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Cutting Edge: Telomerase Activation in Human T
Lymphocytes Does Not Require Increase in
Telomerase Reverse Transcriptase (hTERT) Protein
But Is Associated with hTERT Phosphorylation and
Nuclear Translocation
Kebin Liu, Richard J. Hodes and Nan-ping Weng
J Immunol 2001; 166:4826-4830; ;
doi: 10.4049/jimmunol.166.8.4826
http://www.jimmunol.org/content/166/8/4826
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References
●
Cutting Edge: Telomerase Activation in
Human T Lymphocytes Does Not
Require Increase in Telomerase Reverse
Transcriptase (hTERT) Protein But Is
Associated with hTERT Phosphorylation and Nuclear Translocation
Kebin Liu,* Richard J. Hodes,† and Nan-ping Weng1*
T
elomerase, a complex ribonucleoprotein enzyme, functions to synthesize telomere repeats, compensating for the
telomere loss that accompanies cell division and chromosomal replication, and thus prolonging telomere length-restricted
replicative life span of cells (1–3). Telomerase activity is constitutively expressed in germline cells and in the majority of malignant tumor cells and is repressed in most human normal somatic
cells (4, 5). Strikingly, however, telomerase activity is expressed in
*Laboratory of Immunology, National Institute on Aging, National Institutes of
Health, Baltimore, MD 21224; and †Experimental Immunology Branch, National
Cancer Institute, and National Institute on Aging, National Institutes of Health, Bethesda, MD 20892
Received for publication November 9, 2000. Accepted for publication February
26, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint request to Dr. Nan-ping Weng, Laboratory
of Immunology, National Institute on Aging, National Institutes of Health, 5600
Nathan Shock Drive, Box 21, Baltimore, MD 21224. E-mail address: wengn@grc.
nia.nih.gov
Copyright © 2001 by The American Association of Immunologists
●
a highly regulated manner in certain somatic cell populations such
as lymphocytes and hemopoietic stem cells (6 –10). This selective
expression of telomerase provides a molecular basis for the mortality and immortality of cells and is in addition an important consideration for development of telomerase-based therapeutics for
extending the replicative life span of normal cells and for limiting
the growth of malignant tumor cells.
Studies of telomerase regulation in normal somatic cells have
focused on expression of the two essential components of telomerase, telomerase RNA template (hTER)2 (11) and telomerase
reverse transcriptase (hTERT) (12, 13). hTER appears to be ubiquitously present in all cells regardless of telomerase enzymatic
activity (11). In contrast, it has been reported that hTERT mRNA
is detected only in telomerase-positive germline and malignant tumor cells but not in telomerase-negative fibroblasts or other somatic cells (3). These findings have led to the conclusion that
telomerase activity is determined at the level of hTERT transcription in normal somatic cells. Recent studies have suggested that
telomerase activity can also be regulated by alternative splicing of
hTERT transcripts, at times with resulting loss of enzymatic activity, as observed during fetal kidney development (14, 15) and in
some tumor cells (15, 16).
It has been well documented that telomerase activity is expressed in a highly regulated fashion during human lymphocyte
development, differentiation, and activation (7, 17–20). Activation
of peripheral blood T lymphocytes can increase both the levels of
hTERT transcripts and telomerase activity. However, we recently
reported that hTERT transcripts are present at similar levels in
human thymocytes and tonsil and peripheral blood T and B cells
independent of the status of telomerase activity in these cells (21).
These results indicate that transcriptional regulation of hTERT alone
does not determine telomerase activity in human lymphocytes. We
report here that hTERT protein, like its transcript, is present in all
subsets of lymphocytes isolated from thymus and peripheral blood
regardless of the status of telomerase activity. Furthermore, activation
of telomerase in peripheral blood CD4⫹ T lymphocytes after stimulation does not require an increase of hTERT protein. We further
demonstrate that phosphorylation and nuclear translocation of hTERT
are induced by activation of human CD4⫹ T cells. These findings
2
Abbreviations used in this paper: hTER, telomerase RNA template; hTERT, telomerase reverse transcriptase; TRAP, telomeric repeats amplification protocol.
