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The Journal of Immunology, 2005, 174: 5261-5269.
Copyright © 2005 by The American Association of Immunologists

IL-2 Increases Human Telomerase Reverse Transcriptase Activity Transcriptionally and Posttranslationally through Phosphatidylinositol 3'-Kinase/Akt, Heat Shock Protein 90, and Mammalian Target of Rapamycin in Transformed NK Cells

Kiyotaka Kawauchi1,*, Kimiko Ihjima*,{dagger} and Osamu Yamada1,2,{dagger}

* Department of Medicine, Daini Hospital, Tokyo Women’s Medical University, Arakawa-ku, Tokyo, Japan; and {dagger} Medical Research Institute and Department of Hematology, Tokyo Women’s Medical University, Shinjuku-ku, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human telomerase activity is induced by Ag receptor ligation in T and B cells. However, it is unknown whether telomerase activity is increased in association with activation and proliferation of NK cells. We found that telomerase activity in a human NK cell line (NK-92), which requires IL-2 for proliferation, was increased within 24 h after stimulation with IL-2. Levels of human telomerase reverse transcriptase (hTERT) mRNA and protein correlated with telomerase activity. ERK1/2 and Akt kinase (Akt) were activated by IL-2 stimulation. LY294002, an inhibitor of PI3K, abolished expression of hTERT mRNA and protein expression and abolished hTERT activity, whereas PD98059, which inhibits MEK1/2 and thus ERK1/2, had no effect. In addition, radicicol, an inhibitor of heat shock protein 90 (Hsp90), and rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), blocked IL-2-induced hTERT activity and nuclear translocation of hTERT but not hTERT mRNA expression. hTERT was coimmunoprecipitated with Akt, Hsp90, mTOR, and p70 S6 kinase (S6K), suggesting that these molecules form a physical complex. Immunoprecipitates of Akt, Hsp90, mTOR, and S6K from IL-2-stimulated NK-92 cells contained telomerase activity. Furthermore, the findings that Hsp90 and mTOR immunoprecipitates from primary samples contained telomerase activity are consistent with the results from NK-92 cells. These results indicate that IL-2 stimulation induces hTERT activation and that the mechanism of IL-2-induced hTERT activation involves transcriptional or posttranslational regulation through the pathway including PI3K/Akt, Hsp90, mTOR, and S6K in NK cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Telomeres at the ends of linear mammalian chromosomes consist of arrays of the TTAGGG repeat and associated proteins that protect the telomere from shortening during cell division (1). The human telomere is maintained by a reverse transcriptase called telomerase that is composed of a catalytic subunit, human telomerase reverse transcriptase (hTERT)3; a template RNA, hTR; and an associated protein, TEP1 (1, 2). Telomerase activity is observed primarily in germ cells, stem cells, and cancer cells. Stimulation of cells with epidermal growth factors (EGFs) (3), fibroblast growth factors (4), and insulin-like growth factors (5) increases telomerase activity. Therefore, telomerase activity is required for rapid expansion of cell populations, immortalization of various cell types, and maintenance of cell proliferation in response to growth stimuli. The hTERT catalytic subunit of telomerase plays a central role in regulation of telomerase activity. Telomerase activity is correlated with expression of hTERT mRNA (6). Transcription factors such as myc, Sp1, estrogen receptor, E2F-1, WT-1, NF-{kappa}B, and MZF-2 may be involved in regulation of hTERT gene expression (2). hTERT activity can also be regulated by signaling intermediates through posttranlational modification. In vitro assembly experiments revealed that hTERT forms a complex with chaperone proteins such as heat shock proteins (Hsp) (7). Moreover, hTERT protein was shown to be phosphorylated at serine/threonine residues by protein kinase C and recombinant protein kinase B/Akt in an in vitro reconstitution assay (8). However, it remains unclear how telomerase activity is regulated differentially in various cell systems, including the immune system.

Human NK cells are a small subpopulation of lymphoid cells and are capable of lysing tumor cells without prior stimulation. Two subsets of NK cells can be recognized on the basis of expression of CD56 and CD16 on the cell surface (9). CD56bright NK cells express high- and intermediate-affinity IL-2 receptor (IL-2R) and proliferate in response to IL-2, which was originally identified as a growth factor for T cells. The IL-2R is composed of {alpha}, {beta}, and {gamma} chains. Heterodimers of the {beta} and {gamma} chains have an intermediate affinity for IL-2 that is sufficient to transduce IL-2 signals, and association of an {alpha}-chain with these {beta}{gamma} heterodimers yields an IL-2R with high affinity for IL-2 (10). IL-2R signaling involves Lck, Jak, Fyn, Lyn, Syk, Ras, MAPK, and PI3K in T cells (10, 11). The MAPK and PI3K pathways play central roles in cell growth, differentiation, and survival. ERK, a major member of the MAPK family, transduces mitogenic signals from the Ras/Raf/MEK pathway to the nucleus by activating transcription factors such as Elk-1. PI3K, which plays an important role in cell survival, induces activation of phosphatidylinositol-dependent kinase 1/2 and then activates Akt kinase by phosphorylating the Ser473 and Thr308 residues. Akt prevents apoptosis by disrupting the interaction between Bad and Bcl-2 or by activating the mammalian target of rapamycin (mTOR), which then phosphorylates p70 S6 kinase (S6K) and leads to progression of the cell cycle. In contrast to the situation in T cells, intracellular signals, including telomerase, triggered by IL-2 in NK cells are not well understood.

