Quantification Of Telomerase Activity Research Articles

A target-triggered fluorescence-SERS dual-signal nano-system for real-time imaging of intracellular telomerase activity

Telomerase (TE) is a promising diagnostic and prognostic biomarker for many cancers. Quantification of TE activity in living cells is of great significance in biomedical and clinical research. Conventional fluorescence-based sensors for quantification of intracellular TE may suffer from problems of fast photobleaching and auto-fluorescence of some endogenous molecules, and hence are liable to produce false negative or positive results. To address this issue, a fluorescence-SERS dual-signal nano-system for real-time imaging of intracellular TE was designed by functionalizing a bimetallic Au@Ag nanostructure with 4-p-mercaptobenzoic acid (internal standard SERS tag) and a DNA hybrid complex consisted of a telomerase primer strand and its partially complimentary strand modified with Rhodamine 6G. The bimetallic Au@Ag nanostructure serves as an excellent SERS-enhancing and fluorescence-quenching substrate. Intracellular TE will trigger the extension of the primer strand and cause the shedding of Rhodamine 6G-modified complimentary strand from the nano-system through intramolecular DNA strand displacement, resulting in the recovery of the fluorescence of Rhodamine 6G and decrease in its SERS signal. Both the fluorescence of R6G and the ratio between the SERS signals of 4-p-mercaptobenzoic acid and Rhodamine 6G can be used for in situ imaging of intracellular TE. Experimental results showed that the proposed nano-system was featured with low background, excellent cell internalization efficiency, good biocompatibility, high sensitivity, good selectivity, and robustness to false positive results. It can be used to distinguish cancer cells from normal ones, identify different types of cancer cells, as well as perform absolute quantification of intracellular TE, which endows it with great potential in clinical diagnosis, target therapy and prognosis of cancer patients.

Ratiometric Fluorescent Biosensor for Visual Discrimination of Cancer Cells with Different Telomerase Expression Levels.

Telomerase is inactive in normal somatic cells but highly activated in tumor cells to maintain their indefinite proliferation and immortal phenotype. As a specific marker for the generation and progress of almost all tumors, the detection of telomerase activity by classical PCR techniques has served in the biological research of tumors. However, the detection of in situ telomerase activity in cell extracts to evaluate the malignancy, progress, and metastasis of tumors remains a daunting challenge. Here, a precisely designed FRET-based ratiometric fluorescent oligonucleotide probe has achieved high-fidelity detection of telomerase activity for accurate discrimination of different cancer cells toward advanced diagnosis of tumors. Our method is superior to other methods in its capabilities to quantify telomerase activity in cell extracts and visualize various tumor cell extracts with different telomerase expression levels by the naked eye for clinical diagnosis. In particular, the ratiometric fluorescent probe used in the assay could exclude other experimental factors influence, and further avoid false positive signal generation. The method reported here could provide a reliable, accurate, and convenient way in medical diagnostics and therapeutic response assessment.

Quantification of telomerase activity in normal oral mucosal tissue and oral squamous cell carcinoma

Background and Objective:The role of telomeres and telomerase in oral cancer is an area of much recent interest. The understanding of the role of telomere biology, the end replication problem leading to genomic instability and the reactivation of telomerase, is absolutely critical to our understanding of oral cancer, and more so, to our ability of early diagnosis and developing novel therapies and cancer prevention approaches. The aim of the present study was to quantify telomerase activity (TA) in oral squamous cell carcinoma (OSCC) and normal oral mucosa and assess the role of telomerase as diagnostic and prognostic marker of oral malignancy.Materials and Methods:We quantified TA in 45 patients with OSCC and 20 normal oral mucosal specimens using polymerase chain reaction-based telomeric repeat amplification protocol assay and compared it with the clinical status and grade of malignancy.Results:TA was detected in 89% of malignant and 5% of normal oral mucosal tissue. The TA levels ranged from 0.28 to 6.91 (mean 2.05, standard deviation [SD] 1.33) in OSCC and 0.21 to 1.09 (mean 0.54, SD 0.27) in normal oral mucosa. There was no relationship between TA levels and clinical stages, site of the lesion, history of adverse habits, or sex of the patient. However, under the WHO classification, there were significant differences (P < 0.00) between Grades I, II, and III. Furthermore, increasing age of the patient significantly correlated with TA.Interpretation and Conclusion:The results of the present study indicate that activation of TA is frequent in OSCC. Statistically significant difference in quantified telomerase levels of OSCC and normal oral mucosa (P < 0.00) demonstrates the significant clinical usefulness of telomerase activation as a valuable marker for diagnosis while significant correlation of TA with grades of malignancy indicates its effectiveness as marker for prognosis of OSCC.

