Detection of telomerase activity by combination of telomeric repeat amplification protocol and electrochemiluminescence assay

A highly sensitive telomerase detection method that combines telomeric repeat amplification protocol （TRAP） and magnetic beads based electrochemiluminescence （ECL） assay has been developed. Briefly, telomerase recognizes biotinylated telomerase synthesis primer （B-TS） and synthesizes extension products, which then serve as the templates for PCR amplification using B-TS as the forward primer and tris-（2′2′-bipyridyl） ruthenium （TBR） labeled ACX （TBR-ACX） as the reversed primer. The amplified product is captured on streptavidin-coated paramagnetic beads and detected by ECL. Telomerase positive HeLa cells were used to validate the feasibility of the method. The experimental results showed down to 10 cancer cells can be detected easily. The method is a useful tool for telomerase activity analysis due to its sensitivity, rapidity, safety, high throughput, and low cost. It can be used for screening a large amount of clinical samples.     

Magnetic beads-based electrochemiluminescence assay for rapid and sensitive detection of telomerase activity

Telomerase is a potential cancer marker. We developed a new and robust telomerase activity assay which combines the modified telomere repeat amplification protocol (TRAP) with magnetic beads-based electrochemiluminescence (ECL) detection. The high performance of this assay is related to the determination of telomerase activity from single cell levels, and ECL intensity is linear over the range of 1–1000 HeLa cell equivalents. The proposed telomerase assay offers a highly cost- and time-effective alternative to presently available telomerase assays, which are limited by tedious and complicated post-PCR detection.     

Magneto-mechanical detection of nucleic acids and telomerase activity in cancer cells

The ultra-sensitive magneto-mechanical detection of DNA, single-base-mismatches in nucleic acids, and the assay of telomerase activity are accomplished by monitoring the magnetically induced deflection of a cantilever functionalized with magnetic beads associated with the biosensing interface. The analyzed M13phi DNA hybridized with the nucleic acid-functionalized magnetic beads is replicated in the presence of dNTPs that include biotin-labeled dUTP. The resulting beads are attached to an avidin-coated cantilever, and the modified cantilever is deflected by an external magnetic field. Similarly, telomerization of nucleic acid-modified magnetic beads in the presence of dNTPs, biotin-labeled dUTP, and telomerase from cancer cell extracts and the subsequent association of the magnetic beads to the cantilever surface results in the lever deflection by an external magnetic field. M13phi DNA is sensed with a sensitivity limit of 7.1 x 10(-20) M by the magneto-mechanical detection method.     

A new approach to amplified telomerase detection with polyvalent oligonucleotide nanoparticle conjugates

We report a new assay for human telomerase activity that relies on polyvalent oligonucleotide nanoparticle conjugates as diagnostic probes and amplification units. Gold nanoparticles functionalized with specific oligonucleotide sequences can efficiently capture telomerase enzymes and subsequently be elongated. Both the elongated and unmodified oligonucleotide sequences are simultaneously measured. The two strands not only serve as internal positive controls for each other but also provide a way of amplifying signal. At high concentrations, both elongated and unmodified strands exhibit measurable responses. At low telomerase concentrations (e.g., from 10 HeLa cells), elongated strands cannot be detected, but the unmodified sequences, which come from the same probe particles, can be detected because their concentration is higher, providing a novel form of amplification. This new assay rivals the sensitivity of the conventional PCR-based method of telomerase detection.     

PCR-free electrochemical assay of telomerase activity

In this paper, we report a non-PCR-based electrochemical assay that can detect telomerase activity. Telomerase from HeLa cells may induce telomerization of thiolated primers immobilized on a gold electrode surface. With the telomerization reaction, more and more guanine-rich telomeric repeats are formed, so the electrochemical oxidation signal of guanine at about 1.00 V, which is utilized to indicate the elongated guanine-rich telomeric repeats tethered to the primers, will be increased. This assay method can detect the telomerase activity originated from 3000 HeLa cells and thus holds promise as a simple and sensitive approach in clinical diagnosis of cancer.     