We and others have previously shown that stimuli, such as PHA, phorbol ester plus ionomycin, and anti-CD3 Ab cross-linking, increase telomerase activity in PBMC (12, 13, 14). We report here that IL-2 stimulation induces telomerase activity in IL-2-responsive NK cell line and that the PI3K/Akt/Hsp90/mTOR/S6K pathway is involved in regulation of IL-2-induced telomerase activity at both the transcriptional and posttranslational levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture and reagents

The human NK cell line NK-92 (15), which requires IL-2 for proliferation, was obtained from American Type Culture Collection and maintained in RPMI 1640 supplemented with 10% FBS containing 100 U/ml IL-2 (kindly provided by Takeda Pharmaceutical and Shionogi Pharmaceutical). The viability of NK-92 cells was markedly reduced when cells were cultured in the absence of IL-2 for >5 days, which was consistent with previously reported findings (15). For analysis of cell surface markers, cells were stained with FITC- or PE-conjugated mouse mAbs against CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD14, CD16, CD19, CD20, CD33, CD45, CD56, CD57, TCR{alpha}{beta}, and HLA-DR and analyzed by flow cytometry (FACS; Coulter Electronics). NK cell killing activity was measured by 51Cr-release cytotoxicity assay with NK-sensitive K562 target cells. Cell viability was assessed by trypan blue exclusion. For cell starvation and restimulation, NK-92 cells were cultured in the absence of IL-2 for 4 days followed by culture in the presence of 100 U/ml IL-2 for 24 to 48 h at 5% CO2 and 37°C. For assessment of the effects of inhibitors, NK-92 cells were treated with various concentrations of specific agents for 60 min before the addition of IL-2 to IL-2-starved cell cultures, and cells were cultured for an additional 24 h in the presence of inhibitors. Mononuclear cells containing tumor cells in bone marrow (patient 1) or lymph node (patient 2) from patients with NK cell lymphoma/leukemia were separated by Ficoll density gradient centrifugation, washed in PBS, and immediately frozen in liquid nitrogen after informed consent was given, as previously described (16). Although patient 1 remains in hematological remission after chemotherapy, patient 2 developed progressive disease and died despite chemotherapy (16). K562, a cell line derived from a patient with chronic myelogenous leukemia blast crisis, was maintained in RPMI 1640 with 10% FBS.

LY294002, PD98059, geldanamycin, radicicol, staurosporine, herbimycin A, and rapamycin were all purchased from Calbiochem and reconstituted in DMSO. Rabbit Akt, phospho-(Ser/Thr)Akt substrate, ERK1/2, and p70 S6K antisera were obtained from Cell Signaling. Rabbit Abs against mTOR were kindly provided by Dr. Peter J. Houghton (St. Jude Children’s Research Hospital, Memphis, TN) and purchased from Sigma-Aldrich. Rabbit polyclonal Abs against Hsp90 were purchased from StressGen. Rabbit polyclonal Abs against hTERT were purchased from Calbiochem and Abcam. Rabbit {alpha}-tubulin antisera were purchased from Sigma-Aldrich. Mouse mAb against nucleolin (4E2) was purchased from Medical and Biological Laboratory.

Cell cycle analysis

Cells (2 x 106) were washed once in PBS, fixed in 1 ml of 70% ethanol, and stored at –20°C until staining. For staining, fixed cells were removed from ethanol by centrifugation, and resuspended in 1 ml of propidium iodide staining solution (Wako Pure Chemicals) in the presence of RNase A. After 30 min of incubation at room temperature, cells were analyzed immediately. DNA content, which reflected the cell cycle distribution, was determined by EPICS-XL flow cytometer (Coulter Electronics). Cell cycle distribution was determined with cell cycle analysis software WinCycle.

Nonradioisotopic telomerase repeat amplification protocol (TRAP) assay

Fluorescence-based telomerase activity was assayed by TRAP method in a total volume of 50 µl. Aliquots of either untreated or heat-treated (10 min, 85°C) cell extracts were incubated with 0.1 µg of FAM-labeled TS oligonucleotide (5'-AATCCGTCGAGCAGAGTT-3') for 20 min at 22°C as described previously (17). ITAS was included as an internal standard. Following elongation of the [F]-TS primer by telomerase, the elongated products were then amplified in the presence of 0.1 µg of CX primer (5'-CCCTTACCCTTACCCTTACCCTTA-3') and Taq polymerase by PCR (27 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 60 s). Fluorescent amplification products were detected by electrophoresis through 4.5% polyacrylamide/6% urea sequencing gels on a 377 Automated Sequencer with GeneScan software (PerkinElmer Life and Analytical Sciences). Telomerase activity was expressed in arbitrary units as reported elsewhere (17). Alternatively, we used a more convenient method instead of the fluorescence-based TRAP assay. Briefly, TS oligonucleotide was used in place of [F]-TS primer, and amplification products were detected by electrophoresis on 12.5% polyacrylamide nondenaturing gels in 1x Tris-borate-EDTA (TBE), and enzymatic activity was quantified with NIH image software as described (12). Extract of Namalva cells was used as a positive control, and extraction buffer instead of cell extract was used as a negative control.

Analysis of telomere length

Telomere length was assessed by Southern blotting with a telomere probe labeled with digoxigenin as described previously (18). Briefly, high m.w. DNA was extracted by standard phenol/chloroform extraction. Five micrograms of HinfI-digested DNA was fractionated by 0.7% agarose gel electrophoresis and then transferred to Hybond N filters (Amersham Biosciences). Filters were hybridized with digoxigenin-labeled telomere probe (TTAGGG)4 according to the manufacturer’s instructions (Roche). The filters were rinsed twice at 50°C in wash solution (0.1x SSC, 0.1% SDS) and exposed to x-ray film (Fujifilm). The hybridized probe was detected by chemiluminescence method (Tropix).

RT-PCR assay

Expression of hTERT mRNA was analyzed by RT-PCR assay as described previously (17). Total cellular RNA was isolated with the single-step acid guanidinium-isothiocyanate/phenol-chloroform extraction method with Isogen (Nippongene). DNase treatment and a repurification step were added to remove contaminating genomic DNA. Contamination by genomic DNA was examined by direct PCR of mRNA in each sample. cDNA was synthesized from 1 µg of total RNA with random primers and Moloney murine leukemia virus transcriptase (Clontech). cDNA from 50 ng of RNA was subjected to PCR with primers for hTERT (sense, 5'-ACGGCGACATGGAGAACAA-3' and antisense, 5'-CACTGTCTTCCGCAAGTTCAC-3') and {beta}-actin (sense, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and antisense, 5'-CTAGAAGCATTGCGGTGGACGATGGAGGG-3'). After an initial denaturation for 3 min at 94°C, cDNA was amplified in a final volume of 50 µl with 2.5 units TaqDNA polymerase (AmpliTaq; PerkinElmer Life and Analytical Sciences). Amplification consisted of 25 cycles ({beta}-actin) or 30 cycles (hTERT) of 30 s at 94°C, 30 s at 60°C, and 60 s at 72°C. PCR products were separated by 2% agarose gel electrophoresis, and bands were visualized with ethidium bromide and UV translumination. Gels were photographed, and bands were analyzed by computerized densitometry.