Quantification of Telomerase Activity in Colonial Ascidians

Colonial ascidians are marine invertebrate chordates that reproduce sexually and asexually, and are also capable of whole body regeneration in response to damage. Telomerase is required to maintain chromosome length in all eukaryotic cells that divide indefinitely; it is therefore expressed in stem cells and is hypothesized to be necessary for both continuous asexual budding and for regeneration. Here, we investigate telomerase activity in the colonial ascidian Botrylloides. Telomerase activity was determined using a qPCR‐based assay and normalized by total protein. Results so far show that adult ascidian tissue has high levels of telomerase activity; this may be related to stem cell abundance and regeneration abilities of Botrylloides. Work in progress is examining telomerase activity in different tissues, including probable stem cell niches, as well as in different developmental and regeneration stages.Support or Funding InformationCSU Council on Ocean Affairs, Science &amp; Technology (COAST); Biological Sciences Department, California Polytechnic State University

Distribution of TTAGG-specific telomerase activity in insects.

In most eukaryotes, telomeres consist of tandem arrays of a short repetitive DNA sequence. Insect telomeres are generally constituted by a (TTAGG)n repeat motif. Usually, telomeres are maintained by telomerase, a specialized reverse transcriptase that adds this sequence to chromosome ends. We examined telomerase activity in 15 species across Insecta. Telomerase activity was revealed in Isoptera, Blattaria, Lepidoptera, Hymenoptera, Trichoptera, Coleoptera, and Sternorrhyncha. In contrast, we were not able to detect telomerase activity in Orthoptera, Zygentoma, and Phasmida. Because we found telomerase activity in phylogenetically distant species, we conclude that a distribution pattern of (TTAGG)n sequence in Insecta is generally consistent with that of telomerase activity. Thus, the TTAGG-telomerase system is functional across the Insecta. Using real-time quantitative telomeric repeat amplification protocol (RTQ-TRAP) system, we quantified telomerase activity in different developmental stages and different tissues of a cockroach, Periplaneta americana. We show that telomerase is upregulated in young instars and gradually declines during development. In adults, it is most active in testes and ovaries. Thus, the telomerase activity of hemimetabolous insects seems to be associated with cell proliferation and organismal development.

Abstract LB-253: TRAPing telomerase activity using droplet digital PCR.