0022-1767/01/$02.00
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Capacity for cellular replication is critically important for
lymphocyte function and can be regulated by telomerase-dependent maintenance of telomere length. In contrast to most
normal human somatic cells that do not express telomerase
due to the failure to transcribe telomerase reverse transcriptase (hTERT), lymphocytes express telomerase in a highly regulated fashion yet constitutively transcribe hTERT during development and activation. Here, we report that hTERT protein
is present in both thymocytes and blood T cells at equivalent
levels despite their substantial differences in telomerase activity, and that induction of telomerase activity in resting CD4ⴙ
T cells is not dependent on net hTERT protein increase. Moreover, hTERT is phosphorylated and translocated from cytoplasm to nucleus during CD4ⴙ T cell activation. Thus, human
T lymphocytes regulate telomerase function through novel
events independent of hTERT protein levels, and hTERT phosphorylation and nuclear translocation may play a role in regulation of telomerase function in lymphocytes. The Journal of
Immunology, 2001, 166: 4826 – 4830.
The Journal of Immunology
suggest that human lymphocytes use novel mechanisms in regulating
telomerase activity.
Materials and Methods
T cell subsets isolation and in vitro stimulation conditions
Peripheral blood samples were obtained with informed consent from normal donors of the National Institutes of Health Blood Bank, and thymi
were obtained during elective pediatric cardiac surgery at Fairfax County
Hospital (Fairfax, VA) following National Institutes of Health guidelines.
The procedures for isolation and stimulation of thymocytes, peripheral
blood CD4⫹ and CD8⫹ T cells, and naive (CD45RA⫹) and memory
(CD45RO⫹) CD4⫹ T cells were previously described (18).
Cell proliferation and flow cytometry
The measurement of lymphocyte proliferation after in vitro stimulation was
previously described (22). The purity of isolated CD4⫹ T cells and naive
and memory CD4⫹ T cells was analyzed by FACScan (Becton Dickinson,
Mountain View, CA) as described (23).
Total cell lysate was prepared by standard protocol. Cytosol and nuclear
extract were prepared as previously described (24). Cellular proteins from
⬃1 ⫻ 107 cells were separated by 6% SDS-PAGE, transferred to Immobilon-P membranes (Millipore, Bedford, MA), probed with anti-hTERT Ab
K-370 (Calbiochem, La Jolla, CA) (25) at 1:2000 dilution, and detected
using the ECL Plus Western detecting kit (Amersham Pharmacia Biotech,
Piscataway, NJ). The membranes were stripped and probed again with
anti-ZAP70 Ab (a gift from Dr. Ronald Wange, National Institute on Aging/National Institutes of Health). As a negative control, human fibroblasts
(26) were used, and anti-␣-tubulin Ab (Sigma-Aldrich, St. Louis, MO) was
used at a 1:2000 dilution as a loading control. Immunoprecipitation of
hTERT was conducted with anti-hTERT K-370. Immunoprecipitates were
separated by 6% SDS-PAGE, transferred to Immobilon-P membranes
(Millipore), and probed with either K-370 or H-231 (Santa Cruz Biotechnology, Santa Cruz, CA) anti-hTERT Ab. The specificity of Ab K-370 was
determined by preincubating Ab with the immunizing peptide (PEPT-1,
FQKNRLFFYRKSVWC) or with a nonspecific peptide of the same length
and amino acid composition (PEPT-2, WFVQNLRYFKFKRSC), and then
assaying remaining Ab activity by immunoblotting.
Telomerase activity assay
Telomerase activity was measured by a modified telomeric repeats amplification protocol (TRAP) assay as described (21, 27).
Phosphorylation analysis of hTERT by radiolabeling
Based on a previously described protocol for determining phosphorylation
by radiolabeling (28), we cultured freshly isolated CD4⫹ T cells (7.5 ⫻
107) with either IL-2 (100 U/ml; Roche Molecular Biochemicals, Burlington, NC) or anti-CD3/CD28 Abs for 1.5 days and pulsed with 5 mCi
[32P]orthophosphate (NEN Life Science, Boston, MA) in a phosphate-free
RPMI 1640 medium for 4 h. Cell lysates were prepared, immunoprecipitated with anti-hTERT Abs and agarose-immobilized protein A, and separated by 12% SDS-PAGE. Autoradiography and Western blot were then
conducted.
Results
hTERT protein expression in human T lymphocyte lineage
In an effort to assess the levels at which telomerase activity is
regulated in human lymphocytes, we measured hTERT protein in
thymocytes and peripheral blood T lymphocytes by Western blot.