Immunoprecipitation and immunoblotting

Cells (2 x 107) were lysed in RIPA buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 0.2 mM Na3VO4, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin for 30 min on ice. Equal amounts of cell lysates (200 µg of total protein per sample) precleared with protein G-Sepharose were incubated with appropriate Abs for 2 h or overnight. Immune complexes were separated by incubating with protein G-Sepharose beads for 1 h at 4°C. Protein G-Sepharose beads were washed extensively in ice-cold RIPA buffer and boiled in 2x Laemmli sample buffer for 10 min at 100°C. For Western blotting, cells (2 x 107) were lysed in RIPA buffer and boiled in 2x Laemmli sample buffer for 10 min at 100°C. Equal amounts of total cell lysates (25 µg of total protein per sample) were used. Proteins were resolved by 5% linear or 5–20% gradient SDS-PAGE (ATTO) and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked with buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 0.01% Tween 20, and 3% BSA. Blots were incubated with primary Abs overnight at 4°C followed by goat anti-mouse or rabbit IgG-HRP (both Ab used at 1:3000) for 60 min at room temperature. Immune complexes were visualized with ECL reagent according to the manufacturer’s protocol (Amersham Biosciences). To analyze subcellular localization of hTERT, cytosolic and nuclear fractions were isolated from 6 x 107 cells according to the method described by Dignam et al. (19). Briefly, cells were resuspended in 0.5 ml of buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM DTT). After incubation for 10 min on ice, cells were centrifuged at 1000 x g for 10 min at 4°C, and the supernatant was saved as the cytosolic fraction. The precipitate was washed twice with buffer A and homogenized with a Dounce homogenizer. The cell pellet collected by centrifugation was resuspended in 200 µl of buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 25% glycerol, 0.5 mM PMSF), homogenized with a Dounce homogenizer, and incubated with agitation at 4°C for 30 min. After centrifugation at 25,000 x g, the supernatant was saved as the nuclear fraction. Total protein (10 µg) from the cytosol and nuclear fractions was subjected to immunoblotting with Abs against hTERT, {alpha}-tubulin, or nucleolin.

Immunoprecipitation-TRAP assay

Equal amounts of cell lysates were precleared with purified preimmune mouse or rabbit IgG and protein G-agarose for 1 h at 4°C. Cell lysates were incubated with rabbit antisera against hTERT, Akt, phospho-(Ser/Thr)Akt substrate, Hsp90, mTOR, or S6K for 1 h or overnight at 4°C and then incubated with protein G-agarose for 1 h at 4°C. Agarose beads were washed five times in wash buffer containing 10 mM Tris-HCl (pH 7.5), 20% glycerol, 50 mM KCl, 5 mM MgCl2, 0.01% Nonidet P-40, 1 mM PMSF, 2 mM DTT, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Agarose beads were resuspended in 16 µl of wash buffer, and 2 µl of each immunoprecipitate was subjected to TRAP assay as described earlier.

In vitro kinase assay

Cells (2 x 107) were lysed in buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM Na3VO4, 1 mM PMSF, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerol phosphate, and 10 µg/ml leupeptin for 20 min at 4°C before centrifugation at 16,000 x g for 20 min. Equal amounts of lysates containing 200 µg of total protein were incubated overnight at 4°C with 15 µl of immobilized phospho-p44/42 MAPK (Thr202/Tyr204) mAb for ERK1/2 assay and 20 µl of immobilized Akt Ab for Akt assay (New England Biolabs). Immobilized beads were washed with lysis buffer followed by kinase buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM {beta}-glycerol phosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2. Immobilized beads were mixed with 2 µg of Elk-1 fusion protein and 1 µg of GSK-3 fusion protein (New England Biolabs) in the presence of 200 µM ATP. Kinase reactions were incubated at 30°C for 30 min and then stopped by the addition of SDS sample buffer. Samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was performed overnight at 4°C with rabbit antiphospho-Elk-1 (Ser383) for the ERK1/2 assay and rabbit antiphospho-GSK-3{alpha}/{beta} (Ser21/9) for the Akt assay (both Ab used at 1:1000) (New England Biolabs). Immune complexes were detected with the Phototope-HRP Western detection kit (New England Biolabs).

Statistics

Differences were analyzed statistically by Student’s t test, and p < 0.01 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-2 activates ERK, Akt, and telomerase in a human NK cell line

