Flow cytometric measurement of telomere length

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The regulation of telomere length may be involved in the cellular physiology of senescence, reproduction, cancer, immune response to infection, and possibly ...
Cytometry (Communications in Clinical Cytometry) 42:159 –164 (2000)

Review Article

Flow Cytometric Measurement of Telomere Length Wallace Lauzon,1 Jaime Sanchez Dardon,2 D. William Cameron,1,2,3 and Andrew D. Badley

1,2,3

*

1

Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Canada 2 Ottawa General Hospital Research Institute, Ottawa, Canada 3 Division of Infectious Disease, Ottawa Hospital, General Campus, Ottawa, Canada

The regulation of telomere length may be involved in the cellular physiology of senescence, reproduction, cancer, immune response to infection, and possibly immune deficiency. The measurement of telomere length, critical to research in this area, has traditionally been performed by Southern blot analysis, which is cumbersome and time consuming. Several alternative methods have been described in recent years. Some, such as pulsed-field electrophoresis, slot blots, and centromere-to-telomere ratio measurements are essentially improvements to the Southern blot technique. However, other methods such as fluorescent in situ hybridization on metaphase chromosome spreads and flow cytometry– based fluorescent in situ hybridization represent a completely new technical approach to the problem. In this review, we compare methods, with particular emphasis placed on flow cytometric techniques for measuring telomere length in situ and identifying potential areas where improvements may still be made. Cytometry (Comm. Clin. Cytometry) 42: 159 –164, 2000. © 2000 Wiley-Liss, Inc. Key terms: telomere length; flow cytometry; fluorescent in situ hybridization; peptide nucleic acid; flow fluorescent in situ hybridization

In recent years, there has been a growing interest in telomere biology. The telomere is composed of an array of repetitive DNA sequence (TTAGGG in the human) and associated proteins found at the ends of linear eukaryotic chromosomes (1). Telomeres are believed to be involved in positioning the chromosomes during mitosis (2), maintenance of chromosomal integrity, and the protection of unique DNA sequences (reviewed in 3,4). Interest in telomeres lies in the absolute requirement of cellular DNA polymerase to use RNA primers to initiate lagging strand DNA synthesis (5,6). As the RNA primers are removed, they are filled in by the DNA polymerase in the 5⬘33⬘ direction, which leaves a gap at the 5⬘ end of the chromosome. Thus, there is a net loss of chromosome length that is composed of telomeric sequence each time the cell enters the S phase of cell cycle. Telomere loss may be repaired by the action of the enzyme telomerase. Telomerase is expressed in germ-line cells (7), stem cells (8), and in some activated T cells (9). There is a strong association between telomere length stabilization, whether by telomerase or some telomerase-independent mechanism, and immortalization of human cells (10, reviewed in 11). Thus, telomere biology may be a key component to understanding tumor formation, aging, infections, and immune diseases. Perhaps an aspect of telomere biology of more fundamental interest is the consequence of the natural erosion of telomere length. When the number of telomere repeats

© 2000 Wiley-Liss, Inc.

reaches a critical (shortened) length, the cell no longer proliferates and it takes on a senescent phenotype characterized by an accumulation of morphologic and biochemical changes within the cell (reviewed in 12). Senescent cells tend to be larger and flatter than normal cells; they express increased p21, and senescence-associated ␤-galactosidase activity, decreased nucleolar RNA content (13), and have decreased rates of protein synthesis. The process of senescence has been referred to as a “mitotic clock” that limits the proliferative capacity of somatic cells (14), also called the Hayflick limit. At the level of the organism, telomere shortening may be responsible for the progressive functional impairments of organ systems that rely on cellular proliferation and replicative capacity, including the immune system. It has recently been reported, in a large cross-sectional study, that granulocyte and Tlymphocyte telomere length decreases with age in a biphasic manner, reflecting increased telomere erosion in early childhood (15). Thus, theoretically, it may become possible to delay the onset of certain age-related pathologies by “healing” telomeres or preventing telomere eroGrant sponsor: Ontario HIV Treatment Network and Medical Research Council of Canada; Grant sponsor: Ontario Ministry of Health. *Correspondence to: Andrew D. Badley, Division of Infectious Diseases, Ottawa Hospital, 501 Smyth Road, Ottawa, Ontario, K1H 8L6, Canada. E-mail: [email protected] Received 11 January 1999; Accepted 13 March 2000

