From the Huffington Center on Aging, Baylor College of Medicine, Houston, Texas 77030
Received for publication, December 19, 2002
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ABSTRACT |
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Expression of the catalytic subunit of
human telomerase (hTERT), in normal human fibroblasts allows them to
escape replicative senescence. However, we have observed that
populations of hTERT-immortalized human fibroblasts contain 3-20%
cells with a senescent morphology. To determine what causes the
appearance of these senescent-like cells, we used flow cytometry to
select them from the population and analyzed them for various
senescence markers, telomere length, and telomerase activity. This
subpopulation of cells had elevated levels of p21 and
hypophosphorylated Rb, but telomere length was similar to that of
the immortal cells in the culture that was sorted. Surprisingly,
telomerase activity in the senescent-like cells was significantly
elevated compared with immortal cells from the same population,
suggesting that high telomerase activity may induce the senescent
phenotype. Furthermore, transfection of normal fibroblasts with a
hTERT-expressing plasmid that confers high telomerase activity led to
the induction of p21, a higher percentage of
SA- Normal human somatic cells undergo a limited number of
divisions before entering an irreversible growth-arrest state defined as senescence (1). Replicative senescence is thought to provide a
barrier against the unlimited proliferation and formation of cancer.
The best described counting mechanism for replicative senescence
involves the telomere-shortening hypothesis. Telomerase is not
expressed in most human tissues; therefore, telomeres shorten with
every cell division. When telomeres reach a critical length, irreversible growth arrest is activated.
It has been demonstrated that ectopic expression of the catalytic
subunit of human telomerase
(hTERT)1 can immortalize
fibroblasts (2, 3), retinal pigment cells (2), and endothelial cells
(4). Whether reconstitution of telomerase activity alone is sufficient
for immortalization of cell types other than fibroblasts has been a
subject of debate. Kiyono et al. (5) have shown that
inactivation of Rb/p16INK4A is required in addition to telomerase for
immortalization of epithelial cells. O'Hare et al. (6) have
found that telomerase was not sufficient to immortalize adult mammary
fibroblasts and endothelial cells. However, subsequently,
Ramirez et al. (7) demonstrated that telomerase activity
alone could immortalize epithelial cells if they were grown on
feeder layers, and they proposed that the results of the earlier
report (5) were because of inadequate growth conditions (7).
Although immortalization of normal human fibroblasts by ectopic
expression of hTERT has been convincingly demonstrated, it is becoming
apparent that immortality is not the universal outcome of hTERT
expression. It has been observed that cultures of hTERT-transduced fibroblasts may undergo a crisis-like stage or acquire a senescent phenotype (8, 9). It was found that senescence was not related to the
loss of telomerase activity, because there was no correlation between
telomere length and immortalization, and among the clones that entered
growth arrest, some had long and others short telomeres. The
mechanism(s) that induces senescence, despite the presence of
telomerase activity, remains unclear. One possibility is that in some
cells senescence is triggered by the activation of a stress response
pathway. Another explanation for the apparent lack of immortalization
in some fibroblasts is that immortalization is achieved only at a
certain level of telomerase activity, which may not be always
reproduced by ectopic expression of hTERT.
We have observed that in three different lines of human fibroblasts,
3-20% of the cells in the population immortalized by expression of
hTERT and grown for >70 PDs had a senescent-like morphology. In this
study, we aimed to examine the mechanism(s), that caused the
senescent-like growth arrest in telomerase-expressing cells. To this
end, we developed a flow cytometry-based method to isolate these cells
from the population of actively growing immortalized cells. We then
compared them to the actively growing cells from the same population.
The isolated cells had features typical of senescent cells.
Unexpectedly, we found that telomerase activity in the senescent-like
cells was significantly elevated compared with immortal cells from the
same population. Furthermore, the overexpression of hTERT from a CMV
promoter from a plasmid induced a senescent-like phenotype in a number
of the newly transfected young cells. These results suggest that
excessive telomerase activity may induce the senescent-like phenotype.
We discuss the potential mechanism of telomerase-induced senescence and
its similarities to premature senescence induced by oncogenes or
proliferative signals.
