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Telomere Without End, Amen: Looking Into Longevity with Telomere Detection Kits

Date: March 30, 1998 Author: Laura DeFrancesco T he excitement over telomerase continues to mount as evidence accumulates that makes the connection between telomere length and cell lifespan likely to be more than a coincidence. The most recent findings show that the age span of cultured cells, normally limited to around 50 cell doublings--the so-called Hayflick limit, named for the scientist who first observed that the lifespan of cultured cells was finite--can be more than doubled by transfec

By | March 30, 1998

Date: March 30, 1998
Author: Laura DeFrancesco

T he excitement over telomerase continues to mount as evidence accumulates that makes the connection between telomere length and cell lifespan likely to be more than a coincidence. The most recent findings show that the age span of cultured cells, normally limited to around 50 cell doublings--the so-called Hayflick limit, named for the scientist who first observed that the lifespan of cultured cells was finite--can be more than doubled by transfecting them with telomerase genes (A.G. Bodnar et al., Science, 279:349-52, 1998). These findings come on the heels of a series of observations correlating the loss of telomerase activity and/or the shortening of the ends of chromosomes (telomeres) with the loss of proliferative capacity, an observation that holds true in a number of situations: somatic (limited proliferative capacity) as compared to germ cells (larger proliferative capacity); normal tissue (limited) versus malignant tumors (unlimited); and normal T cells versus HIV-infected T cells, whose telomeres resemble those of aged individuals. And the list goes on. What exactly is telomerase and what has it to do with aging? Telomerase is a novel ribonucleoprotein, or reverse transcriptase (RT), that adds nucleotides to the ends of chromosomes during DNA replication. Unlike other known reverse transcriptases, all of which are associated with viruses, the telomeric RT is the only one to date associated with a normal genome.

Early in the study of the mechanism of DNA replication, replicating the ends of chromosomes was recognized as a potential problem due to what James Watson termed the "lagging end problem." (Consider what happens when the DNA polymerase, which works off primers, reaches the last primer and that primer, as is the case with all primers, is removed). Not surprisingly, a special enzyme, telomerase, appears to exist solely for the purpose of maintaining the telomeres by adding to the ends of the chromosomes a six-nucleotide piece of DNA complementary to its RNA component. In the absence of such an enzyme, it was predicted that the chromosomes would get progressively shorter with each cell division, and this in fact appears to be the case; as telomerase activity lessens with age (or disease state), so do the telomeres themselves, going from 15-20 kilobase in size in germ cells to <5 kilobases in length in senescing cells.

As if aging, cancer, and AIDS weren't enough, connections between telomerase activity (and/or telomere length) and a variety of other diseases and developmental processes have been made: arteriosclerosis, progeria, Down's syndrome, and failed bone marrow transplants, to name a few.

Given this, you might expect that companies would be falling over themselves to provide tools for researching such a hot topic. But surprisingly, LabConsumer discovered only a handful of kits in the market for telomerase detection. Why would that be? Perhaps learning from the experiences of other companies in their patent and licensing practices, the company holding patent rights to telomerase (Geron Corporation of Menlo Park, Calif.) is taking pains to see that the products entering the marketplace meet its standards for quality and its desired marketing strategy.

Furthermore, the purification of telomerase had proven to be quite difficult, since even in what would be considered an actively expressing cell type, such as cultured cells or tumor cells, there is something on the order of only 100 molecules of telomerase per cell. Last August, however, two groups of scientists (one from Geron and the University of Colorado, Boulder, and the second at MIT) reported the cloning and isolation of the protein component of human telomerase, a feat that should open the way for the development of a new generation of research tools--antibodies, inhibitors, novel substrates, and the like.


Serially diluted extracts of telomerase-positive control cells subjected to the TRAP assay. Reaction products were run on a native polyacrylamide gel and stained with SYBER™ Green. Lanes 1 and 6 are negative controls.
Presently, two kinds of kits have been developed for assessing telomerase activity or telomere length. The first is a PCR-based assay, termed TRAP or telomerase repeat amplification protocol by the inventors and patent-holders of the technology (N.W. Kim et al., Science , 266:2011-2015, 1994). TRAP technology is used in kits offered by three companies: Telomerase PCR ELISA by Boehringer Mannheim; TRAPeze® offered by Oncor, available in a gel-based and an ELISA-based form; and TRAP Assay Module by PharMingen. These kits utilize a primer that serves as a substrate for the addition of the telomeric sequence (TTAGGG) by telomerase, which adds six (and multiple of six) base extensions to the substrate primer. Following this elongation step, the PCR is used to amplify the elongated substrates using an anchored reverse primer which eliminates primer-dimers and other PCR artifacts, a particular problem when amplifying repeated sequences. The products can then be either visualized on a gel as a characteristic ladder of fragments or detected by ELISA.

