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Department of Immunology, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, and
Department of Pathology, Section Molecular Pathology, University Hospital Free University, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A correlation between telomere shortening and life span has also been found in cells of the immune system. It was reported that the average telomeric length and the replicative potential are higher in naive T cells as compared with memory T cells from the same donor, in both CD4+ cells (11, 12) and CD8+ cells (13, 14). These findings are consistent with two ex vivo studies with peripheral blood leukocytes, indicating a correlation between the loss of telomere repeat fragments and the age of the donor (15, 16). Ongoing shortening of telomeres and subsequent induction of replicative senescence in cells of the immune system occur despite the presence of endogenous hTERT in T and B cells (17, 18). The levels of telomerase activity in peripheral blood T and B lymphocytes are regulated at the level of hTERT transcription (19, 20, 21, 22), but posttranscriptional mechanisms may also play an important role in the control of the function of the enzyme (23). Activation of T cells by strong stimuli like PMA and ionomycin (20), but also milder stimulation by a combination of CD3 and CD28 Abs (19, 24) or by the cognate Ag presented by the appropriate target cell (25), can induce a transient expression of telomerase. Recently, it was shown that telomerase is up-regulated and telomere length is preserved after virus-induced clonal expansion of CD8+ T cells (26). Despite the endogenous expression and activation induced up-regulation of hTERT in subsets of human T cells, presumably resulting in maintenance of replicative potential in vivo (18), Ag-specific T cell clones cannot be expanded in vitro beyond 20–25 population doublings (PD; reviewed in Ref. 17). This finding raises the question whether the replicative life span of T cells is solely regulated by hTERT. This issue is investigated in the present study.
We examined the effect of ectopic expression of hTERT on the life span of two CD8+ CTL clones, derived from a melanoma patient, which are specific for the Mart-1/Melan-A27–35, the Tyrosinase368–376 epitopes, respectively, and restricted by HLA-A2. Ectopic expression of hTERT led to a dramatic extension of the life span of these T cell clones, without altering the phenotype, the specificity, and the function of the cells. T cells ectopically expressing hTERT remained dependent on cytokines and Ag stimulations for their in vitro expansion. These findings indicate that constitutive expression of hTERT is necessary and sufficient to extend the life span of CD8+ memory T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The virus-producing cell line Phoenix, the melanoma cell line Mel-AKR, and the EBV-transformed B cell lines JY and EBV-AKR, were grown in medium consisting of Iscove’s medium (Life Technologies B.V., Breda, The Netherlands) supplemented with 5–10% FCS (BioWhittaker, Verviers, Belgium), penicillin, and streptomycin (Boehringer Mannheim, Mannheim, Germany).
T cell blasts and T cell clones
T cell blasts were prepared by incubation of 5 x 105 PBMC per ml in Yssel’s medium supplemented with 1% normal human serum and 2 µg/ml PHA. T cell blasts and Ag-specific T cell clones were cultured as previously described (27). Briefly, 3 x 105 cells/well were stimulated weekly with a mixture of 1 x 106 irradiated (80 Gy instead of the previously used 30–50 Gy) allogeneic PBMC/ml and 1 x 105 irradiated EBV-B cells (JY), supplemented with 100 ng/ml PHA and 20 IU/ml IL-2 (Chiron, Amsterdam, The Netherlands) in Yssel’s medium. In some experiments, T cell clones were cultured with cytokines without weekly stimulations. The following concentrations of cytokines were used: 20 IU/ml IL-2, 10 ng/ml IL-7 (PeproTech, Rocky Hill, NJ), and 10 ng/ml IL-15 (Peprotech). Cell cultures were kept in incubators at 37°C in humidified air containing 5% CO2.
Construction of the retroviral hTERT vector
The full-length coding sequence of hTERT was isolated from pGRN145 (kindly provided by Geron, Menlo Park, CA) as a 3.5-kb EcoRI-NotI fragment. The hTERT fragment was subsequently ligated into the polylinker of LZRS-linker-internal ribosomal entry site-GFP (28). Correct cloning of hTERT was confirmed by restriction enzyme analysis. This construct designated LZRS-hTERT-IRES-GFP was used to produce retroviral supernatant as previously described (28, 29). As control, we used LZRS-polylinker-IRES-GFP.