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sion. Conversely, disease states or medical interventions that serve to accelerate telomere erosion may result in premature onset of these pathologic processes. In this context, it is interesting to note that children receiving hematopoietic stem-cell transplants from older donors had significantly shorter telomeres than did children receiving stem cells from younger donors (16). The difference was a function of the donor stem-cell telomere length. Similarly, it has been reported that Dolly, the cloned Finn Dorset sheep, has significantly shorter mammary-gland tissue cell telomeres than age-matched controls (17). The investigators suggested that the difference can be explained by the telomere length of the “mother” and the loss during in vitro tissue culture. A natural extension of the telomere theory of aging suggests that T-cell depletion that follows infection with the human immunodeficiency virus (HIV) may be due in part from premature exhaustion of the proliferative capacity of T cells, or accelerated immunosenescence. The concept that the replicative exhaustion of cells of the immune system contributes to the immune deficiency of AIDS is attractive in light of the massive CD4 T-cell turnover predicted by Ho et al.(18) and the lymphoproliferation associated with HIV disease. Several recent studies have reported significant reduction in telomere length in CD8⫹ T lymphocytes in HIV infection (19 –22), suggesting a role for replicative exhaustion in the accumulation of senescent CD8⫹ T cells (22) and deficiency in this compartment. This takes place in the presence of potential, continuous production of T lymphocytes from the thymus (23) by exhausting memory/reactive cells, leading eventually to the observed oligoclonality in the CD8⫹ T-cell compartment. Telomere dynamics in the CD4⫹ T-cell compartment have been much more difficult to assess. This difficulty may be explained in part by the recent challenges to the original assumption of massive CD4⫹ T-cell destruction in HIV infection (24,25) by directly measuring T-cell turnover. However, the complex interactions between the virus and the host during HIV infection and our incomplete understanding of T-cell homeostasis have led to conflicting conclusions. The telomeres of CD4⫹ T cells have been shown to increase (19), remain stable (20), or decrease (21) in length. It is likely that these discrepancies result from the numerous factors affecting T-cell proliferation and telomere length in the various HIV⫹ patient populations examined. An improved methodology for the assessment of telomere length that permitted high throughput processing of relatively small samples and, ideally, subpopulations of lymphocytes would greatly facilitate the understanding of the telomere dynamics in HIV disease. Telomere biology has had perhaps an even greater impact on the fundamental understanding of cell immortalization and tumor formation. It has been estimated that approximately 90% of primary tumors and perhaps up to 98% of immortal cell lines possess short telomeres and express increased amounts of telomerase activity as opposed to the negligible expression in normal somatic cells and benign tissues (26). However, it has been reported

that telomerase activity is an indicator of cell proliferation rather than of transformation (27) and that expression of telomerase in somatic cells induces an extended lifespan (28) but not phenotypic changes consistent with tumorigenesis (29,30). Furthermore, those cell lines that do not express detectable levels of telomerase are often found to possess alternative methods for maintaining telomere length (31). Therefore, telomere maintenance seems to be a key component in the multistep process of tumorigenesis. It is conceivable that monitoring telomere length may be predictive, in conjunction with other factors, in tracking malignant tumor formation. Research into telomere biology has been hampered by the relatively complex and cumbersome methods currently available. The introduction of rapid methods that permit high throughput processing of samples would greatly enhance the ability of researchers to address questions of telomere dynamics that are as yet unfeasible and bring the study of telomeres into the clinical laboratory. The gold standard for measurement of telomere length is Southern blotting (14,32–34), which, although accurate and reproducible, is relatively cumbersome; thereby precluding its use for large-scale analysis (35–37). This method requires a relative large number of cells (at least 5 ⫻ 105 to produce ⱖ2 ␮g of DNA) that are lysed to release the DNA. The DNA is phenol-chloroform extracted and digested with a combination of frequent cutting restriction enzymes (such as RsaI and HinfI) that cleave nontelomeric DNA into small fragments. These digested DNA samples are run on an agarose gel and hybridized to a P32-labeled (CCCATT)3 telomeric probe. The blot is exposed to photographic film, and the telomere length is estimated from the resultant autoradiograph by densitometric analysis. The telomere restriction fragment length (TRFL) represents the average telomere length of all the chromosomes in all of the nucleated cells in the sample. Thus, the results provide information only at the population level, with each chromosome end in the sample contributing to the overall telomere length measurement, and is limited by the ability to purify cells of one distinct type. It has been proposed that significant variations may exist in the location of subtelomeric restriction enzyme cleavage sites between individuals, which may contribute further to variable results in telomere length measurement (38). Several improvements to the Southern blot technique have been proposed. Pulsed-field electrophoresis has been used to increase the resolution of the TRFL differences on the autoradiographs (24). Slot blots have been employed to measure telomere-specific DNA content (39). This method permits reproducible telomere measurements from relatively few cells and is capable of measuring DNA fragments and whole genomic DNA (35). Another refinement of this technique uses the relatively constant centromeric DNA content as an internal comparison for the telomeric slot-blot values and can be performed in whole cells (36). However, each of these refinements to the Southern blot method increases rather than decreases the complexity of the assay.

