence was detected by sequencing.
Results The results showed that there was no difference in the NLS sequence of TRF1 between cancer tissues and paired noncancerous tissues.
Conclusions There was no base mutation in TRF1 NLS sequence of NSCLC.
引文
1 Hideki I. High Telomerase Activity Correlates With the stabilities of Genome and DNA Ploidy in Renal cell. Carcinoma Neoplasia, 2002(4:2), 103-111
2 Gollahon LS. Alternative methods of extracting telomerase activity from human tumor samples. Cancer Lett, 2000(159): 141-149
3 Wu XF, Amos CI., Zhu Y, Zhao H, et al. Telomere Dysfunction: A Potential Cancer Predisposition Factor. J Nati Cancer Inst, 2003, 95(16): 1211- 218
4 Hackett JA, Feldser DM, Greider CW. Telomere dysfunction increases mutation rate and genomic instability. Cell 2001; 106: 275-86.
5 Morin GB. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell, 1989, 59: 521-529.
6 杨义军，郑晓飞．人端粒酶催化亚基(TERT)基因及其表达调控．生命的化学：2002(1)：32-34
7 Shao R, Guo X. Human microvascular endothelial cells immortalized with human telomerase catalytic protein: a model for the study of in vitro angiogenesis. Biochem Biophys Res Commun, 2004, 321: 788-794
8 Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cell and cancer. Science, 1994, 266: 2011
9 Chong L, Van Steenscl B, Broccoli D, et al. A human telomeric protein. Scinece, 1995, 270(5242): 1663-1667
10 Van Steensel B, de Lange T. Control of telomere length by the human telomeric protein TRF1. Nature, 1997, 385: 740-743
11 Crifith J, Bianchi A, de Lange T. TRF1 promotes parallel pairing of telomeric tracts in vitro. J Mol Biol, 1995, 278: 79-88
12 Smith S, Giriat I, Schmitt A. et al. Tankyrase, a poly(ADP-ribose)polymerase at human telomeres. Science, 1998, 282(5393): 1484-1487
13 Smith S, de Lange T. Tsnkyrase promotes telomere elongation in human cells. J curr Biol, 2000, 10(20): 1299-1302
14 Finnon P, Silver ARJ, Bouffler SD. Upregulation of telomerase activity by X-irradiation in mouse Leukaemia cells is independent of Tert, Terc, Tnks and Myc transcription. Carcinogenesis, 2000; 21(4): 573-78.
15 Smogorzewska A, de Lange T. Regulation of telomerase by telomeerc proteins. Annu Rev Biochem. 2004; 73: 177-208
1 Van Steensel B, de Lange T. Control of telomere length by the human telomefic protein TRF1. Nature, 1997, 385: 740-743
2 Smith S, de Lange T. Tsnkyrase promotes telomere elongation in human cells. J curr Biol, 2000, 10(20): 1299-1302
3 Kuniaki N, Toshiaki K, Fumiyuki K, et al. Expression of mRNAs for Telomeric Repeat Binding Factor (TRF)-land TRF2 in Atypical Adenomatous Hyperplasia and Adenocarcinoma of the Lung. Clinical Cancer Research; 2003, 9: 1105-1111
4 Wei C, Price M. Protecting the terminus: t-loops and telomere end-binding proteins. Cell Mol Life Sci. 2003; 60(11): 2283-94
5 NikitinaT, Woodcock CL, Closed chromatin loops at the ends of chromosomes. J Cell Biol 2004, 160(2): 161-165.
6 蓝建平，来晓瑜，朱园园，等．人端粒结合因子在端粒酶阳性和阴性细胞中的定位与周期性表达的研究．浙江大学学报(医学版)．2004，33(6)：475-480
7 胡坚，屠政良，孙鹂，等．端粒酶活性在非小细胞肺癌不同组织细胞中表达的研究．浙江医学，2003，25(10)：583-584
8 Hastie ND, Demster M, Dunlop MG, et al. Telomere reduction in human colorectal carcinoma. and with aging. Nature. 1990; 346: 866-868.
