Genetic Variation of <i>hTERT</i>, Leukocyte Telomere Length and the Risk of Breast Cancer: A Case-Control Study in Egyptian Females

Background: hTERT is a key player in telomere biology and its activity is directly related to cell senescence and development of many health-related problems including cancer. Although previous studies investigated this association, the results greatly vary among populations. This study aimed to investigate the association of hTERT gene SNPs and the risk of breast cancer (BC) in Egyptian females and their impact on telomere length (TL). Methods: 218 BC patients and 178 age-matched healthy females were genotyped for hTERT variants rs2736098G > A, rs2735940C > T using PCR-RFLP and for MNS16A tandem repeat using PCR to determine their association with breast cancer risk. Telomere length was measured using qPCR. Results: hTERT rs2736098G > A results indicated that both AG and GG genotypes and G allele were associated with an increased risk of BC. The rs2735940 TT genotype was significantly associated with BC risk, however, the MNS16A tandem repeat region polymorphism didn’t show any correlation with the risk of developing BC. TL showed a significant reduction in BC patients with age 40 years compared with controls. However, it didn’t show a significant difference above the age of 40 years. Conclusions: hTERT rs2736098 and rs27365940, not MNS16A may be associated with an increased risk of developing BC in Egyptian females. Also, telomere length can be a promising screening marker of BC especially in young population.

Clinical significance of telomerase and its associate genes expression in the maintenance of telomere length in squamous cell carcinoma of the esophagus

AIM: To observe the interaction between the expression of telomerase activity (TA) and its associate genes in regulation of the terminal restriction fragment length(TRFL) in esophageal squamous cell carcinoma (SCC).METHODS: Seventy-four specimens of esophageal SCC were examined. The TA was measured by telomeric repeat amplification protocol (TRAP) assay, and the associated genes [human telomerase-specific reverse transcriptase (hTERT), hTERC, TP1, c-Myc, TRF1,and TRF2] were detected using RT-PCR method. The TRFL was measured by Telomere Length Assay Kit and Southern blotting. The correlations between the expression of telomerase and its associated genes with the TRFL and survivals were examined.RESULTS: Expressions of the TA, hTERT, hTERC, TP1,c-Myc, TRF1, and TRF2 genes were observed in 85.1%,64.9%, 79.7%, 100.0%, 94.6%, 82.4%, and 91.9% of the tumor tissues, respectively. The TRFL of the tumor and normal esophageal tissues were 2.70±1.42 and 4.93±1.74 kb, respectively (P＜0.0001). The TRFL of the telomerase positive and telomerase negative tumor tissues were 2.72±1.44 and 2.58±1.32 kb, respectively (P = 0.767).The TRFL ratios (TRFLR) of the telomerase positive and telomerase negative tumor tissues were 0.55±0.22 and 0.59±0.41, respectively (P = 0.742). The expression rates of h-TERT (P = 0.0002), hTERC (P＜0.0001), and TRF1(P = 0.002) in the tumor tissues are higher than those of the normal paired tissues. Though TA is markedly activated in tumor tissues (P＜0.0001), its expression is not related to clinicopathological parameters including gender, tumor differentiation, and TNM stages. The cumulative 4-year survival rates of telomerase positive and telomerase negative cases were 35.86% and 31.2%,respectively (P = 0.8442). The cumulative 4-year survival rates of patients with their TRFLR ≤85% and ＞85%were 38.7% and 15.7%, respectively (P = 0.1307).CONCLUSION: Though telomerase expression is not related to tumor stages and prognosis, our data support that the TA increased as the TRFL decreased,probably under the control of hTERT, hTERC, and TRF1.When telomerase expression was activated, only TRF2overexpression persisted to stabilize T-loop formation.Furthermore, as the TRFLR decreased to 85%, a trend of better prognosis was observed. Cox model analysis indicates a higher t/n TRFLR and distant metastasis are independent poorer prognostic factors (P = 0.035 and P = 0.042, respectively).

