Little is known about the long-term consequences of overexpression of the human telomerase reverse transcriptase (hTERT) gene in T lymphocytes. To address this issue, we transduced polyclonal as well as clonally derived populations of naive and memory CD4+ T cells from 2 healthy donors (aged 24 and 34 years) with retroviral vectors encoding green fluorescence protein (GFP) and hTERT (GFP-hTERT) or GFP alone. After transduction, cells were sorted on the basis of GFP expression and cultured in vitro until senescence. T cells transduced with hTERT exhibited high stable telomerase activity throughout the culture period. Relative to GFP controls, minor changes in overall gene expression were observed yet the proliferative lifespan of the hTERT-transduced populations was significantly increased and the rate of telomere loss was lower. Nevertheless, hTERT-transduced cells showed progressive telomere loss and had shorter telomeres at senescence than controls (2.3 ± 0.3 kilobase [kb] versus 3.4 ± 0.1 kb). Furthermore, a population of cells with 4N DNA consisting of binucleated cells with connected nuclei emerged in the hTERT-transduced cells prior to senescence. We conclude that overexpression of hTERT in CD4+ T cells provides a proliferative advantage independent of the average telomere length but does not prevent eventual genetic instability and replicative senescence.

Introduction

Telomeres are specialized structures at the end of eukaryotic chromosomes, which consist of tandem repeats of the sequence T2AG31  and associated proteins2  folded into a telomere loop structure.3  Telomeres are required to maintain chromosomal integrity and prevent end-to-end fusions of chromosomes. When telomeric ends become too short, DNA damage signals from telomeres4  can induce apoptosis or a state of replicative senescence and telomere shortening has been proposed to limit the proliferation of most human cells.5  Telomeres shorten with each round of cell division as a result of failure to completely replicate the 3′ end of chromosomes6,7  as well as other causes.8  The average telomere length in cells from most human tissues decreases with age in vivo and with culture in vitro.9-11  The loss of telomeric DNA with age in T lymphocytes exceeds that in other cell types.12  Limitations in the proliferative potential of the cells of the immune system are consistent with the recent finding that the telomere length in human nucleated blood cells is significantly correlated with mortality caused by infectious diseases.13 

The ribonucleoprotein enzyme telomerase is able to synthesize terminal T2AG3 telomeric repeats de novo and thus extend telomeres (for reviews, see Collins and Mitchell14  and Smogorzewska and de Lange2 ). In contrast to most somatic cells with little or no telomerase activity, the cells of the immune system show transient telomerase activity allowing mitogenic and antigen stimulation.15-17  Nevertheless, T lymphocytes maintained in culture have a finite lifespan and undergo replicative senescence typically after 30 to 50 population doublings (PDs) of in vitro culture (for a review, see Effros and Pawelec18 ). Difficulties in propagating T lymphocytes may limit their use for clinical applications that require large cell numbers such as adoptive transfer therapy of infections and malignancies.

Telomerase activity can be stably reconstituted in cells by ectopic overexpression of the hTERT gene, which encodes the catalytic reverse transcriptase subunit of the telomerase enzyme complex. The ectopic expression of hTERT is sufficient to permit some cell types, such as fibroblasts, retinal pigment epithelial cells, and endothelial cells19,20  to overcome the senescence checkpoint and to proliferate indefinitely without detectable changes characteristic of malignant transformation.21,22 

We have recently shown that inhibition of endogenous telomerase activity (by expressing a dominant-negative hTERT gene) shortens the lifespan of both human CD8+ and CD4+ T cells.23  These findings support the idea that endogenous telomerase activity is essential for cytogenetic stability and telomere function in human T cells. However, expression of endogenous hTERT requires mitogenic stimulation and does not prevent telomere loss in vivo and in vitro.24,25  In contrast, ectopic expression of hTERT is able to reconstitute a constant high level of telomerase activity and extend the lifespan of T cells.23,26-28 

In the present study, we show that ectopic hTERT expression extends the replicative lifespan of CD4+ T cell without preventing overall loss of telomeric DNA. Interestingly, the telomere length in late passage hTERT-transduced cells was shorter than was ever measured in controls. Furthermore, many binucleated cells with chromatin bridges between the nuclei were observed in such late-passage hTERT-transduced CD4+ T cells. We speculate that the observed failure to separate chromosomes and nuclei in such cells reflects loss of telomere function that occurs despite high levels of telomerase. Our results indicate that telomere length in CD4+ T cells is very tightly regulated and that overexpression of hTERT provides only a modest replicative advantage, does not prevent telomere erosion, and does not protect against eventual replicative senescence and chromosomal abnormalities.

Materials and methods

Isolation of naive and memory CD4+ T cells from peripheral blood

Density centrifugation with Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) was used to obtain peripheral blood mononuclear cells (PBMCs) from healthy male and female individuals who gave informed consent. The PBMCs (2-4 × 107 cells) were stained with CD4-phycoerythrin (PE; Becton Dickinson, San Jose, CA) and CD45RA-cyanine 5 (Cy5). Naive CD4+CD45RA+ and memory CD4+CD45RA T lymphocytes were sorted by fluorescence-activated cell sorting (FACStar Plus, Vantage SE; Becton Dickinson). Cultures were initiated with large numbers (> 100) of cells as well as with single cells and expanded as described (see “T-cell cloning and culture”).

T-cell cloning and culture

Single T cells were deposited by single-cell sorting (FACStar Plus, Vantage SE; Becton Dickinson) into 96 U-bottom plates (Falcon, Becton Dickinson). Cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY) containing 10% human serum (HS) supplemented with phytohemagglutinin (PHA; Gibco), 100 U/mL recombinant interleukin 2 (rIL-2, Roche, Nutley, NJ), and 1 × 106/mL irradiated (3000 rad) allogeneic mononuclear feeder cells. After 12 to 14 days of culture, positive wells were transferred into 24-well plates in RPMI 1640 medium containing 10% HS and 100 U/mL rIL-2. After one additional round of stimulation with PHA, irradiated feeder cells (1 × 106/mL), and rIL-2 (100 U/mL), enough cells (1-2 × 107) were obtained for transduction and for telomere length analysis. Population doublings (PDs) were calculated from the average cell count, using the following equation: PDs = 10log (number of cells counted after expansion) – 10log (number of cells seeded)/10log2.

Retrovirus-mediated transduction of the hTERT gene into T cells

Gene transfer was achieved using retrovirus-mediated gene transduction. We constructed murine stem-cell virus (MSCV)–based retroviral vectors29  containing the gene for enhanced green fluorescent protein (GFP; Clontech, Palo Alto, CA) under the control of phosphoglycerate kinase (PGK) promoter with or without an internal ribosomal entry site (IRES) element and the full-length hTERT. Helper-free retrovirus pseudotyped with the Gibbon ape leukemia virus envelope for efficient infection of human cells was generated using PG13 packaging cells.30 

T cells were stimulated with PHA (Gibco) and irradiated allogeneic mononuclear feeder cells prior to transduction. After 2 to 3 days, the cells were harvested and counted. A total of 0.5 to 1 × 106 T cells of either cloned or polyclonal cultures were then incubated on nontissue culture 24-well plates (Falcon) coated with 6 to 10 μg/cm2 RetroNectin (Takara Shuzo, Otsu, Japan) preloaded with 2 mL retrovirus-containing supernatant supplemented with 100 U/mL rIL-2 (Roche). Plates were coated either at 4°C overnight or at 37°C for 2 hours, subsequently blocked with 2% bovine serum albumin (BSA) for 30 minutes at 37°C, and then washed once with phosphate-buffered saline (PBS) prior to use. After 4 hours, cells were harvested from the wells by vigorous pipetting and washed once with PBS. Cells were resuspended in RPMI 1640 medium (Gibco) containing 10% HS supplemented with 100 U/mL rIL-2 (Roche) and kept in 24-well plates (Falcon) overnight. The transduction procedure was repeated on 2 consecutive days.31  After transduction, cells were expanded for 5 to 6 days and then sorted for GFP expression. The efficiency of transduction was estimated by the percentage of cells expressing GFP to be around 1% to 40%.