Abstract The aim of this work was to develop a more sensitive and high throughput assay for measuring telomerase activity. Telomeres are the protective structures at the ends of chromosomes consisting of 6 bp repeat sequences. In young cells, these regions can be as long as 15kb and act as caps which protect the DNA ends. These ends naturally degrade with each passing cell division, usually losing 25-200 base pairs per division. Once they are shortened below a critical length (estimated to be 200-300 bp) the cells arrest and become senescent, or “old”. Telomeres can be thought of as a cellular or mitotic clock. Once the clock has wound down, the cells either die or pass through crisis and become immortal. One of the mechanisms of immortality is the activation of the enzyme, telomerase. Telomerase is the endogenous reverse transcriptase responsible for adding repeats to telomeres, rewinding the clock and enabling a cell to continuously divide. Abundant telomerase activity is found in fetal and adult stem cells, germ cells, and cancer. It is also present at much lower levels in non-stem cells, such as immune cells, but these levels can be difficult to measure reliably. The telomerase repeat amplification protocol (TRAP) measures the presence of active telomerase by measuring the activity of the enzyme on a starting DNA template, which is then amplified by PCR. For samples with abundant telomerase activity, SYBR qPCR assays provide high throughput. However, the current most sensitive method of detection still uses radioactivity, laborious PAGE sequencing gels, and densitometry to quantify telomerase. Here we explore using droplet digital™ PCR (ddPCR™) to provide absolute quantification of telomerase activity. This was achieved through single-molecule counting of telomerase-extended templates that were partitioned into droplets, amplified by PCR and detected by fluorescence droplet flow cytometry. Analysis of control samples suggests ddPCR is at least an order of magnitude more sensitive than TRAP radiography and is more amenable to higher throughput analysis. We extend throughput, sensitivity, and the range of biological samples that can be analyzed for telomerase activity. Citation Format: Dawne Shelton, Jack Regan, George Karlin-Neumann, Jue Lin, Elizabeth Blackburn. TRAPing telomerase activity using droplet digital PCR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-253. doi:10.1158/1538-7445.AM2013-LB-253

Quantification of Alternative Splicing Variants of Human Telomerase Reverse Transcriptase and Correlations with Telomerase Activity in Lung Cancer

Telomerase plays important roles in the development and progression of malignant tumors, and its activity is primarily determined by transcriptional regulation of human telomerase reverse transcriptase (hTERT). Several mRNA alternative splicing variants (ASVs) for hTERT have been identified, but it remains unclear whether telomerase activity is directly associated with hTERT splicing transcripts. In this study, we developed novel real-time PCR protocols using molecular beacons and applied to lung carcinoma cell lines and cancerous tissues for quantification of telomerase activity and three essential hTERT deletion transcripts respectively. The results showed that lung carcinoma cell lines consistently demonstrated telomerase activity (14.22–31.43 TPG units per 100 cells) and various hTERT alternative splicing transcripts. For 165 lung cancer cases, telomerase activity showed significant correlation with tumor differentiation (poorly->moderately->well-differentiated, P<0.01) and with histotypes (combined small cell and squamous cell carcinoma>squamous cell carcinoma>adenosquamous carcinoma>adenocarcinoma, P<0.05). Although the overall hTERT transcripts were detected in all the samples, they were not associated with telomerase activity (r = 0.092, P = 0.24). Telomerase activity was significantly correlated with the transcriptional constituent ratio of α-deletion (r = -0.267, P = 0.026), β-deletion (r = -0.693, P = 0.0001) and γ-deletion (r = –0.614, P = 0.001). The positive rate and average constituent ratio of β-deletion transcripts (92.12%, 0.23) were higher than those of α-deletion (41.82%, 0.12) or γ-deletion (16.36%, 0.18) transcripts. The combined small-cell and squamous cell carcinomas expressed less deletion transcripts, especially β-deletion, than other histotypes, which might explain their higher telomerase activity. In conclusion, the molecular beacon-based real-time PCR protocols are rapid, sensitive and specific methods to quantify telomerase activity and hTERT ASVs. Telomerase activity may serve as a reliable and effective molecular marker to assist the evaluation of histological subtype and differentiation of lung carcinomas. Further studies on hTERT deletion splicing transcripts, rather than the overall hTERT transcripts, may improve our understanding of telomerase regulation.

Abstract A58: Wnt signaling pathway components are associated with telomerase activity in breast cancer