IL-2 is required for activation and proliferation of NK cells. We examined whether ERK and Akt, critical components of mitogenic and survival signals in many cell systems, are activated by IL-2 in the NK-92 cell, which was established from a patient with progressive NK cell lymphoma (15). The NK-92 cells used in the present study express high levels of surface CD2 (92.5%) and CD56 (99.8%) but not CD16, CD3, CD4, CD8, TCR{alpha}{beta}, CD19, or CD14. NK-92 cells were cytotoxic against NK-sensitive K562 cells (effector/target cells = 20:1, 43%), a feature consistent with activated CD56bright NK cells (9, 20). When NK-92 cells were cultured in the presence of 100 U/ml recombinant IL-2 after 4 days of IL-2 starvation, in vitro kinase assays revealed that ERK and Akt were activated within 30 min (Fig. 1A). Akt activation was sustained for 2 days after IL-2 stimulation, whereas ERK activity had decreased by 2 days (Fig. 1A). ERK and Akt were expressed at similar levels in NK cell lines before and after IL-2 stimulation. ERK and Akt activities were both detected in K562 control cells as previously reported (21). Because IL-2 stimulation is associated with cell cycle progression (Fig. 1B), it is possible that activation of ERK and Akt is involved in IL-2 mitogenic signaling. We next examined whether IL-2 stimulation increases telomerase activity, which is thought to be associated with the cell cycle. Cells were cultured in the absence of IL-2 for 4 days and then stimulated with IL-2. Telomerase activity was then measured at various time points by TRAP assay. As shown in Fig. 2A, telomerase activity was detected at 24 h and reached a peak by 48 h. Because telomerase activity correlates with expression of hTERT mRNA (6), we examined expression of hTERT mRNA by RT-PCR. hTERT mRNA was detectable at 4 h and higher levels were detected at 24 h after IL-2 stimulation (Fig. 2B). Our observation of full activation of telomerase activity in NK-92 cells >24 h after stimulation with IL-2 is compatible with previous findings in PBLs stimulated with mitogen (5, 12, 13, 18). Consistent with the finding of hTERT mRNA expression, we detected hTERT protein by immunoblotting at 24 h after IL-2 stimulation (Fig. 2C). The fold changes in telomerase activity as measured by TRAP assay appear to be smaller than those of hTERT mRNA and protein levels. Although the exact reason for this difference is not clear, one possibility is that the proportion of activated hTERT in total hTERT is low. Nevertheless, the level of activated hTERT after stimulation with IL-2 for 24 h was comparable to the level in Namalva control cells. Therefore, the increase in telomerase activity caused by IL-2 stimulation was associated with increased transcription and translation of hTERT.



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  		FIGURE 1. IL-2-induced activation of ERK and Akt kinase accompanied by cell cycle progression in NK-92 cells. A, NK-92 cells were cultured for 4 days in IL-2-free medium and then stimulated with 100 U/ml IL-2 for 30 min or 48 h. ERK (right panel) and Akt kinase (left panel) activities were measured with in vitro kinase assays with Elk-1 or GSK-3, respectively. Expression of ERK, Akt, and {alpha}-tubulin (loading control) in total cell lysates was analyzed by immunoblotting with rabbit-specific antisera against each protein. ERK and Akt activities were also examined in K562 cells as a positive control. The values from in vitro kinase assay were normalized to those of Akt or ERK kinase and are the mean ± SD of more than two experiments (bottom panel). B, IL-2-starved NK-92 cells (left panel) were stimulated with 100 U/ml IL-2 for 48 h (middle panel) or longer (right panel), and DNA contents were measured by flow cytometry. Cell cycle distributions were determined with WinCycle cell cycle analysis software as described in Materials and Methods.

 


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  		FIGURE 2. IL-2-induced telomerase activity and expression of hTERT mRNA and protein in NK-92 cells. IL-2-starved NK-92 cells were stimulated with 100 U/ml IL-2 for various time periods. A, Telomerase activities measured by TRAP assay at each time point. PC, positive control (Namalva cells); NC, negative control (left panel). Telomerase activity is expressed as the value relative to that of IL-2-starved cells (0 h) that was normalized to 1.0 (right panel). Data are the mean ± SD of at least three separate experiments. B, Expression of hTERT mRNA as assessed by RT-PCR (top panel). {beta}-Actin mRNA was analyzed as a positive internal control (middle panel). The level of expression of hTERT mRNA is normalized to that of {beta}-actin and is the mean ± SD of at least three separate experiments (bottom panel). C, Expression of hTERT protein as measured by immunoblotting with specific Ab against hTERT (top panel). {alpha}-Tubulin was examined as an internal control (middle panel). The level of hTERT protein expression is normalized to that of {alpha}-tubulin and is the mean ± SD of at least three separate experiments (bottom panel).

 
IL-2-induced telomerase activity is inhibited by LY294002, radicicol, and rapamycin but not PD98059

To clarify the mechanism by which IL-2 increases telomerase activity, we treated cells with various reagents that block the critical signaling pathway involved in cell proliferation and cell survival. Reagents were herbimycin A, staurosporine, LY294002, PD98059, geldanamycin, radicicol, and rapamycin, and incubation was for 1 h before stimulation with IL-2. IL-2 induction of telomerase activity was abolished in a dose-dependent manner by treatment with LY294002, a specific inhibitor of PI3K; radicicol, an inhibitor of Hsp90; and rapamycin, an inhibitor of mTOR that is a downstream effector of Akt (Fig. 3, A and B, left panel). In addition, geldanamycin, another Hsp90 inhibitor; staurosporine, an inhibitor of phospholipid/calcium-dependent protein kinases; and herbimycin A, an inhibitor of protein tyrosine kinases; also prevented IL-2-induced telomerase activity (Fig. 3, A and B, left panel). In contrast, PD98059, a specific inhibitor of MEK1/2 that blocks ERK1/2, did not affect IL-2-induced telomerase activity (Fig. 3, A and B). Cell viability was not reduced significantly by 24 h of treatment with the inhibitors used in this study (Fig. 3B, right panel).



