Am. J. Hum. Genet. 52:661-667, 1993

Loss of Telomeric DNA during Aging of Normal and Trisomy21 Human LymphocytesHomayoun Vaziri,* Franpois Schachter,4 Irene Uchida,t Lan Weit Xiaoming Zhu, Rita Effros,Daniel Cohen,4 and Calvin B. Harley*Departments of Biochemistry and tPediatrics, McMaster University, Hamilton, Ontario; tCentre d'Etude du Polymorphisme Humain, Paris; andDepartment of Pathology, University of California School of Medicine, Los Angeles

SummaryThe telomere hypothesis of cellular aging proposes that loss of telomeric DNA (TTAGGG) from humanchromosomes may ultimately cause cell-cycle exit during replicative senescence. Since lymphocytes have alimited replicative capacity and since blood cells were previously shown to lose telomeric DNA during aging invivo, we wished to determine (a) whether accelerated telomere loss is associated with the prematureimmunosenescence of lymphocytes in individuals with Down syndrome (DS) and (b) whether telomeric DNA isalso lost during aging of lymphocytes in vitro. To investigate the effects of aging and trisomy 21 on telomereloss in vivo, genomic DNA was isolated from peripheral blood lymphocytes of 140 individuals (age 0-107 years),including 21 DS patients (age 0-45 years). Digestion with restriction enzymes Hinfl and RsaI generated terminalrestriction fragments (TRFs), which were detected by Southern analysis using a telomere-specific probe (32P-(C3TA2)3). The rate of telomere loss was calculated from the decrease in mean TRF length, as a function of donorage. DS patients showed a significantly higher rate of telomere loss with donor age (133 15 bp/year) comparedwith age-matched controls (41 7.7 bp/year) (P < .0005), suggesting that accelerated telomere loss is abiomarker of premature immunosenescence of DS patients and that it may play a role in this process. Telomereloss during aging in vitro was calculated for lymphocytes from four normal individuals, grown in culture for10-30 population doublings. The rate of telomere loss was -120 bp/cell doubling, comparable to that seen inother somatic cells. Moreover, telomere lengths of lymphocytes from centenarians and from older DS patientswere similar to those of senescent lymphocytes in culture, which suggests that replicative senescence couldpartially account for aging of the immune system in DS patients and in elderly individuals.

IntroductionThe dependence of DNA polymerases on primers andthe unidirectional 5'-to-3' direction of synthesis poses aproblem for complete replication of the ends of eukary-otic chromosomes (Olovnikov 1971, 1973; Watson1972). This problem is circumvented in eukaryotes bytelomeres, the specialized nucleoprotein structurescontaining highly conserved repeats (reviewed in Black-burn and Szostak 1984). In vertebrates, telomeric DNA

Received September 8, 1992; revision received December 3, 1992.Address for correspondence and reprints: Dr. Calvin B. Harley,

Department of Biochemistry, McMaster University, Hamilton, On-tario L8N 3Z5, Canada.) 1993 by The American Society of Human Genetics. All rights reserved.0002-9297/93/5204-0002$02.00

terminates in TTAGGG repeats (Moyzis et al. 1988;Allshire et al. 1989). Telomeric repeats are synthesizedby telomerase, a ribonucleoprotein that is capable ofelongating telomeres de novo and hence of overcomingloss of telomeric DNA due to incomplete replication ordegradation of ends. Telomerase activity was first de-tected in Tetrabymena (Greider and Blackburn 1985)and later in extracts from immortalized human cells(Morin 1989; Counter et al. 1992).

