Real-time quantitative telomeric repeat amplification protocol assay for the detection of telomerase activity

BACKGROUND: Telomerase is a ribonucleoprotein enzyme associated with immortalization and transformation of human cells. The telomeric repeat amplification protocol (TRAP) is widely used for the detection of telomerase activity. The TRAP method, although highly sensitive and specific because it includes PCR amplification, is laborious and does not provide precise quantitative information. METHODS: We developed a real-time quantitative TRAP (RTQ-TRAP) system by combining a real-time PCR technique with the conventional TRAP method. Telomerase activity in human tumor cell lines and in 13 lymphoma samples was measured using the RTQ-TRAP assay, and the results obtained from the samples using the RTQ-TRAP method were compared with the conventional TRAP method. RESULTS: The RTQ-TRAP method was both accurate and reproducible in measuring telomerase activity in a dilution series of protein extracts from HL60 cells. Telomerase activity in 13 lymphoma samples, as determined by the RTQ-TRAP method, was ninefold lower than that measured by the conventional TRAP method. The half-life of telomerase activity in human tumor cells, as determined using RTQ-TRAP, was much shorter than the half-life reported previously. CONCLUSIONS: Our results suggest that the conventional TRAP assay frequently overestimates telomerase activity in tumor samples. The RTQ-TRAP method is thus a useful tool to rapidly and precisely quantify telomerase activity.     

Improved TRAP-silver staining versus conventional radioactive TRAP assays: quantification of telomerase activity during immortalization and in pathological human endometrium

OBJECTIVES: To develop a sensitive telomeric repeat amplification protocol (TRAP)-silver staining assay for telomerase activity quantification. DESIGN AND METHODS: TRAP assays were performed by using a TRAPeze telomerase kit with or without [alpha-32P]-dCTP. Amplification products were electrophoresed in polyacrylamide gels and detected by autoradiography or a modified silver staining protocol. Telomerase activity was quantified from radioactive counts or optical density of telomerase products from test extracts and controls. RESULTS: TRAP-silver staining assay was at least as sensitive as radioactive TRAP assay and quantified telomerase activity within linearity from 10 to 3,000 cell equivalents. Both methods quantified a weak telomerase activity in normal endometrial glandular epithelial cells (GEC) and a strong increase in immortalized GEC. In human pathologic endometria (n=24), telomerase activity was correlated with lesion seriousness and distinguished simple hyperplasias from nonhyperplasic or cancerous lesions. CONCLUSIONS: TRAP-silver staining assay is suitable for cell and tissue telomerase activity routine quantification.     

Real-time telomeric repeat amplification protocol using the duplex scorpion and two reverse primers system: the high sensitive and accurate method for quantification of telomerase activity

BACKGROUND: Real-time quantitative TRAP assays for detection of telomerase activity have been recently developed to eliminate complex post-PCR procedures. However, all of them use the conventional TRAP assay that possesses an unpredictable cascade of events in PCR amplification caused by stagger annealing, which may affect the accuracy of quantitation. METHODS: A novel RTQ-TRAP method was developed by combining the duplex scorpion with modified TP-TRAP assay that has high fidelity PCR amplification of the telomerase product (DS/TP-TRAP). The synthesized oligonucleotide that represents telomerase products is used to set up a standard curve. RESULTS: The DS/TP-TRAP method gives the standard curve a dynamic range of 6 orders of magnitude (R(2)=0.9992). It optimizes PCR amplification efficiency and determines telomerase activity in a lower threshold cycle number (Ct value). The method is both accurate and reproducible to measure telomerase activity in human tumor cell lines, and linearity from 1 to 1000 cells could be obtained (R(2)=0.9926). For tumor samples, the results determined by the DS/TP-TRAP assay are comparable to the data obtained with the conventional TRAP method. CONCLUSIONS: The DS/TP-TRAP assay provides a high sensitive and accurate method for real-time quantitative detection of telomerase activity. It is thus a potential robust tool for application in cancer molecular diagnostics.     