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  		FIGURE 3. Inhibition of IL-2-induced telomerase activity and hTERT mRNA and protein expression by various reagents. IL-2-starved NK-92 cells were stimulated with 100 U/ml IL-2 for 24 h in the presence or absence of LY294002, PD98059, geldanamycin, radicicol, rapamycin, herbimycin A, and staurosporine at indicated concentrations. A, Representative example of telomerase activity as measured by TRAP assay in the presence or absence of various inhibitors is shown. Lane 1, IL-2 starvation; lane 2, IL-2 stimulation alone; lane 3, IL-2 + DMSO; lane 4, IL-2 + 50 µM PD98059; lane 5, IL-2 + 20 µM LY294002; lane 6, IL-2 + 100 nM radicicol; lane 7, IL-2 + 1 µM geldanamycin; lane 8, IL-2 + 100 nM rapamycin; lane 9, IL-2 + 5 nM staurosporine; lane 10, IL-2 + 1 µM herbimycin A; lane 11, positive control; and lane 12, negative control. B, Dose-dependent effect of various inhibitors on IL-2-induced telomerase activity (left panel). Column 1, IL-2 starvation; column 2, IL-2 stimulation alone; column 3, IL-2 + DMSO; column 4, IL-2 + 50 µM PD98059; column 5, IL-2 + 100 µM PD98059; column 6, IL-2 + 10 µM LY294002; column 7, IL-2 + 20 µM LY294002; column 8, IL-2 + 40 µM LY294002; column 9, IL-2 + 1 nM radicicol; column 10, IL-2 + 10 nM radicicol; column 11, IL-2 + 100 nM radicicol; column 12, IL-2 + 1 nM rapamycin; column 13, IL-2 + 10 nM rapamycin; column 14, IL-2 + 100 nM rapamycin; column 15, IL-2 + 1 nM staurosporine; column 16, IL-2 + 5 nM staurosporine; column 17, IL-2 + 10 nM staurosporine; column 18, IL-2 + 500 nM herbimycin A; column 19, IL-2 + 1 µM herbimycin A; column 20, IL-2 + 2 µM herbimycin A; column 21, positive control; column 22, negative control. Telomerase activity is expressed as the value relative to that of IL-2 stimulation + DMSO, which was set as 1.0. Values shown represent the mean ± SD of at least three separate experiments. Cell viability of NK-92 cells cultured in the presence of various inhibitors was evaluated by trypan blue dye exclusion (right panel). Column 1, IL-2 + DMSO; column 2, IL-2 + 50 µM PD98059; column 3, IL-2 + 100 µM PD98059; column 4, IL-2 + 10 µM LY294002; column 5, IL-2 + 20 µM LY294002; column 6, IL-2 + 40 µM LY294002; column 7, IL-2 + 1 nM radicicol; column 8, IL-2 + 10 nM radicicol; column 9, IL-2 + 100 nM radicicol; column 10, IL-2 + 1 nM rapamycin; column 11, IL-2 + 10 nM rapamycin; column 12, IL-2 + 100 nM rapamycin; column 13, IL-2 + 1 nM staurosporine; column 14, IL-2 + 5 nM staurosporine; column 15, IL-2 + 10 nM staurosporine; column 16, IL-2 + 500 nM herbimycin A; column 17, IL-2 + 1 µM herbimycin A; column 18, IL-2 + 2 µM herbimycin A. Values shown represent the mean ± SD of at least three separate experiments. C, Effects of inhibitors on expression of hTERT mRNA as determined by RT-PCR (top panel). {beta}-Actin was included as an internal control (middle panel). Expression of hTERT mRNA was quantified by normalizing the hTERT level to that of {beta}-actin (bottom panel). Values are the mean ± SD of three separate experiments. Lane 1, IL-2 stimulation alone; lane 2, IL-2 + DMSO; lane 3, IL-2 + 50 µM PD98059; lane 4, IL-2 + 20 µM LY294002; lane 5, IL-2 + 100 nM radicicol; lane 6, IL-2 + 10 nM rapamycin. D, Effect of inhibitors on expression of hTERT protein as determined by immunoblotting with Ab specific for hTERT (top panel). Expression of {alpha}-tubulin was examined as a loading control (middle panel). Expression of hTERT protein was quantified by dividing the density of hTERT by the corresponding density of {alpha}-tubulin (bottom panel). Values represent the mean ± SD of three separate experiments. Lane 1, IL-2 starvation; lane 2, IL-2 + DMSO; lane 3, IL-2 + 20 µM LY294002; lane 4, IL-2 + 100 nM radicicol; lane 5, IL-2 + 10 nM rapamycin. E, Changes in telomere length after IL-2 stimulation in the absence or presence of LY294002. Cells were either cultured for 4 days in medium without IL-2 and then stimulated with IL-2 in the presence or absence of LY294002, or they were cultured in medium without IL-2 for 24 h and then incubated with LY294002 for 24 h. Telomere length was estimated as described in Materials and Methods. Lane 1, IL-2 starvation for 4 days; lane 2, IL-2 stimulation for 24 h after IL-2 starvation for 4 days; lane 3, IL-2 stimulation for 24 h in the presence of LY294002 after IL-2 starvation for 4 days; lane 4, IL-2 starvation for 1 day; lane 5, IL-2 starvation for 1 day followed by incubation with LY294002; lane 6, IL-2 starvation for 1 day followed by incubation with DMSO. *, p < 0.01.

 
We next examined whether IL-2-induced expression of hTERT mRNA was affected by LY294002, radicicol, rapamycin, or PD98059. When cells were treated with 20 µM LY294002, IL-2-induced expression of hTERT mRNA and protein was blocked completely, suggesting that the PI3K pathway is involved in transcriptional regulation of telomerase activity (Fig. 3, C and D). Treatment of cells with 0.1 µM radicicol, 10 nM rapamycin, or 50 µM PD98059 had no effect on IL-2-induced hTERT mRNA expression (Fig. 3C), and IL-2-induced expression of hTERT protein was not influenced by treatment with radicicol or rapamycin (Fig. 3D). To determine whether IL-2-induced telomerase activity was correlated with telomere length, we examined the telomere length by Southern blotting before and after stimulation of IL-2-starved cells with IL-2. As a result, there was no difference in telomere length between IL-2-starved cells (Fig. 3E, lanes 1 and 4) and IL-2-stimulated cells (Fig. 3E, lane 2). In addition, LY294002 did not affect telomere length in either IL-2-starved cells (Fig. 3E, lane 5) or IL-2-stimulated cells (Fig. 3E, lane 3).