Association between Physical Activity and Telomere Length in a North Chinese Population： A China Suboptimal Health Cohort Study

Several studies have demonstrated an association between physical activity and telomere length; however, the association remains inconsistent. A cross-sectional study consisting of 588 participants （375 females, median age of 33.8 years） was carried out to investigate the association between telomere length and physical activity in a general population from North China. The results show that relative telomere length is not significantly different in participants in the northern Chinese population with different levels of physical activity, either in the model only adjusted for age （F = 2.127, P = 0.120） or in the model adjusted for demographics and lifestyle （F = 1.227, P = 0.294）. The gender-stratified analysis also produced insignificant results. Our study confirmed a non-significant association between physical activity and telomere length in the northern Chinese population, which adds to the inconsistent association between physical activity and telomere length across different ethnic populations.

Determination of Telomere Length by the Quantitative Fluorescence <i>in Situ</i>Hybridization (Q-FISH) Method

Telomeres are nucleoprotein complexes located at the ends of eukaryotic chromosomes and are shorten with aging or various causes. Shortened telomere plays an important role for chromosomal instability in carcinogenesis, or a number of diseases in relation to aging. FISH using peptide-nucleic acid (PNA) probe is suitable for analyzing telomeres, because telomere is a part of DNA molecule and localized in chromosomal end in loop shape structure (T-loop). We introduce our telomere analysis for tissue sections and cultured cells in this paper. On the tissue sections, we analyze telomere length with telomere intensity to centromere intensity ratio (TCR), centromere as an inner control. In the cultured cells, we analyze telomere lengths on each chromosome with karyotyping. Those Q-FISH methods by PNA probe are an accurate and reliable tool for research on telomere lengths in various conditions in biology.

Association between Dietary Collagen Consumption and Telomere Length: National Health and Nutrition Examination Survey

Background: Bioactive peptides derived from hydrolyzed collagen have broad physiological functions and beneficial effects on human health, ranging from reducing skin aging to modifying lipid metabolism. Telomere length shortening is an established biomarker of cellular aging. It is not known if collagen consumption is associated with telomere length protection. Our purpose was to investigate the relationship between dietary collagen consumption and telomere length in a nationally representative US adult population. Methods: We analyzed the data of 6173 adults aged 20 - 84 from the National Health and Nutrition Examination Survey (NHANES) 1999-2002. Multivariable linear regression and a generalized additive model with smoothing plot were used to assess the association between the total collagen consumption and log-transformed leukocyte telomere length (LTL). Results: Compared with the lowest quartile of total collagen (Q1), we found that the second quartile of collagen (Q2) consumption (1.36 - 3.40 g/1000kcal) was positively associated with telomere length (β: 0.017;95% CI: 0.006, 0.028;P = 0.022) in the females while no association in the males (β: −0.003;95% CI: −0.019, 0.012;P = 0.678) and overall population (β: 0.008;95% CI: −0.002, 0.018;P = 0.141). The association in the females is nonlinear with an inflection point of 2.5 g/1000kcal (P for non-linearity: Conclusion: In conclusion, moderate dietary collagen has a positive and nonlinear association with telomere length in US females, while no significant associations were found in the males and the overall population.

Antipsychotics preserve telomere length in peripheral blood mononuclear cells after acute oxidative stress injury

Antipsychotics may prolong or retain telomere length,affect mitochondrial function,and then affect the metabolism of nerve cells.To validate the hypothesis that antipsychotics can prolong telomere length after oxidative stress injury,leukocytes from healthy volunteers were extracted using Ficoll-Histopaque density gradient.The mononuclear cells layer was resuspended in cell culture medium.Oxidative stress was induced with hydrogen peroxide in cultured leukocytes.Four days later,leukocytes were treated with aripiprazole,haloperidol or clozapine for 7 days.Real-time PCR revealed that treatments with aripiprazole and haloperidol increased the telomere length by 23%and 20%in peripheral blood mononuclear cells after acute oxidative stress injury.These results suggest that haloperidol and aripiprazole can reduce the damage to telomeres induced by oxidative stress.The experiment procedure was approved by the Ethics Committee of Faculty of Medicine of the University of Sao Paulo(FMUSP/CAAE approval No.52622616.8.0000.0065).