9 Takagi S, Kinouchi Y, Hiwatashi N, et al. Telomere shortening and the clinicopathologic characteristics of human colorectal carcinomas. Cancer 1999; 86; 1431-1436.
10 Lantuejoul S, Sofia JC, Morat L, et al. Telomere shortening and telomerase reverse transcriptase expression in preinvasive bronchial lesions. Clin Cancer Res 2005; 11: 2074-82.
11 Hyeong RK, Young JK, Hyun JK, et al. Telomere Length Changes in Colorectal Cancers and Polyps, J Korean Med Sci 2002; 17: 360-365
12 Griffith JD. Comeau L, Rosenfield S, et al. Mammalian telomeres end in a large duplex loop. Cell, 1999; 97(4): 503-514.
13 La Torre D, De Divitiis O, Conti A, et al. Expression of telomeric repeat binding factor-1 in astroglial brain tumors. Neurosurgery. 2005; 56(4): 802-810.
14 孙洁，黄河，朱园园，等．端粒结合蛋白TRF1和TANK1在急性白血病细胞中的表达及与端粒酶活性关系的研究．中华血液学杂志，2006，27(1)：48-50
15 Gelmini S, Poggesi M, Distante V, Tankyrase, et al. A positive regulator of telomere elongation, is over expressed in human breast cancer. Cancer Lett. 2004; 216(1): 81-87.
16 Seimiya H, Muramatsu Y, Ohishi T, et al. Tankyrase 1 as a target for telomere-directed molecular cancer therapeutics. Cancer Cell. 2005; 7(1): 25-37.
1 La Torre D, De Divitiis O, Conti A, et al. Expression of telomeric repeat binding factor-1 in astroglial brain tumors. Neurosurgery. 2005; 56(4): 802-810.
2 施继敏，丁伟，黄河，等．端粒重复序列结合因子在肾脏肿瘤中的表达水平。浙江大学学报(医学版)，2004，33(6)：496-499
1 Broccoli D, Chong L, Oelmann S, et al. Comparison of the human and mouse genes encoding the telomeric protein, TRF1: chromosomal localization, expression and conserved protein domains. Hum Mol Genet. 1997; 6(1): 69-76.
2 Iwano T, Tachibana M, Reth M, et al. Importance of TRF1 for functional telomere structure. J Biol Chem; 2004; 279(2): 1442-1448.
3 Aragona M, De Divitiis O, La Torte D, et al. Immunohistochemical TRF1 expression in human primary intracranial tumors. Anticancer Res. 2001; 21(3C): 2135-2139
4 Aragona M, Buda CA, Panetta S, et al. Immunohistochemical telomedc-repeat binding factor-1 expression in gastrointestinal tumors. Oncol Rep. 2000; 7(5): 987-990.
5 Lazads ACh, Rigopoulou A, Tseleni-Balafouta S. et al. Immunodetection and clinico-pathological correlates of two turnout growth regulators in laryngeal carcinoma. Histol Histopathol. 2002; 17(1): 131-8.
6 Oh BK, Kim YJ, Park C, et al. Park YN. Up-regulation of telomere-binding proteins, TRF1, TRF2, and TIN2 is related to telomere shortening during human multistep hepatocarcinogenesis. Am J Pathol. 2005; 166(1): 73-80.
1 Kalderon D, Richardson WD, Markham AF, et al. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature, 1984, 311: 33-38.