Telomeric repeat amplification protocol assay

Telomerase activity was measured using the telomeric repeat amplification protocol (TRAP) assay with an end-labeled telomerase substrate (TS) primer as described.32,33  Cell extracts were obtained from a positive control cell line (K562) and from 1 × 105 quiescent T cells present in cultures 10 days after initial stimulation with PHA (Gibco), 100 U/mL rIL-2 (Roche), and 1 × 106/mL irradiated feeder cells. Extension of the TS primer by telomerase was performed for 30 minutes at room temperature, and the products generated were amplified by 30 cycles of polymerase chain reaction (PCR) at 95°C for 60 seconds, 50°C for 45 seconds, and 72°C for 60 seconds using the ACX-anchored return primer. Half of the amplified products were resolved on a 12% polyacrylamide gel and visualized by a phosphoimaging system (Storm 820; Molecular Dynamics, Sunnyvale, CA).

Analysis of GFP expression

Cells (2-4 × 105) were washed once with PBS (StemCell Technologies, Vancouver, BC, Canada) and resuspended in Hanks balanced salt solution (StemCell Technologies) containing 2% fetal calf serum (FCS; StemCell Technologies) and propidium iodide at 1 μg/mL. Samples were analyzed on a FACSSort flow cytometer (Becton Dickinson).

Telomere fluorescence in situ hybridization and flow cytometry

The average length of telomere repeats at chromosome ends in individual cells from T lymphocyte clones was measured by automated fluorescence in situ hybridization (FISH) and flow cytometry (flow-FISH) as described previously.34 

Cell-cycle analysis and cell sorting

Cells (1 × 106) were washed once with PBS (StemCell Technologies) and resuspended in permeabilizing buffer (100 mM Tris [tris(hydroxymethyl)-aminomethane], pH 7.4, 154 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% Nonidet P-40) containing 5 μM propidium iodide. Samples were analyzed and sorted on the basis of DNA content on a FACStar Plus (Becton Dickinson) cell sorter.

To sort viable lymphocytes on the basis of DNA content, cells were stained with Hoechst 33342. Cells (1 × 106) were washed once with PBS (StemCell Technologies) and resuspended in prewarmed medium. Hoechst 33342 was added to a final concentration of 5 μg/mL. Cells were mixed well and placed in a 37°C water bath. After 30 minutes, cells were spun down in the cold and resuspended in cold Hanks balanced salt solution (StemCell Technologies) containing 2% FCS (StemCell Technologies) and sorted on a FACStar Plus (Becton Dickinson).

Cytospin preparation

Sorted cells were resuspended in 100 μL RPMI 1640 medium (Gibco) containing 10% BSA (Calbiochem-Novabiochem, San Diego, CA) and spun at 200g for 1 minute with high acceleration in a cytocentrifuge (Cytospin-2, Thermo Shandon, Pittsburgh, PA). The slides were air-dried, stained with Wright-Giemsa stain (Hematek 2000; Bayer Diagnostics, Tarrytown, NY) or DAPI (4,6 diamidino-2-phenylindole; Vectashield, Vector Laboratories, Burlingame, CA), and analyzed with an Axioplan 2 light and fluorescence microscope (Zeiss Canada, Toronto, ON, Canada) equipped with an Axiocam MRM digital camera (both from Zeiss Canada, Toronto, ON, Canada). Digital images of cells observed with a 63×/1.4 oil objective lens were acquired using Isis software (Metasystems, Altlussheim, Germany).

Figure 1.

Telomerase activity in transduced T lymphocytes. Telomerase activity in cultured clones (cl.) and polyclonal (p.c.) populations of CD4+ T cells transduced with hTERT or a GFP control vector. After transduction, cells were sorted for GFP expression and further expanded. Two thousand transduced cells were analyzed for telomerase activity using the TRAP assay. T lymphocytes transduced with hTERT exhibited high telomerase activity 10 days after stimulation. At this time point no telomerase activity was detected in GFP control cells. Negative control (n.c.) indicates no cell extract; positive control, extracts obtained from K562 cell line. Internal control shows PCR products amplified from an internal control primer to ensure equal loading of the samples.33  Results shown are representative of 3 separate experiments.

Figure 1.

Telomerase activity in transduced T lymphocytes. Telomerase activity in cultured clones (cl.) and polyclonal (p.c.) populations of CD4+ T cells transduced with hTERT or a GFP control vector. After transduction, cells were sorted for GFP expression and further expanded. Two thousand transduced cells were analyzed for telomerase activity using the TRAP assay. T lymphocytes transduced with hTERT exhibited high telomerase activity 10 days after stimulation. At this time point no telomerase activity was detected in GFP control cells. Negative control (n.c.) indicates no cell extract; positive control, extracts obtained from K562 cell line. Internal control shows PCR products amplified from an internal control primer to ensure equal loading of the samples.33  Results shown are representative of 3 separate experiments.

Affymetric gene-expression analysis

RNA from 1 to 2 × 108 expanded T cells was extracted 10 days after initial stimulation, purified, and used for cDNA synthesis using standard procedures. The cDNA was used to synthesize biotinylated cRNA using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY). Labeled cRNA was purified using the RNeasy mini kit (Qiagen, Hilden, Germany). Fragmentation of cRNA, hybridization to HuGeneFL microarrays (Affymetrix, Santa Clara, CA), and washing, staining, and scanning of the arrays in a GeneArray scanner (Agilent, Palo Alto, CA) were performed as recommended in the Affymetrix Gene Expression Analysis Technical Manual. Signal intensities (MAS5 signal) and detection calls for statistical analysis were determined using the Microarray Suite (MAS 5.0) software (Affymetrix). Scaling across all probe sets of a given array to an average intensity of 1000 U was included to compensate for variations in the amount and quality of the cRNA samples and other experimental variables.

Results

hTERT expression increases the lifespan of human CD4+ T cells

Progressive telomere loss limits the expansion of human T lymphocytes in vitro and probably in vivo. Overexpression of hTERT in CD8+ human T lymphocytes results in constitutive telomerase activity and extends their replicative lifespan.23,27,28  To test whether the replicative lifespan of human CD4+ T lymphocytes could also be extended, naive and memory CD4+ T-cell clones as well as naive and memory polyclonal populations of such cells from different donors were purified and transduced with MCSV-based retroviral vectors encoding wild-type hTERT (GFP-hTERT) or GFP as control. After transduction cells were sorted for GFP expression and further expanded by stimulation with PHA and irradiated feeders.