IL-2-induced telomerase activity is associated with Akt, Hsp90, mTOR, and S6K

IL-2-induced telomerase activity was inhibited by LY294002, radicicol, and rapamycin, indicating that the PI3K/Akt pathway, Hsp90, and mTOR/S6K pathway regulate IL-2-induced telomerase activity in NK-92 cells. To confirm the association of these molecules with telomerase, we performed TRAP assays of immunoprecipitates (IP-TRAP) with specific Abs against hTERT, Akt, phospho-(Ser/Thr)Akt substrate, Hsp90, mTOR, and S6K. IL-2-starved NK-92 cells were stimulated with IL-2 for 24 h and then lysed. When cell lysates were subjected to immunoprecipitation with rabbit Ab against hTERT followed by TRAP assay, telomerase activity was detected in the immunoprecipitates (Fig. 4A). When rabbit control IgG or rabbit ERK Ab was used for immunoprecipitation, no telomerase activity was detected in immunoprecipitates (Fig. 4A). Akt, phospho-Akt substrate, and Hsp90 immunoprecipitates contained telomerase activities that were reduced significantly by treatment of cells with 20 µM LY294002 or 0.1 µM radicicol (Fig. 4, A and B). Similarly, mTOR and S6K immunoprecipitates contained IL-2-induced telomerase activity that was inhibited significantly by treatment with 10 nM rapamycin (Fig. 4, A and B).



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  		FIGURE 4. Telomerase activity in immunoprecipitates of lysates from IL-2-stimulated NK-92 cells reacted with Abs against hTERT, Akt, Hsp90, mTOR, and S6K. IL-2-starved NK-92 cells were stimulated with 100 U/ml IL-2 for 24 h in the presence or absence of LY294002, radicicol, and rapamycin. Cell lysates were immunoprecipitated with Abs against rabbit IgG (control), ERK (control), hTERT, Akt, phospho-(Ser/Thr)Akt substrate, Hsp90, mTOR, and S6K and then subjected to TRAP assay as described in Materials and Methods. A, Representative example of at least three separate experiments is shown. Lanes 1, 3, and 5 are supernatants after immunoprecipitation and lanes 2, 4, and 6-22 are immunoprecipitates. Lane 1, IgG IL-2 stimulation+; lane 2, IgG IL-2+; lane 3, anti-hTERT IL-2–; lane 4, anti-hTERT IL-2–; lane 5, anti-hTERT IL-2+; lane 6, anti-hTERT IL-2+; lane 7, anti-Akt IL-2–; lane 8, anti-Akt IL-2+; lane 9, anti-Akt IL-2 + 20 µM LY294002; lane 10, anti-(Ser/Thr)phospho-Akt substrate IL-2–; lane 11, anti-(Ser/Thr)phospho-Akt substrate IL-2+; lane 12, anti-(Ser/Thr)phospho-Akt substrate IL-2 + 20 µM LY294002; lane 13, anti-Hsp90 IL-2–; lane 14, anti-Hsp90 IL-2+; lane 15, anti-Hsp90 IL-2 + 100 nM radicicol; lane 16, anti-mTOR IL-2–; lane 17, anti-mTOR IL-2+; lane 18, anti-mTOR IL-2 + 10 nM rapamycin; lane 19, anti-S6K IL-2–; lane 20, anti-S6K IL-2+; lane 21, anti-S6K IL-2 + 10 nM rapamycin; lane 22, anti-ERK IL-2+. B, Quantification of telomerase activity in immunoprecipitates with or without inhibitors shown in A. The relative telomerase activity of IL-2-stimulated cells in the absence of inhibitors (defined as 1.0) was compared with that in the presence of inhibitors or after IL-2 starvation. Values represent the mean ± SD for at least three separate experiments. *, p < 0.01.

 
We then examined whether hTERT forms physical complexes with Akt, Hsp90, mTOR, and S6K. NK-92 cells were stimulated with IL-2, lysed, immunoprecipitated, and immunoblotted with Abs against ERK, hTERT, Akt, phospho-(Ser/Thr)Akt substrate, Hsp90, mTOR, and S6K. When cells were stimulated with IL-2, hTERT was coimmunoprecipitated with Akt, Hsp90, mTOR, and S6K (Fig. 5A). In addition, Hsp90 was coimmunoprecipitated with Akt in IL-2-stimulated NK-92 cells (Fig. 5A). In contrast, hTERT was not coimmunoprecipitated with ERK (Fig. 5A). hTERT was detected with anti-hTERT in immunoprecipitates of anti-phospho-(Ser/Thr)Akt substrate, suggesting that hTERT is a substrate of Akt (Fig. 5A). Phosphorylation of hTERT with Akt detected by anti-phospho-(Ser/Thr)Akt Ab was abolished by LY294002, radicicol, and rapamycin (Fig. 5A). These findings suggest that hTERT forms a functional complex with Akt, Hsp90, mTOR, and S6K after IL-2 stimulation and that hTERT activity is regulated posttranslationally by the Akt/Hsp90/mTOR/S6K pathway in a NK cell line. To examine the effect of inhibitors on subcellular localization of hTERT protein in IL-2 stimulated cells, we performed immunoblotting with anti-hTERT Ab of the cytosolic and nuclear fractions before and after IL-2 stimulation. As shown in Fig. 5B, hTERT protein was detected in both cytosolic and nuclear fractions after IL-2 stimulation, whereas hTERT was not detected in the cytosol or the nucleus in IL-2-starved cells. LY294002 suppressed expression of hTERT protein in the cytosolic and nuclear fractions (Fig. 5B). In contrast, radicicol and rapamycin prevented translocation of hTERT from the cytosol to the nucleus (Fig. 5B).



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  		FIGURE 5. hTERT forms complexes with Akt, Hsp90, mTOR and S6K in IL-2-stimulated NK-92 cells. IL-2-starved NK-92 cells were either unstimulated or stimulated with 100 U/ml IL-2 for 24 h in the presence or absence of specific inhibitors, including 20 µM LY294002, 100 nM radicicol, and 10 nM rapamycin. A, Cell lysates were immunoprecipitated with either rabbit IgG (control) or Abs against ERK1/2, hTERT, Akt, phospho-(Ser/Thr)Akt substrate, Hsp90, mTOR, and S6K and then immunoblotted with the same or different Abs as indicated. B, Cells untreated or treated with inhibitors were fractionated to determine subcellular localization of hTERT. Whole cell lysates (WCL), cytosol fractions (C), and nuclear fractions (N) were subjected to immunoblotting with either anti-hTERT Ab or control Abs against {alpha}-tubulin or nucleolin.

