Microarray TRAP--a high-throughput assay to quantitate telomerase activity

Telomeric repeat amplification protocol (TRAP)--a sensitive, PCR-based assay to detect telomerase activity was quintessential to the evaluation of telomerase role in telomere maintenance, cell proliferation, tumour development, and cell immortalization. The assay, however, suffers from many limitations. The most significant are: lack of telomerase activity quantification, changes of the enzyme activity product size and/or ratio, and complex post-amplification procedures which limit the assay throughput. Here we report the development of the microarray TRAP (MTRAP) assay which combines advantages of microarray technology with a modified TRAP assay. The MTRAP was designed and optimized on rice cell suspension telomerase extract to enable telomerase specific, reliable, and linear quantification in high throughput mode, with sensitivity comparable to those of radioisotope-based TRAP assays. The MTRAP has a built-in system guaranteeing the amplification of telomerase activity products unchanged in length and/or ratio and built-in control for false negatives. Thus, our MTRAP assay provides new reliable tool for experiments requiring massive quantitation of telomerase activity.     

Increased sensitivity and reproducibility of TRAP assay by avoiding direct primers interaction.

Telomeric Repeat Amplification Protocol (TRAP) is a sensitive procedure to measure telomerase activity in small samples of cell or tissue extracts. Due to the strict correlation between high levels of telomerase activity and neoplastic transformation, TRAP assay could provide an important diagnostic marker of malignancy. Although the original TRAP assay is very sensitive and some improvements have been described, occasional artifacts still persist in the modified procedures. Here we describe how changes in the sequence of the primer used for the amplification step enhance the reproducibility and sensitivity in the TRAP assay.     

Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol

The telomeric repeat amplification protocol (TRAP) is a two-step process for analyzing telomerase activity in cell or tissue extracts. Recent modifications of this sensitive assay include elimination of radioactivity by using a fluorescently labeled primer instead of a radiolabeled primer. In addition, the TRAP assay has been modified for real-time, quantitative PCR analysis. Here, we describe cost-effective procedures for detection of telomerase activity using a fluorescent-based assay as well as by using real-time PCR. These modified TRAP assays can be accomplished within 4 h (from lysis of samples to analysis of telomerase products).     

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.Compared to the traditional TRAP assay that utilize ³²P labeled radioactive dCTP''s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.     

Detection of telomerase activity in Plasmodium falciparum using a nonradioactive method

A simple, quick and sensitive method was used to detect telomerase activity in Plasmodium falciparum. The telomeric repeat amplification protocol (TRAP assay) was modified using electrophoresis and staining with SYBR-green I to detect telomerase activity in a range of 10 to 10(7) parasites. This might be a useful way to ascertain telomerase activity in different types of nontumor cells.     

A quantitative method to measure telomerase activity by bioluminescence connected with telomeric repeat amplification protocol.

Telomerase is expected to be a new biomarker for cancer diagnosis. The telomeric repeat amplification protocol (TRAP) is a sensitive method to detect telomerase activity. However, TRAP and its modified protocols are not always suitable for measuring telomerase activity of a large number of clinical samples to diagnosis cancer because these methods generally require a time-consuming detection step such as gel electrophoresis. To improve the procedure for mass diagnosis, we applied bioluminescence to replace the detection step. Telomerase activity is measured by evaluating the amount of inorganic pyrophosphate generated in PCR amplification of telomerase elongation product, with use of the sensitive enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA). TRAP connected with ELIDA (TRAP-ELIDA) can quantitatively detect telomerase activity within linearity from 2 to 1000 cell equivalents. The ELIDA signals accorded with results of TRAP-SYBR green staining, and the results of ELIDA were significantly correlated to those of TRAP connected with an enzyme-linked immunosorbent assay (TRAP-ELISA) (r(2) = 0.992, P < 0.001). TRAP-ELIDA is a simple and sensitive method to quantify telomerase activity without time-consuming gel electrophoresis. Because TRAP-ELIDA measures telomerase activity with a luminometer, it could be applied to a large number of clinical samples at the same time.     