2 Robbins, J., Dilworth, S. M., Laskey, R. A., and Dingwall, C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 1991, 64: 615-623
3 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NC_000008
4 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=9257245
5 Gorlieh D and Kutay U. Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol 1999, 15: 607-660
1 Wei C, Price M. Protecting the terminus: t-loops and telomere end-binding proteins. Cell Mol Life Sci. 2003; 60(11): 2283-94
2 Yamada M, Tsuji N, Nakamura M,. Down-regulation of TRF1, TRF2 and TIN2 g enes is important to maintain telomeric DNA for gastric cancers. Anticancer Res 2002; 22(6A): 3303-7
3 Okabe J, Eguchi A, Wadhwa R, et al. Limited capacity of the nuclear matrix to bind telomere repeat binding factor TRF1 may restrict the proliferation of mortal human fibroblasts. Ham Mol Genet. 2004; 3(3): 285-93
4 Yanez GH, Khan SJ, Locovei AM, et al. DNA structure-dependent recruitment of telomeric proteins to single-stranded/double-stranded DNA junctions. Biochem Biophys Res Commun. 2005; 328(1): 49-56
5 Nikitina T, Woodcock C. L. Closed chromatin loops at the ends of chromosomes. J Cell Biol. 2004; (166)1: 61-65.
6 Fouladi B, Sabatier L, Milller D, et al. The relationship between spontaneous telomere loss and chromosome instabilityin a human tumor cell line. Neoplasia. 2000; 2(6): 540-45
7 Kim NW, Piatyszek MA, Prowse KR, et al. Specific assosiation of human telomerase activity with immortal cells and cancer. Science. 1994; 266: 2011-15
8 Ferreira MG, Miller KM, Cooper JP. Indecent exposure: when telomeres become uncapped. Mol Cell. 2004; 13(1): 7-18
9 Shay J, Bacchetti S. A survey of telomerase activity in human cancer. Ear J Cancer. 1997; 33: 787-91
10 Damm K, Hemmann U, Garin-Chesa P, et al. A highly selective telomerase inhibitor limiting human cancer cell proliferation. EMBO. 2001; 20(24): 6958-68
11 Geng ZH, Zhang DH, Liu YK. Expression of telomerase hTERT in human non-small cell lung cancer and its correlation with c-myc gene. Chin Med J. 2003; 116(10): 1467-70
12 Blasco MA, Lee HW, Hande MP, et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell. 1997; 91(1): 25-34
13 Colgin LM, Baran K, Baumann P, et al. Human POT1 facilitates telomcre elongation by telomerase. Curr Biol. 2003; 13(11): 942-46
14 Chong L, van Stcensel B, Broccoli D, et al. A human telomeric protein. Science. 1995; 270: 1663-73
15 van Steensel B, de Lange T. Control of telomere length by the human telomeric protein TRF1. Nature. 1997; 385(6618): 740-43
16 Dantzer F, Giraud-Panis MJ, Jaco I, et al. Functional Interaction between Poly(ADP-Ribose) Polymerase 2 (PARP-2) and TRF2: PARP Activity Negatively Regulates TRF2. Mol Cell Biol. 2004; 24(4): 1595-607
17 Karlseder J. Telomere repeat binding factors: keeping the ends in check. Cancer Lett. 2003; 194(2): 189-97
18 Smogorzewska A, van Steensel B, Bianchi A. Control of human telomere length by TRF1 and TRF2. Mol Cell Biol. 2000; 20(5): 1659-68
19 Cook BD, Dynck JN, Chang W. Role for the related poly(ADP-Ribose) polymerases tankyrase 1 and 2 at human telomeres. Mol Cell Biol. 2002; 22(1): 332-42
20 Smith S, Ginat I, Schmitt A, et al. Tankyrase, a poly (ADP-ribose) polymerase at human telomere. Science. 1998; 282(5393): 1484-87
21 Smith S, de Lange T. Tankyrase promotes telomere elongation in human cell. Curr Biol. 2000; 10(20): 1299-302
22 Finnon P, Silver A R J, Bouffler S D. Upregulation of telomerase activity by X-irradiation in mouse Leukaemia cells is independent of Tert, Terc,Tnks and Myc transcription. Carcinogenesis. 2000; 21(4): 573-78
23 Kaminker PG, Kim SHTD. TANK2, a New TRFl-associated Poly(ADP-ribose) Polymerase, causes rapid induction of cell death upon overexpression. J Biol Chem. 2001; 276(38): 35891-99
24 Ye JZ, Hockemeyer D, Krutchinsky AN, et al. POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex. Genes Dev. 2004; 18: 1649-54
25 Rodier F, Kim SH, Nijjar T, et al. Cancer and aging: the importance of telomeres in genome maintenance[J]. Int J Biochem Cell Biol. 2005; 37(5): 977-90
2 Gollahon LS. Alternative methods of extracting telomerase activity from human tumor samples. Cancer Lett, 2000(159): 141-149
3 Wu XF, Amos CI., Zhu Y, Zhao H, et al. Telomere Dysfunction: A Potential Cancer Predisposition Factor. J Nati Cancer Inst, 2003, 95(16): 1211- 218
4 Hackett JA, Feldser DM, Greider CW. Telomere dysfunction increases mutation rate and genomic instability. Cell 2001; 106: 275-86.