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An alternative method of telomere measurement that circumvents the problem of subtelomeric DNA is quantitative fluorescent in situ hybridization (Q-FISH) performed on metaphase spreads (40 – 42). This procedure directly hybridizes a fluorescent-labeled telomere probe to telomeric repeats on the intact metaphase chromosome. The resulting fluorescent signal is captured by using a fluorescent microscope equipped with a digital camera and dedicated computer. Metaphase spreads provide an internal control of hybridization efficiency in that the sister chromatids necessarily have identical telomere lengths. Although the results have shown a good correlation with Southern blot methodology (43), Q-FISH is a labor-intensive, time-consuming procedure that requires a high degree of technical expertise, thus limiting its general applicability. The principles of this technique could be simplified and the throughput increased by performing the hybridization within nuclei and measuring the fluorescence by flow cytometry, as has been done for other repetitive DNA sequences (44,45). There are advantages and disadvantages to examining the telomere length at specific chromosome ends. Although this information may be particularly important in determining when a particular chromosome is approaching a critical degree of shortening, no information can be determined about the original cell type. Furthermore, Q-FISH is performed on cells that are undergoing mitosis on metaphase spreads. Because cells that are approaching replicative senescence may be less likely to enter the S phase (33) than are cells with longer telomeres, there may be biased results. Therefore, Q-FISH may not be suitable for the examination of cell populations approaching senescence. However, it is the method of choice to examine and track changes in telomeric sequences on specific chromosomes. An alternative method for examining telomere length is required to provide information at the cellular level, which may distinguish critical changes in telomere length in cellular subpopulations. An ideal telomere-length assay must be rapid, permit the examination of small numbers of cells, require few steps, and produce reliable, reproducible and accurate results. Furthermore, it would ideally require relatively few cells, discriminate between small changes in telomere length (i.e., be quantitative), measure telomere length in situ, and allow for the simultaneous detection of cell phenotype. FISH and flow cytometry hold a great deal of promise is these respects. There have been recent reports from Rufer et al. (36) and Hultdin et al. (37) describing flow cytometric methods for the measurement of telomere length in the whole cell that approaches this ideal in several key respects. Both protocols provide quantitative, highly reproducible results that correlate well with the Southern blot “gold standard.” In each case, the number of preparation steps of the samples is minimized and the procedure is rapid in comparison with other techniques. The original description of flow FISH by Rufer et al. (36) has recently been revised (15) and clearly shows the utility of the methodology in the examination of a very large number of samples.