Analysis of telomerase activity and detection of its catalytic subunit,hTERT

The discovery of the enzyme telomerase and its subunits has led to major advances in understanding the mechanisms of cellular proliferation, immortalization, aging, and neoplastic transformation. The expression of telomerase in more than 85% of tumors provides an excellent tool for the diagnosis, prognosis, and treatment of cancer. However, the techniques employed in its detection appear to play a significant role in the interpretation of the results. The telomeric repeat amplification protocol (TRAP assay) has been the standard assay in the detection of telomerase activity and many variations of this technique have been reported. Recent advances in the development of the TRAP assay and the incorporation of techniques that provide a quantitative and qualitative estimate of telomerase activity are assessed in this review. In addition to histological and cytological examination of tissues, distribution patterns of the catalytic subunit of telomerase, hTERT, are frequently used in the prognosis of tumors. The methods involved in the detection of hTERT as a biomarker of cellular transformation are also analyzed.     

SYBR Green real-time telomeric repeat amplification protocol for the rapid quantification of telomerase activity

The sensitive telomeric repeat amplification protocol (TRAP) permits telomerase detection in mammalian cell and tissue extracts with very low telomerase activity levels. Unfortunately, conventional TRAP assays require complex post-amplification procedures, such as polyacrylamide gel electrophoresis and densitometry, to measure telomerase products. Therefore, a real-time quantitative TRAP assay (RQ-TRAP) was optimized in the present study and evaluated in comparison with a commercially available quantitative TRAP kit and by monitoring telomerase activity in human hepatocyte cultures, human hepatoma cell lines and telomerase reconstitution experiments. The novel real-time telomerase detection method has many advantages. Other than sample extraction and real-time cycling, no additional time-consuming steps have to be performed for telomerase quantification; reliable and linear telomerase quantification is possible down to single-cell dilutions without the interference of primer-dimer artifacts, and the costs are less. Moreover, the precision is similar to other amplification-based telomerase quantification assays and the results are comparable to data obtained with two commercially available assays. The closed-tube system reduces the risk of carryover contamination and supports high throughput. In conclusion, RQ-TRAP provides a new tool for the rapid and reliable quantification of telomerase activity.     

荧光定量PCR 法检测肿瘤细胞端粒酶活性的变化

目的：利用荧光定量PCR法检测端粒酶抑制剂作用于人肝癌细胞SMMC-7721后端粒酶活性的变化,探讨其抑制端粒酶活性的可能机制,为端粒酶抑制剂的临床应用提供理论依据。方法：利用荧光染料SYBR—Green I建立一种新的端粒酶活性检测方法：FQ—TRAP法。利用FQ—TRAP法检测端粒酶抑制剂作用后肿瘤细胞端粒酶活性变化。结果：端粒酶抑制剂作用后,肝癌细胞端粒酶活性都有变化,其中以ASODN,EGCG,AZT抑制效果较明显。结论：端粒酶FQ—TRAP法是一种特异性、灵敏度、重复性都较好,可快速、简便及定量检测人端粒酶活性的方法,端粒酶抑制剂作用后癌细胞端粒酶活性的变化,为端粒酶抑制剂的临床应用提供理论依据。     

Synthesis and evaluation of cationic phthalocyanine derivatives as potential inhibitors of telomerase

A series of water-soluble cationic phthalocyanine derivatives (1-10) were designed and synthesized to develop novel and potent telomerase inhibitors. These phthalocyanine derivatives as inhibitors of telomerase were investigated via modified telomerase repeat amplification protocol (TRAP) assay. The TRAP assay indicates that these cationic compounds had strong telomerase inhibitory activity (IC(50)<1.65 microM). To determine whether the phthalocyanine derivatives binding to G-quadruplex enhance the block to DNA synthesis, primer extension reactions were carried out in the presence of phthalocyanines. The interaction of the G-quadruplex of telomerase DNA with these molecules was examined by CD melting and PCR stop assay. These cationic phthalocyanine derivatives can stabilize G-quadruplex, which is demonstrated by the increased T(m) values. All these results indicate that the phthalocyanine derivatives might be potential lead compounds for the development of new telomerase inhibitor.     

Development patterns of telomerase activity in barley and maize

Eukaryotic chromosomes terminate with specialized structures called telomeres. Maintenance of chromosomal ends in most eukaryotes studied to date requires a specialized enzyme, telomerase. Telomerase has been shown to be developmentally regulated in man and a few other multicellular organisms, while it is constitutively expressed in unicellular eukaryotes. Recently, we demonstrated telomerase activity in plant extracts using the PCR-based TRAP (Telomeric Repeat Amplification Protocol) assay developed for human cells. Here we report telomerase activities in two grass species, barley and maize, using a modified, semi-quantitative TRAP assay. Telomerase was highly active in very young immature embryos and gradually declined during embryo development. The endosperm telomerase activity was detectable, but significantly lower than in the embryo and declined during kernel development with no detectable activity in later stages. Telomerase activity in dissected maize embryo axis was several orders of magnitude higher than in the scutellum. Telomerase activity was not detected in a range of differentiated tissues including those with active meristems such as root tips as well as the internode and leaf base. The role of telomerase repression during differentiation and the relationship between chromosome healing and telomerase activity is discussed.     