Several lines of evidence suggest that telomere short-ening plays a causal role in cellular aging. We have previ-ously shown that, in human fibroblasts, telomeresshorten as a function of cell doublings in vitro and invivo and that initial telomere length predicts the repli-cative capacity of these cells (Harley et al. 1990; Allsoppet al. 1992). Loss of telomeric DNA during aging in vivo

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has also been observed in peripheral blood cells andcolon mucosa epithelia (Hastie et al. 1990). Telomereshortening in normal (nonimmortalized) human cellstrains is associated with the inability to detect telo-merase in extracts from these cells (Counter et al. 1992).In contrast, immortal cells express telomerase, and theirtelomeres do not progressively shorten (Counter et al.1992). A causal link between telomere loss and cell-cy-cle exit has not been proved, but the sudden increase inthe number of dicentric chromosomes in senescing fi-broblasts (Saksela and Moorhead 1963; Benn 1976;Sherwood et al. 1989) and the significant age-relatedincrease of these abnormalities in human peripheralblood lymphocytes (Bender et al. 1989) suggest that theintegrity of chromosome ends is compromised duringreplicative aging. The above observations are the basisof the telomere hypothesis of cellular aging and immor-talization (Harley 1991).

Aging of the immune system could account for someof the morbidity of elderly individuals and DS patients.If this were true and if telomere shortening plays a rolein cellular aging, then one might predict critically short-ened telomeres in the lymphocytes of these individuals.To investigate this, we measured the length of terminalrestriction fragments (TRFs) that contain the terminalTTAGGG repeats in peripheral blood lymphocytes(PBLs) of 140 individuals (age 0-107 years), including18 centenarians (age >99 years) and 21 Down syn-drome (DS) patients. Here we report that mean TRFlength shortens as a function of age in these cells invitro and in vivo and that cells from DS patients have ahigher rate of telomere loss with age, compared withage-matched controls.

Subjects, Material, and MethodsAll blood samples were obtained with informed con-

sent. Normal subjects (age 0-90 years) were primarilyhealthy volunteers from the Hamilton (southwesternOntario) region who were ostensibly free from anyblood disorder. DNA from all centenarians (age >99years) was from the Centre d'Etude du PolymorphismeHumain (CEPH) collection, Paris. DS individuals wereall trisomy 21 by cytogenetic analysis and were alsoprimarily from southwestern Ontario.

Culture ofHuman Peripheral Blood T LymphocytesAdult peripheral blood samples were collected, and

mononuclear cells were isolated by Ficoll-Hypaquegradient centrifugation and then were cryopreserved inliquid nitrogen. Cultures were initiated by mixing 106

mononuclear cells with 106 irradiated (8,000 Rad) lym-phoblastoid cells (Epstein-Barr virus-transformed Bcells) or by mixing 106 mononuclear cells with 10 jgphytohemagglutinin (PHA-P; Difco)/ml in each well ofa 48-well cluster plate (Costar). After 8-11 d, cells werewashed and plated in 2-ml wells of 24-well clusterplates at a concentration of 2-4 X 105 cells/ml. Cul-tures were passaged every 3-4 d or whenever viable cellconcentration (determined by trypan blue exclusion)reached >8 x 105 cells/ml. Cultures were terminatedwhen they showed no proliferative response to irra-diated lymphoblastoid cells or when there were no via-ble cells present in the entire visual field of the hemocy-tometer. Once transferred to the 2-ml wells, cells werecontinuously exposed to 25 U of recombinant interleu-kin-2 (Amgen)/ml in RPMI (Irvine Scientific) supple-mented with 10%-20% FCS, 2 mM glutamine, and 1mM Hepes or in AIM V1', a DMEM/F-12 basal me-dium containing purified human albumin, transferrin,and recombinant insulin (Gibco), supplemented with25% Ex-cyte (an aqueous mixture of lipoprotein, cho-lesterol, phospholipids, and fatty acids [Miles Diagnos-tics]).At each cell passage, the number of population dou-

blings (PDs) was calculated according to the followingformula: PD = In(final no. of viable cells/initial no. ofcells)/ln 2.Isolation of CellsPBLs (including 15% monocytes) were isolated us-

ing Ficoll-Hypaque gradient centrifugation (Boyum1968). Purification of human B and T cells (Gutierrez etal. 1979) and neutrophils (Dooley et al. 1982) was per-formed on a discontinuous percoll gradient. Densitieswere monitored using Density Marker Beads (Pharma-cia). T-cell identification was confirmed by erythrocyterosette testing, essentially as described by Gutierrez etal. (1979), and by Wright and Giemsa staining. All cellpopulations consisted of >98% viable cells, as judgedby trypan blue exclusion.Isolation ofDNA