Abstract Telomerase is a ribonucleoprotein complex that adds TTAGGG repeats to telomeres in a highly regulated fashion. Telomerase activity is absent in normal somatic cells, thus limiting their number of cell divisions due to telomere shortening. In contrast, the large majority of tumor cells upregulate telomerase activity, consistent with their ability to maintain stable telomere lengths and to divide indefinitely. Telomerase activity is detectable in over 90% of breast tumors, and it is well established that telomerase overexpression is an important step in the progression of normal epithelium to invasive breast cancer. In addition to telomerase upregulation, deregulation of various signaling pathways promotes tumor progression and metastasis. To better understand in which cellular pathway telomerase and telomeres are involved, we comprehensively quantified telomerase activity and telomere length in a panel of 52 extensively characterized breast cancer cell lines and correlated these metrics with the expression profiles of the cell lines. Across the entire panel of breast cancer cell lines, expression of 530 genes was significantly correlated with telomerase activity (FDR-corrected p-val &amp;lt; 0.2), while expression of 585 genes was significantly correlated with telomere length. Secondary G0-term analysis revealed that telomere length was significantly associated with genes involved in Alzheimer's disease, apoptosis regulation and negative regulation of transcription. Genes significantly correlated with telomerase activity were associated with apoptosis regulation, DNA repair, nucleotide metabolism and Wnt signaling. Several Wnt target genes, including MYC, KLF5, and FGF18 were also correlated with telomerase activity. We also used the network analysis tool PARADIGM (1) to identify cellular pathways associated with telomerase activity and telomere length. Notably, Wnt signaling components were significantly correlated with telomerase activity. Given the recent evidence that the telomerase component TERT can act as a transcriptional modulator of the Wnt/beta-catenin signaling pathway (2), we are currently testing how Wnt signaling is altered due to telomerase perturbation in breast cancer cells. (1) Vaske, C. J. et al. Inference of patient-specific pathway activities from multi-dimensional cancer genomics data using PARADIGM. Bioinformatics 2010; 26:i237–245 (2) Park, J. I. et al. Telomerase modulates Wnt signaling by association with target gene chromatin. Nature 2009; 460:66–72 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A58.

Rapid Quantification of Telomerase Activity Employing an Improved Real-time Telomeric Repeat Amplification Protocol in Clinical Tissue Samples Eliminates Interference by PCR Inhibitors

Rapid Quantification of Telomerase Activity Employing an Improved Real-time Telomeric Repeat Amplification Protocol in Clinical Tissue Samples Eliminates Interference by PCR Inhibitors

Telomere deregulations possess cytogenetic, phenotype, and prognostic specificities in acute leukemias

Telomeres are protected by tightly regulated factors and elongated by telomerase. Short and/or deprotected chromosomes are recombinogenic and thereby cancer prone. Together with the quantification of telomerase activity (TA), measuring telomere length (TL) and expression of the genes that govern telomere protection and elongation are useful for assessing telomere homeostasis. By these means we demonstrate that TL, hTERT, and TA are in the order acute myelogenous leukemia (AML) > T-cell acute lymphoblastic leukemia (T-ALL) > B-cell acute lymphoblastic leukemia (B-ALL) > T-ALL > AML, and B-ALL > AML > T-ALL. AML0 and AML3 display the lowest amounts of hTERT transcripts, and ALL and AML cells with cytogenetic abnormalities possess the shortest telomeres. hTERT expression includes phenotype-specific RNA maturation and correlates with TA but not with TL. A wide ratio of TA to hTERT expression between leukemia subtypes suggests phenotype-specific hTERT post-transcriptional deregulations. B- and T-ALL overexpress Ku70 and Pinx1, T-ALL PTOP and RAP1, and B-ALL TRF2, the expression of which is significantly higher in cases with abnormal karyotype. hTERT transcription and TL correlate with response to intensive chemotherapy, and hTERT and RAD50 are independent prognostic factors for survival. Each leukemia subtype possesses specific telomere dysregulations that rely on phenotype, karyotype, response to treatment, and survival.