While the TRAP kits have been engineered to provide fast and easy tests for telomerase activity, a limitation to the usefulness of these kits is that they all require active enzyme to be present. This means that only fresh samples or samples frozen appropriately (fresh frozen at -80°C) can be used in these assays. For some kinds of studies in which only archival (fixed) material is available, the telomerase assay kits simply won't work. While products for detecting the telomerase RNA or the telomerase RT protein or its corresponding mRNA are in development, according to Geron, currently only one product fills this niche: PharMingen's TeloQuant™ Telomere Length Assay Kit. This kit is designed to provide telomere length measurements of isolated chromosomal DNA using standard blotting and hybridization technology. This gives a slightly different but related piece of information on the remaining proliferative capacity of a cell.

Long before the present excitement over telomerase erupted, a series of telomeric probes, specific for individual chromosomes, had been designed for fluorescence in situ hybridization (FISH) (NIH/IMM Collaboration, Nature Genetics , 14:86-89, 1996). In conjunction with centromeric probes, the telomeric probes define the terminal boundaries of chromosomes, which can be useful in the research of chromosomal rearrangements and deletions related to cell aging or other genetic abnormalities. Also, these chromosome-specific telomeric probes have application in cytogenetics, as they can detect submicroscopic deletions and cryptic translocations of genes, associated with unexplained mental retardation and miscarriages. In addition, with the recent upsurge in interest in telomeres and telomerase, telomere probes, in general, have found use outside the clinic (P.M. Landsdorp et al., Human Molecular Genetics, 5:685-691, 1996).


TelVysion™ DNA FISH probes for telomere regions from Vysis
Telomere probes are chromosome specific by virtue of the inclusion of sequence from chromosomal DNA adjacent to telomeres in the probes. Commercial versions of these probes are currently available from two companies in this country, Oncor and Vysis, Inc. (Downers Grove, Ill.), and several European companies. Oncor offers human and mouse digoxigenin-labeled probes, both for individual chromosomes (1-20, X and Y), at a cost of $193 for material to process ten slides, and an all-human telomere probe, based on the TTAGGG repeat present on all human telomeres ($138 for material for 33 slides). Vysis markets 82 direct labeled telomere probes for all human chromosomes (available in two different fluors, SpectrumOrange or SpectrumGreen) for human telomere research use ($175 for 20 assay volume).

Boehringer-Mannheim
Telomerase PCR ELISA is a photometric immunoassay for the detection of telomerase activity utilizing the TRAP assay. Using a single tube, a biotinylated synthetic telomerase substrate primer is first reacted with a cell extract to create extension products, and the elongated substrates are then amplified by PCR using a set of telomerase primers. The elongated substrates are denatured and hybridized to a digoxigenin labeled telomeric repeat-specific probe. The hybridization products are immobilized via the biotin-labeled primer to a streptavidin-coated microplate, and once immobilized, detected with anti-digoxigenin antibody conjugated to peroxidase using the color-generating substrate TMB. If desired, the amplified products can also be run on gels, blotted, and detected using a biotin detection system.

The advantages of this kit, according to company literature, are that both the extension and amplification steps can be done in a single tube and that the 96 well ELISA format can be completed in a few hours without gels or radioactivity. In addition, through judicious design of primers, the conditions for PCR have been optimized such that there is no need for hot-start PCR or wax barriers, and amplification artifacts are eliminated.

The kit comes with lysis buffer, reaction mixture, denaturation reagent, hybridization buffer, anti-Dig-peroxidase buffers, reagents, and a microplate for ELISA, as well as a positive control in the form of a cell extract. Company literature claims that the kit is as sensitive as home-grown, radioactive versions of the TRAP assay and can detect telomerase in small numbers (tens to hundreds) of embr- yonic (that is, expressing telomerase) cells. For a 96-sample kit, the cost is $505. An overview of the kit can be viewed on Boehringer Mann-heim's web site: http://biochem. boehringer-mann-heim. com/ techserv/ telom_ nw. htm.