T cells transduced with hTERT showed high and stable telomerase activity 10 days after stimulation (Figure 1). At that time point, no telomerase activity was detectable in GFP control samples. In most of the transduced cells, GFP expression (results not shown) as well as telomerase activity were stably expressed throughout the entire culture period (Figure 2). Populations of CD4+ T cells transduced with the GFP control vector could not be expanded beyond 25 to 60 PDs. In contrast, the proliferative lifespan of hTERT-transduced populations was significantly increased in some cases to over 120 PDs (Figure 3A-B). However, the majority of hTERT-transduced cells eventually died. Similar results were found for naive as well as memory CD4+ T-cell populations (Figure 3C). We also transduced the progeny of a CD4+ T-cell clone with hTERT and GFP to test the significance of transgene expression levels on proliferation. Cells were sorted for high and low GFP expression and subclones were generated. Two subclones of each group were selected and expanded until senescence. No difference in PDs was found for the GFP control-transduced T lymphocytes with high or low GFP expression and the clones reached senescence after 24.9 ± 2.9 PDs and 22.7 ± 2.3 PDs, respectively. Subclones transduced with hTERT and high GFP expression exhibited a significantly higher proliferative potential compared to hTERT-transduced subclones with low GFP expression (49.9 ± 2.9 PDs versus 29.9 ± 1.9 PDs; Figure 4). Interestingly, no differences in telomerase activity between the hTERT-transduced clones were detectable using the TRAP assay. However, analysis of hTERT expression by reverse transcription–PCR (RT-PCR) revealed significant higher expression levels of the transgene in T lymphocytes sorted on the basis of high GFP expression (data not shown). These observations suggest that GFP expression levels correlate with expression levels of the transgene as expected and that high expression levels of exogenous hTERT are required for extending the proliferative capacity of hTERT-transduced CD4+ T lymphocytes.

Figure 2.

Telomerase activity in hTERT-transduced T lymphocytes during long-term culture. Telomerase activity in cultures of CD4+ naive T-cell clones as well as polyclonal populations transduced with hTERT was analyzed using the TRAP assay. After transduction cells were sorted for GFP expression and further expanded. Two thousand cells of each population were analyzed at different time points during long-term expansion. High stable telomerase activity was detected for all the analyzed samples 10 days after stimulation as indicated throughout the whole culture period (day 64, 119, and 212 after transduction). Negative control (n.c.) indicates no cell extract; positive control, extracts obtained from K562 cell line.

Figure 2.

Telomerase activity in hTERT-transduced T lymphocytes during long-term culture. Telomerase activity in cultures of CD4+ naive T-cell clones as well as polyclonal populations transduced with hTERT was analyzed using the TRAP assay. After transduction cells were sorted for GFP expression and further expanded. Two thousand cells of each population were analyzed at different time points during long-term expansion. High stable telomerase activity was detected for all the analyzed samples 10 days after stimulation as indicated throughout the whole culture period (day 64, 119, and 212 after transduction). Negative control (n.c.) indicates no cell extract; positive control, extracts obtained from K562 cell line.

Figure 3.

Overexpression of hTERT increases the lifespan of CD4+ T lymphocytes. Polyclonal populations as well as cells derived from a single sorted naive and memory CD4+ lymphocyte from 2 different donors (aged 34 years [A] and 24 years [B]) were transduced with hTERT (right panels) and a GFP control vector (left panels) on the indicated days and cultured until no further growth was obtained. Population doublings (PDs) were determined by counting viable cells once a week. The proliferative lifespan of the hTERT-transduced populations was increased to over 120 PDs in polyclonal as well as clonally derived naive CD4+ cells (A and B) as well as CD4+ memory T cells (C, results obtained with cells from donor B) transduced with hTERT compared with GFP controls that stopped proliferation after 25 to 60 PDs. Accumulated total PDs are shown in panel C, whereas the number of PDs relative to the days in culture are shown in panels A and B.

Figure 3.

Overexpression of hTERT increases the lifespan of CD4+ T lymphocytes. Polyclonal populations as well as cells derived from a single sorted naive and memory CD4+ lymphocyte from 2 different donors (aged 34 years [A] and 24 years [B]) were transduced with hTERT (right panels) and a GFP control vector (left panels) on the indicated days and cultured until no further growth was obtained. Population doublings (PDs) were determined by counting viable cells once a week. The proliferative lifespan of the hTERT-transduced populations was increased to over 120 PDs in polyclonal as well as clonally derived naive CD4+ cells (A and B) as well as CD4+ memory T cells (C, results obtained with cells from donor B) transduced with hTERT compared with GFP controls that stopped proliferation after 25 to 60 PDs. Accumulated total PDs are shown in panel C, whereas the number of PDs relative to the days in culture are shown in panels A and B.

Figure 4.

PDs and GFP expression of hTERT-transduced T lymphocytes. The progeny of one naive CD4+ T-cell clone was transduced with hTERT and a GFP control vector. After transduction cells were sorted for GFP expression and subclones with high and low GFP expression were obtained. Two subclones of each were selected and further expanded. GFP expression was analyzed and PDs were determined by counting viable cells once a week. The subclone transduced with hTERT showing high GFP expression exhibited a significantly higher proliferative potential compared to the subclone with low GFP expression (49.9 ± 2.3 PDs versus 29.9 ± 1.9 PDs). Dotted and solid vertical lines are shown for comparison; left of dotted line indicates no GFP expression, between dotted and solid lines indicates low levels of GFP expression, and right of solid line indicates high GFP expression. No difference in PDs was found for the GFP control-transduced T-lymphocyte clones with high or low GFP expression (24.9 ± 2.9 PDs versus 22.7 ± 2.3 PDs). Error bars indicate mean ± standard deviation of duplicate measurements.

Figure 4.

PDs and GFP expression of hTERT-transduced T lymphocytes. The progeny of one naive CD4+ T-cell clone was transduced with hTERT and a GFP control vector. After transduction cells were sorted for GFP expression and subclones with high and low GFP expression were obtained. Two subclones of each were selected and further expanded. GFP expression was analyzed and PDs were determined by counting viable cells once a week. The subclone transduced with hTERT showing high GFP expression exhibited a significantly higher proliferative potential compared to the subclone with low GFP expression (49.9 ± 2.3 PDs versus 29.9 ± 1.9 PDs). Dotted and solid vertical lines are shown for comparison; left of dotted line indicates no GFP expression, between dotted and solid lines indicates low levels of GFP expression, and right of solid line indicates high GFP expression. No difference in PDs was found for the GFP control-transduced T-lymphocyte clones with high or low GFP expression (24.9 ± 2.9 PDs versus 22.7 ± 2.3 PDs). Error bars indicate mean ± standard deviation of duplicate measurements.

Figure 5.

Telomere length analysis of transduced CD4+ T lymphocytes. Representative results of polyclonal CD4+ T cells transduced with GFP control (A) and hTERT (B) as well as a clone transduced with hTERT (C) measured by automated flow-FISH. The GFP control-transduced polyclonal population exhibited a decline of telomere length (111 bp/PD) and stopped at 54.0 PDs as indicated at a telomere length of approximately 3.4 ± 0 kb. There was also a decline of telomere length for the hTERT-transduced polyclonal population. The telomere loss was less over time compared to the GFP control (59 bp/PD [B], 58 bp/PD [C]). At later passages, hTERT-transduced polyclonal populations and clones exhibited shorter telomeres than were ever measured in the GFP controls (2.9 ± 0.25 kb [B], 2.3 ± 0.3 kb [C] versus 3.4 ± 0 kb [(A]). Error bars indicate mean ± standard deviation of duplicate measurements.

Figure 5.