Transduction method
The recombinant human fibronectin fragments transduction procedure (RetroNectin; Takara, Otsu, Japan) was based on a method originally developed by Hanenberg et al. (30) with the modifications described by Heemskerk et al. (28). T cells were prestimulated with PHA (31) or with a feeder cell mixture containing PHA and IL-2 for 32–48 h before transduction. Subsequently, the target cells were plated on RetroNectin-coated dishes (maximum 5 x 106 cells/petri dish with a diameter of 3 cm) in 0.5 ml of complete medium mixed with 1 ml of thawed retroviral supernatant. Cells were cultured at 37°C for 6 h or overnight, washed, and transferred to 24-well culture plates (Falcon plastics; Becton Dickinson Labware, Mountain View, CA). The capability of hTERT retrovirus to induce telomerase activity was determined in cultures of primary human keratinocytes devoid of detectable telomerase activity (data not shown).
Flow cytometric analysis
CD2, CD3, CD4, CD8, CD25, CD27, CD28, CD38, CD45, CD45RA,
CD45RO, CD54, CD69, CD80, anti-TCR
ß, anti-HLA-DR, and
anti-HLA-class I mAbs (all from Becton Dickinson) directly labeled
with PE were used for flow cytometric analysis. HLA-A2 tetramers
containing the Mart-1/Melan-A27–35, the
Tyrosinase368–376, or the
influenza-A58–66 epitope labeled with PE were
prepared as previously described (28). Stained cells were
analyzed using a FACScan (Becton Dickinson), and the data were
processed with CellQuest computer software.
Chromium release assays
Cytotoxicity of T cell clones was determined using a standard chromium release assay previously described (32). All assays were performed in the presence of a 50-fold excess of unlabeled K562 cells to block nonspecific lysis of the target cells. The spontaneous release varied between 10 and 25% of the maximum. SD of triplicate determinations never exceeded 10% of the mean.
Measurement of telomerase activity and hTERT mRNA levels
Cell lysates for analysis of telomerase activity were prepared
from 
1 x 106 cells using the CHAPS
(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) detergent
lysis method as described before (33, 34). Briefly, cells
were suspended in 50 µl lysis buffer. After homogenization, the
suspension was placed on ice for 30 min. Subsequently, the cells were
spun down at maximum speed in a microcentrifuge for 30 min at 4°C.
The supernatant was transferred to a new tube and snap-frozen in liquid
nitrogen. The amount of protein was determined using the Bio-Rad
protein detection kit (Bio-Rad Laboratories, Veenendaal, The
Netherlands). From the resulting pellet, RNA was isolated using RNAzolB
(Campro Scientific, Veenendaal, The Netherlands) for subsequent hTERT
mRNA analysis.
Telomerase activity was determined in samples containing various amounts of protein (representative for 100–10,000 cells used) by the telomeric repeat amplification protocol (TRAP). The TRAPeze kit (Oncor, Gaithersburg, MD) was used according to instructions of the manufacturer. To determine the specificity of the assay, the protein samples were preheated for 10 min at 70°C to inactivate telomerase and tested in parallel experiments. No stepladder patterns were observed after this preheating step. Following separation of TRAP products on polyacrylamide gels, autoradiography was performed overnight at -80°C using intensifying screens. Relative telomerase activities were quantified by densitometric evaluation of the TRAP ladder bands relative to the corresponding internal controls. Semiquantitative RT-PCR for hTERT was performed essentially as previously described (34) except that 25 instead of 30 PCR cycles were run to ensure linearity of the amplification reaction. First-strand cDNA synthesis was performed on 100 ng of total RNA using antisense primers for both hTERT and the housekeeping gene snRNP U1A in a single reaction. Subsequent RT-PCR for hTERT and snRNP U1A were performed in a single reaction as well, using primers previously described (34). Resulting PCR products were run in duplicates on the same agarose gel and blotted to the same nylon membrane, followed by hybridization with radiolabeled hTERT and snRNP U1A-specific oligonucleotide probes, respectively. Signal intensities were measured after exposure of the hybridized filters to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Relative levels of hTERT mRNA were calculated according to the following formula: intensity ratio hTERT/snRNP U1A.
Telomere length assay
For measurement of telomere length, genomic DNA was extracted
from 1.5 x 106 cells using the PureGene
DNA extraction kit (Gentra Systems, Minneapolis, MN). Five micrograms
of DNA were digested with BglII (Boehringer Mannheim) and
electrophoresed through a 0.7% agarose gel before Southern blotting.