Real-time RT-PCR for the measurement of prostate-specific antigen mRNA expression in benign hyperplasia and adenocarcinoma of prostate.

Since PSA is supposed to play an active role in the progression of prostate cancer, we applied a quantitative RT-PCR to measure the absolute levels of prostate-specific antigen (PSA) mRNA expression in benign and malignant prostatic tissue. Consecutive fine needle prostate biopsy material from 59 patients (43 with prostate adenocarcinoma and 16 with benign prostatic hyperptrophy; BPH) was used for the measurement of PSA mRNA expression. In addition, we evaluated the correlation between PSA synthesis and PSA circulating levels in the same patients. The relationship between PSA mRNA expression and histological grade was also evaluated. PSA mRNA was measured with a quantitative RT-PCR, based on the use of fluorogenic probes, according to the TaqMan reaction system. The mRNA expression for PSA in prostate adenocarcinoma biopsies was highly variable, ranging from 2 x 10(4) to 2.1 x 10(8) molecules/microg total RNA with a mean value of 2.5 x 10(7) and significantly higher (p = 0.006) than that found in BPH patients (mean: 1.3 x 10(6) and range: 6.9 x 10(2) to 8 x 10(6)). The mRNA PSA expression in needle biopsy material did not seem to be related to PSA circulating levels in prostate cancer patients (r = 0.281), whereas in BPH patients the two parameters correlated significantly (r = 0.667, p < 0.01). A reduction of PSA mRNA expression in samples with a lower grade of differentiation (Gleason score 9-10) was also observed. Even though a mean increase of PSA expression was demonstrated in cancer samples, this small difference does not confirm a significant role of PSA proteolytic activity in prostate cancer progression. In conclusion, the assay procedure we proposed represents a reliable basis for more extensive study of PSA physiopathology in prostate cancer.     

Telomerase detection in body fluids.

BACKGROUND: Telomerase is a ribonucleoprotein that maintains chromosomal telomere length. Telomerase is not active in nonmalignant somatic cells, but is activated in most human cancers. Telomerase activity in easily obtainable body fluids that bathe tumors may be a useful cancer marker, especially when used in conjunction with conventional cytology. APPROACH: Results from studies that assayed telomerase activity in easily obtainable body fluids are reviewed. CONTENT: The telomerase repeat amplification protocol (TRAP) assay has been used to measure telomerase activity in body fluids, including ascites, pleural effusions, pelvic washes, bronchial washings, bronchial lavage, urine, bladder washings, oral rinses, and plasma. Telomerase activity has sensitivities of 60-90% as a tumor marker with clinical specificities for cancer of approximately 90%. Telomerase activity is more sensitive than conventional cytology, the sensitivity of which was 40-65% in various studies. SUMMARY: Telomerase activity in body fluids, as measured by the TRAP assay, is a sensitive potential tumor marker that might help increase the cancer detection rate and the cancer treatment success rate when combined with conventional cytology.     

抗端粒酶反义寡核苷酸对人胃癌细胞增殖的影响

目的：观察抗端粒酶反义寡脱氧核苷酸（ASODN）对人胃癌细胞系端粒酶活性的抑制作用及其对癌细胞增殖的影响。方法：合成与人类端粒酶RNA模板区碱基序列互补的ASODN，并用阳离子脂质体转染剂DOSPER介导，作用于人胃癌细胞系FGC85，空白组和正义寡脱氧核苷酸（SODN）作为对照，用以PCR为基础的端粒重复序列扩增法（TRAP）结合ELISA方法检测胃癌细胞的端粒酶活性，台盼蓝拒染法细胞计数，噻唑蓝（MTT）试验观察癌细胞增殖情况，免疫组化方法检测细胞增生抗原Ki-67。结果：脂质体介导的抗端粒酶ASODN对胃癌细胞的端粒酶活性具有抑制作用；细胞坟数和MTT试验显示胃癌细胞增殖速度明显减慢；细胞Ki-67染色阳性率显著降低。结论：特异性抗端粒酶ASODN可运用于抗肿瘤实验性治疗和相关研究。     