Cell Lines and Culture Conditions--
IMR-90
and LF1 are normal human lung fibroblasts. WI-38 fibroblasts and IMR-90
cells were from the Coriell Institute for Medical Research, and
LF1 fibroblasts were a kind gift from J. Sedivy (10). HCA2 human
foreskin fibroblasts were isolated in our laboratory. IMR-90-hTERT and
HCA2-hTERT were kindly provided by J. Campisi. LF1-hTERT was kindly
provided by J. Sedivy (11, 12). Cells were grown in Hank's minimal
essential medium or Earle's minimal essential medium supplemented with
10% fetal calf serum, nonessential amino acids, and sodium pyruvate.
Isolation of Senescent Cells by Flow Cytometry--
In a typical
experiment, 107-108 cells were collected by
trypsinization, resuspended in 2 ml of fresh EMEM medium containing 10% fetal calf serum, placed on ice, and immediately used for sorting.
Cells were sorted by a Beckman-Coulter EPICS Altra using Expo 32 MultiComp software (Applied Cytometry Systems).
The sorting parameters for selection of "large-senescent" and
"small-young" cells were determined for each cell line using the
corresponding young and senescent cultures without hTERT as standards
(Fig. 1). The following two areas were selected: area A
contained the majority of young cells (>70%); and area B
covered the large-senescent cells and contained <1% of the dividing
cells. Small and large cells were sorted simultaneously using two-way casting. Following sorting, cells were aliquoted and frozen for subsequent experiments, and a small aliquot (105 cells) was
plated into a 35-mm tissue culture dish for SA- SA- Comparison of Telomere Length by Flow Cytometry--
Cells were
hybridized in situ with a fluorescent telomere-specific
peptide nucleic acid probe as described previously (14-17). Frozen
cells (105) were thawed, washed in phosphate-buffered
saline, and resuspended to 105 cells/100 µl of a
hybridization mixture containing 70% dimethylformamide, 20 mM Tris, pH 7.0, 1% bovine serum albumin, and 0.3 µg/µl telomere-specific (CCCTAA)3 fluorescein
isothiocyanate-conjugated peptide nucleic acid probe (Applied
Biosystems). After 10 min at 82 °C, samples were incubated overnight
at room temperature in the dark. Following hybridization, cells were
spun down, washed twice with phosphate-buffered saline at 40 °C for
10 min, and finally resuspended in phosphate-buffered saline containing
0.1% bovine serum albumin, 10 µg/ml RNase A, and 0.1 µg/ml
propidium iodide. After 4 h at room temperature in the dark, cells
were analyzed by FACS on Beckman-Coulter EPICS XL-MCL using System II
version 3.0 software. Events were gated according to propidium iodide
fluorescence to restrict the analysis to cells with diploid DNA content.
Western Blot Analysis--
Adherent fibroblasts were harvested
and lysed in protein sample buffer and boiled for 10 min, and equal
numbers of cells were loaded on SDS-polyacrylamide gels. The proteins
were transferred to nitrocellulose membrane using a semidry transfer
cell (Bio-Rad). Membranes were hybridized with the following
antibodies: anti-p21(WAF1(Ab1) (Oncogene) and anti-Rb (Rb Ab1(1F8)
(LabVision). Equivalent loading of lanes was verified by hybridization
with anti-actin antibodies (Calbiochem).
Colony Size Distribution--
Cells were transfected with
pcDNA3.1- hTERT or pcDNA3.1 plasmids plated at a density of
200 cells/100-mm dish, and they were incubated undisturbed in the
presence of 1 mg/ml G418 for 2 weeks. Dishes were then fixed and
stained with 1% crystal violet. The number of cells in individual
colonies was determined by microscopy.