The blot was subsequently hybridized to a
[
-32P]ATP end-labeled telomeric oligoprobe
(TTAGGG)4. Autoradiography was performed for 5
days. To assess the median telomere length, the peak signal intensity
was determined by PhosphorImager analysis at a shorter exposure time to
avoid problems of overexposure of the signal.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We introduced hTERT cDNA in a human CD8+ T cell clone to test the effect of ectopic expression of hTERT on expansion and extension of the life span of human T cells. The T cell clone (AKR-IL7-clone no. 4) has been obtained after stimulation of patient-derived peripheral T cells with autologous melanoma cells genetically engineered to produce IL-7 (E. Hooijberg, J. J. Ruizendaal, and H. Spits, manuscript in preparation). This T cell clone is specific for the HLA-A2-restricted Mart-1/Melan-A27–35 epitope. After establishment of this T cell clone, the cells were cultured for 6–8 wk before transduction. In this period, the cells went through 2 PD per week, giving a total number of 12–16 PD.
To address the question whether the introduction of hTERT had an effect
on T cell expansion, we followed the expression of green fluorescent
protein (GFP) upon further culturing of hTERT-IRES-GFP and
control-GFP-transduced T cells. The level of GFP expression in
hTERT-IRES-GFP-transduced cells showed an exponential increase over
time in two independent experiments with T cells from the same clone
from different frozen batches (Fig. 1
).
In a period of 7 wk, the percentage of hTERT-IRES-GFP-positive cells
increased from the initial 5% and 3%, respectively, measured 5 days
after retroviral transduction, to 95% on day 53 (Fig. 1). The
percentage of hTERT-IRES-GFP-positive cells remained stable (95%)
for another period of 5 wk, after which this experiment of coculturing
hTERT-IRES-GFP-positive and -negative cells was terminated. Based on
weekly counting of the cells and the percentages of GFP-positive and
-negative cells, we calculated the average weekly expansion of
hTERT-IRES-GFP-positive T cells and of the untransduced cells to be
10-fold (3 PD) and 5-fold (2 PD), respectively.
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). The level of GFP expression remained stable in this T cell
culture for a period of 53 days, after which the cells went into crisis
and died. Thus, the maximum number of PD of the control-transduced
cells was 30–35, which is comparable to that of the untransduced cells
of this clone (data not shown). These data indicate that retroviral
transduction per se does not lead to enhanced expansion or extension of
the life span of T cells. Extension of the life span of human T cells upon ectopic expression of hTERT
The experiments shown in Fig. 1 strongly suggest that ectopic
expression of hTERT results not only in an increased expansion but also
in an extension of the life span of the cells. To obtain more
information on the degree of life span extension of the
hTERT-transduced cells, we performed subcloning experiments. The
maximum number of PD of established CD8+ human T
cell clones is in the order of magnitude of 20–30 (for a review see
Ref. 17), consistent with experiences in our lab. As
mentioned above, the
Mart-1/Melan-A27–35-specific T cells went
through 12–16 PD as an established T cell clone before they were
transduced with hTERT. On day 32 after the transduction, we sorted part
of the hTERT-transduced cells into a GFP-positive and a GFP-negative
fraction. These cells were cloned by single cell disposition using a
FACStarPlus. We observed growing cells in the
wells seeded with cells from the GFP-negative fraction (12/480) and the
GFP-positive fraction (61/480). The clones derived from the positive
fraction were, as expected, all GFP-positive, and the clones from the
negative fraction were all negative for GFP (data not shown). All
subclones, either GFP-negative or GFP-positive, were shown to be
positive for staining with HLA-A2 tetramers containing the
Mart-1/Melan-A27–35 epitope, indicating that all
subclones obtained were indeed derived from the same parental clone
(data not shown). A second single cell cloning was performed with two
randomly selected subclones from the GFP-positive pool and the two best
growing clones from the GFP-negative pool. This cloning experiment only
yielded subclones from the hTERT-IRES-GFP-positive T cell fraction
(10/386 and 5/386). Three clones were isolated for further studies:
A11, D8, and F6 (all subclones from subclone no. 1 of clone AKR-IL7 no.
4). The finding that the hTERT transduced no. 4
CD8+ T cell clone could be successfully subcloned
twice confirms the notion that ectopic expression of telomerase leads
to a dramatic extension of the life span.