Telomerase inhibitors identified by a forward chemical genetics approach using a yeast strain with shortened telomere length

Telomerase has been proposed as a selective target for cancer chemotherapy. We established a forward chemical genetics approach using a yeast strain with shortened telomere length. Since this strain rapidly enters cell senescence in the absence of active telomerase, compounds that induce selective growth defects against telomere-shortened yeast could be candidates for drugs acting on telomeres and telomerase. We screened our microbial products library and identified three structurally unrelated antibiotics, chrolactomycin, UCS1025A, and radicicol, as active compounds. Detailed analysis showed that chrolactomycin inhibited human telomerase in a cell-free assay as well as in a cellular assay. Long-term culture of cancer cells with chrolactomycin revealed population-doubling-dependent antiproliferative activity accompanied by telomere shortening. These results suggest that chrolactomycin is a telomerase inhibitor, and that the yeast-based assay is useful for discovering the small molecules acting on human telomerase.     

Telomerase activity measurement in magnetically captured epithelial cells: comparison of slab-gel and capillary electrophoresis

We have compared telomerase activity measurements by slab-gel and capillary electrophoresis in cultured cells (A549 and H125 human cancer cell lines) and in cells isolated from clinical peripheral blood specimens epithelial cells of patients with lung and esophageal cancer. Telomerase activity was determined using the telomerase repeat amplification protocol (TRAP) assay with phosphoimager scanning of slab-gels and by laser-induced fluorescence capillary electrophoresis (LIF-CE). Experiments using A549 and H125 cells were performed to determine the reproducibility of each method and to identify the contribution of each stage of the TRAP/polymerase chain reaction (PCR) assay to the variability. In these experiments, it was found that more than half of the overall variability (coefficient of variation, CV = 35%) of the slab-gel method and almost all of the overall variability (CV = 20%) of the CE method was due to the PCR stage of the TRAP assay. In the clinical samples, classification as positive or negative was by visual inspection of the slab-gel and CE electropherograms for the presence of the characteristic 6 base-pair TRAP ladder and by GeneScan analysis of the CE. We examined several criteria including the use of 3, 4, or 5 TRAP bands as the definition of a positive test. Using the slab-gel method, the 5-band criterion gave 40% sensitivity with 100% specificity (no false positives in inactive controls). The CE method yielded a comparable 38% sensitivity and 100% specificity using this criterion. These data indicate that detection of telomerase activity in epithelial cells isolated from peripheral blood has a useful level of sensitivity and specificity and may be useful in the detection and monitoring of aerodigestive cancers. However, analysis by slab-gel is cumbersome and the precision is poor (inter-replicate CV = 20%) compared to LIF-CE (CV = 5%). A high-throughput CE-LIF detection platform will be indispensable for validation studies of telomerase activity measurements.     

Facile assay of telomerase activity utilizing a DNA-detectable chemiluminogenic reagent.

Telomerase shows increased activity in most human cancers and germ line cells, but not in normal human somatic cells. We describe a novel chemiluminescence method for the facile assay of telomerase activity in human cells. The telomerase substrate was incubated with the cell lysate containing various amounts of telomerase, and then the telomerase product was amplified by the polymerase-chained reaction (PCR). The PCR products were separated from the excess substrate, primer and deoxyribonucleotide triphosphates by a centrifugal filter, which distinguished different molecular sizes. The isolated products were reacted with a DNA-detectable chemiluminogenic reagent, 3,4,5-trimethoxyphenylglyoxal. The proposed assay method gave linearity for the telomerase activity in 100 to 10000 cells (r2=0.997), and allowed the assay not only of lower activity, but also of higher activity of telomerase without the requirement of any special labeled-PCR primers in the assay system.     

DNAzyme‐Based Probes for Telomerase Detection in Early‐Stage Cancer Diagnosis

Human telomerase is a polymerase enzyme that adds tandem repeats of DNA (TTAGGG) in the telomeric region to the ends of chromosomes. Since telomerase can be detected in immortalized, but not normal, somatic cells, it has been considered a selective target for cancer chemotherapy. Here, we describe a DNAzyme‐based probe to detect the presence of telomerase in cell lysates. Telomerase elongates the primer site on the probe. Subsequent addition of the Pb^II cofactor activates the DNAzyme, which cleaves the elongated fragment at the RNA site, releasing the probe for repetitive cycling and signal amplification. The cleaved fragment is detected by a reporter molecular beacon. Enzymatic amplification with rapid turnover allows detection of telomerase in the range of 0.1 to 1 μg cell lysate, with a fivefold increase in signal level for cancer cells over normal cells. This probe design can provide a simple, yet rapid and sensitive, measurement of telomerase activity.     