Experimental System--
The introduction of telomerase activity
into normal human fibroblasts allows them to escape replicative
senescence (2, 3). However, we have observed that immortal cultures of
human fibroblasts contain some cells with a senescent morphology. In order to examine whether this was associated with other senescent cell
markers and related to changes in telomere length or telomerase activity, we analyzed three lines of human fibroblasts that have been
frequently used to study senescence: fetal lung fibroblasts IMR-90 and
LF1 and foreskin fibroblasts HCA2. The fibroblasts were immortalized by
infection with a retroviral vector containing an hTERT expression
cassette. IMR-90+hTERT and HCA2+hTERT cells were propagated as mass
cultures following infection, whereas LF1+hTERT was a clonal isolate.
Cells were grown for at least 70 PDs after infection to confirm the
immortalized phenotype. Although the cultures were actively
proliferating, we observed some enlarged flat cells with a senescent
morphology in the population. To quantify the percentage of senescent
cells, we used SA- Isolation of Senescent-like Cells from the Populations of
hTERT-expressing Cultures and Analysis of Senescence
Markers--
The biochemical analysis of the senescent cells
that arise within populations of proliferating cells has been limited
by the inability to isolate a sufficient number of living senescent
cells from the growing mass culture. To overcome this limitation, we developed a method to purify senescent cells by flow cytometry using a
FACS cell sorter. Senescent cells are characterized by increased cell
volume; therefore, we have sorted cells according to size (Fig.
1). To allow for subsequent biochemical
analysis and to avoid fluctuations in cell volume that might result
from the use of fixatives, we sorted live unfixed cells. To determine the cut-off parameters for selection of large-senescent and small-young cells for each cell line, we first analyzed the size distribution of
young (PD 18-24) and senescent (PD 55-75) cultures of normal IMR-90,
HCA2, and LF1 fibroblasts that were not expressing hTERT. Two areas
were selected: area A contained the majority of young cells
(>70%); and area B contained the large-senescent cells
with <1% of young cells (Fig. 1). We observed that in normal
senescent cultures, a larger percentage of cells was in area
B when compared with young cultures. In addition, the percent of
SA-
Following sorting, aliquots of cells were plated and stained for
SA-
To test whether isolated cells had other senescent features, we
analyzed the level of p21 and Rb status in the sorted cells. Large-senescent cells had elevated levels of p21 and hypophosphorylated Rb compared with the small-young cells from the same culture (Fig. 3), similar to what has been observed in
normal replicative senescent cells.
Telomere Length Is Unchanged in Senescent-like Cells Isolated from
Immortal Cultures--
To determine whether telomere shortening or
excessive telomere elongation was responsible for the appearance of the
senescent-like cells, we compared telomere length in these cells with
proliferating cells isolated from the same hTERT-immortalized culture.
The initial assessment of telomere length using terminal restriction
fragment polymorphism and Southern blotting did not show any
significant differences in telomere length between old and young cells
(data not shown). However, because it was difficult to obtain a
sufficient number of sorted cells for the accurate comparison of
telomere length by Southern analysis, we used a flow-fluorescent
in situ hybridization technique to compare the
telomere lengths. This technique quantifies by flow cytometry the
average fluorescent signals from individual cells after hybridization
with a fluorescent probe specific for telomere repeats. Following
sorting, cells were fixed, hybridized with the fluorescent probe, and
stained with propidium iodide. Telomere fluorescence was measured only in G1 cells. The telomere length in the large
senescent-like cells was not different from that in proliferating cells
from the same population (Fig. 4),
indicating that telomere shortening is not responsible for the
induction of the senescent-like growth arrest in this case.
Senescent-like Cells Isolated from the Immortal Cultures Have
Elevated Telomerase Activity--
A simple explanation for the
appearance of the subpopulation of senescent-like cells in the
hTERT-immortalized cultures was the loss of telomerase activity due to
silencing of the transgene or by some other mechanism. Our system for
isolation of these cells allowed us to directly test this possibility
as we could compare telomerase activity in the senescent-like and
immortal cells from the same population.
Telomerase activity in the sorted cells was analyzed using the
telomeric repeat amplification protocol (TRAP) assay. Protein extracts
were prepared from the samples of sorted cells, and telomerase activity
was determined in the samples containing equal amounts of protein.