Ectopic expression of hTERT in human T cells does not alter functional and phenotypic characteristics
After having established that ectopic expression of hTERT leads to
an extended life span, we examined whether it affected the cell surface
phenotypes, the specificities, and the functions of the transduced T
cells. No differences between the original T cell clone and the
hTERT-transduced subsubclones were found in the expression of any of
the cell surface markers tested (Table I). It is of note that the original clone
as well as the hTERT-transduced T cells were negative for CD27 and
CD28, and positive for CD45RO, which is the typical phenotype of memory
T cells (35).
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A).
Consistent with this observation, the parental clone and the three
hTERT transduced subsubclones lysed autologous melanoma cells, and
peptide (Mart-1/Melan-A27–35) loaded EBV-B cells
to the same extent (Fig. 2B). As expected, none of the
clones lysed untreated EBV-B cells.
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An extension of the life span of somatic cells may lead to abnormal growth characteristics. We examined the growth characteristics of the three isolated subsubclones (A11, D8, and F6) in the absence and presence of cytokines. We cultured the hTERT-transduced T cells in medium without cytokines, or with added IL-2, IL-7, IL-15, or mixtures of these cytokines. IL-7 alone was not capable of supporting survival of either of the subsubclones, nor of the original clone. The hTERT-transduced cells could be maintained in medium containing IL-2 or IL-15 for a period up to 3 mo (longer periods were not tested) without significant expansion. Like the original, untransduced clone, the subsubclones remained highly dependent on periodic activation for their growth. The expansion of T cell cultures in a feeder cell mixture containing IL-2 and PHA was about 5 for the original clone (2 PD per week) and about 10 for the subsubclones (3 PD/wk). These findings demonstrate that T cells expressing ectopic hTERT maintained normal growth characteristics for prolonged periods of time.
Increased hTERT mRNA levels, telomerase activity, and stabilization of telomeres in T cells ectopically expressing hTERT
We next examined the hTERT mRNA levels, telomerase activity, and
telomere lengths in untransduced and hTERT-transduced T cells. Ten days
after sorting GFP-negative and -positive cells, the levels of hTERT
mRNA and of telomerase activity were assayed in the sorted cells.
Semiquantitative RT-PCR for measurement of hTERT mRNA levels, relative
to a household gene, was performed on day 42 after retroviral
transduction of the wild-type T cell clone no.4, 1 wk after the last
stimulation with a feeder cell mixture. At that time point, the feeder
cells were completely cleared. The hTERT mRNA levels were about 5-fold
increased in GFP-positive cells compared with the GFP-negative
counterparts (Fig. 3A).
Telomerase activity was high in both cell fractions and could be
detected in as little as 5 ng of protein lysate, which is equivalent to
100 cells (Fig. 3B). Densitometric scanning of TRAP
ladder bands compared with the internal control yielded a two to three
times higher OD in the lanes corresponding to the GFP-positive cells,
indicating a 2- to 3-fold increased telomerase activity. Moreover, a
slight decrease in mean telomere length (median length of 6–6.5 kb vs
7 kb) and a smear indicating loss of telomere integrity was observed in
the GFP-negative cells compared with the GFP-positive T cell fraction
(Fig. 3C). At the time of analysis, 12 PD had occurred in
the GFP-negative and 18 in the GFP-positive cell populations since the
transduction. A larger difference in telomere length between wild-type
and the hTERT-transduced cells may be expected near the point of
senescence of the wild-type clone no. 4. Unfortunately, this could not
be verified because it is difficult to obtain enough material from the
GFP-negative fraction or of wild-type T cells close to the point of
senescence. Accurate measurement of hTERT mRNA levels, telomerase
activity, and telomere lengths is also severely hampered by the
presence of contaminating feeder cells, in such near-senescent
cultures. In cultures with well-growing T cell clones, these irradiated
feeder cells are rapidly cleared. To obtain independent proof for the
biological activity of the transduced hTERT in the T cell clones, we
compared the telomere lengths of untransduced wild-type T cells of the
same clone with the isolated subsubclones A11, D8, and F6. Fig. 3D shows that stabilization of telomere length had occurred
in the hTERT-IRES-GFP-positive T cell clones, which were subcloned
twice (A11, D8, and F6). After being subcloned twice, these clones were
maintained in in vitro culture for another 11 wk, which involves on
average 3 PD per week. At the time of telomere length analysis,
these cells differed 60–70 PD from the cells analyzed 10 days after
sorting and of the wild-type cells used in this analysis. It is
apparent that the telomeres in these clones (A11, D8, F6) had not
eroded and were maintained at an average length of 7 kb (Fig. 3D).
