proliferation dynamics in cultured skin fibroblasts from down syndrome subjects
TRANSCRIPT
www.elsevier.com/locate/freeradbiomed
Free Radical Biology & M
Original Contribution
Proliferation dynamics in cultured skin fibroblasts
from Down syndrome subjects
Masayuki Kimuraa, Xiaojian Caoa, Joan Skurnickb,
Michael Codyc, Patricia Soteropoulosc, Abraham Aviva,*
aHypertension Research Center, MSB, University of Medicine & Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103, USAbDepartment of Preventive Medicine and Community Health, University of Medicine & Dentistry of New Jersey,
New Jersey Medical School, Newark, NJ 07103, USAcCenter for Applied Genomics, Public Health Research Institute, Newark, NJ 07103, USA
Received 21 October 2004; revised 10 January 2005; accepted 21 March 2005
Available online 8 April 2005
Abstract
With a view to better understanding the role of oxidant/antioxidant variables in proliferation dynamics of somatic cells, we explored the
relationships among superoxide dismutase (SOD) activity, glutathione peroxidase (Gpx) activity, reactive oxygen intermediates (ROI), and
indices of cellular proliferation and senescence in cultured fibroblasts from Down syndrome and normal donors. We found that Down
syndrome cells had a significantly slower proliferative rate, but attain replicative senescence at similar population doubling (PD) as control
cells. Irrespective of donor origin, the number of PD until replicative senescence was positively correlated with Gpx activity (r = 0.784, P =
0.007). In addition, the presence of exogenous catalase in the growth medium significantly extended the number of PD until replicative
senescence (P = 0.011). The loss of telomere repeats per PD was not different between Down syndrome cells and controls. However, SOD
activity was inversely correlated with the loss of telomere repeats per PD. Collectively, these findings suggest that replicative senescence
ultimately relates to mechanisms downstream to SOD (i.e., Gpx and catalase) and confirmed previous observations about inverse
relationships between SOD activity and telomere repeat loss per cellular replication.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Down syndrome; SOD; Glutathione peroxidase; Catalase; Telomere; Reactive oxygen intermediates; Fibroblast
Introduction
An increase in oxidative stress in cultured somatic cells
from humans is associated with an accelerated rate of
telomere attrition per cellular replication and premature
0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2005.03.023
Abbreviations: DTT, dithiothreitol; DHR, dihydrorhodamine 123; EC-
SOD, extracellular superoxide dismutase; Gpx, glutathione peroxidase;
PBS, phosphate-buffered solution; PD, population doubling; ROI, reactive
oxygen intermediate; ROS, reactive oxygen species; SDS, sodium dodecyl
sulfate; SOD, superoxide dismutase; TRF, terminal restriction fragment.
* Corresponding author. Hypertension Research Center, Room F-464,
MSB, University of Medicine and Dentistry of New Jersey, New Jersey
Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA. Fax:
+1 (973) 972 5576.
E-mail address: [email protected] (A. Aviv).
replicative senescence [1,2]. In addition, a higher anti-
oxidant capacity and increased expression of the extra-
cellular superoxide dismutase (EC-SOD), a powerful
antioxidant gene, are associated with a slower rate of
telomere attrition per cellular replication [2]. The question
then is whether SOD activity is a determinant in cellular
proliferation and what is the impact of other antioxidant
enzymes such as glutathione peroxidase (Gpx) and catalase on
the proliferation dynamics of cultured human somatic cells.
A body of research has suggested that some of the
manifestations of premature aging in Down syndrome arise
from an oxidant/antioxidant imbalance due to a higher
expression of SOD-1 (reviewed in [3]), the gene of which
has been mapped to chromosome 21 [4]. Superoxide
radicals are dismutated by SOD-1 to generate hydrogen
edicine 39 (2005) 374 – 380
M. Kimura et al. / Free Radical Biology & Medicine 39 (2005) 374–380 375
peroxide, which is then further metabolized by Gpx and
catalase [5,6]. Without the upregulation of Gpx and catalase
activity, a higher activity of SOD-1 would promote the
interaction between hydrogen peroxide and transition
metals. This reaction, referred to as the Harber-Weiss or
Fenton reaction, generates the hydroxyl radical—an
extremely potent oxidant [7,8]. Some tissues of patients
with Down syndrome exhibit an imbalance between
activities of SOD-1 vs Gpx and catalase [3], which, in
principle, can increase oxidative stress and accelerate not
only tissue breakdown but also telomere attrition. This is
because the GGG triplets on the telomeric repeats are
sensitive to oxidative stress [9]. In fact, Vaziri et al. [10]
have reported that age-dependent telomere attrition in
peripheral lymphocytes is considerably higher in patients
with Down syndrome than in normal subjects. This may
arise from an increase in telomeric repeats lost per cell
replication, increased lymphocyte turnover rate, or both. For
these reasons we focused in this study on the relationships
between oxidant/antioxidant variables, cellular and telomere
dynamics in cultured skin fibroblasts form Down syndrome
and normal subjects.