Cells were washed three times in PBS, and the pelletwas resuspended at a density of 106_107 cells/ml inproteinase K digestion buffer (100 mM NaCl, 10 mMTris pH 8,5 mM EDTA, and 0.5% SDS) containing 0.1mg proteinase K/ml. The lysate was incubated at 48Covernight and then was extracted twice with phenol/chloroform/isoamyl alcohol (25:24:1 [v/v/v]) andonce with chloroform. Nucleic acid was precipitatedwith 95% ethanol and was dissolved in TE (10 mM Tris

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and 1 mM EDTA, pH 8). In some experiments, RNAwas first degraded with pancreatic RNase A, but thishad no effect on measurements of telomeric DNA.

Analysis of Telomeric DNADNA (10 jig) was digested with Hinfl and RsaI (BRL)

(20 U each), reextracted as described above, precipi-tated with 95% ethanol, washed with 70% ethanol, dis-solved in 50 pJ TE, and quantified by fluorometry. Onemicrogram of digested DNA was resolved by electro-phoresis in 0.5% [w/v] agarose gels poured on GelBound (FMC Bioproducts) for -700 V-h. Gels weredried at 60'C for 30 min, denatured, neutralized, andprobed with 5' end-labeled 32P-(C3TA2)3, as describedby Allsopp et al. (1992). Autoradiograms exposedwithin the linear range of signal response were scannedwith a Hoefer densitometer. The signal was digitizedand subdivided into 1-kbp intervals between 2 kbp to21 kbp, for calculation of the mean TRF length (L) byusing the formula L = X(ODi Li)/XODi, where ODi =integrated signal in interval i and Li = TRF length at themidpoint of interval i.

Results

When measured as a function of donor age, meanTRF length in PBLs of 119 unrelated normal individ-uals (age 0-107 years) declined at a rate of 41 2.6bp/year (P < .00005; r = .83) (fig. 1). This rate of TRFloss for PBLs is close to that previously found for pe-ripheral blood cells by Hastie et al. (1990). When ourdata were separated according to gender, it was noticedthat males lost telomeric DNA at a rate slightly fasterthan that of females (50 4.2 vs. 40 3.6 bp/year,respectively), but this difference did not reach statisti-cal significance (P = .1). The 18 centenarians (age 99-107 years) among our population of normal individualshad a mean TRF length of 5.28 0.4 kbp (fig. 1). TRFlength in these long-lived individuals is predicted byextrapolation of the line for individuals 0-80 years ofage, suggesting that centenarians did not have an unusu-ally long initial telomere length or an unusually slowrate of telomere loss. Interestingly, the SD of mean TRFvalues for the centenarians (0.4 kbp) was much smallerthan that for other age groups (- 1 kbp). It is possiblethat this represents selection of a more homogeneouspopulation of cells with age or that there exists somemechanism for homogenization of telomere lengthwith time. However, it is also possible that the group ofcentenarians was less genetically diverse than theyounger populations in our study.

Mean TRF length was also analyzed in PBLs of 21 DSindividuals (age 2-45 years), and the rate of loss wascompared with that of 68 age-matched controls (age0-43 years). Cells from DS patients showed a signifi-cantly greater rate of telomere loss (133 15 bp/yearvs. 41 7.7 bp/year, respectively; P < .0005) (fig. 2).The rate of telomere loss in DS individuals 20 PDs in vitro, was 5.5 0.8kbp (fig. 4, right). The observed TRF values in vivo forPBLs of centenarians (5.3 0.4 kbp) and old DS pa-tients (4.9 0.6 kbp) were not significantly differentfrom this value, suggesting that a fraction of the cellsfrom these individuals were at the limit of their replica-tive capacity.

DiscussionPeripheral blood lymphocytes, like fibroblasts and

other replicating somatic cells, have a limited divisioncapacity (Effros et al. 1990). Thus, our results showingthat telomeres in PBLs from normal individuals shortenduring aging in vivo and in vitro (a) extend similar ob-servations on human fibroblasts (Harley et al. 1990;Allsopp et al. 1992) and (b) support the hypothesis thattelomere loss may be involved in replicative senescence.