Telomere length analysis of transduced CD4+ T lymphocytes. Representative results of polyclonal CD4+ T cells transduced with GFP control (A) and hTERT (B) as well as a clone transduced with hTERT (C) measured by automated flow-FISH. The GFP control-transduced polyclonal population exhibited a decline of telomere length (111 bp/PD) and stopped at 54.0 PDs as indicated at a telomere length of approximately 3.4 ± 0 kb. There was also a decline of telomere length for the hTERT-transduced polyclonal population. The telomere loss was less over time compared to the GFP control (59 bp/PD [B], 58 bp/PD [C]). At later passages, hTERT-transduced polyclonal populations and clones exhibited shorter telomeres than were ever measured in the GFP controls (2.9 ± 0.25 kb [B], 2.3 ± 0.3 kb [C] versus 3.4 ± 0 kb [(A]). Error bars indicate mean ± standard deviation of duplicate measurements.

Lifespan extension without net telomere lengthening in hTERT-transduced T lymphocytes

The average telomere length was measured in serial passages of transduced T-cell clones by automated flow-FISH. As shown in Figure 5, a progressive decline in telomere length was observed in all populations that were analyzed indicative of telomere loss over time. The control population of GFP+ polyclonal CD4+ T cells reached senescence after 54.0 PDs at a telomere length of approximately 3.4 ± 0 kilobase (kb) (Figure 5A) with a calculated telomere attrition rate of 111 base pair (bp)/PD. Despite high and stable telomerase activity, loss of telomere repeats over time was also observed for all hTERT-transduced populations. However, the calculated telomere loss/PD was lower than in the GFP controls (59 bp/PD [Figure 5B] and 58 bp/PD [Figure 5C] versus 111 bp/PD [Figure 5A]) and the lifespan of most of the hTERT-transduced populations was increased. Interestingly, hTERT-transduced populations at later passages exhibited shorter telomeres than were ever measured in the GFP controls (2.9 ± 0.3 kb and 2.3 ± 0.3 kb versus 3.4 ± 0.1 kb in panels B, C, and A, respectively).

Binucleated cells in late passages of hTERT-transduced CD4+ T lymphocytes

Telomere length measurement of hTERT-transduced CD4+ T lymphocytes revealed a progressive decline of telomere length during expansion in long-term culture. Surprisingly, telomere fluorescence measurements of late-passage hTERT-transduced cells revealed a population of cells with twice average the telomere fluorescence once the average telomere length reached approximately 3.3 kb. Moreover, the size of this population increased with further population doublings and continued shortening of the average telomere length (Figure 6A). Cells with double the amount of telomere fluorescence were never observed in control cells transduced with GFP alone. DNA analysis of late-passage hTERT-transduced cells showed a high percentage of cells with 4N DNA, which were sorted and analyzed by microscopy. Many binucleated cells with chromatin bridges between nuclei were observed (Figure 6B). Similar results were obtained with live cells sorted on the basis of staining with Hoechst 33342 (Figure 6C).

Figure 6.

Telomere length analysis by automated flow-FISH of CD4+ T-cell clones transduced with hTERT. Naive CD4+ T-cell clones were transduced with hTERT and a GFP control vector. After transduction cells were sorted for GFP expression and further expanded. Telomere length was measured by automated flow-FISH. (A) Telomere fluorescence histograms at different PDs. Once the average telomere length reached approximately 3.3 kilobase (kb), a population of cells with double the telomere signal was observed. Interestingly, this population also increased in size on further culture. (B) Cell-cycle analysis also revealed an increasing fraction of cells with 4N DNA with further increase of PDs. The cells were sorted and cytospins were prepared. DNA was stained with DAPI and analyzed with a Zeiss Axioplan II fluorescence microscope. Chromatin bridges between nuclei were observed, suggesting that cells failed to complete anaphase. (C) To analyze live cells, samples were also stained with Hoechst 33342. Cells were sorted for their DNA content, cytospins were prepared and stained with Wright-Giemsa stain. Again, binucleated cells with chromatin bridges between nuclei were observed.

Figure 6.

Telomere length analysis by automated flow-FISH of CD4+ T-cell clones transduced with hTERT. Naive CD4+ T-cell clones were transduced with hTERT and a GFP control vector. After transduction cells were sorted for GFP expression and further expanded. Telomere length was measured by automated flow-FISH. (A) Telomere fluorescence histograms at different PDs. Once the average telomere length reached approximately 3.3 kilobase (kb), a population of cells with double the telomere signal was observed. Interestingly, this population also increased in size on further culture. (B) Cell-cycle analysis also revealed an increasing fraction of cells with 4N DNA with further increase of PDs. The cells were sorted and cytospins were prepared. DNA was stained with DAPI and analyzed with a Zeiss Axioplan II fluorescence microscope. Chromatin bridges between nuclei were observed, suggesting that cells failed to complete anaphase. (C) To analyze live cells, samples were also stained with Hoechst 33342. Cells were sorted for their DNA content, cytospins were prepared and stained with Wright-Giemsa stain. Again, binucleated cells with chromatin bridges between nuclei were observed.

Gene expression profiles in early- and late-passage transduced CD4+ T cells

Ideally, the consequences of hTERT overexpression should be studied using relevant functional T-cell assays. We did observe that both GFP- and hTERT-GFP–transduced CD4+ T cells maintained a strict requirement for IL-2 and irradiated allogeneic cells to stimulate their proliferation. To explore possible differences in gene expression resulting from the hTERT overexpression, we compared the gene expression in the clonal progeny of a CD4+ T cell before and after transduction with GFP and hTERT-GFP vectors. The overall gene expression pattern was strikingly similar between the 4 cell populations tested (National Center for Biotechnology Information GSE2230) and of 20 000 transcripts tested fewer than 200 were significantly increased or decreased between the different cell types. The most striking differences in gene expression between cells that overexpress hTERT and untransduced or GFP-transduced cells are shown in Tables 1 and 2. However, no major differences between hTERT or control cells or between early or late-passage hTERT-transduced cells were observed in the expression of some of the genes known to be involved in DNA repair or cell-cycle control (Table 3). This observation does not exclude differences in the activity of the proteins encoded by these genes because in many cases this activity is known to be controlled at the protein level.

Table 1.

Genes significantly up-regulated in cells that overexpress hTERT


Accession no.

Gene description

Symbol

Affymetrix ID

GFP PD32

GFP PD47

hTERT PD44

hTERT PD80
NM_024628  Solute carrier family 12 (potassium/chloride transporters)  SLC12A8  219874_at   240   190*  752   708  
NM_016323  Hect domain and RLD 5  HERC5  219863_at   1641   1644   3193   2931  
NM_017912  Hect domain and RLD 6  HERC6  219352_at   435   477*  700   727  
NM_006417  Interferon-induced protein 44  IFI44  214453_s_at   1988   1792   3534   3721  
AI337069  Viperin  cig5  213797_at   666   1522   2817   4442  
AA121502  HEG homolog  HEG  212822_at   1558   1712   2267   2483  
AF089087  G protein-coupled receptor 35  GPR35  210264_at   576   931   473   1160  
M68874  Phospholipase A2, group IVA (cytosolic, calcium-dependent)  PLA2G4A  210145_at   120   187   627   344  
NM_003733  2′-5′-oligoadenylate synthetase-like  OASL  205660_at   1155   1001   2094   2265  
NM_001548  Interferon-induced protein with tetratricopeptide repeats 1  IFIT1  203153_at   555   385*  1313   1533  
NM_001549  Interferon-induced protein with tetratricopeptide repeats 3  IFIT3  204747_at   772   393   1107   1180  
NM_006820  Chromosome 1 open reading frame 29  C1orf29  204439_at   377   416   2247   3029  
AI824012  Nuclear receptor interacting protein 1  NRIP1  202600_s_at   620   519   1224   1061  
NM_003489  Nuclear receptor interacting protein 1  NRIP1  202599_s_at   1024   910   2052   1517  
NM_002462
 
Myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse)
 
MX1
 
202086_at
 
2905
 
2988
 
5537
 
6459
 

Accession no.