Materials and methods
Cell origins
Human skin fibroblast cell strains were obtained from the
Coriell Institute for Medical Research (Camden, NJ). Cell
strain characteristics are presented in Table 1. All cell
donors in this study were males. Karyotype analysis,
performed in our laboratory, confirmed the presence of
trisomy 21 in cell strains designated as originating from
Down syndrome donors and a normal karyotype control
strains. Cell strains from normal donors were matched with
those from Down syndrome patients, according to age, race,
Table 1
Cell stain characteristicsa
Pair Coriell
Repository
Numberb
Karyotype Age Race Passagec
frozen
1 GM05658 46XY 1 year African
Americans
2
AG07438A 47XY,+21 9 months African
Americans
2
2 GM05659D 46XY 1 year Caucasian 5
AG05397 47XY,+21 1 year Caucasian 2
3 GM05565 46XY 3 years Caucasian 2
AG06922A 47XY,+21 2 years Caucasian 4
4 GM03523A 46XY 21 years Caucasian 3
AG08942 47XY,+21 21 years Caucasian 6
5 GM00498B 46XY 3 years Caucasian 9
AG04823A 47XY,+21 5 years – 9
a All cell strains were from male subjects.b Repository at Coriell Institute For Medical Research, Camden, NJ.c Cell culture passage at time of freezing.
and the passage number (documented in the data base of the
Coriell Institute for Medical Research). Pairs of Down
syndrome and matched normal cell strains were processed
and studied in parallel.
Cell culture
Cells were cultured in DMEM with and without 5000 U/
mL catalase (Calbiochem, San Diego, CA; Cat. No. 219001)
plus 10% FBS and 1% glutamine pen-strep (Irvine Scientific,
Santa Ana, CA; L-glutamine (292 Ag/mL), penicillin (100 U/
mL), and streptomycin (100 Ag/mL)) in 5% CO2:95% air at
37oC. Growth medium was changed twice a week and cells
were subcultured at confluence every 7 to 10 days. Cells were
cultured until replicative senescence, defined as <10%
increase in cell number in 2 weeks.
Metaphase analysis
Colcemid (Sigma; Cat. No. D-1925) was added during
the exponential growth phase at a final concentration of
10 ng/ml and cells were incubated overnight. Cells were
harvested and dropped on clean wet slides, using minor
modifications of the standard protocol [11]. The slides were
aged at 60-C overnight, and banded by a modification of the
trypsin-Giemsa method [12]. Standard karyotypes were
prepared using the Cytovision Ultra Karyotyping and
Imaging workstation (Applied Imaging, Pittsburgh, PA).
b-Galactosidase staining
h-Galactosidase staining was performed at 2- to 4-week
intervals as previously described [13]. Briefly, cells were
seeded in a 25 mm2 dish. Staining was performed at 50–
75% confluence. Cells were washed twice with phosphate-
buffered solution (PBS) (pH 7.5), fixed for 3–5 min (at
room temperature) in 2% formaldehyde/0.5% glutaralde-
hyde, washed twice with PBS, and incubated with 1 mg/ml
of 5-bromo-4-chloro-3-indolyl h-D-galactopyranoside sus-
pended in 20mg/ml dimethylformamide solution, 40mmol/L
citric acid/sodium phosphate (pH 6.0), 150 mmol/L NaCl,
2 mmol/L MgCl2, 5 mmol/L K3Fe(CN)6, and 5 mmol/L
K4Fe(CN)6 (Sigma) at 37-C for 18 h. Cells were washed
twice with PBS and scored (for a positive blue color) at 100�magnification. At least 200 cells were scored in each flask.