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Figure I Loss of telomeric DNA as a function of donor age in PBLs. Seven milliliters of peripheral blood was obtained from 119 donors(age 0-107 years), and genomic DNA from PBLs was isolated and digested with restriction enzymes for Southern analysis, as described inSubjects, Material, and Methods. Left, Autoradiogram showing a subset of the normal individuals, including most of the centenarians. Donorage and size markers (in kbp) are indicated. In this experiment, sperm DNA (lane S), which has long TRFs, was included as a control. The bandsappearing at '-2, 1.6, and - 1 kbp are nontelomeric repetitive sequences that hybridize to the probe, even at high stringency. They serve as aconvenient control for DNA integrity and quality of the gel. Right, Mean TRF length from quantitative analysis of autoradiograms, as describedin Subjects, Material, and Methods, plotted as a function of donor age. The slope (-41 2.6 bp/year) of the linear regression line is significantlydifferent from 0 (P < .00005).

We also found that, in DS, the rate of telomere loss inPBLs as a function of donor age was three times higherthan that in age-matched normal donors. Since DS ischaracterized by immune dysfunction, including thy-mus abnormalities, derangements of both lymphoidand myeloid cell compartments (reviewed by Ugazio etal. 1990), and premature T-cell aging (Rabinowe et al.1989), the accelerated loss of telomeres in PBLs couldreflect a generalized early senescence of immune cells inthese individuals.

It is possible that the decrease in telomere lengthwith age in vivo reflects, in part, changes in the subpop-ulations of cells, which are known to occur in bothnormal individuals (reviewed in Thoman and Weigle1989) and DS patients (Cossarizza et al. 1991). How-ever, the absolute and relative changes for the majorT-cell subpopulations are relatively small in normal indi-viduals (Murasko and Goonewardene 1990). In addi-tion, our measurements in B and T cells, as well asneutrophils, showed similar TRF-length distributionsbetween the subpopulations in young and old normaland DS individuals and confirmed the loss of telomeric

DNA observed in total PBLs (fig. 3). These data indi-cate that aging of the lymphoid and myeloid lineages ischaracterized by similar rates of telomeric DNA loss.Although we have not yet been able to further subdi-vide these subpopulations for telomere analysis, itseems unlikely that differences in the major subpopula-tions within each lineage (e.g., CD4 vs. CD8 lympho-cytes) would be greater than the differences in the ma-jor leukocyte subdivisions and that these differencescould account for the systematic and parallel loss thatwe see with age in both myeloid and lymphoid lineages.Finally, within cultured fibroblasts, variation in intitialTRF length exists between clones from a single massculture, but all clones lose telomeric DNA during repli-cative aging (R. C. Allsopp and C. B. Harley, unpub-lished data). Thus, telomere loss due to cell divisionwith age is the simplest interpretation of our data.

If we accept that telomere length is a biomarker ofthe replicative history of normal somatic cells (Harleyet al. 1990; Hastie et al. 1990; Harley 1991; Levy et al.1992) and of their future replicative potential (Allsoppet al. 1992), then the increased rate of telomere loss in

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Telomere Loss in Aging and Trisomy 21

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DONOR AGE (YEARS)Figure 2 Accelerated telomere loss in DS patients. GenomicDNA isolated from PBLs of DS patients was analyzed according tothe method described in fig. 1 and Subjects, Material, and Methods.Mean TRF length is shown as a function of donor age, for DS pa-tients (U) and age-matched controls (U). The slopes of the linearregression lines (-133 15 bp/year for trisomy 21, vs. -43 7.7bp/year for normal) are significantly different (P < .0005). Two DSsamples, at ages 24 and 47 years, (E), were analyzed a second time, 2years after the first analysis. These points indicate the reproducibilityof data from separate experiments but were not included in the statis-tical analysis.

