Research Article: Assays for Detection of Telomerase Activity

Date Published: , 2011

Publisher: A.I. Gordeyev

Author(s): D.A. Skvortsov, M.E. Zvereva, O.V. Shpanchenko, O.A. Dontsova.

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Abstract

Progressive loss of the telomeric
ends of chromosomes caused by the semi-conservative mechanism of DNA replication is an
important timing mechanism which controls the number of cells doubling. Telomerase is an
enzyme which elongates one chain of the telomeric DNA and compensates for its shortening
during replication. Therefore, telomerase activity serves as a proliferation marker.
Telomerase activity is not detected in most somatic cells, with the exception of embryonic
tissues, stem cells, and reproductive organs. In most tumor cells (80–90%), telomerase is
activated and plays the role of the main instrument that supports the telomere length, which
can be used for the diagnostics of neoplastic transformation. This is the primary reason why
assays regarding the development of telomerase activity have attracted the attention of
researchers. Telomerase activity testing may be useful in the search for telomerase
inhibitors, which have the potential to be anti-cancer drugs. Moreover, telomerase
activation may play a positive role in tissue regeneration; e.g., after partial removal of
the liver or cardiac infarction. All telomerase activity detection assays can be divided
into two large groups: those based on direct detection of telomerase products, and those
based on different systems of amplification of the signals from DNA that yield from
telomerase. The methods discussed in this review are suitable for testing telomerase
activity in different samples: in protozoa and mammalian cells, mixed cellular populations,
and tissues.

Partial Text

In 1961, Hayflick and Moorhead demonstrated that a somatic cell culture has a limited life period (the Hayflick limit) [1]. In 1973, Olovnikov postulated that the number of cell divisions can be determined by the shortening of the telomeric ends of chromosomes [2], which play the role of a timing mechanism in cells. Telomeres protect the cell genome against degradation and participate in the meiotic pairing of chromosomes and the regulation of the transcription of the genes located in the pretelomeric region [3, 4]. Present in immortalized cells (with the ability of infinite division) is a mechanism that compensates for telomere shortening. In 1985, Blackburn and Greider discovered telomerase, an enzyme that elongates one of the telomere chains [5].

The most common methods for detecting telomerase activity are TRAPs (telomeric repeat amplification protocols) [11], which allow one to perform semi-quantitative and quantitative analyses, using some of their modifications. Such modifications include: increase of the analysis rate, replacement of the radioactive label by nonlabeled compounds, the reduction of the amount of side products, etc. Among these methods are the scintillation proximity assay, hybridization protection assay, transcription amplification assay, and the magnetic bead-based extraction assay [12]. Some modifications even enable to detect telomerase activity within a single cell [13].

Detection of telomerase activity using direct incorporation of a radioactively labeled substrate

Determination of telomerase activity using digoxigenin-labeled oligonucleotides complementary to telomeres

At the time of publication of this article, there were a number of methods for the determination of telomerase activity in various specimens: extracts of cells, tissues, and mixed cell populations. All the aforementioned methods can be divided into two groups: those with direct detection of telomerase-synthesized DNA and those with various amplification schemes; each method has its advantages and drawbacks. A comparison of all these methods is provided in Table 2 .

 

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