水稻端粒酶活性定量分析研究

摘要：端粒酶活性直接与细胞的分生能力、生活力、生命力有着密不可分的关系, 因此对其活性测定有着重要意义。本文参照人类端粒酶体外检测的原理和方法,设计了特别的先导引物和反向引物,采用不同的退火温度将PCR循环分成两步进行,再结合DNA凝胶电泳成像定量分析系统,以模式植物水稻为研究对象,对端粒酶活性定量检测方法和反应条件进行了探索。结果显示,水稻端粒酶活性的最佳反应条件为：温度19℃,反应时间13分钟,总蛋白浓度0.28μg/μl,与人端粒酶最佳反应条件有明显差异,建立了一种有效测定植物端粒酶活性的定量检测方法。应用该方法对6个水稻品种的根、幼叶及幼穗三个不同组织器官的端粒酶活性进行了定量测定,结果显示幼穗最高,其次为幼叶,根最弱。说明植物端粒酶活性与细胞、组织的生活力有着密切关系。     

Limitations of detection of malignancy in pleural effusions using ELISA-based TRAP assay: comparison with cytological examination

OBJECTIVE: Telomerase is active in almost all cancers from various organs but is not detectable in most normal cells. Thus, telomerase activity might be a universal and specific marker for diagnosing malignancy. The aim was to evaluate the potential use of the ELISA-based TRAP assay to detect malignancy in pleural effusion, and to compare it with conventional cytological examination. METHODS: Using the ELISA-based TRAP assay, telomerase activity was examined in 94 consecutive pleural effusions submitted for cytological examination. RESULTS: According to the results of cytology, the 94 samples were divided into two groups: group I, 79 non-malignant pleural effusions, including group IA, no association with a malignant tumour, a control group (n = 63), and group IB, associated with a malignant tumour (n = 16); and group II, 15 malignant pleural effusions. Telomerase activity was detected in five of 63 samples in group IA (7.9%), four of 16 samples in group IB (25%), and six of 15 samples in group II (40%). All five false-positive effusions were from patients with tuberculosis. Comparing group II with group IA, the TRAP assay showed 40% sensitivity, 92.1% specificity, 54.5% positive and 86.6% negative predictive value, and 82.1% accuracy. However, the detection rate of the TRAP assay (88.9%) was higher than that of the cytological examination (66.7%) in lung cancer-inflicted pleural effusions. CONCLUSION: The ELISA-based TRAP assay is relatively insensitive; therefore, it is unsuitable as a routine diagnostic tool for pleural effusion. False-positive telomerase activity due to lymphocytic contamination may weaken its diagnostic value for malignant effusions in a tuberculosis-endemic area.     

Molecular basis of artifacts in the detection of telomerase activity and a modified primer for a more robust 'TRAP' assay.

Human somatic cells have essentially no telomerase activity. Telomerase is linked to tumor genesis and is a valuable marker for malignant growth. Extreme paucity of the enzyme neccessitated development of a PCR-based assay, 'telomeric repeat amplification protocol' (TRAP). Unfortunately, this method is not without difficulties.Amplification products are not related to the size of the amplified telomerase products. Furthermore, false positive results can occur, and careful control of reaction conditions is crucial. We analyzed in detail the molecular basis of artifacts. Based on these data, reverse PCR primer was changed and both problems in the TRAP assay were eliminated.     