First, we characterized the specificity of an anti-hTERT Ab,
K-370, which was generated against a synthetic peptide corresponding to amino acids 568 –581 of hTERT (25). When lysates
prepared from resting CD4 T cells were analyzed by electrophoresis and immunoblotting, the anti-hTERT Ab K-370 recognized a
polypeptide with a molecular mass of ⬃130 kDa, as expected for
the hTERT protein. Moreover, immunoblotting with K-370 was
blocked by preincubation of K-370 with the antigenic hTERT peptide, but not by a control peptide with the same amino acid composition (Fig. 1A). In addition, a second anti-hTERT Ab H231,
which was raised against a recombinant protein corresponding to
amino acids 900-1130 of hTERT, recognized the polypeptide immunoprecipitated with anti-hTERT K-370 (Fig. 2B). These results
confirmed the hTERT specificity of Ab K-370, which was used in
subsequent experiments.
Despite the fact that telomerase enzymatic activity was detected
only in thymocytes and not in peripheral blood T cells, we found
that hTERT protein is present in both cell populations. Moreover,
there was no significant difference in quantity of hTERT protein
between thymocytes and peripheral blood T cells despite the difference in expression of telomerase activity in these populations
(Fig. 2). No hTERT protein was detected in human fibroblasts,
consistent with previous reports (Fig. 2B). Thus, the regulation of
telomerase activity in human T lymphocytes is not controlled at
the level of total cellular hTERT protein.
Telomerase induction does not require increase of hTERT
protein in activated CD4⫹ T cells
Stimulation of peripheral blood T cells through the TCR/CD3
complex alone or in combination with costimulatory receptor results in induction of telomerase activity (7, 17, 18). To determine
whether hTERT protein is regulated in stimulated T cells, we
treated freshly isolated peripheral blood naive and memory CD4⫹
T cells with anti-CD3 alone or anti-CD3 plus anti-CD28 (antiCD3/CD28) mAbs. Anti-CD3 stimulation induced significant telomerase activity in both naive and memory CD4⫹ T cells in the
absence of any detectable increase in hTERT protein and without
detectable cellular proliferation (Fig. 3). In contrast, hTERT protein, telomerase activity, and cellular proliferation were all signif-
Subcellular localization of hTERT by confocol microscopy
Freshly isolated and stimulated CD4⫹ T cells were fixed in 3.7% formaldehyde in PBS at 4°C overnight (or up to 1 wk), then permeablized with
0.1% Triton X-100 in PBS for 3 min. After three washes with PBS, cells
were blocked with PBS containing 1% BSA for 30 min at room temperature followed by incubation with a 1:1000 dilution of anti-hTERT Ab
K-370 for 1 h at room temperature in PBS containing 1% BSA. The stained
cells were washed three times with PBS and incubated with a 1:400 dilution of Alexa Fluor 568 goat anti-rabbit IgG conjugate (Molecular Probes,
Eugene, OR) for 1 h at room temperature. 4⬘,6⬘-diamidino-2-phenylindole
(2.5 ␮g/ml) was then added to the cell-Ab suspension and incubated at
room temperature for another 10 min. The cells were washed three times
with PBS and examined by confocal microscopy (Zeiss, Oberkochen,
Germany).
FIGURE 1. Identification of hTERT by immunoprecipitation and immunoblogtting. A, Western blot analysis of total cell lysates from CD4⫹ T
cells using K-370 Abs alone (control) or in the presence of the antigenic
peptide (PEPT-1) or a nonspecific peptide (PEPT-2). B, Immunoprecipitation of lysates from CD4⫹ T cells with anti-hTERT Ab K-370 specific for
hTERT peptide 568 –581 and immunoblotting with anti-hTERT Ab H231
specific for hTERT peptide 900-1130.
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Western blot and immunoprecipitation analysis
4827
4828
CUTTING EDGE
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FIGURE 2. hTERT protein is present in human thymocytes and peripheral blood T lymphocytes independent of their expression of telomerase
activity. A, Western blot images of hTERT protein levels in thymocyte
subsets and peripheral CD4⫹ and CD8⫹ T cells are shown. B, Western blot
images of hTERT protein level in human peripheral blood CD4⫹ T cells
and fibroblasts. The blot was also probed with anti-tubulin Ab (as a loading
control). C, The relative abundance of hTERT protein levels in thymocytes
and blood T cells is shown. The hTERT level shown in A was quantified
by densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA), and relative hTERT level was normalized on the basis of cellequivalents. D, Telomerase activity was measured from the same cells by
the TRAP assay normalized by cell equivalents. The internal control (IC)
is indicated at the right.
icantly increased in both naive and memory CD4⫹ T cells after
anti-CD3/CD28 stimulation (Fig. 3). Therefore, induction of telomerase activity in CD4⫹ T cells does not require a net increase
in hTERT protein.