5 Morin GB. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell, 1989, 59: 521-529.
6 杨义军，郑晓飞．人端粒酶催化亚基(TERT)基因及其表达调控．生命的化学：2002(1)：32-34
7 Shao R, Guo X. Human microvascular endothelial cells immortalized with human telomerase catalytic protein: a model for the study of in vitro angiogenesis. Biochem Biophys Res Commun, 2004, 321: 788-794
8 Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cell and cancer. Science, 1994, 266: 2011
9 Chong L, Van Steenscl B, Broccoli D, et al. A human telomeric protein. Scinece, 1995, 270(5242): 1663-1667
10 Van Steensel B, de Lange T. Control of telomere length by the human telomeric protein TRF1. Nature, 1997, 385: 740-743
11 Crifith J, Bianchi A, de Lange T. TRF1 promotes parallel pairing of telomeric tracts in vitro. J Mol Biol, 1995, 278: 79-88
12 Smith S, Giriat I, Schmitt A. et al. Tankyrase, a poly(ADP-ribose)polymerase at human telomeres. Science, 1998, 282(5393): 1484-1487
13 Smith S, de Lange T. Tsnkyrase promotes telomere elongation in human cells. J curr Biol, 2000, 10(20): 1299-1302
14 Finnon P, Silver ARJ, Bouffler SD. Upregulation of telomerase activity by X-irradiation in mouse Leukaemia cells is independent of Tert, Terc, Tnks and Myc transcription. Carcinogenesis, 2000; 21(4): 573-78.
15 Smogorzewska A, de Lange T. Regulation of telomerase by telomeerc proteins. Annu Rev Biochem. 2004; 73: 177-208
1 Van Steensel B, de Lange T. Control of telomere length by the human telomefic protein TRF1. Nature, 1997, 385: 740-743
2 Smith S, de Lange T. Tsnkyrase promotes telomere elongation in human cells. J curr Biol, 2000, 10(20): 1299-1302
3 Kuniaki N, Toshiaki K, Fumiyuki K, et al. Expression of mRNAs for Telomeric Repeat Binding Factor (TRF)-land TRF2 in Atypical Adenomatous Hyperplasia and Adenocarcinoma of the Lung. Clinical Cancer Research; 2003, 9: 1105-1111
4 Wei C, Price M. Protecting the terminus: t-loops and telomere end-binding proteins. Cell Mol Life Sci. 2003; 60(11): 2283-94
5 NikitinaT, Woodcock CL, Closed chromatin loops at the ends of chromosomes. J Cell Biol 2004, 160(2): 161-165.
6 蓝建平，来晓瑜，朱园园，等．人端粒结合因子在端粒酶阳性和阴性细胞中的定位与周期性表达的研究．浙江大学学报(医学版)．2004，33(6)：475-480
7 胡坚，屠政良，孙鹂，等．端粒酶活性在非小细胞肺癌不同组织细胞中表达的研究．浙江医学，2003，25(10)：583-584
8 Hastie ND, Demster M, Dunlop MG, et al. Telomere reduction in human colorectal carcinoma. and with aging. Nature. 1990; 346: 866-868.
9 Takagi S, Kinouchi Y, Hiwatashi N, et al. Telomere shortening and the clinicopathologic characteristics of human colorectal carcinomas. Cancer 1999; 86; 1431-1436.