Senescence evasion by MCF-7 human breast tumor-initiating cells

IntroductionA subpopulation of cancer cells, tumor-initiating cells, is believed to be the driving force behind tumorigenesis and resistance to radiation and chemotherapy. The persistence of tumor-initiating cells may depend on altered regulation of DNA damage and checkpoint proteins, as well as a reduced propensity to undergo apoptosis or senescence.MethodsTo test this hypothesis, we isolated CD24-/low/CD44+ tumor-initiating cells (as mammospheres) from MCF-7 breast cancer cells grown in adherent monolayer culture, and carried out a comprehensive comparison of cell death and DNA damage response pathways prior to and after exposure to ionizing radiation in mammospheres and monolayer MCF-7 cells. Single and double-strand break repair was measured by single-cell gel electrophoresis. The latter was also examined by phosphorylation of histone H2AX and formation of 53BP1 and Rad51 foci. Apoptosis was quantified by flow-cytometric analysis of annexin V-binding and senescence was analyzed on the basis of cellular β-galactosidase activity. We employed the telomeric repeat amplification protocol to quantify telomerase activity. Expression of key DNA repair and cell cycle regulatory proteins was detected and quantified by western blot analysis.ResultsOur data demonstrate that in comparison to the bulk population of MCF-7 cells (predominantly CD24+/CD44+), the MCF-7 mammosphere cells benefit from a multifaceted approach to cellular protection relative to that seen in monolayer cells, including a reduced level of reactive oxygen species, a more active DNA single-strand break repair (SSBR) pathway, possibly due to a higher level of expression of the key SSBR protein, human AP endonuclease 1 (Ape1), and a significantly reduced propensity to undergo senescence as a result of increased telomerase activity and a low level of p21 protein expression. No significant difference was seen in the rates of double-strand break repair (DSBR) between the two cell types, but DSBR in mammospheres appears to by-pass the need for H2AX phosphorylation.ConclusionsEnhanced survival of MCF-7 tumor-initiating cells in response to ionizing radiation is primarily dependent on an inherent down-regulation of the senescence pathway. Since MCF-7 cells are representative of cancer cells that do not readily undergo apoptosis, consideration of senescence pathways may play a role in targeting stem cells from such tumors.

Abstract 3334: Telomerase activity corresponds to the level of aggressiveness of ovarian cancer stem cells and are a potential site of targeted therapy

Abstract Objective: Cancer stem cells possess the important property of self-renewal. Telomerase activity (TA) is often used as a molecular marker for cancer aggressiveness and allows propagation of cancer cells by adding telomere repeats which can result in unlimited cell replication. As such, the potential of telomerase in maintaining self-renewal properties of cancer stem cells have been proposed. Our objectives were to determine the TA of ovarian cancer stem cells (OSC) and the effectiveness of targeting telomerase for cancer therapy. Methods Ovarian cancer cell lines (ES2, TOV112D, and SKOV3) were grown in non-adherent growth conditions to form spheroid forming cells for numerous successive spheroid generations. Flow cytometry determined the CD44+/CD24- phenotype consistent with cancer stem-cells. Histologic subtypes were chosen to correspond to decreasing levels of aggressiveness: ES2- clear cell; SKOV3- papillary serous; and TOV112D- endometrioid. TRAPEZE RT Telomerase Detection Kit utilized flourescently labeled primers to detect and quantify telomerase activity by RT-PCR. Results were confirmed with luciferase reporter plasmid containing promoter of human telomerase reverse transcriptase (hTERT). Each cancer cell line was treated with 10, 20, 50, and 100uM of a small molecule telomerase inhibitor BIBR1532 measured by cell proliferation survival assay for a total of 72 hours. Results All ovarian cancer cell lines readily formed spheroids in non-adherent growth conditions with flow cytometry demonstrating the enrichment of cancer stem cell phenotype: CD44+/CD24-. TA was significantly increased in spheroid forming cells enriched for OSC in both ES2 (5-fold increase) and SKOV3 (6-fold increase) (p &amp;lt; 0.001), however TOV112D showed a decrease in TA. Similarly, ES2 and SKOV3 showed significant TA by luciferase assay with 400% and 1,700%, respectively (p &amp;lt; 0.001), while TOV112D had a decrease in TA. In all cell lines studied, treatment with BIBR1532 demonstrated significant cell death in a dose-dependent fashion with a 5-fold cell kill and only 20% of cancer cell survival at 72 hours. Conclusions Telomerase activity appears to increase with ovarian cancer stem cells and supports the hypothesis that it could be important in the self-renewal properties of cancer stem cells. Inhibition of telomerase with a competitive small molecule demonstrated significant response in-vitro and could represent novel targeted therapy for ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3334.

