Quantitative assays for telomerase: means for studying the end.

The ends of human chromosomes consist of several kilobases of simple telomeric repeats (TTAGGG)n (1). Telomeres are important for maintaining chromosome structure by protecting the chromosomes from DNA degradation, end-to-end fusions, rearrangements, and chromosome loss. Because DNA polymerases synthesize DNA in the 59 to 39 direction and require an RNA primer for initiation, telomeric DNA may be lost at chromosome ends after cell division unless the termini are specifically extended by an alternative mechanism. This loss of telomeric DNA has been postulated to be one of the bases of cellular and possibly organismal senescence (1). The known mechanism for preventing this loss of telomeric DNA is based on a ribonucleoprotein polymerase called telomerase, which contains an integral RNA with a short template element that directs the synthesis of telomeric repeats at chromosome ends (2). In humans, telomerase activity is present in the germ line but is not detected in many nondiseased adult tissues (3). In contrast to nondiseased tissues, telomerase activity is found in many human tumors (4). It is thought that the reactivation of telomerase activity, which maintains telomere length, plays an important role in the immortalization of cancer cells. Thus, considerable interest is focused on the potential use of assays for telomerase activity for cancer diagnosis and antitelomerase drugs as a strategy for cancer chemotherapy (1). In 1994, Kim et al. (4) described a sensitive assay for telomerase activity, which they termed the telomeric repeat amplification protocol (TRAP). In the TRAP assay, the telomerase is extracted and allowed to synthesize extension products, which then serve as the templates for PCR amplification. As described originally, the TRAP assay suffers from several shortcomings, including the use of radioisotopes, the requirement for time-consuming polyacrylamide gel electrophoresis, the susceptibility to effects of PCR inhibitors, and the relative difficulty in accurate quantification. Since its inception, numerous improvements have been made to the original TRAP protocol, making the assay more robust and reliable for potential clinical application (5, 6). In this issue of the Journal, Hirose et al. (7) describe a new quantitative and nonisotopic assay for telomerase activity. Unlike most of the other telomerase assays, the method of Hirose et al. is not based on PCR but uses an isothermal amplification protocol called transcription-mediated amplification (TMA). After isothermal amplification, the amplified telomerase products are detected using a nonisotopic detection system termed the hybridization protection assay (HPA) (8). HPA utilizes an acridinium ester-labeled probe, which is much more resistant to hydrolysis when hybridized to a target sequence compared with the free state (9). The results of the assay are then obtained easily by chemiluminescence measurement in a luminometer. The data from Hirose et al. (7) demonstrate the rapidity and linearity of this TMA/HPA assay. Of particular significance is the improved resistance of the new assay to inhibitors that are a source of inaccuracy for the conventional TRAP assay (5). This feature is especially useful for the analysis of clinical specimens. There is, however, still a risk that the method of Hirose et al. (7) may expose the laboratory environment to potential contamination from the TMA amplicons, because additional downstream detection of the amplicons is necessary. In this regard, it is an advantage of the TMA protocol that a substantial proportion of the TMA-amplified product is RNA, which is considerably more labile than DNA and thus may be expected to constitute a lesser risk for carryover contamination. Recent work has led to the cloning of the genes encoding the various components of the human telomerase complex, including the telomerase RNA component (TERC; alternative symbol, hTR) (2), telomerase protein component 1 (TEP1; alternative symbol, TP1) (10), and telomerase reverse transcriptase (TERT; alternative symbols, TCS1 or EST2) (11). These advances have enabled the analysis of mRNA expression of these various components as an alternative assessment of telomerase function, in addition to the direct assaying of telomerase activity. In a second paper in this issue of the Journal, Yajima et al. (12) describe the development of a quantitative reverse transcription (RT)-PCR assay for TERC expression. Yajima et al. used a recently developed real-time PCR assay that utilizes the 59 to 39 exonuclease activity of the Taq DNA polymerase, which leads to the liberation of a fluorescent reporter during PCR amplification (13). The continuous optical monitoring of the increase in fluorescence, using a combined thermal cycler-fluorescence detector allows accurate quantification of the starting template copy number (14). The data from Yajima et al. (12) demonstrate the linearity and dynamic range of this system. This system is also relatively resistant to carryover contamination of PCR amplicons because no post-PCR sample processing is required. This feature makes this type of system very suitable for routine clinical use. Yajima et al. used this quantitative RT-PCR system to study two human pancreatic cancer cell lines and showed that their assay can demonstrate the difference in TERC expression between the cell lines. For clinical use it should be noted that previous work has shown that TERC expression does not parallel telomerase activity, suggesting that the RNA component of telomerase is not the limiting factor for telomerase activation in cellular immortalization (2, 15). A good correlation between TERT expression and telomerase activity, however, has been reported (11). On the basis of the work by Yajima et al. (12), it would be relatively easy to develop an analogous real-time RT-PCR system for quantifying TERT expression, which may provide an alternative to direct measurement of telomerase activity. Editorial