Measuring Telomere Length in Proliferating Cells by Flow-FISH Method

The purpose of present work is a measurement of telomere length dynamic in proliferating cells in vitro by modified flow-FISH method. This method is a combination of two modifications： telomere length measurement in differentiated cells by surface antigen and analysis of cells divisions＇ number by vital dye dilution. Lymphocytes were activated by anti-CD3 Abs with IL-2 presents and grown in vitro for 7 days. Cells division＇s number was measured by dilution of CFSE vital dye which cells were stained previously activation. For telomere length measurement we used flow-FISH method with Cy3 labeled telomere PNH probe. Using this method we evaluated the dynamic of telomere length in CD4＋ and CD8＋ T-cells after 7 days culturing in vitro and revealed the difference in telomere lengthening and shortening versus division rounds in cell subsets. In CD8＋ cells telomeres start lengthen on a second division with the maximum on 4th division round becoming more that 20% longer compared with undividing cells. In CD4＋ cells telomeres did not have any length peculiarities through all division rounds demonstrating different telomere regulation in subsets. This probably occurs due to the higher level ofhTERT protein expression in CD8＋ than CD4＋ cells do.

Hematopoietic stem cell transplantation of aplastic anemia by relative with mutations and normal telomere length: A case report

BACKGROUND Immunosuppressive therapy and matched sibling donor hematopoietic stem cell transplantation(MSD-HSCT)are the preferred treatments for aplastic anemia(AA).CASE SUMMARY In this report,we describe a 43-year-old male patient with severe AA who carried BRIP1(also known as FANCJ),TINF2,and TCIRG1 mutations.Screening of the family pedigree revealed the same TINF2 mutation in his mother and older brother,with his older brother also carrying the BRIP1 variant and demonstrating normal telomere length and hematopoietic function.The patient was successfully treated with oral cyclosporine A,eltrombopag,and acetylcysteine,achieving remission 4 years after receiving MSD-HSCT from his older brother.CONCLUSION This case provides a valuable clinical reference for individuals with suspected pathogenic gene mutations,normal telomere length,and hematopoietic function,highlighting them as potential donors for patients with AA.

端粒长度与10种常见肌肉骨骼疾病的关系孟德尔随机化分析

背景:多项观察性研究表明,端粒长度与肌肉骨骼疾病之间存在潜在的关联,然而它们之间的潜在机制仍不清楚。目的:利用两样本孟德尔随机化分析来探索端粒长度与肌肉骨骼疾病之间的遗传因果关系。方法:从英国生物银行中获得端粒长度的全基因组关联研究汇总数据。从FinnGen财团中获得了关于10种常见肌肉骨骼疾病(骨坏死、骨髓炎、骨质疏松、类风湿关节炎、腰痛、椎管狭窄、痛风、肩周炎、强直性脊柱炎和下肢深静脉血栓)的全基因组关联研究汇总数据。使用逆方差加权、孟德尔随机化-Egger和加权中位数方法评估端粒长度与10种肌肉骨骼疾病的因果关系,逆方差加权作为主要的孟德尔随机化分析方法,并进行敏感性分析探讨结果稳健性。结果与结论:①逆方差加权法结果表明,遗传预测的端粒长度与类风湿关节炎(OR=0.78,95%CI:0.64-0.95,P=0.015)和骨坏死(OR=0.56,95%CI:0.36-0.90,P=0.016)风险之间存在负向因果关系,但未发现端粒长度与其他8种肌肉骨骼疾病之间存在因果关系(P均>0.05)。②敏感性分析结果表明因果关系稳健,孟德尔随机化-Egger截距分析未检测到潜在的水平多效性(P均>0.05)。③此项孟德尔随机化研究支持端粒长度对类风湿关节炎和骨坏死的保护作用的结论,然而,未来将需要更多的基础和临床研究来验证。