卵巢良性和恶性上皮性肿瘤中端粒酶蛋白表达及活性的研究

目的　研究卵巢良性和恶性上皮性肿瘤中端粒酶蛋白的表达水平及其活性 ,探讨其作为卵巢癌肿瘤标记物的可能性。方法　应用免疫组织化学方法检测了 30例卵巢癌和 10例卵巢腺瘤组织中端粒酶相关蛋白 1(TP1)和人端粒酶蛋白催化亚基 (hTRT)的表达水平 ,并采用TRAP -ELISA方法检测了 30例卵巢癌和 10例卵巢腺瘤组织中的端粒酶活性。结果　免疫组织化学结果显示 ,30例卵巢癌和 10例卵巢腺瘤组织中TP1和hTRT表达阳性率均为 10 0 %,其中卵巢癌的强阳性表达率高于卵巢囊腺瘤 (P <0 0 1) ,TP1和hTRT的表达强度无明显差别。TRAP ELISA方法检测发现 30例卵巢癌组织中 ,2 8例检测到端粒酶活性 ,阳性率为 93.3%;10例卵巢腺瘤中 ,仅 1例浆液性乳头状腺瘤检测到端粒酶活性 ,阳性率为 10 %(P <0 0 0 1)。结论　本研究的结果表明卵巢良性和恶性上皮性肿瘤中端粒酶蛋白TP1和hTRT的表达水平与端粒酶活性之间无明显相关性 ,端粒酶活性的检测与卵巢癌的发生发展密切相关 ,有望成为卵巢癌的一种肿瘤标记物。     

特异性人端粒酶催化亚单位基因小干扰RNA对HeLa细胞生长的抑制作用

通过设计并化学合成人端粒酶催化亚单位(hTERT)特异性siRNA,观察其对hTERT表达水平及肿瘤细胞生长的影响。将hTERT-siRNA以脂质体法转染入HeLa细胞,应用RT-PCR、实时定量TRAP、Western印迹、软琼脂克隆形成实验、荷瘤裸鼠肿瘤内注射等方法检测细胞内hTERTmRNA、蛋白质表达水平及对肿瘤细胞生长的影响。RT-PCR、实时定量TRAP和Western印迹的结果显示hTERT-siRNA明显降低了HeLa细胞内hTERT的mRNA及蛋白质表达水平并伴随有端粒酶活性的下降。克隆形成实验表明hTERT-siRNA组的体外肿瘤形成能力受到抑制。荷瘤裸鼠肿瘤内注射hTERT-siRNA使肿瘤平均体积显著小于对照组。TUNEL凋亡检测表明hTERT-siRNA转染组的凋亡率明显高于对照组。研究表明hTERT特异性siRNA可以明显抑制HeLa细胞内hTERT的表达水平,对其生长有明显抑制作用,是一种有前途的肿瘤治疗新方法。     

A rapid biosensor chip assay for measuring of telomerase activity using surface plasmon resonance

Considerable interest has been focused on telomerase because of its potential use in assays for cancer diagnosis, and for anti-telomerase drugs as a strategy for cancer chemotherapy. A number of assays based on the polymerase chain reaction (PCR) have been developed for evaluation of telomerase activity. To overcome the disadvantages of the conventional telomerase assay [telomeric repeat amplification protocol (TRAP)] related to PCR artifacts and troublesome post-PCR procedures, we have developed a telomeric repeat elongation (TRE) assay which directly measures telomerase activity as the telomeric elongation rate by biosensor technology using surface plasmon resonance (SPR). 5′-Biotinylated oligomers containing telomeric repeats were immobilized on streptavidin-pretreated dextran sensor surfaces in situ using the BIACORE apparatus. Subsequently, the oligomers associated with the telomerase extracts were elongated in the BIACORE apparatus. The rate of TRE was calculated by measuring the SPR signals. We examined elongation rates by the TRE assay in 18 cancer and three normal human fibroblast cell lines, and 12 human primary carcinomas and matching normal tissues. The elongation rates increased in a concentration- and time-dependent manner. Those of cancer cells were two to 10 times higher than fibroblast cell lines and normal tissues. Telomerase activities and its inhibitory effects of anti-telomerase agents as measured by both the TRE and TRAP assays showed a good correlation. Our assay allows precise quantitative comparison of a wide range of human cells from somatic cells to carcinoma cells. TRE assay is suitable for practical use in the assessment of telomerase activity in preclinical and clinical trials of telomerase-based therapies, because of its reproducibility, rapidity and simplicity.     

Interaction of an acridine dimer with DNA quadruplex structures.

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt an intramolecular G-quadruplex structure in vitro, which has been shown to inhibit telomerase activity. The C-rich sequence can also adopt a quadruplex (intercalated) structure (i-DNA). Two acridine derivatives were shown to increase the melting temperature of the G- quadruplex and the C-quadruplex at 1 microM dye concentration. The increase in Tm value of the G-quadruplex was associated with telomerase inhibition in vitro. The most active compound, "BisA", showed an IC(50) value of 0.75 microM in a standard TRAP assay.