hTERT is phosphorylated during CD4⫹ T cell activation
It has been reported that constitutive phosphorylation of hTERT
is found in a tumor cell line (29), but the regulation of hTERT
phosphorylation has not previously been characterized in normal somatic cells. To determine whether phosphorylation of
hTERT is regulated during T cell activation, we cultured freshly
isolated peripheral blood CD4⫹ T cells with anti-CD3 alone,
with anti-CD3/CD28, or with IL-2 for 1.5 days and pulsed with
[32P]orthophosphate for 4 h. Subsequent immunoprecipitation
with anti-hTERT Ab revealed phosphorylation of hTERT in
CD4⫹ T cells cultured with either anti-CD3 alone or anti-CD3/
CD28 but not in cells cultured with IL-2 alone (Fig. 4A), indicating that phosphorylation of hTERT is a regulated event after
T cell activation. hTERT in the tumor cell line 293 was constitutively phosphorylated (Fig. 4A). The intensity of 32P-la-
FIGURE 3. Expression of hTERT protein, telomerase activity, and proliferation in naive (N) and memory (M) CD4⫹ T cells after in vitro stimulation. A, Expression of hTERT protein in naive and memory CD4⫹ T
cells before and after stimulation with anti-CD3 alone or with anti-CD3
plus anti-CD28. B, The relative abundance of hTERT protein levels in
resting and stimulated naive and memory CD4⫹ T cells is shown. The
hTERT level shown in A was quantified by densitometer and ImageQuant
software and was normalized on the basis of cell equivalents. Telomerase
activity (C) and proliferation (D) of the same cells before and after stimulation. Telomerase was measured by the TRAP assay. Cellular proliferation was measured by the incorporation of [3H]thymidine in the presence
or absence of stimulation.
beled hTERT relative to total hTERT detected by immunoprecipitation-Western blot was ⬃3-fold higher in CD4⫹ T cells
stimulated by anti-CD3/CD28 (relative signal intensity ⫽
1.48 ⫾ 0.47) than in cells stimulated by anti-CD3 alone (relative signal intensity ⫽ 0.50 ⫾ 0.16) (Fig. 4B). No detectable
phosphorylation was observed in control cells cultured with
IL-2 (relative signal intensity ⫽ 0.02 ⫾ 0.03).
The Journal of Immunology
4829
Nuclear translocation of hTERT is associated with telomerase
activation in CD4⫹ T cells
The physiological role of telomerase in synthesis of telomeric repeats occurs in the cell nucleus. However, it is unknown whether
the cellular distribution of telomerase components is regulated during cell activation or differentiation in normal somatic cells. To
address the subcellular localization of hTERT protein and to determine whether telomerase activation is accompanied by changes
in hTERT localization, we stained freshly isolated or activated
CD4⫹ T cells with anti-hTERT Ab and analyzed cellular localization of hTERT by immunofluorescence confocal microscopy.
Interestingly, we observed a dramatic change in distribution of
hTERT protein in CD4⫹ T cells after activation (Fig. 5A). In nonactivated freshly isolated CD4⫹ T cells, hTERT protein was found
only in the cytoplasm and not detectably in the nucleus. In contrast, hTERT was present in the nucleus and cytoplasm of activated
T cells. The translocation of hTERT protein to the nucleus was
observed in anti-CD3-stimulated T cells in the absence of a net
increase of total cellular hTERT. Consistent with Western blot
results, total hTERT protein was most abundant in T cells after
stimulation with anti-CD3/CD28.
To further confirm the subcellular localization of hTERT in resting and activated cells, we isolated cytoplasmic and nuclear fractions and analyzed hTERT in these fractions by Western blotting.
Consistent with the confocal microscopic observations, hTERT
was detected only in the cytoplasmic fraction of resting CD4⫹ T
cells and was translocated to the nuclear fraction after stimulation
of cells with either anti-CD3 or anti-CD3/CD28 (Fig. 5B). The
redistribution of hTERT from cytoplasm to nucleus was correlated
with the presence of telomerase activity in both the cytoplasm and
the nucleus (Fig. 5B).