 
Telomerase activity in primary NK cell tumors

We examined whether the same components are involved in regulation of telomerase activity in primary tumors. NK cell tumors are rare lymphoid neoplasms, and the difficulty in obtaining tumor samples from patients limits detailed experiments. We conducted IP-TRAP analyses using anti-hTERT, anti-Hsp90, and anti-mTOR Abs of a limited number of samples from two patients with NK cell lymphoma/leukemia. In both samples, we detected telomerase activity in immunoprecipitates of hTERT Abs and remaining supernatants (Fig. 6A). Interestingly, Abs against Hsp90 and mTOR coimmunoprecipitated telomerase activities, suggesting that the same components involved in up-regulation of telomerase activity in NK-92 cells are involved in regulation of telomerase activity in primary NK cell tumors (Fig. 6B).



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  		FIGURE 6. Telomerase activities in immunoprecipitates of lysates from primary NK cell tumor samples with Abs against control IgG, hTERT, Hsp90, and mTOR. A, Cell lysates from patients with NK cell tumors (patients 1 and 2) were immunoprecipitated with control IgG or hTERT antibodies. Telomerase activities in immunoprecipitates (p) or remaining supernatants (s) were measured by TRAP assay. PC, positive control; NC, negative control. B, Cell lysates from a patient with an NK cell tumor (patient 2) were immunoprecipitated with control IgG or Abs against hTERT, Hsp90, and mTOR and subjected to TRAP assay. Telomerase activity is expressed as the value relative to that of IgG immunoprecipitate, which was set at 1.0.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Lectins such as PHA, PWM, Staphylococcus aureus Cowan, and PMA plus ionomycin induce telomerase activity in PBLs, including T and B cells, but there is no detectable telomerase activity in quiescent lymphocytes (12, 13, 14, 22). In addition, cross-linking by anti-CD3, anti-CD3 plus anti-CD28, or by anti-IgM, anti-IgM plus anti-CD40 results in activation of telomerase in CD4+ T cells or IgM+ B cells, respectively (23, 24, 25). In the present study, we showed that IL-2 stimulation of NK-92 cells induces entrance into the cell cycle and telomerase activity within 24 h. Moreover, IL-2-induced telomerase activity is accompanied by expression of hTERT mRNA and hTERT protein. Thus, IL-2 stimulation causes transcriptional activation of hTERT in a human NK cell line.

IL-2 signal transduction has been investigated extensively in T cells (10, 11). In contrast, signaling pathways triggered by IL-2 in NK cells have been explored only recently. Stimulation of NK cells with IL-2 causes tyrosine phosphorylation of Jak, STAT, Syk, p120Cbl, CrkL, and Shc as well as activation of RAS, MAPK, PI3K, and transcription factors such as AP-1 or SP-1 (26, 27, 28, 29, 30). In the present study, we found that ERK and Akt are activated by IL-2 within 30 min in NK-92 cells. Because IL-2 promotes entry of NK-92 cells into the cell cycle, activation of the telomerase by IL-2 should be associated with cell proliferation. Therefore, ERK and Akt appear to participate in regulation of IL-2-induced telomerase activity in NK-92 cells. However, PD98059, a specific inhibitor of MEK1/2 that inhibits ERK1/2, does not block IL-2-induced increases in telomerase activity or expression of hTERT mRNA. Although IL-2 activates NK cell functions such as lymphokine-activated killer activity and cytokine production via ERK (31), IL-2-triggered proliferation of a mouse pro-B cell line expressing IL-2R is independent of MAPK activity (32). This is consistent with our finding that telomerase activity associated with cell proliferation is not sensitive to MEK inhibitor. In contrast, LY294002, a specific inhibitor of PI3K that can prevent PI3K-dependent activation of Akt, blocked IL-2-induced telomerase activity and expression of hTERT mRNA, indicating that the PI3K pathway plays a crucial role in transcriptional regulation of IL-2-induced activation of hTERT in NK cells. Furthermore, LY294002 inhibited existing telomerase activity in NK-92 cells after IL-2 deprivation, suggesting involvement of the PI3K pathway in the regulation of hTERT enzyme activity (data not shown). Our results are consistent with those of recent reports that NK cell survival is promoted by the PI3K/Akt pathway (33) and that estrogen induces telomerase activity through transcriptional and posttranscriptional regulation via the PI3K/Akt pathway in ovarian cancer cells (34). However, this differs from the mechanism underlying EGF-induced and progesterone-induced transactivation of hTERT mRNA expression in which EGF and progesterone activate telomerase via ERK (3, 35). Although Akt was shown to regulate telomerase activity through phosphorylation of hTERT in melanoma (8) and myeloma cells (36), telomerase activation was not accompanied by increased expression of hTERT mRNA or protein in these systems. Moreover, telomerase activation induced by anti-CD3 Ab cross-linking in T cells does not require de novo synthesis of hTERT and instead correlates with hTERT phosphorylation and nuclear translocation (37). These data indicate that induction of human telomerase activity by various stimuli is regulated in a cell-dependent manner.