Immunodetection of human telomerase reverse-transcriptase (hTERT) re-appraised: nucleolin and telomerase cross paths

The involvement of telomerase in cellular immortalization and senescence has often been assessed by means of telomerase expression at the RNA level and quantification of telomerase activity by the telomeric repeat amplification protocol assay. However, these methods either neglected the existence of various telomerase splice variants, or ignored the nonconventional functions of telomerase independent of its ability to elongate and maintain telomere length. Immunodetection of telomerase is now being recognized as a necessary approach to precisely elucidate its roles in oncogenesis and senescence. A few antibodies directed against the catalytic subunit of the human telomerase (hTERT) are currently used but their specificity is not always demonstrated. A survey of the literature showed inconsistencies and led us to comparatively re-evaluate the most frequently used antibodies. Surprisingly, mass spectrometry, two-dimensional gel analysis and immunofluorescent experiments revealed that the most frequently used hTERT immunoprobe, a mouse monoclonal antibody that was claimed to be directed against an hTERT protein epitope, in fact recognizes nucleolin rather than telomerase. Our findings have interesting implications regarding the biology of nucleolin and telomerase in the context of pathophysiological investigations recently carried out.

Rapid quantification of telomerase activity in liver tissue samples to detect human hepatocellular carcinoma

Rapid quantification of telomerase activity in liver tissue samples to detect human hepatocellular carcinoma

Clinical significance and usefulness of quantification of telomerase activity in oral malignant and nonmalignant lesions

We quantified telomerase activity (TA) in patients with oral and maxillofacial malignant and nonmalignant lesions, and compared it with their clinical status and grade of malignancy. Fifty-two malignant and 52 nonmalignant lesions were analyzed. All malignant lesions were pathologically diagnosed as oral squamous cell carcinoma (OSCC). Normal gingival tissue served as a control. These specimens were obtained by biopsy or surgical resection, and stored at −80 °C until use. TA was quantified by a fluorescence-based TRAP method. TA levels ranged from 0.00 to 95.24 (average 33.24) U/μgP in 52 malignant lesions, and from 0.00 to 79.35 (average 11.91) U/μgP in 52 nonmalignant lesions ( P<0.0001). TA was detected in 96.2% of malignant and 65.4% of nonmalignant lesions. There was no relationship between TA levels and clinical stages or YK classification. However, under WHO classification, there were significant differences ( P<0.05) between Grades I and III or II+III. Among nonmalignant lesions, epithelial dysplasia showed a significantly higher TA level than that of oral lichen planus ( P<0.05) and other benign lesions ( P<0.0001). Oral lichen planus also significantly differed from other benign lesions ( P<0.05). These results suggest that TA is related to the histological grade of malignancy, and is also useful as a prognostic predictor for precancerous lesions and conditions.

Real-time detection of telomerase activity utilizing duplex scorpion primers and hairpin-like reverse primers

Telomerase, a ribonucleoprotein enzyme, is expected to be a new marker for cancer diagnosis. The development of the telomeric repeat amplification protocol (TRAP) and its modified versions have facilitated the detection of telomerase activity in small tissue and tumour samples. But most of these techniques require complex post-PCR procedures. As for the two real-time quantitative methods (SYBR Green and Amplifluor methods) reported so far, both use fluorogenic probes without specificity. To overcome these problems we developed a new real-time method for the detection of telomerase activity. In this method a duplex scorpion primer and reverse primers with hairpin-like structures were used. The use of duplex scorpion primers shows a series of advantages: the target-specific probe sequence provides higher specificity than the SYBR Green and Amplifluor methods; the unimolecular probing mechanism allow the assay to be conducted under fast cycling conditions and a single operation can be completed in 1.5 h; the closed-tube system reduces the risk of carryover contamination and supports high throughput. This method may be a useful tool to rapidly, specifically 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 R telomerase kit with or without [α- 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.