Discussion
Previous reports have described mechanisms of telomerase regulation by control of hTERT transcription (13, 30, 31), alternative
FIGURE 5. Translocation of hTERT protein from cytoplasm to nucleus
after in vitro activation. A, Confocal images of peripheral blood CD4⫹ T
cells freshly isolated, or after 3 days in vitro stimulation with anti-CD3 Ab
or anti-CD3/CD28 Abs. hTERT protein was in the cytosol of freshly isolated resting CD4⫹ T cells and was translocated into the nucleus after
activation. DNA counterstaining (4⬘,6⬘-diamidino-2-phenylindole (DAPI),
blue) of the same cells is shown in the bottom panel. B, Western blot
images (upper panel) and telomerase activity (lower panel) of cytoplasm
(c) and nuclear (n) fractions of resting and activated CD4⫹ T cells, representative of three independent donors.
splicing of hTERT (14, 16), and assembly of telomerase holoenzyme (32). Recently, it has been demonstrated that telomerase activity can be induced in multiple cell types (30, 33), including
human CD8⫹ T cells, through transfection of hTERT (33, 34). The
findings presented here are consistent with previous studies in that
stimulation of CD4 T cells with anti-CD3/CD28 does result in
increased levels of hTERT protein in association with increased
telomerase activity. However, in the present report, we describe
evidence that human T lymphocytes also regulate telomerase activity by novel mechanisms distinct from those previously described. First, we demonstrate that telomerase activity in CD4⫹ T
cells is regulated by mechanisms other than the expression of
hTERT protein. This is supported by the finding that hTERT protein is present in both immature (thymocytes) and mature (peripheral blood) T cells regardless of their telomerase activity status,
and by the demonstration that induction of telomerase activity in
CD4⫹ T cells after in vitro stimulation does not require an increase
of hTERT protein. Second, we observe that T cell activation results in hTERT phosphorylation coincident with telomerase activation. Third, we present evidence that hTERT nuclear translocation is a regulated process in response to CD4⫹ T cell activation.
The mechanism underlying regulation of telomerase activity in
human T lymphocytes during development and activation appears
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FIGURE 4. Phosphorylation of hTERT during telomerase activation in
CD4⫹ T cells. A, hTERT phosphorylated in activated CD4⫹ T cells. The
experiment shown is representative of three independent experiments. Tumor cell line 293 was used as control. Immunoprecipitates of cell lysates
with anti-hTERT Ab (⫹) or normal rabbit serum (–) were separated by
12% SDS-PAGE. Displayed are autoradiograph images (upper panel). The
location of phosphorylated hTERT was confirmed by Western blot (lower
panel). The location of hTERT is indicated at the right. B, Quantitation of
phosphorylated hTERT in culture CD4⫹ T cells. The relative intensity of
phosphorylated hTERT was calculated as the intensity of [32P]hTERT signal normalized to total hTERT as determined by Western blot. The quantitation represents means and SDs of results from three independent
experiments.
4830
Acknowledgments
We thank Drs. Stephen Shaw (National Cancer Institute) and Carl June and
Bruce Levine (University of Pennsylvania) for providing valuable Abs,
Drs. Magda Juhaszova and Steven Sollott (National Institute on Aging) for
help in confocal microscopy, and Fairfax County Hospital and National
Institutes of Health Blood Bank for assistance in obtaining thymus and
blood. We thank Drs. David Schlessinger and Nikki Holbrook for comments on the manuscript.
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different from that previously described in other normal somatic
cells and tumor cells. The reported mechanisms of regulating telomerase in other normal somatic cells are transcriptional repression and alternative splicing of hTERT. In contrast, tumor cells
that constitutively express high levels of telomerase activity express hTERT protein in phosphorylated form (29, 35) that is located predominantly in the nucleus (25, 36, 37). Our results indicate that the presence of hTERT protein in resting T cells is not
sufficient to determine telomerase activity. Indeed, we have identified two regulated events that are associated with telomerase activation in CD4⫹ T lymphocytes independent of total levels of
hTERT protein: phosphorylation and nuclear translocation of
hTERT. Although the precise role of hTERT phosphorylation in
regulation of telomerase activity remains to be elucidated, it is
conceivable that nuclear translocation of telomerase from a presumably nonfunctional cytosolic location to a physiologically relevant nuclear compartment, where its activity can mediate functions such as telomere elongation, is an important regulatory
process for telomerase function in CD4⫹ T cells.
Regulation of telomerase activity in human lymphocytes appears to be a complex process. Determination of how the signals
resulting from engagement of TCR/CD3 and costimulatory receptors on the surface of T cells lead to the phosphorylation and nuclear translocation of hTERT, and ultimately lead to telomerase
activation will require further studies. Lymphocytes, normal somatic cells that express telomerase in a highly regulated manner,
provide a valuable model system in which to study the physiological regulation and functions of telomerase in human cells. Information gained from the study of telomerase regulation and function in lymphocytes will not only enhance our understanding of
lymphocyte replication but is also likely to have broad applications
for somatic cell biology.
CUTTING EDGE