10 Lantuejoul S, Sofia JC, Morat L, et al. Telomere shortening and telomerase reverse transcriptase expression in preinvasive bronchial lesions. Clin Cancer Res 2005; 11: 2074-82.
11 Hyeong RK, Young JK, Hyun JK, et al. Telomere Length Changes in Colorectal Cancers and Polyps, J Korean Med Sci 2002; 17: 360-365
12 Griffith JD. Comeau L, Rosenfield S, et al. Mammalian telomeres end in a large duplex loop. Cell, 1999; 97(4): 503-514.
13 La Torre D, De Divitiis O, Conti A, et al. Expression of telomeric repeat binding factor-1 in astroglial brain tumors. Neurosurgery. 2005; 56(4): 802-810.
14 孙洁，黄河，朱园园，等．端粒结合蛋白TRF1和TANK1在急性白血病细胞中的表达及与端粒酶活性关系的研究．中华血液学杂志，2006，27(1)：48-50
15 Gelmini S, Poggesi M, Distante V, Tankyrase, et al. A positive regulator of telomere elongation, is over expressed in human breast cancer. Cancer Lett. 2004; 216(1): 81-87.
16 Seimiya H, Muramatsu Y, Ohishi T, et al. Tankyrase 1 as a target for telomere-directed molecular cancer therapeutics. Cancer Cell. 2005; 7(1): 25-37.
1 La Torre D, De Divitiis O, Conti A, et al. Expression of telomeric repeat binding factor-1 in astroglial brain tumors. Neurosurgery. 2005; 56(4): 802-810.
2 施继敏，丁伟，黄河，等．端粒重复序列结合因子在肾脏肿瘤中的表达水平。浙江大学学报(医学版)，2004，33(6)：496-499
1 Broccoli D, Chong L, Oelmann S, et al. Comparison of the human and mouse genes encoding the telomeric protein, TRF1: chromosomal localization, expression and conserved protein domains. Hum Mol Genet. 1997; 6(1): 69-76.
2 Iwano T, Tachibana M, Reth M, et al. Importance of TRF1 for functional telomere structure. J Biol Chem; 2004; 279(2): 1442-1448.
3 Aragona M, De Divitiis O, La Torte D, et al. Immunohistochemical TRF1 expression in human primary intracranial tumors. Anticancer Res. 2001; 21(3C): 2135-2139
4 Aragona M, Buda CA, Panetta S, et al. Immunohistochemical telomedc-repeat binding factor-1 expression in gastrointestinal tumors. Oncol Rep. 2000; 7(5): 987-990.
5 Lazads ACh, Rigopoulou A, Tseleni-Balafouta S. et al. Immunodetection and clinico-pathological correlates of two turnout growth regulators in laryngeal carcinoma. Histol Histopathol. 2002; 17(1): 131-8.
6 Oh BK, Kim YJ, Park C, et al. Park YN. Up-regulation of telomere-binding proteins, TRF1, TRF2, and TIN2 is related to telomere shortening during human multistep hepatocarcinogenesis. Am J Pathol. 2005; 166(1): 73-80.
1 Kalderon D, Richardson WD, Markham AF, et al. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature, 1984, 311: 33-38.
2 Robbins, J., Dilworth, S. M., Laskey, R. A., and Dingwall, C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 1991, 64: 615-623
3 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NC_000008
4 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=9257245
5 Gorlieh D and Kutay U. Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol 1999, 15: 607-660
1 Wei C, Price M. Protecting the terminus: t-loops and telomere end-binding proteins. Cell Mol Life Sci. 2003; 60(11): 2283-94
2 Yamada M, Tsuji N, Nakamura M,. Down-regulation of TRF1, TRF2 and TIN2 g enes is important to maintain telomeric DNA for gastric cancers. Anticancer Res 2002; 22(6A): 3303-7
3 Okabe J, Eguchi A, Wadhwa R, et al. Limited capacity of the nuclear matrix to bind telomere repeat binding factor TRF1 may restrict the proliferation of mortal human fibroblasts. Ham Mol Genet. 2004; 3(3): 285-93
4 Yanez GH, Khan SJ, Locovei AM, et al. DNA structure-dependent recruitment of telomeric proteins to single-stranded/double-stranded DNA junctions. Biochem Biophys Res Commun. 2005; 328(1): 49-56