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The methods described by Rufer et al. (36) and Hultdin et al. (37) resemble in many respects the earlier Q-FISH protocols. A significant advance to facilitate the transition of Q-FISH protocols to in situ flow cytometry is the use of peptide nucleic acid (PNA)–labeled probes. PNA probes contain an uncharged glycine backbone rather than the charged phosphate-ribose/deoxyribose backbone of traditional oligonucleotide probes (reviewed in 46). These probes overcome the problem of the genomic DNA strands reannealing and excluding the oligonucleotide probe. The PNA probe hybridizes to complementary DNA in low ionic strength solutions that do not favor reannealing of the target strands. PNA–DNA interactions are more stable than DNA–DNA or DNA–RNA interactions under hybridization conditions (reviewed in 46). This property may yield more reproducible results and a stronger fluorescent signal than can be obtained with conventional DNA oligonucleotides. Indeed, the fluorescent intensity of the telomeres using PNA probes (15,36,37) appears to be significantly greater than that using a fluorescein-labeled (CCCTAA)3 DNA probe (W. Lauzon et al., unpublished data). Flow cytometry methods for quantitative FISH (flow FISH) on whole cells pose a unique problem related to providing the probe access to the target DNA while maintaining cell structure. The first obstacle is getting the probe inside the cell. This is generally a delicate balance between fixing the cell sufficiently to maintain cellular structure without cross-linking the proteins to such an extent that the probe can no longer penetrate the cell or irreversibly bind the DNA-associated proteins to the chromosome. Hultdin et al. (37) employed the standard in situ hybridization method, of fixation followed by permeabilization, with the Caltag Fix & Perm kit™. Rufer et al. (36) did without these steps entirely, relying instead on the denaturation conditions to provide access of the probe into the cell. It is somewhat surprising that the probe penetration occurs without the permeabilization of the cell membrane. However, the small size of the probe and the increased fluidity of the cell membrane at the temperatures used to denature the DNA must permit sufficient permeability to the PNA probe. This strategy avoids the potential probe-accessibility problems associated with excessive fixation (36). This benefit, however, comes at the price of diminishing the likelihood of efficient simultaneous cell-surface immunophenotyping (see below). Once the probe is inside the cell, the DNA must be denatured to expose the telomere target sites. The denaturation step, in each case, takes place with the cells suspended in formamide-based hybridization solution at 87°C for 10 min (37) or at 80°C for 10 min (36). We found that it was essential to denature the DNA in the presence of the DNA probe to provide access to the telomere before the chromosome strands could reanneal (W. Lauzon et al., unpublished data). This may not be as critical for PNA probes; however, Rufer et al. (36) depended on this heating step to permeabilize the cell to the probe. The requirement to denature the DNA introduces another complication in that proteins also are readily

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denatured. This limits the selection of available fluorochromes to be conjugated to the PNA probe to include only nonproteinaceous molecules. This feature also creates difficulty in designing efficient strategies to allow for concurrent surface (or intracellular) marker analysis. Flow cytometric analysis of telomere length in whole cells avoids the selection of proliferating cells inherent in conventional Q-FISH on metaphase spreads, thus permitting the examination of all cells, including senescent cells. However, this too poses a potential pitfall. Many cell populations of interest in the study of telomere dynamics, such as tumor cells and peripheral blood mononuclear cells from HIV-infected patients, contain cells in all stages of the cell cycle. The number of chromosomes and thus the number of telomeres present per cell will have a direct effect on the fluorescent signal detected by flow FISH. Because telomere replication takes place in early S phase (37), this theoretical concern also may be of practical concern. To address this problem, Hultdin et al. described a technique to standardize the number of telomeres per cell by analyzing telomere fluorescent signal only in cells in G0/G1 phase as determine by propidium iodide (PI) staining (37). Rufer et al. (36) do not seem to have corrected for this possible source of error with little apparent effect, perhaps because the normal lymphocytes that they analyzed were uniformly in G0/G1. Both procedures were designed to limit the number of steps required to obtain meaningful, reproducible data. However, flow FISH involves many steps where relatively minor human or systematic error can translate into large shifts in fluorescent readout. Therefore, a great deal of effort was spent addressing controls necessary to negate or at least assess the contribution of these potential errors in the procedures. The method of Rufer et al. (36) focuses primarily on controlling for variations in the cytometer and monitoring intra- and interexperimental errors, probably because the lack of fixation and permeabilization steps limits some of the inherent tube-to-tube variations. In this procedure, fluorescein isothiocyanate (FITC)–labeled beads are run at the beginning and end of each experiment, and the resulting calibration curve is used to correct for daily fluctuations in laser intensity, alignment, and linearity. In early experiments, Rufer et al. (36) used an FITC-labeled PNA probe for sequences of an X-chromosome marker as a negative control. However, no differences were detectable between male and female samples by this technique, so subsequent experiments used hybridizations without probe to measure background fluorescence. Intraexperimental variation in fluorescence was assessed to be ⬍5% by analyzing multiple samples of the same cell line or patient samples in the same experiment. Interexperimental variation was assessed at 0 –20% by evaluating aliquots of the same frozen control cells in each experiment. Hultdin et al. (37) used a different approach. Their method employed an internal control. Each experimental sample was mixed with an equal number of 1301 (T-cell lymphoblastic leukemia cell line) cells before experimental manipulation. The 1301 cells are known to possess extremely long telomeres (⬎25 kb) (37) and thus