端粒重复扩增生物发光分析法定量检测端粒酶活性

目的：建立端粒酶活性定量检测的TRAP-发光分析法。方法：细胞株、组织样品与引物孵浴后，释放的焦磷酸盐由硫酸化酶作用转变为ATP，用荧光素酶生物发光系统检测发光信号；比较TRAP-发光分析法与TRAP—ELISA的结果。结果：TRAP-发光分析法的线性范围在2-1000个细胞之间。检测结果与TRAP—SYBRGreen染色一致，与TRAP—EHSA法显著相关（r^2=0．992，P〈0.001）。结论：TRAP-发光分析法是一种稳定、快速、实际可行的端粒酶活性的定量分析方法。     

Comparison of telomerase activity and GSTP1 promoter methylation in ejaculate as potential screening tests for prostate cancer

New diagnostic tools are needed for the early detection of prostatic cancer. The molecular detection of prostate cancer cells in ejaculates was evaluated using complementary PCR-based methods. LNCaP cells, a cell line derived from prostatic carcinoma, were spiked into normal seminal ejaculates and the prostatic epithelial component of the specimens was isolated by immunomagnetic bead sorting, using a monoclonal antibody to prostate-specific membrane antigen (PSMA). Ejaculates from nine patients with a recent diagnosis of prostate cancer were processed in a similar fashion, using LNCaP-spiked aliquots as an internal positive control. Telomerase expression was evaluated by the telomeric repeat amplification protocol (TRAP) and glutathione S-transferase gene promoter (GSTP1) hypermethylation was evaluated by methylation-sensitive restriction endonuclease digestion and PCR amplification. Telomerase activity was detected in LNCaP cells recovered from normal seminal ejaculates but was not found in all nine samples from patients with prostate cancer. The sensitivity of GSTP1 analysis was similar to telomerase analysis for the detection of LNCaP cells from normal ejaculate samples but was positive in ejaculates from four out of nine patients with prostate cancer. GSTP1 DNA methylation status is more sensitive than telomerase analysis for the detection of malignant cells in seminal ejaculates from patients with prostate cancer.     

胃癌及其癌前病变中端粒酶的表达

目的探讨胃癌及其癌前病变中端粒酶的表达。方法对纤维内窥镜下活检的胃组织采用对TRAP方法加以改进的银染TRAP法,检测了端粒酶活性。结果29例胃癌中阳性率（23／29）82．7％：胃溃疡8例及胃息肉1例皆未检测到端粒酶活性,慢性萎缩性胃炎伴肠上度化生（AGIM）8例中4例有端粒酶表达。胃癌及AGIM端粒酶阳性率与良性病变阳性率比较差异显著（P＜0．05）。结论银染TRAP法具有检测标本用量少,周期短,灵敏度高,特异性强等优点。对胃活检组织进行端粒酶检测在胃癌诊断中具有重要意义。对端粒酶检测阳性者应加强随访。     

Limitations on the quantitative determination of telomerase activity by the electrophoretic and ELISA based TRAP assays

Telomerase is a promising new tumor marker and can be detected using the TRAP (Telomeric Repeat Amplification Protocol) method. To address factors affecting its quantitative determination, we evaluated two commercial TRAP assays, an electrophoretic and an ELISA assay formats, using cultured cells and human tumor samples. We found that both TRAP assays had a limited linearity from 250 to 5000 tumor cells, with a similar intra-assay variation. The quantification of TRAP products was affected by high cell number in sample, the presence of non-tumor cells, and interfering substances in patient specimens. Because both assays have different limitations, determination of telomerase by a combined use of the two may provide more accurate information on the telomerase activity in a specimen. Extracts of specimens should also be tested at several concentrations to insure that the result is not being falsely decreased by an inhibitor. The quantitative results for telomerase activity by the TRAP assays, however, should be interpreted cautiously.     