Gene description

Symbol

Affymetrix ID

GFP PD32

GFP PD47

hTERT PD44

hTERT PD80
NM_024628  Solute carrier family 12 (potassium/chloride transporters)  SLC12A8  219874_at   240   190*  752   708  
NM_016323  Hect domain and RLD 5  HERC5  219863_at   1641   1644   3193   2931  
NM_017912  Hect domain and RLD 6  HERC6  219352_at   435   477*  700   727  
NM_006417  Interferon-induced protein 44  IFI44  214453_s_at   1988   1792   3534   3721  
AI337069  Viperin  cig5  213797_at   666   1522   2817   4442  
AA121502  HEG homolog  HEG  212822_at   1558   1712   2267   2483  
AF089087  G protein-coupled receptor 35  GPR35  210264_at   576   931   473   1160  
M68874  Phospholipase A2, group IVA (cytosolic, calcium-dependent)  PLA2G4A  210145_at   120   187   627   344  
NM_003733  2′-5′-oligoadenylate synthetase-like  OASL  205660_at   1155   1001   2094   2265  
NM_001548  Interferon-induced protein with tetratricopeptide repeats 1  IFIT1  203153_at   555   385*  1313   1533  
NM_001549  Interferon-induced protein with tetratricopeptide repeats 3  IFIT3  204747_at   772   393   1107   1180  
NM_006820  Chromosome 1 open reading frame 29  C1orf29  204439_at   377   416   2247   3029  
AI824012  Nuclear receptor interacting protein 1  NRIP1  202600_s_at   620   519   1224   1061  
NM_003489  Nuclear receptor interacting protein 1  NRIP1  202599_s_at   1024   910   2052   1517  
NM_002462
 
Myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse)
 
MX1
 
202086_at
 
2905
 
2988
 
5537
 
6459
 
*

Call: absent.

Table 2.

Genes significantly down-regulated in cells that overexpress hTERT


Accession no.

Gene description

Symbol

Affymetrix ID

PD32

GFP PD47

hTERT PD44

hTERT PD80
BE968833  Spectrin, β, nonerythrocytic 1  SPTBN1  212071_s_at   6458   5094   2293   2214  
U73531  Chemokine (C-X-C motif) receptor 6  CXCR6  211469_s_at   10091   10122   5645   3568  
NM_006564  Chemokine (C-X-C motif) receptor 6  CXCR6  206974_at   8031   7840   4560   3449  
BC005254  C-type (calcium-dependent, carbohydrate-recognition domain) lectin, superfamily member 2 (activation induced)  CLECSF2  209732_at   5726   4946   2745   2780  
AI421071  Chemokine (C-C motif) receptor 1  CCR1  205098_at   4874   5902   3447   3141  
NM_000648  Chemokine (C-C motif) receptor 2  CCR2  207794_at   2226   1845   765   189* 
NM_000647  Chemokine (C-C motif) receptor 2  CCR2  206978_at   3635   2912   1237   418  
NM_000579  Chemokine (C-C motif) receptor 5  CCR5  206991_s_at   7944   7281   2879   2827  
NM_002260  Killer cell lectin-like receptor subfamily C, member 1/killer cell lectin-like receptor subfamily C, member 2  KLRC1/KLRC2  206785_s_at   1754   1682   447   221* 
NM_002104  Granzyme K (serine protease, granzyme 3; tryptase II)  GZMK  206666_at   8435   1330   431   393* 
BG546884
 
Cysteine-rich motor neuron 1
 
CRIM1
 
202551_s_at
 
469
 
527
 
323
 
245
 

Accession no.

Gene description

Symbol

Affymetrix ID

PD32

GFP PD47

hTERT PD44

hTERT PD80
BE968833  Spectrin, β, nonerythrocytic 1  SPTBN1  212071_s_at   6458   5094   2293   2214  
U73531  Chemokine (C-X-C motif) receptor 6  CXCR6  211469_s_at   10091   10122   5645   3568  
NM_006564  Chemokine (C-X-C motif) receptor 6  CXCR6  206974_at   8031   7840   4560   3449  
BC005254  C-type (calcium-dependent, carbohydrate-recognition domain) lectin, superfamily member 2 (activation induced)  CLECSF2  209732_at   5726   4946   2745   2780  
AI421071  Chemokine (C-C motif) receptor 1  CCR1  205098_at   4874   5902   3447   3141  
NM_000648  Chemokine (C-C motif) receptor 2  CCR2  207794_at   2226   1845   765   189* 
NM_000647  Chemokine (C-C motif) receptor 2  CCR2  206978_at   3635   2912   1237   418  
NM_000579  Chemokine (C-C motif) receptor 5  CCR5  206991_s_at   7944   7281   2879   2827  
NM_002260  Killer cell lectin-like receptor subfamily C, member 1/killer cell lectin-like receptor subfamily C, member 2  KLRC1/KLRC2  206785_s_at   1754   1682   447   221* 
NM_002104  Granzyme K (serine protease, granzyme 3; tryptase II)  GZMK  206666_at   8435   1330   431   393* 
BG546884
 
Cysteine-rich motor neuron 1
 
CRIM1
 
202551_s_at
 
469
 
527
 
323
 
245
 
*

Call: absent.

Table 3.

Expression of selected genes involved in DNA repair and cell-cycle control in hTERT-transduced T cells


Accession no.

