Telomerase Assays

Telomerase is a ribonudeoprotein complex that plays a critical role in cellular mortality [44]. The vertebrate enzyme catalyzes the addition of TTAGGG sequence repeats to the ends of chromosomes. In the absence of telomerase, human telomeres undergo progressive shortening with each round of cell division, an event that may contribute to cell aging and mortahty. Telomerase is known to be asso- 

---

Sun, D., Hurley, H. L., and Hoff, D. D. (1998). Telomerase assay using biotinylated-primer extension and magnetic separation of the products. Biotechniques 25 1046-1051. 

Telomerase activity is typically measured using the Telomeric Repeat Amplification Protocol (TRAP) assay. In the TRAP assay, products of the telomerase reaction are quantified following their PCR amplification [20, 21], The assay is exquisitely sensitive and incorporates an internal standard (ITAS) with which to normalize signals for differences in PCR efficiency. Telomerase activity is calculated as the ratio of the intensity of the telomeric products to that of the ITAS. With this assay, telomerase activity can be measured in a wide range of specimens, from tissue biopsies to cell pellets [22]. High throughput assays have been developed to adapt the telomerase assay to the clinical environment. Many of these new assays take advantage of fluorophores that alleviate the use of radioisotopes and facilitate the quantification of PCR products. 

Numerous bioassays format have been designed using derivatives of the mono-functionalized europium cryptate such as 47 (commercially available from CISbio, see http //www.htrf.com). Since the last reviews [127] some new applications Eu " C [BP.BP.BP] cryptate were developed through the HTRF technology, as caspase assay [128], telomerase assay [129], leukotriene b4 assay [130], inositol-1-phosphate assay [131], minisequencing [132], and MutS-DNA interaction [133]. Recently, the HTRF technology has been used in the study of protein-protein interactions and the authors demonstrated that measured values compare favorably with those calculated from independent experiments [134]. 

Mergny JL et al. (2001) Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay. Proc Natl Acad Sci USA 98(6) 3062-3067... 

Yajima, T. Yagihashi, A. Kameshima, H. Furuya, D. Kobayashi, D. Hirata, K. Watanabe, N. Establishment of quantitative reverse transcription-polymerase chain reaction assays for human telomerase-associated genes. Clin. Chim. Acta 2000, 290(2), 117-127. 

A variety of assays are being reported in the literature which measure the interaction of cells with biomaterials, be they surfaces of various textures or micro or nano-particulates. While considerable attention has been paid to proper preparation of the materials and their surfaces, less has been paid to the cells used as probes in such assays. We suggest that where possible, primary human cell cultures at relatively low PD numbers from isolation are used, of a cell type directly relevant to the material application, or in toxicity studies, the exposed tissue. We suggest very significantly erroneous conclusions may be drawn from assays using inappropriate cells. If sufficient quantities of low PD primaries are not available, it may be possible to use telomerase transfected cells with extended lifespan, but these must first be verified against the non-transfected cells in the assay to be employed. Furthermore, care must be taken to conduct such assays at a controlled temperature, preferably at the temperature the material will be subjected to in the body. 

Figure 12.9 (A) Schematic illustration of overall assay design for ZnO NR-based telomeric repeat elongation assays. TS is an oligonucleotide whose sequence is recognized by telomerase. (B) Fluorescence panel obtained from positive (1) versus negative (2) samples after performing telomeric repeat elongation assays on ZnO NR stripe platforms.

The demonstrated ZnO NR-enabled fluorescence sensitivity is even more remarkable, when considering the fact that such fluorescence enhancement of ZnO NR platforms is achieved without the use of chemical and biological ampliflcation processes, major improvements of detection apparatus and analysis software, or application of specially designed fluorophores. Although telomerase and cytokine assays are discussed in this section as example cases, the versatility and general applicability of ZnO NR-based assays can be extended to other disease-marker or biomarker systems. 

Lackey, D. B. (1998). A homogeneous chemiluminescent assay for telomerase. y4na/. Biochem. 263 57-61. 

Maesawa, C., Inaba, T., Sato, H., lijima, S., Ishida, K., Terashima, M., Sato, R., Suzuki, M., Yashima, A., Ogasawara, S., Oikawa, H., Sato, N., Saito, K., and Masuda, T. (2003). A rapid biosensor chip assay for measuring of telomerase activity using surface plasmon resonance. Nucleic Acids Res. 31 e4. 

The TRAP (telomeric repeat amplification protocol) assay is a widely used method for detection of telomerase activity. This technique measures the telomerase activity present in cell extracts. Briefly, cellular extract containing telomerase activity is incubated with a telomeric substrate (a short strand of DNA onto which the telomerase wiU. attach the telomeric repeats) followed by polymerase chain reaction (PCR) amplification of the elongated telomere. Detection of the PCR product is by a number of methods, including gel electrophoresis, radiometric detection, ELISA, and real-time PCR detection. ... 

Argemone subfusiformis dried leaves, flowers and seeds were extracted with ethanol-water (70-30%) for 48 h, the aqueous-solution obtained was evaporated under vacuum and the resdue was directly assayed. The antiplasmodial activity was good (24), and extracts were rich in isoquinolleinic Akaloids. In above ground parts, protopine, berberine and allocryptopine were identified (27). According to Sriwilaijaron et al. (28) berberine prevents the development of Plasmodium falciparum by inhibition of its telomerase activity. 

Three classes of proteins in addition to TERT are now known to be associated with various telomerases. Estlp was first found in yeast [21] it is essential for activity in vivo, but seems entirely dispensable for enzyme activity per se as judged by in vitro assays. Estlp interacts directly with the yeast telomere DNA end binding protein, Cdcl3p [22] this interaction appears to recruit telomerase to the chromosome end... 

Silicon nanowires incorporated into arrays provide label-free, multiplexed electrical detection of cancer protein biomarkers such as prostate-specific antigen at femtomolar concentrations with high sensitivity in clinically relevant serum samples [7]. Real-time assays of the binding, activity, and small-molecule inhibition of telomerase could be performed with this technique using unamplified extracts from as few as 10 tumor cells. This opens up substantial possibilities for diagnosis and treatment of cancer. 

---