Telomerase activity in gastrointestinal, bladder and breast carcinomas and their clinical applications

Telomere ends are known to be shortened at every division of the cells. Telomerase is a ribonucleoprotein which compensate for the telomere ends and indispensable for the immortalization of the cells. It is reported that the enzyme is activated in a variety of cancer cells. In the present report, the enzyme was activated in more than 70% of gastrointestinal, bladder and breast cancer by TRAP assay. Moreover, in order to elucidate whether the enzyme activity is useful for the non-invasive detection of exfoliated cancer cells in the colon bowel washings, urine and fine needle aspirate of the breast tumors, TRAP assay was performed. In most of the cases, the positive findings were observed which support that the enzyme activity is useful for the clinical application. The expression of mRNA and protein for hTERT was detected in cancer cells, however, the assay contains the pitfall including the Taq inhibitor or telomerase inhibitor. Moreover, the activity is also detected although it is weak, in the benign or premalignant lesions. Further analysis is required for the clinical application of the method.     

脑胶质瘤端粒酶活性表达的研究

目的：探讨端粒酶在脑胶质瘤发生、发展中的作用，并研究端粒酶活性是否与其恶性程度相关。方法：应用端粒重复序列扩增技术(TRAP)对48例胶质瘤标本和8例正常脑组织的端粒酶活性进行检测。结果：8例正常脑组织端粒酶活性均为阴性表达，48例胶质瘤标本中23例为表达阳性(47．92％)，低度恶性组与高度恶性组胶质瘤之间端粒酶活性阳性率有明显差异(P=0．001)。结论：端粒酶活性与胶质瘤恶性程度明显相关，端粒酶在胶质瘤发生发展过程中可能具有重要作用。     

Telomerase activity in fine needle aspiration biopsy samples: application to diagnosis of human thyroid carcinoma

BACKGROUND: The diagnosis of thyroid follicular carcinoma by fine needle aspiration biopsy is a well known problem in thyroid pathology. METHODS: We evaluated telomerase activity (TA) in 85 fine needle aspiration biopsy (FNAB) samples from patients with thyroid nodules. Surgery samples from patients with tumor or follicular adenomas were also analyzed. RESULTS: Twenty of the FNAB samples corresponded to carcinomas and were positive to telomerase assay (TA >10 Units). Among them, 4 follicular carcinomas and 1 papillary carcinoma were labeled as indeterminate by FNAB cytological examination. Four percent false positive cases and no false negative cases for TA in FNABs were reported. FNAB samples from follicular adenomas were diagnosed as indeterminate by cytological examination, but they showed no detectable TA. Tumor tissues from patients with follicular or papillary thyroid carcinomas presented TA >10 Units, whereas follicular adenoma tissues (benign nodules) showed no TA. CONCLUSION: Our results showed a good correlation between TA in FNAB samples and tumor/nodule thyroid tissue. This suggested that use of TA as a biological marker of malignancy might be a useful tool in the diagnosis of follicular thyroid carcinomas or follicular thyroid adenomas using FNAB samples.     

纤维支气管镜活组织端粒酶活性检测对肺癌的诊断价值

目的探讨肺癌组织端粒酶活性检测对肺癌的诊断价值。方法采用PCR-ELISA法检测了87例纤维支气管镜(纤支镜)活组织的端粒酶活性,其中经病理证实为肺癌病变组织38例,炎症病变组织26例,正常黏膜组织23例。结果肺癌病变组织端粒酶活性水平均值明显高于炎症组织和正常组织(P<0.001),但炎症组织端粒酶活性水平与正常组织比较差异无显著性(P>0.05);不同病理类型肺癌的端粒酶活性水平比较无明显差异(P>0.05)。肺癌病变组织不同病理分级间端粒酶活性比较有显著差异(P<0.05)。结论端粒酶活性检测,对肺癌诊断有重要意义。