Gene description

Symbol

Affymetrix ID

GFP PD32

GFP PD47

hTERT PD44

hTERT PD80
NM_000546  Tumor protein p53 (Li-Fraumeni syndrome)  TP53  201746_at   1784   1501   1752   1709  
U38945   Cyclin-dependent kinase inhibitor 2A  CDKN2A  209644_x_at   630   913   1135   1239  
NM_007295  Breast cancer 1, early onset  BRCA1  204531_s_at   503   415   452   445  
U49844   Ataxia telangiectasia and Rad3 related  ATR  209902_at   421   248   437   373  
NM_004629  Fanconi anemia, complementation group G  FANCG  203564_at   1411   1223   817   901  
AI796269   Nijmegen breakage syndrome 1 (nibrin)  NBS1  202905_x_at   1202   1185   1427   1251  
NM_002853  RAD1 homolog (Schizosaccharomyces pombe) RAD1  204461_x_at   803   731   776   915  
NM_002894  Retinoblastoma-binding protein 8  RBBP8  203344_s_at   2275   2428   1971   3392  
D80000   SMC1 structural maintenance of chromosomes 1-like 1 (yeast)  SMC1L1  201589_at   5093   4578   4021   5308  
NM_001274  CHK1 checkpoint homolog (S pombe) CHEK1  205394_at   1202   1087   810   1231  
NM_001924  Growth arrest and DNA damage-inducible, α  GADD45A  203725_at   5790   4147   6245   3140  
BC006325   G2 and S-phase expressed 1/G2 and S-phase expressed 1  GTSE1  211040_x_at   1989   1788   2203   2035  
AI968626   HUS1 checkpoint homolog (S pombe) HUS1  204883_s_at   851   495   488   613* 
BC004153   Poly(rC)-binding protein 4  PCBP4  209361_s_at   283   356*  645   607* 
U83981   Protein phosphatase 1, regulatory (inhibitor) subunit 15A  PPP1R15A  37028_at   600   790   1183   1285  
AF085736   RAD17 homolog (S pombe) RAD17  211228_s_at   1613   1024   1333   1196  
NM_004584  RAD9 homolog A (S pombe) RAD9A  204828_at   819   777   842   842  
NM_014454  Sestrin 1  SESN1  218346_s_at   1047   1239   1055   742  
U82828   Ataxia telangiectasia mutated  ATM  212672_at   872   938   1017   1019  
NM_002592  Proliferating cell nuclear antigen  PCNA  201202_at   12590   13100   10539   11530  
M19701   Retinoblastoma 1 (including osteosarcoma)  RB1  211540_s_at   770   625   651*  756  
U19599   BCL2-associated X protein  BAX  211833_s_at   2118   2326   2332   1852  
NM_013229  Apoptotic protease-activating factor  APAF1  204859_s_at   783   674   563   743  
NM_000389  Cyclin-dependent kinase inhibitor 1A (p21, Cip1)  CDKN1A  202284_s_at   3126   4148   4935   3732  
NM_002467  v-myc myelocytomatosis viral oncogene homolog (avian)  MYC  202431_s_at   1153   959   1265   682  
NM_000633  B-cell CLL/lymphoma 2  BCL2  203685_at   4220   4929   4231   3746  
AL117381   BCL2-like 1  BCL2L1  212312_at   1015   1507   1498   1067  
NM_005157  v-abl Abelson murine leukemia viral oncogene homolog 1  ABL1  202123_s_at   828   612   786   565  
NM_006265  RAD21 homolog (S pombe) RAD21  200608_s_at   8995   9550   7463   9790  
NM_000249  mutL homolog 1, colon cancer, nonpolyposis type 2 (Escherichia coli)  MLH1  202520_s_at   3214   2736   2182   2810  
NM_014430  Cell death-inducing DFFA-like effector b  CIDEB  221188_s_at   1087   858   1249   541* 
NM_004208  Programmed cell death 8 (apoptosis-inducing factor)  PDCD8  205512_s_at   965   1003   1209   1007  
BF062629
 
Ras-induced senescence 1
 
RIS1
 
213338_at
 
440
 
572
 
1267
 
508
 

Accession no.

Gene description

Symbol

Affymetrix ID

GFP PD32

GFP PD47

hTERT PD44

hTERT PD80
NM_000546  Tumor protein p53 (Li-Fraumeni syndrome)  TP53  201746_at   1784   1501   1752   1709  
U38945   Cyclin-dependent kinase inhibitor 2A  CDKN2A  209644_x_at   630   913   1135   1239  
NM_007295  Breast cancer 1, early onset  BRCA1  204531_s_at   503   415   452   445  
U49844   Ataxia telangiectasia and Rad3 related  ATR  209902_at   421   248   437   373  
NM_004629  Fanconi anemia, complementation group G  FANCG  203564_at   1411   1223   817   901  
AI796269   Nijmegen breakage syndrome 1 (nibrin)  NBS1  202905_x_at   1202   1185   1427   1251  
NM_002853  RAD1 homolog (Schizosaccharomyces pombe) RAD1  204461_x_at   803   731   776   915  
NM_002894  Retinoblastoma-binding protein 8  RBBP8  203344_s_at   2275   2428   1971   3392  
D80000   SMC1 structural maintenance of chromosomes 1-like 1 (yeast)  SMC1L1  201589_at   5093   4578   4021   5308  
NM_001274  CHK1 checkpoint homolog (S pombe) CHEK1  205394_at   1202   1087   810   1231  
NM_001924  Growth arrest and DNA damage-inducible, α  GADD45A  203725_at   5790   4147   6245   3140  
BC006325   G2 and S-phase expressed 1/G2 and S-phase expressed 1  GTSE1  211040_x_at   1989   1788   2203   2035  
AI968626   HUS1 checkpoint homolog (S pombe) HUS1  204883_s_at   851   495   488   613* 
BC004153   Poly(rC)-binding protein 4  PCBP4  209361_s_at   283   356*  645   607* 
U83981   Protein phosphatase 1, regulatory (inhibitor) subunit 15A  PPP1R15A  37028_at   600   790   1183   1285  
AF085736   RAD17 homolog (S pombe) RAD17  211228_s_at   1613   1024   1333   1196  
NM_004584  RAD9 homolog A (S pombe) RAD9A  204828_at   819   777   842   842  
NM_014454  Sestrin 1  SESN1  218346_s_at   1047   1239   1055   742  
U82828   Ataxia telangiectasia mutated  ATM  212672_at   872   938   1017   1019  
NM_002592  Proliferating cell nuclear antigen  PCNA  201202_at   12590   13100   10539   11530  
M19701   Retinoblastoma 1 (including osteosarcoma)  RB1  211540_s_at   770   625   651*  756  
U19599   BCL2-associated X protein  BAX  211833_s_at   2118   2326   2332   1852  
NM_013229  Apoptotic protease-activating factor  APAF1  204859_s_at   783   674   563   743  
NM_000389  Cyclin-dependent kinase inhibitor 1A (p21, Cip1)  CDKN1A  202284_s_at   3126   4148   4935   3732  
NM_002467  v-myc myelocytomatosis viral oncogene homolog (avian)  MYC  202431_s_at   1153   959   1265   682  
NM_000633  B-cell CLL/lymphoma 2  BCL2  203685_at   4220   4929   4231   3746  
AL117381   BCL2-like 1  BCL2L1  212312_at   1015   1507   1498   1067  
NM_005157  v-abl Abelson murine leukemia viral oncogene homolog 1  ABL1  202123_s_at   828   612   786   565  
NM_006265  RAD21 homolog (S pombe) RAD21  200608_s_at   8995   9550   7463   9790  
NM_000249  mutL homolog 1, colon cancer, nonpolyposis type 2 (Escherichia coli)  MLH1  202520_s_at   3214   2736   2182   2810  
NM_014430  Cell death-inducing DFFA-like effector b  CIDEB  221188_s_at   1087   858   1249   541* 
NM_004208  Programmed cell death 8 (apoptosis-inducing factor)  PDCD8  205512_s_at   965   1003   1209   1007  
BF062629
 
Ras-induced senescence 1
 
RIS1
 
213338_at
 
440
 
572
 
1267
 
508
 
*

Call: absent.

Discussion

Our studies show that ectopic expression of hTERT in human CD4+ T cells can be used to increase the replicative lifespan of such cells and obtain large numbers in culture. Nevertheless, none of the hTERT-transduced CD4+ T-cell populations could be expanded for more than 200 PDs in contrast to human fibroblasts transduced with the same vector (results not shown). Furthermore, despite high levels of telomerase activity throughout the culture period, a progressive loss of telomeric DNA was observed and the telomere length in late passages of such cells was extremely short. However, compared to GFP controls, the telomere loss in hTERT-transduced CD4+ T cells was less per PD. These observations suggest that hTERT overexpression in human CD4+ T lymphocytes increases the replicative lifespan of such cells but does not prevent overall loss of telomere length. Of note, the consequences of telomerase overexpression appear to differ between CD4+ T cells (this study) and CD8+ T cells.23,27,28  Whereas hTERT overexpression in CD8+ T cells frequently resulted in net telomere elongation and immortalization, we observed only an increased replicative potential and no net telomere elongation in CD4+ T cells. These observations suggest that hTERT overexpression has different consequences in CD4+ and CD8+ T cells, which warrant further studies in direct comparisons.