Measurements of superoxide dismutase (SOD) and
glutathione peroxidase activities
Activities of SOD and Gpx were assayed using assay kits
(OXIS Health Products, Inc., Portland, OR). For SOD
activity assay, cell protein was extracted by the freeze and
thaw method and the enzymatic activity was expressed as
units per miligram protein. For Gpx activity assay, cells
were homogenized in cold buffer (50 mmol/L Tris, pH 7.5,
5 mmol/L EDTA, 1 mmol/L DTT) and centrifuged at
M. Kimura et al. / Free Radical Biology & Medicine 39 (2005) 374–380376
10,000 g for 15 min at 4-C and the supernatant was
removed for assay. The Gpx activity was expressed as
milliunits per milligram protein.
Measurement of terminal restriction fragment (TRF) length
Cells were washed twice with PBS and immediately fro-
zen at �80-C. Tris buffer composed of NaCl 100 mmol/L,
Tris 10 mmol/L, EDTA 25 mmol/L, sodium dodecyl sulfate
(SDS) 0.5% (pH 8.0) was added and specimens were
thawed at 50-C. Proteinase K (0.2 mg/ml) was added and
mixture digested at 50-C overnight. DNA was extracted
with phenol/chloroform/isoamyl alcohol and with chloro-
form/isoamyl alcohol, precipitated, and dissolved in
10 mmol/L Tris buffer containing 1 mmol/L EDTA
(pH 8.0). TRF length was measured in DNA samples by
a modification of a previous method [14]. Samples were
digested overnight with restriction enzymes HinfI (10 U)
and Rsa I (10 U) (Roche Applied Science). Eighteen DNA
samples (¨5 Ag each) and 4 DNA ladders (1 kb DNA ladder
plus E DNA/HindIII fragments; Invitrogen Corp., Carlsbad,
CA) were resolved on a 0.5% agarose gel (20 � 20 cm) at
50 V (GNA-200 Pharmacia Biotech, Piscataway, NJ). After
16 h, the DNA was depurinated for 15 min in 0.25 N HCl,
denatured 30 min in 0.5 mol/L NaOH/1.5 mol/L NaCl, and
neutralized for 30 min in 0.5 mol/L Tris (pH 8.0)/1.5 M
NaCl. The DNA was transferred for 1 h to a positively
charged nylon membrane (Roche Applied Science) using a
vacuum blotter (Boekel/Appligene, Feasterville, PA). The
membranes were then hybridized at 65-C with the telomeric
probe (dioxigenin 3V-end-labeled 5V-(CCTAAA)3) overnightin 5� SSC, 0.1% Sarkosyl, 0.02% SDS, and 1% blocking
reagent (Roche Applied Science). The membranes were
washed 3 times at room temperature in 2� SSC, 0.1% SDS
each for 15 min and once in 2� SSC for 15 min. The
digoxigenin-labeled probe was detected by the digoxigenin
luminescent detection procedure (Roche Applied Science)
and exposed on X-ray film.
Measurements of reactive oxygen intermediates (ROI)
Dihydrorhodamine 123 (DHR) (Molecular Probes,
Eugene, OR) was used to measure ROI. At 6 weeks in
culture, cells were seeded at density of 105/25 cm2 and
studied when subconfluent. Cells were loaded with DHR
(30 Amol/L in serum-free culture medium) for 30 min at
37-C. The medium was removed and cells were washed
with PBS, trypsinized, and resuspended in the culture
medium. Cells were analyzed by flow cytometry in a
Facscan flow cytometer equipped with a 488-nm laser, 530/
30 and 585/42-nm band pass filters, and a 650-nm long pass
filter (Becton Dickinson, Biosciences, San Jose, CA). Ten
thousand events were acquired and analyzed using Fsc vs
Ssc gating. F11 histograms were displayed and the means of
the relative fluorescence intensity from logarithmically
amplified data expressed as linear values were recorded.
We note that this analysis detects not only hydrogen
peroxide but also peroxynitrite.