Oncor
Oncor offers three products for the detection of telomerase in cell extracts. The gel-based TRAPeze® Telomerase Detection Kit and TRAPeze® Modified Reagent Set are one-buffer, two-enzyme systems for detecting the telomerase activity in cell or tissue extracts. The process re-quires three steps. First, the extract is prepared in a buffer supplied in the kit. Next the TRAP assay itself is performed, in which the primer (TS), (labeled or not) is extended by whatever telomerase is present in the extract and the products are amplified by PCR (single tube, no hot start). Finally, aliquots of the reaction are then run on gels and visualized either by staining in the case of unlabeled primers or by autoradiography in the case of labeled primers.

Oncor touts this kit as being sensitive, usable for both small samples and mixed cell populations (published results using this kit show sensitivity down to a single cell) rapid, with results obtainable in the same day, and quan-titative by virtue of the inclusion of a semi-competitive internal PCR control. This internal control substrate competes for the TS primer, but rather than a ladder, it generates a single, 36-base pair fragment. (The substrate has two parts--an 18-base pair telomere substrate (TS) and a unique 18-base pair segment, termed K1, requiring its own K1 primer.) The presence of an internal control also provides a positive control for each reaction, revealing the presence of any inhibitory substances in individual extracts. As a further refinement, the TRAPeze Modified Reagent Set provides a larger internal control (173 bases), for people concerned about preferential amplification of the smaller substrate.


Chemiluminescent detection of telomere length using Teloquant
L. Harrington, T. McPhail, V. Mar, W. Zhou, R. Oulton, M.B. Bass, I. Arruda, M.O. Robinson, A mammalian telomerase-associated protein, Science 275: (5302) 973-977 Feb14 1997 (Times Cited: 42)

J.I. Nakayama, M. Saito, H. Nakamura, A. Matsuura, F. Ishikawa F,TLP1: A gene encoding a protein component of mammalian telomerase is a novel member of WD repeats family, Cell, 88: (6) 875-884 Mar 21 1997 (Times Cited: 27)

S.E. Holt, W.E. Wright, J.W. Shay, Regulation of telomerase activity in immortal cell lines, Molecular and Cellular Biology, 16:(6) 2932-2939 Jun 1996 (Times Cited: 71)

J.P. Cooper, E.R. Nimmo, R.C. Allshire, T.R. Cech, Regulation of telomere length and function by a Myb-domain protein in fission yeast, Nature, 385: (6618) 744-747 Feb 20 1997 (Times Cited: 27)

D. Gilley, E.H. Blackburn, Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase, Molecular and Cellular Biology, 16: (1) 66-75 Jan1996 (Times Cited: 16)

C. Autexier, C.W. Greider, Telomerase and cancer: Revisiting the telomere hypothesis, Trends in Biochemical Sciences, 21: (10) 387-391 Oct 1996 (Times Cited: 17)

E. Bottius, N.. Bakhsis, A. Scherf , Plasmodium falciparum telomerase: De novo telomere addition to telomeric and nontelomeric sequences and role in chromosome healing, Molecular and Cellular Biology, 18: (2) 919-925 Feb 1998

Oncor also offers an ELISA version of the TRAPeze Telomerase Detection Kit, which affords direct, chromogenic detection of telomerase activity in extracts. In the TRAPeze ELISA assay, active telomerase extends a biotinylated TS primer; the extension products are amplified by PCR using dCTP labeled with DNP. Biotinylated primer is then immobilized on a microplate, followed by detection of the DNP-labeled products with anti-DNP-antibody conjugated with peroxidase. This kit is designed for high-throughput screening for telomerase-positive samples. However, without the positive control of the gel-based assay, Oncor recommends further analysis to eliminate false positives, such as gel visualization, which can be done directly with the ELISA products.

The TRAPeze Telomerase Detection Kit runs $335 for 112 PCR reactions; the Modified Reagent Set, $121 for 112 reactions; and the TRAPeze ELISA, $446 for 96 reactions.