端粒酶hTERT促进肿瘤细胞侵袭与转移及其机制研究

目的观察hTERT基因修饰对人骨肉瘤细胞系U-2OS生物学行为的影响。方法采用脂质体法将克隆有人全长cDNAhTERT的真核荧光质粒(pIRES2-EGFP-hTERT)转染端粒酶阴性的人骨肉瘤U-2OS细胞,经G418筛选,免疫组化和Westorn Blot鉴定后,检测其端粒酶活性的改变、生长周期以及生物力学的变化。结果成功转染人真核荧光表达载体的U-2OS细胞能有效抵抗G418,并可有效表达hTERT蛋白,细胞内端粒酶活性明显增强,G1期细胞比例下降,S期细胞比例升高,并且还明显增强了对细胞外基质的粘附力。进一步采用Transwell小孔迁移实验检测发现,hTERT/U2OS侵袭能力明显增强。结论转染hTERT基因后,U-2OS细胞内可同时通过端粒酶途径延长端粒。hTERT基因修饰可促进细胞周期进程,促使细胞从G1期→S期。通过替代途径延长端粒的肿瘤细胞在hTERT基因修饰后,增殖能力及侵袭能力明显增强。

hTERT和β-catenin在胆囊癌中的表达及其临床意义

目的检测hTERT和-βcatenin在胆囊癌中的表达情况,分析其与临床病理特征及病人生存期之间的关系。方法收集正常及炎性胆囊、胆囊癌非相关息肉、胆囊癌相关息肉和胆囊癌各20、19、16、82例。用免疫组化方法分别检测hTERT、-βcatenin蛋白表达。结果①hTERT在正常及炎性胆囊、胆囊癌非相关息肉、胆囊癌相关息肉和胆囊癌中阳性率分别为0、5.3%、37.5%、84.1%,后两组hTERT表达率显著高于前两组(P<0.05),胆囊癌组hTERT表达率亦显著高于第3组(P<0.001)。-βcatenin在正常及炎性胆囊、胆囊癌非相关息肉、胆囊癌相关息肉和胆囊癌中异位表达率分别为5.0%、10.5%、50.0%、51.2%。后两组-βcatenin表达率显著高于前两组(P<0.05)。②hTERT在T1+T2、T3+T4和淋巴结阳性、阴性组中的表达率分别为72.7%、92.8%(P<0.05)和93.6%、71.4%(P<0.05),差异有显著性。hTERT表达水平与胆囊癌分化程度、浸润深度、淋巴结有无转移的相关系数分别为0.5、0.4、-0.5(P<0.01)。-βcatenin在T1+T2、T3+T4和淋巴结阳性、阴性组中异位表达率分别为33.3%、63.3%(P<0.01)和57.4%、42.9%(P<0.05),差异有显著性。③胆囊癌hTERT阴性、阳性表达累积生存率分别为22.8%、5.9%(P<0.05)。胆囊癌异位-βcatenin阴性、阳性表达累积生存率分别为17.1%、5.7%(P<0.05)。④胆囊癌hTERT蛋白与-βcatenin异位蛋白表达呈正相关(r=0.311,P<0.05)。结论hTERT和-βcatenin蛋白异位表达与胆囊的恶性病变密切相关。hTERT表达与胆囊癌相关息肉及其恶性转化有关。hTERT和-βcatenin蛋白与肿瘤不良预后有关。-βcatenin异位表达增加与胆囊癌hTERT表达增强相关联。

