ReviewAnalysis of telomerase activity and detection of its catalytic subunit, hTERT
Section snippets
Telomeric repeat amplification protocol
The telomeric repeat amplification protocol assay is divided into three main steps that consist of the extension, amplification, and detection of telomerase products (Fig. 1). These steps have been modified, giving rise to TRAP-dependent (two-primer-TRAP) or independent assays (transcription-mediated amplification and hybridization protection assay) (Table 1). In the extension step, telomeric repeats are added to the telomerase substrate (TS), a nontelomeric oligonucleotide, by the telomerase
Two-primer-TRAP
The assay referred to as TP-TRAP utilizes two reverse primers instead of one [63], [64] which leads to more sensitive and accurate detection of telomerase activity. For the analysis of telomerase activity in the standard or conventional TRAP assay, the total products generated are resolved by electrophoresis and quantified by densitometric methods which are time consuming and can lead to inaccuracies. The TP-TRAP eliminates the need for the electrophoretic analysis of the total products
Scintillation proximity assay
Speed and accuracy are two main requirements for an assay to be used in large-scale through-put screening. The conventional TRAP is suitable for telomerase detection, but its application to large-scale screening is unlikely due to the cumbersome radioactive gel-based detection of the TRAP products. The scintillation proximity assay developed by Bosworth and Towers [65] when used in conjunction with TRAP improves the speed and accuracy of the assay in determining telomerase activity [66].
Real-time quantitative TRAP
Amplification of telomerase products by PCR is often inaccurate due in part because the end point of the amplification process cannot be determined. Therefore, optimal conditions for the PCR need to be determined for each cell type being assessed (Table 1). Variances that occur with PCR processing also affect the accuracy of results [67]. The RTQ-TRAP method is a combination of the standard TRAP with real-time PCR. The amplicons generated by PCR are measured exponentially by the intensity of
TRAP-enzyme-linked immunosorbent assay
The TRAP-ELISA assay is similar in many respects to that of conventional TRAP except that the generated products are detected colorimetrically, which provides a qualitative and semiquantitative estimate of telomerase activity [31]. The TS primer is biotinylated, which enables the binding of the products onto streptavidin-coated microtiter plates. The amplification products are denatured, hybridized with a digoxigenin-labeled probe (specific for telomeric repeats), and immobilized on microtiter
Magnetic bead retrieval assay and TRAP
Extraction and detection of telomerase activity in tumor cells or cell lines is relatively simple. However, tumor tissues are composed of several different cell types and contain inhibitory substances that can greatly affect the quantification and accuracy of any assay (Table 3). As a result, false-positive or false-negative results can be obtained that may affect the diagnosis and prognosis of the disease. Telomerase activity is also detected from various sample types (e.g., bladder washings,
Hybridization protection assay-TRAP
The standard TRAP assay requires an electrophoretic step for the analysis and quantification of telomerase activity from amplified PCR products, which at times can be difficult to perform. In the HPA-TRAP assay, the products are amplified by the standard TRAP assay but the detection of the generated products is carried out with the use of the hybridization protection assay. The HPA is a nonradioactive and nonelectrophoretic method for detection of the amplified products that utilizes an
Transcription-mediated amplification and hybridization protection assay
Transcription-mediated amplification was the first method developed that amplified telomerase products without the polymerase chain reaction. Details of the procedure of the transcription-mediated amplification are illustrated in Fig. 2. The amplification of the products autocatalytically prevents the introduction of contaminants encountered with PCR. Unlike the ELISA and PCR-gel-based assays that are more time consuming, the entire procedure for the detection of telomerase activity using
Luminometric hybridization assay
The sensitivity and reproducibility of the assays involved in the detection of transformed cells is extremely important for the proper treatment and diagnosis of cancer. Most of the methods previously described render a qualitative but semiquantitative estimate of telomerase activity. In addition, in some of the methods, PCR-related artifacts affect the analysis of the results. TRAP-ELISA and RTQ-TRAP are examples of two methods that provide a quantitative estimate of telomerase activity.