The finding that ectopic expression of telomerase can extend the replicative lifespan of certain cell types without causing a net elongation of telomere length is not without precedent.19,20,26,35  Furthermore, the telomere length in some human fibroblasts transduced with hTERT were also found to continue to shorten to an average length below those of untransduced cells that entered replicative senescence and crisis.36,37  Our observations support the notion that hTERT and telomerase have other roles in the viability and growth of cells beyond preventing a decline in the average telomere length.38-42  One possibility is that telomerase is required not only for elongation of the 3′ end of chromosomes but has a role in the repair of damage to telomeric DNA as well.43 

Overexpression of hTERT in CD4+ T cells did not result in marked global changes in gene expression and the expression of genes involved in cell-cycle regulation or repair of DNA damage was not notably changed (Table 3). Among the genes that did show an overall change in expression pattern, the HERC5 gene (up-regulated in hTERT-transduced T cells; Table 1) appears of interest. HERC5 plays a role in regulation of cyclin-dependent protein kinase activity and the ubiquitin cycle (like HERC6) and is associated with cell-cycle regulation (G1- to S-phase transition). HERC genes contain a domain that is involved in chromosome condensation (RCC1).44  We further found down-regulation of several chemokine receptors. This might indicate that hTERT-transduced lymphocytes are less dependent on chemokine stimulation. Especially CCR5 was down-regulated. Activation of CCR5 can lead to both the de novo expression of FAS ligand (FASL) and induction of susceptibility to Fas-mediated apoptosis in CD4+ T cells.45 

The average telomere length in a cell will reflect the balance between factors that elongate and those that shorten telomeric DNA at individual chromosome ends in the cells of interest and its immediate progenitors. The level of telomerase activity, subcellular localization of telomerase, recruitment of telomerase to the telomere, the structure of terminal telomeric DNA, levels of telomere-binding proteins, and levels of telomerase RNA all have been proposed to influence the elongation of the telomeres by telomerase.2,14  Likewise, the loss of telomeric DNA is expected to have many causes.8  In antigen-specific CD4+ T cells the loss of telomeres and telomerase activity could be subject to further control by the action of cytokines.46  The data described in this report as well as these previous studies support the notion that telomerase plays a key role in the biology of T cells. Given the many levels at which the telomere length in T cells appears to be controlled, a clear picture of the precise role of exogenous telomerase in the biology of CD4+ T cells is not expected in the near future.

Telomerase activity is believed to promote chromosomal stability primarily by acting at the very ends of chromosomes. Our results suggest that overexpression of hTERT in cells with critically short telomeres does not result in cell-cycle arrest or apoptosis43,47  and allows further proliferation despite chromosomal instability eventually leading to telomere dysfunction and anaphase arrest.48-50  The observed telomeric dysfunction may trigger chromosomal fragmentation through bridge-breakage events and could lead to a transformed phenotype.51  The risk of such transformation events is additive to the risk of insertional mutagenesis inherent in gene transfer strategies.52  These risks of ectopic hTERT expression in T cells have to be balanced against the advantage of increased expansion of T-cell clones that specifically react with tumor or viral antigens. Such cells could be used for adoptive transfer therapy in cancer patients to increase the number of effector cells that may mediate regression of tumors or influence severe viral infections.53-58 

Prepublished online as Blood First Edition Paper, March 1, 2005; DOI 10.1182/blood-2004-10-4144.

Supported by grants from the National Institutes of Health (AI29524) and the National Cancer Institute of Canada with funds from the Terry Fox Run. A.R. was funded by a grant from the Deutsche Forschungsgemeinschaft. G.M.B. was funded by a grant from the Swiss National Science Foundation (31-53774-98) and the Bernese Cancer League (AI29524).

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

We thank Dr Keith Humphries (Terry Fox Laboratory, Vancouver, BC, Canda) for help with the construction of retroviral vectors, Dr Robert Weinberg (Massachusetts Institute of Technology, Boston, MA) for hTERT cDNA, and Dr Ludger Klein-Hitpass (Institute for Cell Biology, Essen, Germany) for help with gene expression analysis.