PBLs from DS patients could reflect a higher turnoverrate of these cells in vivo. There are several possibleexplanations for this. For example, immune cells in DSindividuals could have reduced viability or abnormali-ties in maturation due to trisomy 21, which lead di-rectly to increased cell turnover. However, it is alsopossible that the amount of telomere loss in PBLs fromDS patients is greater per cell doubling than that innormal individuals. In yeast, altered expression of sev-eral genes leads to abnormal regulation of telomerelength (e.g., see Lundblad and Szostak 1989; Conrad etal. 1990). If the expression of genes involved in telo-mere length regulation is altered due to trisomy 21,then the rate of telomere loss could increase in DS.Further work on telomere length and cell turnover andfunction both in vivo and in vitro in DS cells is requiredto determine both the mechanism that accounts for thehigher rate of telomere loss in these individuals andwhether telomere loss might play a causal role in im-mune system failure.

Since lymphocytes are continuously renewed, telo-mere shortening during aging in vivo suggests that telo-merase is either absent or present in low levels in pro-genitor blast cells and primitive stem cells. If there wereno telomerase activity, then the rate of telomere losswith age in circulating cells (-40 bp/year) (see fig. 1and Hastie et al. 1990) might reflect the average rate oftelomere loss in the bone marrow. If we further assumethat the measured rate of telomere loss in lymphocytesin vitro (- 100 bp/cell doubling) (fig. 4) applies to stemcells as well, we could estimate that the stem-cell popu-lation undergoes -0.4 doublings/year, on average.This is derived from the ratio (40 bp/year)/(100 bp/doubling). The comparable values for fibroblasts are(15 bp/year)/(75 bp/cell doubling) 0.2 cell dou-blings/year (Allsopp et al. 1992). The estimate of 0.4cell doublings/year for hematopoietic stem cells maybe low, since partial telomerase activity or other factorscould reduce the rate of telomere loss in vivo in stemcells. Direct measurements of telomere length and telo-merase activity in cells at various points in the hemato-poietic lineage, coupled with independent knowledgeof cell kinetics, will allow us to assess the utility oftelomere length as a biomarker of cell aging in vivo.

21 -

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Figure 3 Loss of telomeric DNA in subpopulations of bloodcells in DS patients and normal individuals. Cells were isolated ac-cording to the protocol described in Subjects, Material, and Meth-ods, and genomic DNA was analyzed for TRF lengths, as described infig. 1. P = PBLs; B = B cells; T = T cells; and N = neutrophils.

665

666 Vaziri et al.

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301------l | | ~PDLFigure 4 Analysis of telomere loss during aging of lymphocytes in vitro. T lymphocytes were cultured for various lengths of time in vitro,and PDs were determined according to the protocol described in Subjects, Material, and Methods. Left, Autoradiogram of terminal restrictionfragments from genomic DNA isolated and prepared for Southern analysis, as described in fig. 1. PDs of a typical T-lymphocyte culture areindicated above each lane; size markers (in kbp) are shown at the left. Right, Decrease in mean TRF length, as a function of PDs, for DNA fromfour normal individuals. Donor ages for these cells were not available. Mean TRF length at terminal passage from a fifth donor, for whichmultiple passages were not available, is also shown (5).

Premature senescence of the immune system in DS ispossibly a major factor in the similarity of DS pathologyand normal aging (Martin 1978). In support of this idea,lymphocytes of old DS patients and old normal individ-uals share several characteristics, including diminishedresponse of T cells to activate and proliferate in re-sponse to antigen, low replicative capacity, and reducedB- and T-cell counts (Cossarizza et al. 1991; Franceschiet al. 1991). Our findings that telomere length in PBLsdecreased faster in DS patients than in normal individ-uals and that the mean TRF length in centenarians andold DS patients in vivo was similar to that of senescentlymphocytes in vitro (-5 kbp) support and extendthose observations. Moreover, our data suggest thatreplicative senescence within the lymphoid and my-eloid lineages in vivo might contribute to the compro-mised immune system of both elderly individuals andDS patients.

AcknowledgmentsWe thank the families of the DS patients for their coopera-

tion and J. Waye for providing fetal blood samples. We alsothank Alexy Olovnikov, Rich Allsopp, Edwin Chang, Silvia

Bacchetti, and Carol Greider for helpful discussions andCharles Epstein for critical comments. This work was sup-ported by the Medical Research Council of Canada (supportto C.B.H.) and by U. S. National Institutes of Health grantsAG09383A (to C.B.H.) and AG05309 (to R.E.).

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