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 CD4+ T cells is not dependent on net hTERT protein increase. Moreover, hTERT is phosphorylated and translocated from cytoplasm to nucleus during CD4+ 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.
Telomerase, 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, 2, 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 a highly regulated manner in certain somatic cell populations such as lymphocytes and hemopoietic stem cells (6, 7, 8, 9, 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, 18, 19, 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 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
Western blot and immunoprecipitation analysis
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-α-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
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.
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 μg/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).
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. 1,A). 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. 2 B). 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. 2 B). 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 (anti-CD3/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 significantly 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. 4,A), indicating that phosphorylation of hTERT is a regulated event after T cell activation. hTERT in the tumor cell line 293 was constitutively phosphorylated (Fig. 4,A). The intensity of 32P-labeled 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. 4 B). No detectable phosphorylation was observed in control cells cultured with IL-2 (relative signal intensity = 0.02 ± 0.03).
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. 5 A). 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. 5,B). The redistribution of hTERT from cytoplasm to nucleus was correlated with the presence of telomerase activity in both the cytoplasm and the nucleus (Fig. 5 B).
Previous reports have described mechanisms of telomerase regulation by control of hTERT transcription (13, 30, 31), alternative 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 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.
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.
Abbreviations used in this paper: hTER, telomerase RNA template; hTERT, telomerase reverse transcriptase; TRAP, telomeric repeats amplification protocol.