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To demonstrate that immortalization by ectopic expression of hTERT
is not restricted to clone no. 4, we transduced another T cell clone
with hTERT-IRES-GFP. This HLA-A2-restricted,
CD8+, T cell clone (AKR-IL7-clone no. 108) is
specific for the Tyrosinase368–376 epitope, and
has been obtained from the same stimulation experiment as AKR-IL7-clone
no. 4. Clone no. 108 was selected from our collection of tumor-specific
T cell clones because it has been very difficult to grow and expand,
with a weekly PD of less than one. Two days after the transduction, the
GFP expression, as an indirect measure for telomerase expression, was
very low (1–2%; Fig. 4). The low
transduction efficiency was most likely due to the low level of
proliferation of this clone. After 4 wk, we observed a significantly
higher percentage of GFP-positive cells in this culture. Following
kinetics similar to those seen with clone no. 4, we observed an
increase to 97% 10 wk after transduction. The transduced cells went
through 2 PD a week. Subclones of hTERT-transduced no. 108 have been
obtained as well. The inset in Fig. 4 shows staining with
control and Tyrosinase368–376 tetramers,
demonstrating that the hTERT-transduced cells have retained their
specificity. Cytotoxicity assays confirmed functionality of these cells
as autologous melanoma cells, and peptide-loaded EBV-B cells were
lysed, whereas EBV-B cells without exogenous peptide were not (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). These observations indicate that the majority of the hTERT-
transduced cells have a growth advantage compared with wild-type cells.
Recently, we have transduced a panel of CD4+ T
cell clones and observed also with these clones an expansion advantage
of all hTERT-transduced cells in comparison with untransduced cells (H.
Yssel and H. Spits, unpublished observations). Another question is
whether all hTERT-transduced cells enter a state of immortalization.
The low cloning efficiency (15%) of hTERT-GFP+
no. 4 cells indicates that not all transduced cells are immortalized
and may suggest that the site of integration of the hTERT DNA is a
determining factor for immortalization. However, the cloning
efficiencies of the no. 4 subclones, which should have hTERT integrated
at a single site in the genome, were even lower (1.5% and 3% in two
experiments with different subclones). A likely explanation for the low
cloning efficiency of these subclones is that a proportion of the
transduced cells lose their hTERT over time either by inactivation of
the inserted DNA or by posttranscriptional inactivation. Recently, it was reported by Liu et al. (23) that low levels of hTERT mRNA are constitutively present in CD4+ T lymphocytes, regardless of the telomerase activity in the cells that was activation-induced and transient, suggesting that telomerase is mainly regulated at a posttranscriptional level. However, it is possible that only a minority of the CD4+ T cells in the study of Liu et al. expressed constitutive telomerase mRNA levels. Moreover, while our data do not exclude posttranscriptional regulation of hTERT activity, they strongly suggest that the level of hTERT transcripts is the main regulator of the life span of T cells. Presently, we analyze the effect of constitutive hTERT expression on a large panel of CD8+ and CD4+ T cell clones to obtain insight into the regulation of telomerase in T cells in general.
It is of note that the stabilization of the telomere length as observed in the subsubclones of clone no. 4 indicates that constitutive hTERT expression does not result in telomere lengthening as has been observed in hTERT-transfected fibroblasts (7, 8) and in germinal center B cells (18, 38). The reasons for this have yet to be determined. The finding that ectopic expression of telomerase extends the replicative life span of certain cell types without net telomere lengthening is not unprecedented. In human fibroblasts transfected with hTERT, the telomeres continue to shorten to average lengths below those of untransfected cells that enter replicative senescence and crisis (39, 40). This suggests that hTERT has a role in life-span extension beyond the prevention of telomere length erosion.