To allow an accurate comparison, the assay was performed on serial
dilutions of cell extract (1,000, 100, and 10 ng of protein,
respectively). The telomerase activity in senescent-like LF1+hTERT, HCA2+hTERT, and IMR-90+hTERT cells was 10-, 5-, and 2.1-fold
higher, respectively, than in the immortal "young" controls from
the same cultures (Fig. 5). Since
senescent cells have 2-3 times larger volume (our observation) and
twice more protein (18) than young cells, the telomerase activity in
the old cells could be underestimated. Because the hTERT cDNA was
expressed from a constitutive LTR promoter, it was unlikely that this
promoter was up-regulated as a result of cells becoming senescent.
However, to rule out this possibility, we introduced a construct with
green fluorescent protein (GFP) under the control of an LTR promoter into mid-passage IMR-90 cells and passaged these cells to senescence. We did not observe any increase in GFP expression or activity in
senescent cells compared with young cells. On the contrary, GFP
activity was down-regulated as cells were passaged to senescence (data
not shown). Therefore, our results suggest that high telomerase activity was what induced loss of proliferation in these cells.
Overexpression of hTERT Induces a Senescent-like Phenotype in
Transfected Cells--
To test directly whether excessive
telomerase activity can induce a senescent-like phenotype, we
transfected young HCA2 fibroblasts with a hTERT expression vector and
examined the newly transfected cells. hTERT cDNA was cloned into
pcDNA3.1( Many years ago, Martinez et al. (19) and Pereira-Smith
and Smith (20) described the fact that in cultures of
tumor-derived and SV40-transformed immortal human cells, there was a
subpopulation of cells with very limited division potential. These
studies involved a clonal analysis of the proliferative capacity of the
cells. The percentage of these cells varied from 1 to 30% depending on the cell line studied, and we have observed this in many other immortalized cell
lines.2 The plausible
explanation at the time was that this was a consequence of the genomic
instability of the cells, causing some cells to drop out of the cell
cycle. However, in this study, we have analyzed the similar phenomenon
in hTERT-immortalized cells that are karyotypically normal.
To examine the factors that trigger senescence in immortalized cell
populations, we have developed a method to purify living senescent
cells from the populations of human fibroblasts immortalized by hTERT.
This allowed us to perform analysis of telomeres and telomerase
activity in these cells. We have found that telomere length in the
senescent cells was not different from the immortal cells of the same
culture. This result corroborates an earlier observation (7) that did
not find a direct correlation between telomere length and
immortalization status. The majority of fibroblast cultures expressing
hTERT have longer than "physiological" telomeres; however, such
long telomeres have not been associated with growth arrest or apoptosis
(8, 21). Surprisingly, we found that telomerase activity was
significantly elevated in senescent cells compared with immortal
proliferating cells from the same culture. Similar results were
obtained with three different cell lines of immortalized human
fibroblasts. hTERT cDNA was expressed from a constitutive LTR
promoter, which is not known to be up-regulated in senescent cells. We
also did not observe any activation of the LTR promoter in senescent
cells in our control experiments with an LTR-GFP construct. When high
levels of telomerase activity were induced in the young cells by
overexpression of hTERT from a plasmid under a CMV promoter, some of
the newly transfected cells acquired senescent-like phenotype and
entered growth arrest. Therefore, these results suggest that excessive
telomerase activity may induce the senescent-like phenotype.
The idea that high telomerase activity may induce senescence seems
reasonable if telomerase is viewed as a proliferative signal. A growing
body of data suggests that hyperproliferative signals may induce a
senescent-like state in normal human cells. Thus, a loss of
proliferative potential is induced by the expression of activated RAS
(22) or RAF (23) or overexpression of E2F1 (24). This type of
premature senescence is thought to be a fail-safe mechanism that
prevents normal cells that experience potentially oncogenic insults
from proliferating.
Telomerase activity is reconstituted by overexpression of hTERT from a
strong promoter, which provides much higher levels of expression than
is normally achieved from the endogenous hTERT promoter. In normal
tissues, telomerase activity is tightly regulated and the hTERT
promoter is turned on in specific cell types only for limited time
periods, for example, during clonal expansion of lymphocytes (25) or in
endometrial cells during the proliferative phase of the menstrual cycle
(26-29). Therefore, it is reasonable to assume that there are some
feedback mechanisms to down-regulate the hTERT promoter when telomerase
activity is no longer needed. When hTERT is expressed ectopically from
a constitutive viral promoter, the cell is unable to down-regulate its
expression and may respond by arresting growth.