Determinants influencing the detection of telomerase activity
The sensitivity and accuracy of the methods involved in the detection of telomerase activity are influenced by the source of the enzyme and the quality of the sample to be assessed. Sources include tissues, whole cells, tissue or cell lysates, and fluids (voided urine, blood, fine needle aspirations, washings or effluents) [68], [69], [70], [71], [72], [73]. For example, most assays employ PCR for the amplification of telomerase-extended products. When the enzyme is extracted from tissues, PCR
Detection of the catalytic subunit of telomerase, hTERT
Although the determination of telomerase activity is a powerful tool in the diagnosis of cancer, it is not always a reliable marker. The advantages and limitations of various techniques involved in the detection of telomerase activity have shown that the source and origin of the tumor plays an important role in the detection. Although the functional telomerase enzyme consists of two main subunits, hTERT and hTR, hTEP1 has also been found to be associated with the enzyme. The expression of
In-situ hybridization
In situ hybridization allows the direct assessment of distribution patterns of the specific mRNA or protein of interest without affecting cellular integrity. Probes that hybridize to the mRNA of interest may be synthetic DNA oligonucleotides, sense or antisense RNAs, or riboprobes. Of the several probes available, the synthetic oligonucleotides (which are about 40–50 nucleotides in length) are the most suitable. The probes are small and can easily enter the cell for hybridization. Moreover, the
Reverse transcriptase-polymerase chain reaction (RT-PCR) detection of hTERT
Three main steps are involved in RT-PCR: (a) extraction or isolation of RNA, (b) cDNA synthesis, and (c) amplification of the cDNA. RNA can be isolated from tissue samples or tumors either by disruption of cells by the use of a sterile pestle [44], by guanidium isothiocyanate extraction [89], [97], or by tri-reagent protocol [98]. The isolated RNA is treated with DNase, and a fraction (0.1–10 μg of total RNA) is used to reverse transcribe cDNA [88], [89], [90]. Finally, the synthesized cDNA is
Immunofluorescence-based flow cytometry detection of hTERT
Immunofluorescence-based flow cytometry is a cellular assay that provides a direct assessment of hTERT within the nucleus in a subpopulation of cells without cell disruption. Telomerase activity, as detected by various methods, is the activity of the entire cell population and not that of specific cells. As tumors contain cells that are telomerase-negative and telomerase-positive, it is essential to determine the cell types expressing telomerase, which can help in determining the prognosis of
Conclusions
Telomerase reactivation has been implicated in the mechanisms of tumor formation, progression, migration, and invasion. The use of sensitive techniques (Fig. 1 and Table 1) has enabled detection of telomerase activity from a variety of sources such as tissue effluents, pleural effluents, juices (pancreas or bile), washings, blood, serum, fine needle aspirations, fresh-frozen and paraffin-embedded tissues, and exfoliations. Based on extensive surveys conducted on different tissues, a strong
Acknowledgements
We thank Dr. Nadejda Lopatina, Dr. Mitchell Pate, Mark Casillas, and Nathaniel Hansen for critical reading of the manuscript. This work was supported by grants from the National Institute on Aging (1 R03 AG20375 01), the American Cancer Society (IRG-60-001-41), the John A. Hartford Foundation (Southeast Center for Excellence in Geriatric Medicine), the Leukemia Research Foundation, and the UAB Center for Aging, Comprehensive Cancer Center, and Department of Biology.
References (109)
- et al.
The serial cultivation of human diploid cell strains
Exp. Cell. Res.
(1961) A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon.
J. Theor. Biol.
(1973)- et al.
Identification of a specific telomere terminal transferase activity in Tetrahymena extracts
Cell
(1985) - et al.
Telomere shortening is proportional to the size of the G-rich telomeric 3′ overhang
J. Biol. Chem.
(2000) Telomeric chromatin: replicating and wrapping up chromosome ends
Curr. Opin. Genet. Dev.
(2001)- et al.
TRF2 protects human telomeres from end-to-end fusions
Cell
(1998) - et al.
Telomere shortening and decline in replicative potential as a function of donor age in human adrenocortical cells
Mech. Ageing Dev.
(2001) Telomeres, telomerase, and myc. An update
Mutat. Res.
(2000)- et al.
In vivo and in vitro analyses of Myc for differential promoter activities of the human telomerase (hTERT) gene in normal and tumor cells
Biochem. Biophys. Res. Commun.
(1999) - et al.
Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT)
Gene
(2001)
Limitations on the quantitative determination of telomerase activity by the electrophoretic and ELISA based TRAP assays
Clin. Chem. Acta
Telomere length dynamics in telomerase-positive immortal human cell populations
Exp. Cell. Res.
Expression of human telomerase subunit genes in primary lung cancer and its clinical significance
Ann. Thorac. Surg.
Immunohistochemical detection of telomerase (hTERT) protein in human cancer tissues and a subset of cells in normal tissues
Neoplasia
The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats
Cell
Alternative methods of extracting telomerase activity from human tumor samples
Cancer Lett.
Modified telomeric repeat amplification protocol: a quantitative radioactive assay for telomerase without using electrophoresis
Anal. Biochem.
Telomerase activity in prostate sextant needle cores from radiation prostatectomy specimens
Urol. Oncol.
Hybridization protection assay: a rapid, sensitive, and specific method for detection of philadelphia chromosome-positive leukemias
Blood
Development of a quantitative luminometric hybridization assay for the determination of telomerase activity
Clin. Biochem.
Enzymatic activity of endogenous telomerase associated with intact nuclei from human leukemia CEM cells
Biochem. Biophys. Res. Commun.