1
Moyzis RK, Buckingham JM, Cram LS, et al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes.
Proc Natl Acad Sci U S A
.
1988
;
85
:
6622
-6626.
2
Smogorzewska A, de Lange T. Regulation of telomerase by telomeric proteins.
Annu Rev Biochem
.
2004
;
73
:
177
-208.
3
Griffith JD, Comeau L, Rosenfield S, et al. Mammalian telomeres end in a large duplex loop.
Cell
.
1999
;
97
:
503
-514.
4
d'Adda dF, Reaper PM, Clay-Farrace L, et al. A DNA damage checkpoint response in telomere-initiated senescence.
Nature
.
2003
;
426
:
194
-198.
5
Campisi J. Cancer and ageing: rival demons?
Nat Rev Cancer
.
2003
;
3
:
339
-349.
6
Olovnikov AM. A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon.
J Theor Biol
.
1973
;
41
:
181
-190.
7
Watson JD. Origin of concatameric T4 DNA.
Nat New Biol
.
1972
;
239
:
197
-201.
8
Verfaillie CM, Pera MF, Lansdorp PM. Stem cells: hype and reality.
Hematology (Am Soc Hematol Educ Program)
.
2002
;
369
-391.
9
Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts.
Nature
.
1990
;
345
:
458
-460.
10
Martens UM, Chavez EA, Poon SSS, Schmoor C, Lansdorp PM. Accumulation of short telomeres in human fibroblasts prior to replicative senescence.
Exp Cell Res
.
2000
;
256
:
291
-299.
11
Chang E, Harley CB. Telomere length and replicative aging in human vascular tissues.
Proc Natl Acad Sci U S A
.
1995
;
92
:
11190
-11194.
12
Rufer N, Brummendorf TH, Kolvraa S, et al. Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood.
J Exp Med
.
1999
;
190
:
157
-167.
13
Cawthon RM, Smith KR, O'Brien E, Sivatchenko A, Kerber RA. Association between telomere length in blood and mortality in people aged 60 years or older.
Lancet
.
2003
;
361
:
393
-395.
14
Collins K, Mitchell JR. Telomerase in the human organism.
Oncogene
.
2002
;
21
:
564
-579.
15
Weng NP, Levine BL, June CH, Hodes RJ. Regulated expression of telomerase activity in human T lymphocyte development and activation.
J Exp Med
.
1996
;
183
:
2471
-2479.
16
Hathcock KS, Weng NP, Merica R, Jenkins MK, Hodes R. Cutting edge: antigen-dependent regulation of telomerase activity in murine T cells.
J Immunol
.
1998
;
160
:
5702
-5706.
17
Liu K, Schoonmaker MM, Levine BL, June CH, Hodes RJ, Weng NP. Constitutive and regulated expression of telomerase reverse transcriptase (hTERT) in human lymphocytes.
Proc Natl Acad Sci U S A
.
1999
;
96
:
5147
-5152.
18
Effros RB, Pawelec G. Replicative senescence of T cells: does the Hayflick Limit lead to immune exhaustion.
Immunol Today
.
1997
;
18
:
450
-454.
19
Bodnar AG, Ouellette M, Frolkis M, et al. Extension of life-span by introduction of telomerase into normal human cells.
Science
.
1998
;
279
:
349
-353.
20
Vaziri H, Benchimol S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span.
Curr Biol
.
1998
;
8
:
279
-282.
21
Morales CP, Holt SE, Ouellette M, et al. Absence of cancer-associated changes in human fibroblasts immortalized with telomerase.
Nat Genet
.
1999
;
21
:
115
-118.
22
Jiang XR, Jimenez G, Chang E, et al. Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype.
Nat Genet
.
1999
;
21
:
111
-114.
23
Röth A, Yssel H, Pene J, et al. Telomerase levels control the lifespan of human T lymphocytes.
Blood
.
2003
;
102
:
849
-857.
24
Bodnar AG, Kim NW, Effros RB, Chiu CP. Mechanism of telomerase induction during T cell activation.
Exp Cell Res
.
1996
;
228
:
58
-64.
25
Pan C, Xue BH, Ellis TM, Peace DJ, Diaz MO. Changes in telomerase activity and telomere length during human T lymphocyte senescence.
Exp Cell Res
.
1997
;
231
:
346
-353.
26
Weng NP, Hathcock KS, Hodes RJ. Regulation of telomere length and telomerase in T and B cells: a mechanism for maintaining replicative potential.
Immunity
.
1998
;
9
:
151
-157.
27
Hooijberg E, Ruizendaal JJ, Snijders PJ, Kueter EW, Walboomers JM, Spits H. Immortalization of human CD8(+) T cell clones by ectopic expression of telomerase reverse transcriptase.
J Immunol
.
2000
;
165
:
4239
-4245.
28
Rufer N, Migliaccio M, Antonchuk J, Humphries RK, Roosnek E, Lansdorp PM. Transfer of the human telomerase reverse transcriptase (TERT) gene into T lymphocytes results in extension of replicative potential.
Blood
.
2001
;
98
:
597
-603.
29
Hawley RG, Lieu FHL, Fong AZC, Hawley TS. Versatile retroviral vectors for potential use in gene therapy.
Gene Ther
.
1994
;
1
:
136
-138.
30
Miller AD, Garcia JV, von Suhr N, Lynch CM, Wilson C, Eiden MV. Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus.
J Virol
.
1991
;
65
:
2220
-2224.
31
Pollok KE, Hanenberg H, Noblitt TW, et al. High-efficiency gene transfer into normal and adenosine deaminase-deficient T lymphocytes is mediated by transduction on recombinant fibronectin fragments.
J Virol
.
1998
;
72
:
4882
-4892.
32
Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells and cancer.
Science
.
1994
;
266
:
2011
-2015.
33
Kim NW, Wu F. Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP).
Nucleic Acids Res
.
1997
;
25
:
2595
-2597.
34
Baerlocher GM, Lansdorp PM. Telomere length measurements in leukocyte subsets by automated multicolor flow-FISH.
Cytometry
.
2003
;
55A
:
1
-6.
35
Weng N, Levine BL, June CH, Hodes RJ. Regulation of telomerase RNA template expression in human T lymphocyte development and activation.
J Immunol
.
1997
;
158
:
3215
-3220.
36
Zhu J, Wang H, Bishop JM, Blackburn EH. Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening.
Proc Natl Acad Sci U S A
.
1999
;
96
:
3723
-3728.
37
Prescott JC, Blackburn EH. Telomerase: Dr Jekyll or Mr Hyde?
Curr Opin Genet Dev
.
1999
;
9
:
368
-373.
38
Gonzalez-Suarez E, Samper E, Ramirez A, et al. Increased epidermal tumors and increased skin wound healing in transgenic mice overexpressing the catalytic subunit of telomerase, mTERT, in basal keratinocytes.
EMBO J
.
2001
;
20
:
2619
-2630.
39
Artandi SE, Alson S, Tietze MK, et al. Constitutive telomerase expression promotes mammary carcinomas in aging mice.
Proc Natl Acad Sci U S A
.
2002
;
99
:
8191
-8196.
40
Mattson MP, Klapper W. Emerging roles for telomerase in neuronal development and apoptosis.
J Neurosci Res
.
2001
;
63
:
1
-9.
41
Young JI, Sedivy JM, Smith JR. Telomerase expression in normal human fibroblasts stabilizes DNA 5-methylcytosine transferase I.
J Biol Chem
.
2003
;
278
:
19904
-19908.
42
Masutomi K, Yu EY, Khurts S, et al. Telomerase maintains telomere structure in normal human cells.
Cell
.
2003
;
114
:
241
-253.
43
Lansdorp PM. Repair of telomeric DNA prior to replicative senescence.
Mech Ageing Dev
.
2000
;
118
:
23
-34.
44
Mitsui K, Nakanishi M, Ohtsuka S, et al. A novel human gene encoding HECT domain and RCC1-like repeats interacts with cyclins and is potentially regulated by the tumor suppressor proteins.
Biochem Biophys Res Commun
.
1999
;
266
:
115
-122.
45
Algeciras-Schimnich A, Vlahakis SR, Villasis-Keever A, et al. CCR5 mediates Fas- and caspase-8 dependent apoptosis of both uninfected and HIV infected primary human CD4 T cells.
AIDS
.
2002
;
16
:
1467
-1478.
46
Reed JR, Vukmanovic-Stejic M, Fletcher JM, et al. Telomere erosion in memory T cells induced by telomerase inhibition at the site of antigenic challenge in vivo.
J Exp Med
.
2004
;
199
:
1433
-1443.
47
Hemann MT, Strong MA, Hao LY, Greider CW. The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability.
Cell
.
2001
;
107
:
67
-77.
48
Zhang X, Mar V, Zhou W, Harrington L, Robinson MO. Telomere shortening and apoptosis in telomerase-inhibited human tumor cells.
Genes Dev
.
1999
;
13
:
2388
-2399.
49
Gisselsson D, Jonson T, Petersen A, et al. Telomere dysfunction triggers extensive DNA fragmentation and evolution of complex chromosome abnormalities in human malignant tumors.
Proc Natl Acad Sci U S A
.
2001
;
98
:
12683
-12688.
50
Röth A, Vercauteren S, Sutherland HJ, Lansdorp PM. Telomerase is limiting the growth in acute myeloid leukemia cells.
Leukemia
.
2003
;
17
:
2410
-2417.
51
Artandi SE, Chang S, Lee SL, et al. Telomere dysfunction promotes non-reciprocal translocations and epithelial cancers in mice.
Nature
.
2000
;
406
:
641
-645.
52
Baum C, von Kalle C, Staal FJ, et al. Chance or necessity? Insertional mutagenesis in gene therapy and its consequences.
Mol Ther
.
2004
;
9
:
5
-13.
53
Latouche JB, Sadelain M. Induction of human cytotoxic T lymphocytes by artificial antigen-presenting cells.
Nat Biotechnol
.
2000
;
18
:
405
-409.
54
Romieu R, Baratin M, Kayibanda M, et al. Passive but not active CD8+ T cell-based immunotherapy interferes with liver tumor progression in a transgenic mouse model.
J Immunol
.
1998
;
161
:
5133
-5137.
55
Rooney CM, Aguilar LK, Huls MH, Brenner MK, Heslop HE. Adoptive immunotherapy of EBV-associated malignancies with EBV-specific cytotoxic T-cell lines.
Curr Top Microbiol Immunol
.
2001
;
258
:
221
-229.
56
Rosenberg SA, Yannelli JR, Yang JC, et al. Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2.
J Natl Cancer Inst
.
1994
;
86
:
1159
-1166.
57
Brentjens RJ, Latouche JB, Santos E, et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15.
Nat Med
.
2003
;
9
:
279
-286.
58
Einsele H, Roosnek E, Rufer N, et al. Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy.
Blood
.
2002
;
99
:
3916
-3922.