5 Nikitina T, Woodcock C. L. Closed chromatin loops at the ends of chromosomes. J Cell Biol. 2004; (166)1: 61-65.
6 Fouladi B, Sabatier L, Milller D, et al. The relationship between spontaneous telomere loss and chromosome instabilityin a human tumor cell line. Neoplasia. 2000; 2(6): 540-45
7 Kim NW, Piatyszek MA, Prowse KR, et al. Specific assosiation of human telomerase activity with immortal cells and cancer. Science. 1994; 266: 2011-15
8 Ferreira MG, Miller KM, Cooper JP. Indecent exposure: when telomeres become uncapped. Mol Cell. 2004; 13(1): 7-18
9 Shay J, Bacchetti S. A survey of telomerase activity in human cancer. Ear J Cancer. 1997; 33: 787-91
10 Damm K, Hemmann U, Garin-Chesa P, et al. A highly selective telomerase inhibitor limiting human cancer cell proliferation. EMBO. 2001; 20(24): 6958-68
11 Geng ZH, Zhang DH, Liu YK. Expression of telomerase hTERT in human non-small cell lung cancer and its correlation with c-myc gene. Chin Med J. 2003; 116(10): 1467-70
12 Blasco MA, Lee HW, Hande MP, et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell. 1997; 91(1): 25-34
13 Colgin LM, Baran K, Baumann P, et al. Human POT1 facilitates telomcre elongation by telomerase. Curr Biol. 2003; 13(11): 942-46
14 Chong L, van Stcensel B, Broccoli D, et al. A human telomeric protein. Science. 1995; 270: 1663-73
15 van Steensel B, de Lange T. Control of telomere length by the human telomeric protein TRF1. Nature. 1997; 385(6618): 740-43
16 Dantzer F, Giraud-Panis MJ, Jaco I, et al. Functional Interaction between Poly(ADP-Ribose) Polymerase 2 (PARP-2) and TRF2: PARP Activity Negatively Regulates TRF2. Mol Cell Biol. 2004; 24(4): 1595-607
17 Karlseder J. Telomere repeat binding factors: keeping the ends in check. Cancer Lett. 2003; 194(2): 189-97
18 Smogorzewska A, van Steensel B, Bianchi A. Control of human telomere length by TRF1 and TRF2. Mol Cell Biol. 2000; 20(5): 1659-68
19 Cook BD, Dynck JN, Chang W. Role for the related poly(ADP-Ribose) polymerases tankyrase 1 and 2 at human telomeres. Mol Cell Biol. 2002; 22(1): 332-42
20 Smith S, Ginat I, Schmitt A, et al. Tankyrase, a poly (ADP-ribose) polymerase at human telomere. Science. 1998; 282(5393): 1484-87
21 Smith S, de Lange T. Tankyrase promotes telomere elongation in human cell. Curr Biol. 2000; 10(20): 1299-302
22 Finnon P, Silver A R J, Bouffler S D. Upregulation of telomerase activity by X-irradiation in mouse Leukaemia cells is independent of Tert, Terc,Tnks and Myc transcription. Carcinogenesis. 2000; 21(4): 573-78
23 Kaminker PG, Kim SHTD. TANK2, a New TRFl-associated Poly(ADP-ribose) Polymerase, causes rapid induction of cell death upon overexpression. J Biol Chem. 2001; 276(38): 35891-99
24 Ye JZ, Hockemeyer D, Krutchinsky AN, et al. POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex. Genes Dev. 2004; 18: 1649-54
25 Rodier F, Kim SH, Nijjar T, et al. Cancer and aging: the importance of telomeres in genome maintenance[J]. Int J Biochem Cell Biol. 2005; 37(5): 977-90