基于杂交链式反应辅助多重信号放大的端粒酶灵敏检测

端粒酶是真核细胞维持端粒长度的关键逆转录酶,其生物活性的高低可以为多种癌症的临床诊断和预后治疗提供有价值的信息.本研究以人宫颈癌细胞(HeLa细胞)裂解液中的端粒酶为研究对象,通过借助杂交链式反应辅助多重信号放大策略,提出了一种新颖、灵敏的检测端粒酶电化学方法.首先将端粒酶的延伸引物自组装在金电极表面,当端粒酶存在时,端粒酶能够催化引物的延伸,产生与发卡环探针H1部分互补的序列,进而引发杂交链式反应,形成由两个发卡环探针(H1和H2)交替杂交而形成的DNA长链.由于H1和H2末端均修饰有生物素,加入链霉亲和素修饰辣根过氧化物酶后,辣根过氧化物酶被被连接到电极表面,催化邻苯二胺氧化生成2,3-二氨基吩嗪,产生显著的电化学信号.实验结果表明,本研究建立的端粒酶电化学检测方法高效、可行,线性范围宽,灵敏度高,可以检测每毫升10个HeLa细胞裂解液中的端粒酶.本方法具有较好的选择性,能有效区分端粒酶和对照蛋白.     

Current literature highlights - April 2002

Telomerase Inhibitors: Telomerase is the enzyme responsible for maintaining telomere length and it has activity not observed in normal somatic cells. In contrast, high expression of telomerase is observed in around 85-90% of human tumour cells and therefore telomerase is regarded as a specific target for development of cancer chemotherapeutic agents. There are several types of inhibitor known. For example antisense oligodeoxynucleotides and related compounds which exhibit potent inhibition of telomerase in the picomolar range. In spite of this research there have been no clinical trials of inhibitors to date, and discovery of novel inhibitors will contribute to evaluation of telomerase inhibitors for cancer chemotherapy. Recent developments have highlighted new telomerase inhibitors based on the bisindole unit (i) (S. Sasaki et. al., Bioorg. Med. Chem. Lett., 11, (2001), 583).     

Aptamer based strategy for cytosensing and evaluation of HER-3 on the surface of MCF-7 cells by using the signal amplification of nucleic acid-functionalized nanocrystals

The electrochemical detection of cell lines of MCF-7 (human breast cancer) has been reported, using magnetic beads for the separation tool and high-affinity DNA aptamers for signal recognition. The high specificity was obtained by using the magnetic beads and aptamers, and the good sensitivity was realized with the signal amplification of DNA capped CdS or PbS nanocrystals. The ASV (anodic stripping voltammetry) technology was employed for the detection of cadmic cation and lead ions, for electrochemical assay of the amount of the target cells and biomarkers on the membrane of target cells, respectively. This electrochemical method could respond to as low as 100 cells mL⁻¹ of cancer cells with a linear calibration range from 1.0 × 10² to 1.0 × 10⁶ cells mL⁻¹, showing very high sensitivity. Moreover, the amounts of HER-3 which were overexpressed on MCF-7 cells were calculated correspond to be 3.56 × 10⁴ anti-HER-3 antibody molecules. In addition, the assay was able to differentiate between different types of target and control cells based on the aptamers and magnetic beads used in the assay, indicating the wide applicability of the assay for early and accurate diagnose of cancers.     

Telomere and Telomerase as Targets for Cancer Therapy

Telomere maintenance and telomerase reactivation is essential for the transformation of most human cancer cells. Telomere shortening to the threshold length, mutations of the telomere-associated proteins, and/or telomerase RNA lead to telomeric dysfunction and therefore genomic instability. Telomerase up-regulation in 85% of human cancer cells has become a hallmark of cancers, hence a promising target for anticancer therapy. In this review, we discuss the mechanism of cancer due to telomere dysfunction and the resulting biological effects, the control of telomerase activity, and the new developments in cancer therapies targeting telomere and telomerase.     

Screening for Oral Cancer Using Electrochemical Telomerase Assay

Electrochemical telomerase assay (ECTA) developed by our group was evaluated in an oral cancer screening using exfoliated oral cells and tissues obtained from patients of oral cancer, mucosa associated disease, or healthy volunteers. Telomerase activity from ECTA is correlated with hTERT mRNA expression level using a real‐time PCR and was increasing in the following order: healthy volunteer group<mucosa associated disease group<oral cancer group. Sensitivity and specificity of ECTA were 88 % and 72 %, respectively when used 17 % of the threshold value based on the receiver operating characteristic curve in ECTA data.     