The level of hTERT expression is not the only determinant of the level
of telomerase activity, because it is also regulated by phosphorylation
(30-32) and nuclear targeting (32). Furthermore, telomerase is not a
classical oncogene, and hTERT-immortalized cells do not display the
characteristics of cancer cells (33). This may explain why
telomerase-induced senescence is not as robust a process as
oncogene-induced senescence and is only induced in a fraction of cells
in which the telomerase activity becomes too high. In summary, we
present the first evidence that high telomerase activity could induce a
senescent-like phenotype. Understanding the mechanisms of this process
will be of great importance in developing applications of hTERT in cell
therapy and tissue engineering.
-galactosidase-positive cells, and a greater number of
cells entering growth arrest compared with controls. These results
suggest that excessive telomerase activity may act as a
hyperproliferative signal in cells and induce a senescent phenotype in
a manner similar to that seen following overexpression of oncogenic
Ras, Raf, and E2F1. Thus, there must be a critical threshold of
telomerase activity that permits cell proliferation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase staining.
-Galactosidase Staining--
Cells were fixed and stained
for SA-
-galactosidase as described previously (13). The percent of
SA-
-galactosidase-positive cells was calculated by counting at least
500 cells/sample.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase staining and determined that the
immortal cultures contained 3-20% SA-
-galactosidase-positive cells (Table
I).
Percent of SA--galactosidase-positive cells
-galactosidase-positive cells corresponded to the percent of
large cells in the population.
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Fig. 1.
Isolation of senescent cells from
immortalized fibroblast cultures using flow cytometry. The
gating for selection of large-senescent and small-young cells was set
up using the corresponding young and senescent cultures without hTERT.
The forward scatter reading (x axis) reflects the cell size.
Area A (green) was selected to contain the
majority of old cells (>70%), and area B (red)
was selected to cover the large cells from senescent culture but <1%
of the cells from the young culture.
-galactosidase activity (Table I) (Fig.
2). The large cells contained 60-95%
SA-
-galactosidase-positive cells and had a typical senescent
morphology, whereas the small cells were SA-
-galactosidase-negative and had a young morphology indicating that the sorting procedure that
we used was capable of selectively isolating senescent cells.
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Fig. 2.
Morphology and
SA- -galactosidase activity of large-senescent
(area A) and small-young (area
B) cell fractions from cell sorting shown in Fig.
1. Following sorting, an aliquot of cells was plated and
stained for SA-
-galactosidase. All of the panels are shown at
equal magnification (×100). A, LF1+hTERT small.
B, LF1+hTERT large. C, IMR-90+hTERT small.
D, IMR-90+hTERT large. E,
HCA2+hTERT small. F, HCA2+hTERT large.
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Fig. 3.
Status of Rb and p21 in senescent cells
isolated from hTERT-immortalized cell cultures. Following sorting,
aliquots of 105 cells were used for Western blot analysis
with the antibodies against Rb (A) and p21 (B).
The membranes were then hybridized with anti-actin antibodies
(C) to demonstrate equal loading of samples.
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Fig. 4.
Comparison of telomere length in senescent
and immortalized cells by flow-fluorescent in situ
hybridization. Telomere fluorescence histograms are shown.
Flow-fluorescent in situ hybridization provides relative
intensities of a telomere-specific fluorescent probe but does not
measure telomere length directly in kilobases. For accurate comparison,
all of the samples were analyzed simultaneously. Events were gated
according to propidium iodide fluorescence to restrict the analysis to
G1 cells. Typically, at least 5,000 events were
acquired.
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Fig. 5.