PharMingen
The TeloQuant Telomere Length Assay Kit allows for the determination of telomere lengths using a nonradioactive, chemiluminescent detection system. After DNA is prepared from cells or tissues by standard methodology, it is digested with a restriction enzyme that does not have a target sequence within the telomere repeat. This digestion will cut the chromosomal DNA into small fragments, leaving the telomeres and subtelomeric regions intact, which together comprise the Terminal Restriction Fragment (TRF). The cleaved DNA is separated on gels and transferred to membranes for blotting and hybridization. The blot is hybridized to a telomere-specific repeat, which consists of a number of the hexameric repeats. Detection of the position of the hybridized bands can be determined using radioactivity or chemiluminescent detection systems. The average TRF length can be calculated from the position of the detected signal relative to the position of known size standards.

The kit comes with DNA- and TRF- size standards, restriction enzyme cocktail containing HinfI/RsaI, hybridization components, a biotinylated telomere probe, and a chemiluminescent detection system. A method for calculating mean TRF length is provided with the kit, which involves densitometry of a scanned autoradiograph.

The claim is that this kit gives good signal with 2.5 µg of chromosomal DNA per lane using chemiluminescence, with higher molecular size TRFs being detectable at a lower copy number than lower molecular size TRF (due to the increased number of binding sites/TRF for the probe). Sensitivity can be improved by increasing the exposure time. A kit good for 50 number of determinations cost $295.

PharMingen's TRAP Assay Module measures telomerase activity from viable or freshly frozen samples. Cell extracts are prepared, incubated with the TRAP buffer for elongation of the telomerase substrate (TS primer) which can be either labeled or not. The products are subsequently subjected to PCR, and analyzed directly by gel electrophoresis. The PharMingen kit comes with buffers for extracting tissue and for performing the TRAP assay, and primers, including an internal, R8 quantitation standard, which generates a six fragment ladder. This module can only be purchased with the Telomere Length Assay Kit and costs $350.

While basic research on aging and chromosome structure (to name just a few areas) will doubtless reap the benefits of these tools, look to the clinical arena for the next wave of excitement in telomerase detection. The connection between telomerase activity and proliferation makes telomerase an obvious target both for cancer diagnostics and therapeutics. However, while the kits currently available are simple in concept, they may not yet be simple enough for direct clinical application, particularly in the quantitation of telomerase activity and the ability to provide standards of measurements that have relevance to particular diseases. Boehringer Mannheim, in an exclusive arrangement with Geron Corporation, is working on the development and commercialization of cancer diagnostics, while Pharmacia, Upjohn, and the Japanese company Kyowa Hakko are working with Geron in the area of cancer treatment.

The author can be reached at ldefrancesco@the-scientist.com

WEB SITEDESCRIPTION
http://resolution.colorado.edu/~nakamut/telomere/telomere.htmlTelomere Club: Telomere & Telomerase Research Information, assembled by Toru Nakamura at the University of Colorado, Boulder, contains information on telomere and related gene sequences, and links to web sites of researcher and companies working in this field.
http://resolution.colorado.edu/nakamut/telomere/telogroup.htmlA listing of telomerase research groups with links to investigators' web pages.
http://www.plattsburgh.edu/biology/bio401/Telomerase.html Animated telomerase demonstration by Donald Slish of SUNY Plattsburgh.
http://vega.crbm.cnrs-mop.fr/bioscience/news/scientis/telomer1.htmlFrontiers in Bioscience:Science News Digest for Physicians and Scientist with links to resource material.
http://ilr.genebee.msu.su/agethry/telomere/tlmrase/ Telomerase Enzyme: references to reviews and research articles
http://www.genlink.wustl.edu/teldb/teldb.html TelDB has information about telomeric and subtelomeric regions of chromosomes and telomere literature references, including a user interface to the searchable telomere literature database.
http://puma.protein.bio.msu.su/biokhimiya/index.htm Special Issue: TELOMERE, TELOMERASE, CANCER, AND AGING Biochemistry (Moscow) On-Line, World Wide Web version of the international edition of this Russian scientific journal, containing recent reviews on telomere biology. The entire text is available on-line.
http://www.arclab.org/Aging Research Centre (ARC) provides a service that allows researchers in this field to find information that is related to the study of the aging process.
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