are unlikely to interfere with the analysis of most human samples. The telomere flow FISH value was then calculated as a ratio between the telomere signal of the experimental sample and the control 1301 cells in the same tube. The daily variations in the cytometer and the differences in fixation, permeabilization, and hybridization were controlled in this simple fashion. This strategy assumes that the 1301 cell telomere fluorescence is constant. It also assumes a constant and linear relationship between flow FISH signal intensity and TRFL. The first assumption is valid for the present study because aliquots of the same batch of 1301 cells, frozen in dimethyl sulfoxide, was used. However, it remains to be determined whether the telomere length stability is sufficient to permit comparison of results from different batches of control cells. In addition, the basis of the second assumption is untested. A potential drawback to the use of flow cytometry for evaluating telomere length is the presence of (T2AG3) repeat sequences in interstitial or centromeric sites. Metaphase spreads from human phytohemaggluanin–stimulated peripheral blood lymphocytes have been reported to contain more than 50 specific sites of intrachromosomal “telomerelike” signals (47). This estimate may, in fact, be an underestimation of the actual intrachromosomal “telomerelike signal” because the resolution of an independent signal with this sort of technology requires a separation of several megabases from the telomere (45). Flow cytometry based in situ hybridization sums the hybridization signal from the entire genome; thus, these nontelomeric signals may inflate the fluorescent telomere signal for the cell. The degree to which this may distort the telomere length calculation will depend on the size and heterogeneity of these sites in the human genome and on the type of investigations being undertaken. Intrachromosomal signals would be predicted to be particularly confounding as the cells approach senescence, where the telomere-specific signal is diminished relative to the intrachromosomal signals. However, within a given cell the amount of intrachromosomal “telomerelike” signals would be predicted to be constant over time. An insight into the contribution of these signals can be gleaned from the correlation of TRFL to telomere fluorescence studies. The TRFL value has been estimated to contain 2.5– 4 kb of subtelomeric DNA (reviewed in 48). Hultdin et al. (37) compared TRFL values against flow FISH values for the same sample and extrapolated a y intercept of ⬃3.2 kb. This value represents the subtelomeric DNA associated with the TRFL measurement but absent from the flow FISH measurement. The agreement between this value and the estimate value for subtelomeric DNA in TRFL indicates that, at least for the samples examined, the intrachromosomal fluorescent signals by flow FISH are substantially lower than the subtelomeric background seen in Southern blots and may, in fact, be negligible. Rufer et al. (36) showed a linear regression that passed through the origin, suggesting that there is no calculated subtelomeric sequence in the Southern-blot data. Furthermore, they suggested that their method of flow FISH could

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detect differences in telomere length of less than 3 kb, whereas the Southern-blot method could not. On the surface, this finding appears to support the concept that the reported flow FISH values contain a background signal roughly equivalent to the subtelomeric signal. However, the correlation curve shows a tendency to a more rapid decrease in telomere fluorescence signal in short telomeres than TRFL. Rufer et al. (36) suggested that this reflects the subtelomeric signal in the Southern blot, but one would expect this difference to remain constant across the various telomere lengths rather than be limited to the shorter samples. The apparent difference may reflect a decrease in specific fluorescent signal in samples with long telomeres. It is not clear whether this skewed correlation is the result of a small number of long TRFL outliers or of systematic error. Hultdin et al. (37) showed that decreased probe concentrations versus those used by Rufer et al. (36) decreases the background signal and that probe concentrations 10-fold lower are optimal in their hands (37). It is unlikely that this would preferentially operate on long telomeres, but, combined with the much shorter hybridization time [2 h (36) versus overnight (37)], it may explain some of the difference. Notwithstanding these inconsistencies, contribution of interstitial and centromeric “telomerelike” signals seem to be minimal in flow FISH. The real potential of flow cytometry in this area is in the ability to simultaneously detect cell phenotype and perhaps intracellular cytokines with telomere length. Both groups of investigators suggested that this is the next step in the development of their respective methods. There are two major obstacles to be overcome in examining telomere length in conjunction with immunophenotyping: permeabilization and denaturation. Heat denaturation of genomic DNA is an absolute requirement of flow FISH. The temperatures used (⬃80°C) in the presence of formamide pose two related problems. First, the cell-surface molecules are denatured and may no longer be recognized by the epitope-specific antibody. Second, proteinaceous fluorochromes, for example, phycoerythrin, are denatured and no longer emit detectable light. Staining the surface before fixation with hapten-conjugated antibodies followed by posthybridization staining with fluorochrome-labeled antihapten antibodies may represent a viable alternative. In this case, the primary antibody is crosslinked to its specific ligand by the fixation process, and the hapten may remain exposed at the surface of the cell despite denaturation of both the antibody and ligand. Moreover, dye-impregnated beads such as Transfluorospheres™ (Molecular Probes, Eugene, OR) may retain their fluorescent properties during flow FISH, thereby supporting their potential use as epitope tags. An additional concern derives from the suggestion of Hultdin et al., that staining for DNA content may an important step to standardize the number of telomeres per cell. The use of PI in this capacity limits the available fluorochromes that are excitable by an argon laser (488 nm) because of its broad emission spectrum. However, 4⬘-6⬘-diamidino-2phenylindole, which is excited in the ultraviolet range and