端粒酶检测新方法研究进展

端粒酶是一种核蛋白逆转录酶,能以自身的RNA为模板添加重复序列(TTAGGG)n至染色体末端,稳定端粒长度。端粒酶在85%～90%的癌细胞中处于激活状态,已经引起了人们的广泛关注,成为肿瘤诊断和临床治疗的重要突破口。因此,准确、高效地测定端粒酶活性具有重要意义。近年来,许多灵敏、准确的体外或原位检测技术在端粒酶活性检测方面得到了发展。该文综述了近年来端粒酶活性检测的最新进展,并对其发展趋势进行了展望。     

High-throughput analysis of telomerase by capillary electrophoresis

The enzyme telomerase is expressed in (85-90)% of all human cancers, but not in normal, non-stem cell somatic tissues. Clinical assays for telomerase in easily obtained body fluids would have great utility as noninvasive, cost-effective methods for the early detection of cancer. The most commonly used method for the detection and quantification of telomerase enzyme activity is the polymerase chain reaction (PCR)-based assay known as the telomerase repeat amplification protocol or TRAP assay. Most of the TRAP assay systems use a slab-gel based electrophoresis system to size and quantify the PCR-amplified extension products. We are developing high-throughput capillary electrophoresis (CE) methods for the analysis of TRAP/PCR products. The TRAP assay was conducted on lysates of the human lung cancer cell line A-549 in reactions containing 5-100 cells. TRAP/PCR products were generated using a fluorescent 4,7,2'4'5'7',-hexachloro-6-carboxyfluorescein(HEX)-labeled TS primer and analyzed on the Applied Biosystems Model 310 CE system using POP4 polymer. After analysis with GeneScan and Genotyper software, the total peak areas of the TRAP ladder extension products were computed using Microsoft Excel. Results were compared with unlabeled TRAP/PCR products analyzed on the Bio-Rad BioFocus 3000 CE system using 6% high molecular weight polyvinylpyrrolidone (HMW PVP) polymer and SYBR Green I dye. Both CE systems were able to resolve the TRAP ladder products with high reproducibility and sensitivity (5-15 cells). With the appropriate robotic sample handling system, these CE methods would enable performing the telomerase TRAP assay with increased sensitivity, reproducibility and automation over slab-gel methods.     

Optimized detection method of telomerase activity in cancer diagnostics

Telomerase is a ribonucleoprotein complex; it uses an internal RNA template to synthesize telomere DNA. Telomerase is active in 90% of cancers and can be used as a diagnostic marker. We have optimized conditions for the extraction from small tissue samples (<0.05 g) of cervical lesions to analyze telomerase activity and selected the optimal concentrations of the tissue extracts. Different concentrations of the extracts were used to determine the presence of possible telomerase inhibitors and Taq-polymerase in the extracts. Using lung and kidney cancer samples it was shown that these conditions are applicable for the estimation of telomerase activity in different cancer types. Many investigations of telomerase activity using different types of TRAP (Telomere Repeat Amplification Protocol) have been performed. The possibility of comparison of TRAP results with radioactive and Sybr green detection remains open. We compared these two types of detection for several samples of cervical intraepithelial neoplasias and conclude that they have similar sensitivities.     

Electrochemical and Electrochemiluminescence Determination of Cancer Cells Based on Aptamers and Magnetic Beads

The electrochemical and electrochemiluminescence (ECL) detection of cell lines of Burkitt’s lymphoma (Ramos) by using magnetic beads as the separation tool and high‐affinity DNA aptamers for signal recognition is reported. Au nanoparticles (NPs) bifunctionalized with aptamers and CdS NPs were used for electrochemical signal amplification. The anodic stripping voltammetry technology employed for the analysis of cadmium ions dissolved from CdS NPs on the aggregates provided a means to quantify the amount of the target cells. This electrochemical method could respond down to 67 cancer cells per mL with a linear calibration range from 1.0×10² to 1.0×10⁵ cells mL^?1, which shows very high sensitivity. In addition, the assay was able to differentiate between target and control cells based on the aptamer used in the assay, indicating the wide applicability of the assay for diseased cell detection. ECL detection was also performed by functionalizing the signal DNA, which was complementary to the aptamer of the Ramos cells, with tris(2,2‐bipyridyl) ruthenium. The ECL intensity of the signal DNA, replaced by the target cells from the ECL probes, directly reflected the quantity of the amount of the cells. With the use of the developed ECL probe, a limit of detection as low as 89 Ramos cells per mL could be achieved. The proposed methods based on electrochemical and ECL should have wide applications in the diagnosis of cancers due to their high sensitivity, simplicity, and low cost.