端粒酶逆转录酶基因hTERT突变表达载体在膀胱癌细胞T24中的表达

目的 构建端粒酶逆转录酶基因 (h TERT)缺失突变的真核表达载体 p EGFP- h TERT,并转入膀胱癌细胞株 T2 4中 ,观察其稳定表达 .探讨其对端粒酶活性调控机制及成为膀胱肿瘤基因治疗新靶点的可能性 .方法 将 PCR扩增的h TERT缺失突变体片段用 Eco RI与 Sal I酶切 ,并连接到真核表达载体 p EGFP- C1上 ,构建成 h TERT缺失突变的真核表达载体 p EGFP- h TERT;利用 DNA-磷酸钙共沉淀法将p EGFP- h TERT导入膀胱癌细胞株 T2 4中 ,应用荧光显微镜、与衰老相关的β-半乳糖苷酶染色等方法观察转染细胞中h TERT- GFP融合蛋白定位表达及对膀胱癌细胞生长的影响 .结果 酶切鉴定证实 h TERT缺失突变体已克隆到p EGFP- C1的 Eco RI与 Sal I位点之间 ,在转染细胞中观察到与其融合的绿色荧光蛋白的稳定表达 ,定位于细胞核内 ;转染细胞 2 wk后与衰老相关 β-半乳糖苷酶表达增加 ,细胞生长受抑制 .结论 构建的端粒酶逆转录酶基因 h TERT缺失突变真核表达载体 p EGFP- h TERT可在膀胱癌细胞 T2 4中稳定表达 .突变型 h TERT蛋白可入细胞核中竞争性影响端粒酶的功能 。

hTERT永生化细胞的研究进展及应用前景

人端粒酶逆转录酶基因(hTERT)诱导的永生化细胞兼具原代细胞的生物学特性和传代细胞系连续传代的能力,是一种理想的细胞来源,在基础研究、临床应用和生物工程领域具有无可比拟的优势。本文主要就hTERT永生化细胞的研究进展及应用前景作一综述。

端粒、端粒酶及hTERT/hTERC基因的研究进展

端粒是真核细胞染色体的末端DNA序列,在维持染色体稳定性中起重要作用。染色体的不完全复制使得端粒随着细胞的分裂而逐渐缩短,快速分裂细胞通过端粒酶合成端粒,以弥补端粒的消耗。端粒酶相关基因突变可导致端粒酶活性的降低和端粒缩短,过短的端粒不再保护基因组稳定性,将引起细胞的老化、凋亡或恶变。端粒酶基因的扩增出现在一些肿瘤细胞中,是癌细胞增殖的重要原因,其扩增的机制及其对端粒酶活性的调节作用尚不完全清楚。近期研究表明,端粒酶基因扩增是基因组不稳定的结果,扩增的hTERT/hTERC基因对端粒酶激活和癌变进展有促进作用。

hPOT1 RNA干扰对人胃癌细胞株BGC-823端粒长度和hTERT表达的影响

背景:人端粒保护蛋白1(hPOT1)的主要作用为保护端粒和调节端粒长度。目的:探讨hPOT1在人胃癌细胞中对端粒长度的调节作用及其可能机制。方法:以RNA干扰(RNAi)技术抑制人胃癌细胞株BGC-823中hPOT1的表达,以实时荧光定量聚合酶链反应(PCR)检测细胞端粒长度,以半定量逆转录聚合酶链反应(RT-PCR)检测人端粒酶逆转录酶(hTERT)mRNA表达。结果:以RNAi技术抑制hPOT1表达后,BGC-823细胞端粒长度明显缩短,反映端粒相对长度的T/S值显著小于亲本BGC-823细胞组和阴性对照组(1.383±0.091对2.758±0.647和3.043±0.548,P<0.05),亲本细胞组与阴性对照组之间则无明显差异。hPOT1 RNAi组细胞hTERT mRNA表达亦明显下调。结论:在人胃癌细胞中,hPOT1能正性调节端粒长度;在hPOT1下调引起端粒缩短的环节中,hTERT表达下降可能起一定作用。

hTERT rs2853676与非贲门胃癌易感性相关联

目的:探讨h TERT基因SNP rs2853676位点与非贲门胃癌遗传易感性的关系。方法:采用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)法检测134例非贲门胃癌组和147例正常对照组h TERT基因rs2853676位点的基因多态性。并用非条件性Logistic回归计算比值比(OR)及其95%可信区间(CI),以评估等位基因、基因型与非贲门胃癌发病风险的关系。结果:h TERT基因SNP rs2853676基因型GG、GA、AA在非贲门胃癌组和正常对照组的频率分别为27. 6%;57. 5%;14. 9%和25. 2%.;46. 9%;27. 9%;携带AA型基因的患者罹患非贲门胃癌的风险显著增加(OR=2. 050,95%CI=1. 102-4. 138)。结论:h TERT基因SNP rs2853676与非贲门胃癌发病风险关联。