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emits at 405– 450 nm, has been used to measure DNA in flow cytometry– based chromosome-specific DNA hybridization in interphase nuclei (9,10) and should be an appropriate substitute for PI. This substitution would interfere less with the available choices of fluorochromes for surface-labeling applications. Flow cytometry provides a powerful tool for examining telomere length in different cell and tissue types. The methods described in this review enable the routine quantitative analysis of telomere length that surpasses other available methods for ease and rapidity. They permit the large-scale screening of samples for changes in telomere length that will be important in the research laboratory in the study of HIV and cancer. This new methodology may be clinically applicable to the screening of stem-cell and bone-marrow samples intended for transplantation to ensure sufficient telomere length to provide hematopoietic reconstitution in the recipient and sufficient residual proliferative potential for a normal lifespan. In conclusion, studies in telomere biology appear to be on the brink of being revolutionized with the introduction of flow FISH technology. ACKNOWLEDGMENTS Wallace Lauzon is the recipient of an Ontario HIV Treatment Network (OHTN) postdoctoral fellowship award. D. William Cameron is a career scientist of the Ontario Ministry of Health. Andrew D. Badley has received a scientist award from the OHTN. LITERATURE CITED 1. Moyzis RK, Buckingham JM, Cram LS, Dani M, Deavan LL, Jones MD, Meyne J, Ratliff RL, Wu J-R. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Natl Acad Sci USA 1988;85:6622– 6626. 2. De Lange T. Human telomeres are attached to the nuclear matrix. EMBO J 1992;11:717–724. 3. Zakian VA. Structure and function of telomeres. Annu Rev Genet 1989;23:579 – 604. 4. Zakian VA. Telomeres: beginning to understand the end. Science 1995;270:1601–1606. 5. Watson JD. Origin of concatameric T4 DNA. Nature 1972;239:197– 201. 6. Olovnikov AM. A theory of marginotomy. J Theor Biol 1973;41:181– 190. 7. Eisenhauer KM, Gerstein RM, Chiu C-P, Conti M, Hsueh AJ. Telomerase activity in female and male rat germ cells undergoing meiosis and in early embryos. Biol Reprod 1997;56:1120 –1125. 8. Chiu CP, Dragowska W, Kim NW, Vaziri H, Yui J, Thomas TE, Harley CB, Lansdorp PM. Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow. Stem Cells 1996;14:239 –248. 9. Weng N-P, Levine BL, June CH, Hodes RJ. Regulation of telomerase RNA template expression in human T lymphocyte development and activation. J Immunol 1997;158:3215–3220. 10. Kim NW, Mieczyslaw A, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Wienrich SL, Shay JW. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011–2015. 11. Rhyu MS. Telomeres, telomerase, and immortality. J Natl Cancer Inst 1995;87:884 – 894. 12. Campis J. Aging and cancer: the double-edged sword of replicative senescence. J Am Geriatr Soc 1997;45:482– 488. 13. Bowman PD, Meek RL, Daniel CW. Decreased synthesis of nucleolar RNA in aging human cells. Exp Cell Res 1976;101:434 – 437. 14. Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai, EV, Futcher AB, Greider CW, Harley CB. Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci USA 1992;89: 10114 –10118.

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