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

https://doi.org/10.1016/S0003-2697(02)00663-2Get rights and content

Abstract

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.

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.

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