Microarray analysis
Total RNA was isolated using TRizol Reagent (Invitro-
gen) followed with an RNeasy kit (Qiagen). Double-
stranded cDNA was synthesized from 10 Ag of total RNA
using the Superscript double-stranded cDNA synthesis
system (Invitrogen). Following phenol/chloroform extrac-
tion and ethanol precipitation, a biotin-labeled in vitro
transcription reaction was carried out using the cDNA
template (Enzo Life Sciences). Ten micrograms of cRNA
was fragmented and added to a hybridization mixture.
Expression profiles were created using the Human Genome
U133A chip (Affymetrix), which contains 18,400 transcripts
and 14,500 well-characterized human genes. Probe sets for
each transcript consist of 11 pairs of 25-mer oligonucleo-
tides. Hybridization was performed overnight at 45-C for
16 h using the Genechip Hybridization Oven 640 (Affyme-
trix). Washing and staining (streptavidin phycoerythrin) was
done using the Genechip Fluidics Station 400 (Affymetrix)
and the EukGE-WS2v4 protocol. Images were acquired
using the Affymetrix GeneArray scanner. Affymetrix
Microarray Suite 5.0 software was used to analyze the
results to obtain the expression level of each transcript.
Statistical analysis
Population doubling of cells in culture was modeled by
the growth curve,
PD ¼ a0 � f1� exp �a1 � dayð Þg; ð1Þ
where PD is population doubling, a0 is maximal PD, and a1is cell division rate constant. The nonlinear regression
parameters were obtained for each individual using the SAS
PROC NONLIN with Marquardts’s algorithm to obtain
maximum likelihood estimates.
The rate of increase per PD in percentage of senescent
cells, as marked by staining for h-galactosidase, was
modeled by the nonlinear curve,
% cells stained ¼ 100= f1þ expðPD50 � PDÞ=slopeg; ð2Þ
where PD50 is population doubling at which 50% of the
cells are positive for h-galactosidase staining, and slope is
the steepness of the curve. To fit the curve, PD values were
interpolated from the fitted growth curves to estimate PD
values on the days of h-galactosidase staining. PD50 and
slope were estimated for each individual using the same
nonlinear regression procedure as for PD growth curve
analyses. The nonlinear regression parameter estimates
converged in all cases. TRF lengths were regressed linearly
on PDs for each individual.
M. Kimura et al. / Free Radical Biology & Medicine 39 (2005) 374–380 377
Growth and senescence parameters of Down syndrome
cells were compared to those of control cells using paired
t tests of matched-paired values. SOD and Gpx values of
Down syndrome cells and control cells were compared
using independent and paired t tests. Means are presented Tstandard error, and P values are two sided; P < 0.05 is
considered as significant.
Each of the microarray chips was normalized to a mean
value of 500. P values were calculated using a paired, two-
tailed t test. The criteria for selecting genes was a P value <
0.05, present in 5 of the 10 samples and had an absolute
average fold change >2.
Fig. 2. Illustration of h-galactosidase staining curves in cultured fibroblasts:
h-galactosidase staining (in %) as a function of PD in Down syndrome cell
strain AG08942 (closed circles) and normal cell strain GM3523A (open
circles).
Results
Fig. 1 presents the fitted curve of the proliferative growth
for the five pairs of Down syndrome and normal cell strains.
The excellent fit is typical of the curves for all cell strains.
Each growth curve of a cell strain was analyzed separately
and data were fitted to the model described by Eq. (1). The
product of a0 � a1 in Eq. (1) represents the initial rate of
PD. Cell strains from Down syndrome donors exhibited a
slower initial rate of PD than that of normal donors (0.46 T0.06 vs 0.55 T 0.03 PD/day, P = 0.013), but at replicative
senescence, there was no difference between the cumulative
number of PD between cell strains from Down vs normal
donors (38.1 T 1.7 vs 37.4 T 4.8; P = 0.900). We note that
Down syndrome cell strain AG05397 was unusual in that it
exhibited the highest number of PD (55.6) at senescence
(inset to Fig. 1).
Fig. 2 depicts the fitted and observed increase in
percentage of cells positive for h-galactosidase staining as
a function of PD for pair No. 4. The good fit is typical for
Fig. 1. Proliferative growth of cultured fibroblasts: Population doubling
(PD, mean T SE) of five pairs of Down syndrome (closed circles) and
normal cell strains (open circles) as a function of time in culture. Vertical
bars denote SE. Inset represents PD from Down syndrome cell strain
AG05397 (closed circles) and normal cell strain GM05659D (open circles)
as a function of time in culture.
other fitted curves. There was no difference in the PD50 for
h-galactosidase between cell strains from Down syndrome
donors vs strains from control donors (37.2. T 2.0 vs 34.7 T5.1; P = 0.602).