Importantly, ectopic expression of hTERT did not affect the phenotype, Ag specificity, or functionality of the T cells. Of equal importance is the observation that the hTERT-transduced T cells are still dependent on cytokines and Ag stimulation for proliferation, suggesting that these T cells were not transformed. These findings are well in agreement with recent publications on the lack of induction of a transformed phenotype in hTERT-transfected normal human fibroblasts and retinal pigment epithelial cells (41, 42). The advantages of immortalized T cell clones are obvious. The availability of immortalized functional T cells with defined specificities will facilitate functional, genetic, and biochemical experiments. The limited in vitro life span of human Ag-specific T cell clones has severely hampered application of these cells in cellular therapies involving adoptive transfer of Ag-specific T cells. Our finding that tumor-specific CTL clones can be immortalized by ectopic expression of hTERT and thus can be expanded to very large numbers may lead to potential application of these cells in the treatment of cancer patients.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Pathology, Section Molecular Pathology, University Hospital Free University, De Boelelaan 1117, NL-1081HV, Amsterdam, The Netherlands.
3 Address correspondence and reprint requests to Dr. Hergen Spits, Department of Immunology, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, NL-1066CX, Amsterdam, The Netherlands.
4 Abbreviations used in this paper: TERT, telomerase reverse transcriptase; hTERT, human TERT; GFP, green fluorescent protein; PD, population doubling; TRAP, telomeric repeat amplification protocol.
Received for publication April 14, 2000. Accepted for publication July 24, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. V. D. Soares, F. J. Plunkett, C. S. Verbeke, J. E. Cook, J. M. Faint, L. L. Belaramani, J. M. Fletcher, N. Hammerschmitt, M. Rustin, W. Bergler, et al. Integration of apoptosis and telomere erosion in virus-specific CD8+ T cells from blood and tonsils during primary infection Blood, January 1, 2004; 103(1): 162 - 167. [Abstract] [Full Text] [PDF]
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M. W. J. Schreurs, K. B. J. Scholten, E. W. M. Kueter, J. J. Ruizendaal, C. J. L. M. Meijer, and E. Hooijberg In Vitro Generation and Life Span Extension of Human Papillomavirus Type 16-Specific, Healthy Donor-Derived CTL Clones J. Immunol., September 15, 2003; 171(6): 2912 - 2921. [Abstract] [Full Text] [PDF]
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A. Roth, H. Yssel, J. Pene, E. A. Chavez, M. Schertzer, P. M. Lansdorp, H. Spits, and R. M. Luiten Telomerase levels control the lifespan of human T lymphocytes Blood, August 1, 2003; 102(3): 849 - 857. [Abstract] [Full Text] [PDF]
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R. M. Luiten, J. Pene, H. Yssel, and H. Spits Ectopic hTERT expression extends the life span of human CD4+ helper and regulatory T-cell clones and confers resistance to oxidative stress-induced apoptosis Blood, June 1, 2003; 101(11): 4512 - 4519. [Abstract] [Full Text] [PDF]
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 M. Dagarag, H. Ng, R. Lubong, R. B. Effros, and O. O. Yang Differential Impairment of Lytic and Cytokine Functions in Senescent Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes J. Virol., March 1, 2003; 77(5): 3077 - 3083. [Abstract] [Full Text] [PDF]
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N. Rufer, M. Migliaccio, J. Antonchuk, R. K. Humphries, E. Roosnek, and P. M. Lansdorp Transfer of the human telomerase reverse transcriptase (TERT) gene into T lymphocytes results in extension of replicative potential Blood, August 1, 2001; 98(3): 597 - 603. [Abstract] [Full Text] [PDF]
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 R. D. M. Steenbergen, D. Kramer, C. J. L. M. Meijer, J. M. M. Walboomers, D. A. Trott, A. P. Cuthbert, R. F. Newbold, W. J. I. Overkamp, M. Z. Zdzienicka, and P. J. F. Snijders Telomerase Suppression by Chromosome 6 in a Human Papillomavirus Type 16-Immortalized Keratinocyte Cell Line and in a Cervical Cancer Cell Line J Natl Cancer Inst, June 6, 2001; 93(11): 865 - 872. [Abstract] [Full Text] [PDF]
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K. Liu, R. J. Hodes, and N.-p. Weng 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 J. Immunol., April 15, 2001; 166(8): 4826 - 4830. [Abstract] [Full Text] [PDF]
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) or the control GFP only (
) over time (given in days). The
cultures were stimulated with a feeder cell mixture every week. On day
32, the cells were sorted in a GFP-positive and a GFP-negative fraction
for further studies. The result of a second hTERT-IRES-GFP transduction
experiment with a different batch of T cells of the same clone is also
indicated (
).
), and
on the autologous melanoma cells (