Telomerase activity in senescent and
proliferating fibroblasts from immortalized cultures determined by TRAP
assay. A, typical telomerase assay gel is shown. Sorted
cells were lysed, and protein concentrations were measured. Serial
dilutions of the protein extract were prepared, and TRAP assay was
performed with 1,000, 100, and 10 ng of protein. TRAP assay was
performed using TRAPeze kit (Interogene) according to manufacturer's
instructions. Telomerase-positive cells provided with the TRAP assay
kit were used as a positive control, and 500 ng of extract was used.
B, quantitation was done using Molecular Dynamics
densitometer and ImageQuant software. The intensity of the positive
control lane was taken as 100%. The experiment was repeated three
times, and error bars represent mean ± S.D.
) vector (Promega) under the control of CMV promoter to
enable strong expression. The resulting plasmid pcDNA3.1-hTERT or
pcDNA3.1 vector as control was transfected into HCA2 normal human
fibroblasts at PD 21. Transfection was done with either FuGENE 6 reagent or Amaxa electroporator using the maximum allowed DNA
concentration in order to introduce a high number of plasmid copies per
cell. Telomerase activity was analyzed by TRAP assay 3 days after
transfection and compared with telomerase activity in
hTERT-immortalized HCA2 cells. Telomerase activity in the newly
transfected cells was significantly stronger than in the immortalized
cell line (Fig. 6A). We then
allowed the cells to divide for 10 days and compared the number of
senescent cells in the pcDNA3.1-hTERT and control transfections
using SA-
-galactosidase staining. pcDNA3.1-hTERT- transfected
cell population contained 10 times more
SA-
-galactosidase-positive cells than the control cells
transfected with the pcDNA3.1 vector (Fig. 6B).
Western blot analysis also showed the induction of p21 in the
pcDNA3.1-hTERT- transfected cells compared with control cells
(Fig. 6C). We also examined the division potential of
pcDNA3.1-hTERT-transfected cells by colony size distribution.
pcDNA3.1-hTERT-transfected cells formed smaller clones than
the control cells, and the majority of the clones stopped proliferation
after 1-4 divisions (Fig. 6D). These results demonstrate
that excessive telomerase activity can induce a senescent-like
phenotype in normal human fibroblasts.
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Fig. 6.
Effect of hTERT overexpression in HCA2 normal
human fibroblasts. HCA2 cells at PD21 were transfected with the
plasmid that contains hTERT cDNA under the CMV promoter.
A, 3 days following transfection, telomerase activity in 100 ng of protein extract from the cells transfected with the control
plasmid (lane 1), hTERT-expressing plasmid (lane
2), and hTERT-immortalized HCA2 cell line was determined by TRAP
assay. B, 10 days after transfection, cells were stained for
SA- -galactosidase (
-gal) activity. C,
Western blot analysis with the antibodies against p21 was performed 10 days after transfection on the protein extract from the cells
transfected with control plasmid (lane 1) and the cells
transfected with hTERT-expressing plasmid (lane 2).
D, colony size distribution of the control (white
bars) and hTERT-transfected (black bars) cells.
Following transfection, cells were seeded at 200 cells/100-mm dish. Two
weeks later, the colonies were fixed and stained with 1% crystal
violet, and the number of cells per individual clone was determined by
microscopy.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank J. Campisi for providing HCA2-hTERT and IMR-90-hTERT cells and J. Sedivy for LF1 and LF1-hTERT cells.
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FOOTNOTES |
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* This work was supported in part by a Human Frontier of Science postdoctoral fellowship (to V. G.) and NIA, National Institutes of Health Grants R37A60533 and P01A620752 (to O. P. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX
77030. Tel.: 713-798-4533; Fax: 713-796-9438; E-mail:
gorbunov@bcm.tmc.edu.
§ Present address: Dept. of Cellular and Structural Biology Sam and Ann Barshop Center on Longevity and Aging, University of Texas Health Science Center, San Antonio, 15355 Lambda Dr., San Antonio, TX 78245.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M212944200
2 O. M. Pereira-Smith, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: hTERT, human telomerase catalytic subunit; PD, population doubling; CMV, cytomegalovirus; FACS, fluorescence-activated cell sorter; LTR, long terminal repeat; GFP, green fluorescent protein; TRAP, telomeric repeat amplification protocol.
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