SOD activity was measured at weeks 0, 2, 6, and 10 and
the average was taken. SOD activity was higher in cells
from Down syndrome donors than normal donors (13.5 T1.6 vs 8.37 T 1.2 U/mg; P = 0.028). Gpx activity was
measured at week 2 and was the same among cells from
Down and normal subjects (27.5 T 2.2 vs 27.3 T 2.8 mU/mg
protein; P = 0.960). However, Down syndrome cell strains
AG05397, which showed the highest PD among all cell
strains, also showed the highest Gpx activity (38.6 mU/mg
protein).
SOD-1 expression was higher in Down syndrome than
control cell strains and statistically significant (5866 T 212
vs 4170 T 288; P = 0.002). However, no significant
differences were observed in the gene expressions of
SOD-2, Gpx, and catalase between Down syndrome and
control cell strains. What’s more, there was strong correla-
tion between SOD activity and SOD-1 expression (Fig. 3),
whereas no significant correlation was observed between
SOD activity and SOD-2 expression.
Significantly higher expressions in Down syndrome cell
strains of multiple genes on chromosome 21 were observed
with a fold change of approximately 1.5 (but less than 2), in
line with the increased gene load of this chromosome in
Down syndrome (not shown). The full data set is available
at the GEO website (http://www.ncbi.nlm.nih.gov/geo/).
No significant difference was observed in the rate of
telomere loss per PD between Down syndrome and control
cell strains (Fig. 4). There was also no significant difference
in ROI between Down syndrome and normal cell strains.
However, there was an inverse correlation between the
Fig. 3. Relation between SOD-1 expression and SOD activity: Open circles,
normal cells; closed circles, Down syndrome cells.
Fig. 5. The relationship between telomere loss per population doubling
(PD) and SOD activity: Open circles, normal cells; closed circles, Down
syndrome cells.
M. Kimura et al. / Free Radical Biology & Medicine 39 (2005) 374–380378
initial rate of PD and ROI, irrespective of the cell strain (r =
�0.790, P = 0.007). In addition, a significant correlation
was observed between telomere loss per PD and SOD
activity (r = �0.744, P = 0.013; Fig. 5), but SOD activity
was not correlated with ROI.
Catalase treatment did not change the initial rate of PD in
strains from Down syndrome or normal donors, but it did
result in a higher final PD for cell strains from both Down
syndrome and normal donors. As no differences between
cell strains from Down syndrome and normal donors were
observed for the effect of catalase, data of cell strains from
both groups were pooled for statistical analysis. The final
PD in cells subjected to catalase treatments was 42.2 T
Fig. 4. TRF length attrition in cultured fibroblasts: Mean (m) TRF length
from 6 pairs of Down syndrome (closed circles) and normal cell strains
(open circles) as a function of PD in culture. Vertical and horizontal bars
denote SE.
3.0 vs 39.3 T 2.8 for cells grown in the absence of catalase
(P = 0.011). Catalase treatment increased the PD50 for h-galactosidase, as compared with no catalase in the growth
medium, indicating that catalase delayed replicative sen-
escence (PD50 = 40.0 T 3.2 vs 37.8 T 1.9, P = 0.038).
Gpx activity exhibited a strong correlation with the final
PD (r = 0.784, P = 0.007; Fig. 6). We note that Down
syndrome cell strain AG05397 contributed considerably to
the overall significance of the relation, although without it
the relation was still of borderline significance (P = 0.080).
Finally, karyotypes of the cell strains were checked
throughout the culture period. There were nonspecific and
presenescent findings, but no significant and consistent
changes (loss or gain of chromosomes) were observed.
Fig. 6. The relationship between population doubling (PD) and GPx
activity: Open circles, normal cells; closed circles, Down syndrome cells.