We also observed that IL-2 induction of telomerase activity is blocked by geldanamycin and radicicol, which are inhibitors of Hsp90 (38). However, radicicol did not inhibit expression of hTERT mRNA and protein, suggesting that Hsp90 may be involved in posttranslational regulation of hTERT. Hsp90 is an abundant cytosolic protein that acts in concert with co-chaperones such as Hsp70 and p23 to prevent aberrant protein folding, which would lead to inactivation and aggregation (39). Proteins that interact with Hsp90 include growth factor receptors, MAPK, cyclin-dependent kinases, and anti-apoptotic kinases (39). Interestingly, Hsp90 prevents inactivation of Akt by phosphatase PP2A (40). In addition, Hsp90 was recently shown to form complexes with active human telomerase in in vitro reconstitution assays (7). Therefore, Hsp90 may act as a chaperone protein for both Akt and hTERT. Indeed, we found that telomerase activity is present in immunoprecipitates of hTERT, Akt, and Hsp90 in IL-2-stimulated NK-92 cells. Moreover, immunoprecipitation and immunoblotting assays revealed that hTERT protein interacts directly with Akt and Hsp90, which also interact with each other, in an IL-2-dependent manner. Furthermore, we showed that hTERT protein is a functional substrate that is phosphorylated by Akt kinase in an IL-2-dependent manner, as already reported in cytokine-responsive myeloma cells (36) or estrogen-responsive ovarian cancer cells (34). These data suggest that IL-2-induced telomerase activity in NK cells is not only regulated transcriptionally through the PI3K pathway but also posttranslationally through formation of a complex between Akt and Hsp90. Rapamycin, an inhibitor of mTOR, blocked telomerase activity but not expression of hTERT mRNA or protein in IL-2-stimulated NK-92 cells. Abs against mTOR and S6K coimmunoprecipitated telomerase activity and hTERT protein, indicating that mTOR and S6K interact directly with active hTERT protein in an IL-2-dependent manner. Our finding that IL-2-stimulated translocation of hTERT from cytosol to nucleus is blocked by radicicol and rapamycin supports posttranslational regulation of IL-2-induced telomerase activity via Hsp90 and mTOR in NK-92 cells. Akt activates mTOR by inhibiting tuberous sclerosis complex proteins (TSC1/2), which isolate mTOR from S6K, and mTOR in turn activates S6K by phosphorylating multiple sites in S6K (41), suggesting that hTERT is regulated posttranslationally through formation of a complex with Akt, Hsp90, mTOR, and S6K (Fig. 7). S6K could accelerate cell cycle progression by activating S6 ribosomal protein that induces translation of cell cycle-related proteins (42). Thus, formation of a complex containing hTERT and S6K might be necessary to synchronize telomere elongation with DNA replication. Importantly, we also observed involvement of Hsp90 and mTOR in human telomerase activity in primary cells from patients with NK cell lymphoma/leukemia, supporting the role of the Akt/Hsp90/mTOR/S6K signaling pathway in the up-regulation of telomerase activity observed in IL-2-stimulated NK-92 cells.



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  		FIGURE 7. Schematic representation of the activation sequence of hTERT through PI3K/Akt/Hsp90/mTOR/S6K signaling pathways after IL-2 stimulation in transformed NK cells.

 
Because telomerase activity is required to maintain the telomere and prevent replicative senescence, telomerase activity appears to be correlated with telomere length. However, in the present study, we could not detect significant shortening of telomere length accompanied by a reduction of telomerase activity in NK-92 cells that were stimulated with IL-2 in the presence of inhibitors such as LY294002. This may be partly explained by the reported findings that somatic cells show significant changes of telomere length after culture for longer periods (43) because the cells lose only 50–100 bp of the TTAGGG repeats per population doubling (44). Indeed, it was reported that long-term incubation (20 population doublings) of K562 leukemia cells with a G-quadruplex-interactive telomerase inhibitor (telomestatin) is required to produce significant shortening of telomere length (45). This indicates that considerable time is needed for telomere shortening. Therefore, down-regulation of telomerase activity by LY294002 may not have affected telomere length in NK-92 cells cultured for shorter periods in the present study. Furthermore, it was recently reported that hTERT abolishes TNF-induced apoptosis independently of telomere maintenance in a human leukemia cell line, suggesting that telomerase has an additional function other than telomere elongation (46). Thus, targeting hTERT activity may not only disrupt telomere maintenance but may also remove protection against apoptosis in tumor cells.

In summary, we found that IL-2 stimulation increases telomerase activity in a human NK cell line, NK-92. IL-2-induced telomerase activity in NK cells is regulated transcriptionally by PI3K and posttranslationally by the complex of Akt, Hsp90, mTOR, and S6K, indicating that the mechanism of telomerase regulation in NK cells differs from that in other cell types. NK cell tumors are typically resistant to chemotherapy, and the prognosis is very poor. Because telomerase activity appears to play a critical role in tumorigenesis, studies clarifying the roles of telomerase in NK cell proliferation may facilitate development of effective and selective therapies for NK cell tumors.


   Acknowledgments
 
We thank Tomohiro Watanabe for assistance in preparing the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 K.K. and O.Y. contributed equally to this study. Back

2 Address correspondence and reprint requests to Dr. Osamu Yamada, Medical Research Institute and Department of Hematology, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail address: yamadao{at}lab.twmu.ac.jp Back

3 Abbreviations used in this paper: hTERT, human telomerase reverse transcriptase; EGF, epidermal growth factor; Hsp, heat shock protein; mTOR, mammalian target of rapamycin; S6K, p70 S6 kinase; TRAP, telomerase repeat amplification protocol; IP-TRAP, immunoprecipitates-TRAP. Back

Received for publication April 12, 2004. Accepted for publication February 11, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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L. Terrin, J. Dal Col, E. Rampazzo, P. Zancai, M. Pedrotti, G. Ammirabile, S. Bergamin, S. Rizzo, R. Dolcetti, and A. De Rossi
Latent Membrane Protein 1 of Epstein-Barr Virus Activates the hTERT Promoter and Enhances Telomerase Activity in B Lymphocytes
J. Virol., October 15, 2008; 82(20): 10175 - 10187.
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 Cancer Res.Home page
U. Svenson, K. Nordfjall, B. Stegmayr, J. Manjer, P. Nilsson, B. Tavelin, R. Henriksson, P. Lenner, and G. Roos
Breast Cancer Survival Is Associated with Telomere Length in Peripheral Blood Cells
Cancer Res., May 15, 2008; 68(10): 3618 - 3623.
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 J. Leukoc. Biol.Home page
O. Yamada, K. Ozaki, M. Nakatake, M. Akiyama, K. Kawauchi, and R. Matsuoka
Multistep regulation of telomerase during differentiation of HL60 cells
J. Leukoc. Biol., May 1, 2008; 83(5): 1240 - 1248.
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K.-y. Kim, J. K. Kim, S. H. Han, J.-S. Lim, K. I. Kim, D. H. Cho, M.-S. Lee, J.-H. Lee, D.-Y. Yoon, S. R. Yoon, et al.
Adiponectin Is a Negative Regulator of NK Cell Cytotoxicity
J. Immunol., May 15, 2006; 176(10): 5958 - 5964.
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