Telomere Protective Effects of a Cyanobacteria Phycocyanin against Blue Light and UV Irradiations: A Skin Anti-Aging and Photo-Protective Agent

Background: Cyanobacteria phycocyanins (Cps) have already shown powerful antioxidant properties. In human cells submitted to oxidative stress the telomeres length decrease, the expression of progerin and the activity of mTOR are increased. At our knowledge, there is no published data on Cps correlated with ultraviolet radiation (UV) and blue light effects in human cells regarding telomeres’ length, progerin expression or mTOR1 complex activity. Objectives: In this study, we sought to assess 1) telomeres’ length in newborn human fibroblasts exposed to UV and blue light;2) progerin production in mature human normal fibroblasts exposed to UV;3) mTOR1 activation in adult human normal keratinocytes exposed to UV, analyzing the activity of a Cyanobacteria phycocyanin (Cp) in these in vitro models. Materials and Methods: Human skin fibroblasts or human normal keratinocytes were cultured—in the absence or in the presence of Cp and submitted to UVB + UVA and blue light irradiations. Telomeres’ length, progerin expression and mTOR1 activity were then assessed by molecular biology and immuno-enzymatic methods. Results: In cultured fibroblasts exposed to irradiations and treated by Cp, telomeres’ shortage and progerin expression were lower compared to irradiated untreated cells. In cultured keratinocytes treated by Cp and exposed to irradiations, the mTOR activity was lower compared to irradiated untreated cells. Conclusions: In these in vitro studies on human skin fibroblasts and on normal human keratinocytes, the cyanobacteria phycocyanin (Cp) showed a decrease of damages induced by UV and blue light expressed by telomeres preservation and downregulation of progerin expression and of mTOR activity, thus showing skin anti-aging and photo-protective potential.

端粒长度与子宫内膜异位症的因果关系:双向孟德尔随机研究

目的:基于大规模人群的全基因组关联研究(GWAS),通过孟德尔随机化(MR)分析探讨端粒长度(TL)与子宫内膜异位症(EMs)是否存在因果关系。方法:采用双向MR研究来自欧洲血统的TL(样本77257例)和EMs(样本472174例)的GWAS汇总统计数据。采用逆方差加权(IVW)、MR-Egger、加权中位数、简单模型和加权模型进行MR分析。结果主要基于IVW,使用随机效应,行敏感性分析。用IVW和MR-Egger检测到异质性。水平检测采用MR-Egger进行分析。采用留一法鉴定单核苷酸多态性(SNPs)。结果:IVW方法正向MR分析结果显示,TL对EMs的影响显著(IVW OR=1.250,P=0.006);反向MR分析中没有发现EMs与TL之间存在明显的因果关系(IVW OR=0.997,P=0.569)。这些发现在几个敏感性分析中都是稳健的。结论:TL与EMs之间存在因果关系,且呈正相关;反向MR分析未发现EMs与TL存在因果关联。

双向两样本孟德尔随机化探究白细胞端粒长度与主动脉瘤发生风险的关系

目的采用双向两样本孟德尔随机化(Mendelian randomization,MR)方法探讨白细胞端粒长度(leukocyte telomere length,LTL)与主动脉瘤(aortic aneurysm,AA)发生风险的潜在因果关系。方法通过使用LTL和AA的最新全基因组关联研究(genome-wide association study,GWAS)公开数据库,找到LTL和AA相关的遗传变异位点作为工具变量(instrumental variable,IV),进行双向两样本MR分析,以逆方差加权法(inverse variance weighted,IVW)为主要方法分析LTL与AA之间的因果关系,应用Cochran's Q检验进行异质性分析,应用MR-Egger截距项探讨工具变量的潜在多效性,应用留一法(Leave-one-out,LOO)进行敏感性分析。结果基因预测更长的LTL对AA发生风险的降低显著相关,并且LOO分析显示结果稳定,而AA与LTL长度改变并无明显因果关系。结论研究结果提示,更长的LTL对AA具有保护作用,对患者进行LTL监测将有助于AA的早期筛查与预防。