M. Kimura et al. / Free Radical Biology & Medicine 39 (2005) 374–380 379
Discussion
Some of the manifestations of accelerated aging in Down
syndrome have been attributed to an imbalance between the
first (dismutation) and second (conversion of hydrogen
peroxide to water by Gpx and catalase) steps in the pivotal
enzymatic pathway defending against reactive oxygen
species (ROS) [3] Although not found in all tissues and
cells of Down syndrome patients, this imbalance is usually
expressed by a higher SOD activity due to the over-
expression of SOD-1 and the lack of sufficient upregulation
of the activity of Gpx and/or catalase, which would lead to
an increase in cellular levels hydrogen peroxide and
hydroxyl radical.
Cultured fibroblasts from Down syndrome donors
demonstrate an imbalance of SOD/Gpx activity [15,16]
associated with diminished rate of replication [16]. More-
over, SOD-1-transfected fibroblasts demonstrate diminished
proliferation analogous to that of fibroblasts from Down
syndrome donors [16]. The present paper confirmed
observations of a diminished rate of cellular proliferation
(expressed by a lower initial rate of PD) in fibroblast cell
strains from Down syndrome than normal donors. However,
in our study neither the cumulative number of PDs until
replicative senescence nor h-galactosidase staining (an
index of replicative senescence in cultured cells) differed
between Down syndrome and control cell strains. These
findings contrast with observations made more than 30
years ago that cultured fibroblasts from Down syndrome
had lower replicative capacity, expressed by reduced
number of population doubling, than control cells [17].
The presence of catalase in the growth medium was
associated with an increase in the cumulative number of PDs
until senescence and a shift to the right in the PD50 for h-galactosidase staining in both Down syndrome and control
cell strains. In addition, Gpx activity was also positively
correlated with the final PD. These observations suggest that
steps downstream to SOD were determinant in replicative
senescence. However, neither the presence of catalase in the
growth medium nor endogenous Gpx activity modified the
initial rate of PD in either Down syndrome or control cell
strains, suggesting that different mechanisms account for
replication rate and replicative senescence in these cells.
We could not find correlation between SOD activity and
ROI in the cultured fibroblasts, indicating that in itself SOD
activity may not be sufficient to determine the overall
amount of ROI, perhaps because different cell strains
produced different amounts of free radicals. Yet, the inverse
relationships between ROI and the initial rate of PD and
between SOD activity and telomere loss per PD suggest that
the redox state has a major role in cellular replication and
telomere dynamics. In this regard, others [2] also found an
inverse relationship between SOD activity and telomere loss
per PD and related this to a higher expression of EC-SOD.
Based on the findings of: (a) an inverse relationship
between SOD and telomere loss per PD, (b) an inverse
relationship between the initial rate of PD and ROI, and (c) a
lower initial rate of PD in Down syndrome fibroblasts, we
anticipated that Down syndrome fibroblasts would show
significantly lower rate of telomere erosion per PD and a
higher level of ROI than control fibroblasts. However,
neither was observed, suggesting that factors other than the
donor (Down vs control) are involved in the dynamics of
cell replication and telomere erosion in cultured fibroblasts.
We would like to underscore two shortcomings of this
work: First, we measured total rather than SOD-1 activity
and in principle, different relationship might have been
observed for SOD-1 activity with proliferative and telomeric
parameters. We note, however, that overall SOD activity
was highly correlated with SOD-1 expression but not SOD-
2 (Mn SOD) expression. Second, we used DHR as a probe
for ROI for the reason that it primarily measures hydrogen
peroxide and ONOO– concentrations. However, other ROI
may also contribute to differences among cell strains.
Nonetheless, these deficiencies do not detract from the
following observations: despite the initially lower replica-
tive rate, the overall replicative capacity of Down syndrome
fibroblasts was equivalent to that of control cells; telomere
attrition was not higher in Down syndrome fibroblasts than
control fibroblasts; regardless of the donor’s status, the
overall replicative capacity positively correlated with Gpx
activity; catalase treatment enhanced the overall replicative
capacity of skin fibroblasts.
Finally, based on our findings we cannot explain in terms
of elevated SOD-1 activity the in vivo observations of Vaziri
et al. [10] of an accelerated telomere erosion in peripheral
blood lymphocytes of Down syndrome patients. Thus,
increased cellular turnover rate or diminished telomerase
activity in progenitor cells may be other explanations for
higher rates of telomere attrition in lymphocytes of Down
syndrome.
Acknowledgments
This work was supported in part by the Healthcare
Foundation of New Jersey and NIH Grant AG-021593.
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