The differentiation status of primary gonadal germ cell tumors correlates inversely with telomerase activity and the expression level of the gene encoding the catalytic subunit of telomerase.

BackgroundThe activity of the ribonucleoprotein enzyme telomerase is detectable in germ, stem and tumor cells. One major component of telomerase is human telomerase reverse transcriptase (hTERT), which encodes the catalytic subunit of telomerase. Here we investigate the correlation of telomerase activity and hTERT gene expression and the differentiation status of primary testicular germ cell tumors (TGCT).MethodsTelomerase activity (TA) was detected by a quantitative telomerase PCR ELISA, and hTERT mRNA expression was quantified by online RT-PCR in 42 primary testicular germ cell tumors. The control group consisted of benign testicular biopsies from infertile patients.ResultsHigh levels of telomerase activity and hTERT expression were detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content. In contrast, differentiated teratomas and testicular control tissue without germ cells (Sertoli-cell-only syndrome) showed no telomerase activity and only minimal hTERT expression.ConclusionsThese findings demonstrate an inverse relationship between the level of telomerase activity and hTERT mRNA expression and the differentiation state of germ cell tumors. Quantification of telomerase activity and hTERT mRNA expression enables a new molecular-diagnostic subclassification of germ cell tumors that describes their proliferation potential and differentiation status.

Bioluminescent Method for Detecting Telomerase Activity

Telomerase is a promising biomarker in cancer diagnosis and therapy. The elongation of telomeric repeats catalyzed by telomerase is accompanied by release of six PP(i) for each TTAGGG repeat (1 pmol PP(i)/310 pg telomeric repeats). We developed a novel method to measure telomerase activity by use of an enzymatic luminometric PP(i) assay (ELIPA). Extracts of cell lines and tissues were incubated with primer at 30 degrees C for 30 min. Released PP(i) was converted to ATP by sulfurylase, and ATP was detected by a luciferase bioluminescence system. The ELIPA results were compared with results obtained with the conventional telomeric repeat amplification (TRAP)-ELISA in 42 lung carcinoma tissues and 27 control tissues without malignancy. The lower detection limits of ELIPA and TRAP-ELISA were 5 and 10 cells, respectively. The within-run imprecision (CV) of ELIPA was < or =12%. When compared with TRAP-ELISA, the correlation coefficient (r) was 0.79. When we used the cutoff value from ROC analysis to distinguish malignant and nonmalignant tissues, the sensitivity and specificity of ELIPA were 83% and 96%, respectively, whereas the sensitivity and specificity of TRAP-ELISA were 71% and 96%, respectively. ELIPA is a simple and sensitive homogeneous method to quantify telomerase activity.

Quantification of telomerase activity, porphobilinogen deaminase and human telomerase reverse transcriptase mRNA in testicular tissue - new parameters for a molecular diagnostic classification of spermatogenesis disorders.

The aim of the study was to evaluate the quantitative detection of human telomerase reverse transcriptase (hTERT) mRNA and telomerase activity as new molecular diagnostic parameters for a subclassification of spermatogenesis disorders. Telomerase activity was detected by a quantitative telomerase PCR ELISA, and hTERT mRNA expression was quantified by fluorescence real-time RT-PCR in a LightCycler in cryopreserved testicular tissue specimens. This was paralleled by a histological workup. The discriminant analysis showed that detection of normalized hTERT expression was able to correctly classify 89.0% of the investigated tissue specimens into the subgroups of full spermatogenesis, maturation arrest or Sertoli-cell-only syndrome. In contrast, discriminant analysis revealed an only 58% accuracy of telomerase activity for the investigated tissue specimens. This study shows that the quantification of hTERT expression in testicular tissue by real-time fluorescence RT-PCR is well suited for correctly classifying spermatogenesis disorders and proved to be markedly superior to the determination of telomerase activity.