Inhibition of human telomerase by 7-Deaza-2′-deoxyguanosine nucleoside triphosphate analogs: potent inhibition by 6-Thio-7-deaza-2′-deoxyguanosine 5′-triphosphate
Bioorg. Chem.
Expression of telomerase components in oral keratinocytes and squamous cell carcinomas
Oral. Oncol.
Telomerase activity and expression of telomerase RNA component and telomerase catalytic subunit gene in cervical cancer
Am. J. Pathol.
Fusion of broken ends of chromosomes following nuclear fusion
Proc. Natl. Acad. Sci. USA
Structure and function of telomeres
Nature
Telomere length predicts the replicative capacity of human fibroblasts
Proc. Natl. Acad. Sci. USA
A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis
Nature
Functional evidence for an RNA template in telomerase
Science
Telomerase catalytic subunit homologs from fission yeast and human
Science
The RNA component of human telomerase
Science
A mammalian telomerase-associated protein
Science
Replicative senescence and cell immortality: the role of telomeres and telomerase
Proc. Soc. Exp. Biol. Med.
At the end of the millennium, a view of the end
Nat. Genet.
Role of the telomeric DNA-binding protein TRF2 in the stability of human chromosome ends
Bioessays
Protection of mammalian telomeres
Oncogene
Many ways to telomere dysfunction: in vivo studies using mouse models
Oncogene
Telomere length maintenance in aging and carcinogenesis
Int. J. Oncol.
Role of telomerase in cell senescence and oncogenesis
Annu. Rev. Med.
Molecular interactions between telomerase and the tumor suppressor protein p53 in vitro
Oncogene
hTERT expression correlates with MYC over-expression in human prostate cancer
Int. J. Cancer
Telomerase activity and telomerase subunits gene expression patterns in neuroblastoma: a molecular and immunohistochemical study establishing prognostic tools for fresh-frozen and paraffin-embedded tissues
Clin. Oncol.
Up-regulation of human telomerase catalytic subunit during gastric carcinogenesis
Cancer
Retinoids down-regulate telomerase and telomere length in a pathway distinct from leukemia cell differentiation
Proc. Natl. Acad. Sci. USA
Specific association of human telomerase activity with immortal cells and cancer
Science
Telomere length and telomerase activity predict survival in patients with B cell chronic lymphocytic leukemia
Cancer Res.
Telomere length and telomerase activity in bladder and prostate cancer cell lines
Int. J. Urol.
Telomeres and telomerase in normal and malignant haematologic cells
Leuk. Lymphoma
Telomere length and telomerase activity in carcinogenesis of the stomach
Jpn. J. Clin. Oncol.
Telomerase activity and telomere length as diagnostic tumor marker for ovarian tumors
Nippon Rinsho
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2021, Urologic Oncology: Seminars and Original InvestigationsCitation Excerpt :Recently, new methods slightly less accurate than TRAP, but more cost-effective were developed, albeit they still require specific reagents and equipment making the detection of TA highly dependent on the locally available facilities [55,56]. Isotope labelling may be replaced by non-radioactive labeling such as in scintillation proximity assay, magnetic particles extraction, fluorescent analysis, TRAP-ELISA, transcription amplification, etc. [57,58]. Some refinements were reported to allow single-cell measurement of TA [59].
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2018, Journal of the Academy of Nutrition and DieteticsAccelerated cellular senescence as underlying mechanism for functionally impaired bone marrow-derived progenitor cells in ischemic heart disease
2017, AtherosclerosisCitation Excerpt :The enzyme telomerase is able to add new telomere repeats onto the chromosome ends and prevents loss of TL. The expression of telomerase, however, is rather low in the majority of mature human somatic cells, whereas it is upregulated in committed progenitor cells, activated immune cells and cancer cells [6,7]. In addition to specific intrinsic genetic factors associated with the process of biological aging, endogenous stimuli such as systemic chronic inflammation and mild oxidative stress may aggravate telomere shortening and promote cellular senescence. [8]
Clinical trials of antioxidants as cancer prevention agents: Past, present, and future
2011, Free Radical Biology and MedicineCitation Excerpt :Each pathway has its own specific biomarkers detectable in colorectal tissue [125,130,135–137] and each pathway affects the cell cycle as reflected in increases in proliferation and decreases in differentiation and apoptosis, for which several markers are available. For example, an informative long-term indicator of proliferation is hTERT, a catalytic subunit of telomerase [138], and a marker of a cell that can no longer proliferate and is differentiated is p21; both of these markers can be measured in tissue biopsy specimens [139–141]. Detection of expression of inhibitors (bcl-2) and promoters (bax) of apoptosis can be readily demonstrated in variety of tissues [142–144], most notably in the in crypts of the normal colon mucosa [139,145–150].