Identification of two distinct elements mediating activation of telomerase (hTERT) gene expression in association with cell growth in human T cells
Yuuki Matsumura-Arioka,
Kiyoshi Ohtani,
Toshifumi Hara,
Ritsuko Iwanaga and
Masataka Nakamura
Human Gene Sciences Center, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
Correspondence to: M. Nakamura; E-mail: naka.gene{at}cmn.tmd.ac.jp
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Abstract
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Lymphocytes are exceptional among normal somatic cells in that they express high telomerase activity like germline and malignant cells. We investigated the induction of telomerase in human T cells in association with cell growth. IL-2 significantly augmented the expression of mRNA for human telomerase reverse transcriptase (hTERT), a rate-limiting component of telomerase, in PHA-activated human peripheral blood leukocytes. An isolated 5'-flanking sequence (3927+51) of the hTERT gene was examined for its promoter activity in an IL-2-dependent human T cell line Kit 225. Addition of IL-2 into quiescent Kit 225 cells induced activation of the hTERT promoter. Reporter assays with mutant fragments of the hTERT promoter further revealed that IL-2-dependent activation was independently mediated by two elements within the +9+51 regions. The two elements showed similar kinetics of activation in response to IL-2, which coincided with the G1 to S phase transition of the cell cycle. Interestingly, introduction of mutation in the elements increased background promoter activities in resting T cells in the absence of IL-2. Our results demonstrate that the hTERT promoter may be suppressed by the elements and IL-2 may signal for de-suppression in association with promotion of cell growth. IL-2-dependent activation of the hTERT promoter may be necessary for prevention from senescence induced by extraordinary cell division during immune reactions.
Keywords: gene regulation, hTERT, IL-2, T lymphocytes
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Introduction
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An extraordinary cell expansion of lymphocytes (105- to 106-fold) occurs during a typical immune response (1). The vigorous cell division is essential for effective immune response. In contrast to malignant cells (2) and cells of the germline lineage, normal somatic cells have a finite capacity for cell division. Among somatic cells, T and B cells are unique in that they seem to adopt a mechanism otherwise used by malignant cells and germline cells to extend the division capacity to achieve relevant immune reactions.
Much attention has been focused on a possible role of telomere in normal somatic cells, which determines the cell division capacity (35). Telomeres are DNAprotein complex structures comprising the physical terminus of eukaryotic linear chromosomes and its binding proteins (6). The structures are thought to contribute to the stabilization of linear chromosomes by protecting from exonucleotic degradation, irregular recombination and end-to-end fusion between chromosomes (6, 7). In humans, the telomeric DNA sequence consists of tandem TTAGGG repeats (6, 8). The length of human telomeric repeats is
515 kb (4, 9, 10) and the sequences are shortened as cells divide (35), due to the 3'-end replication problem (11, 12). The shortening of telomeres results in chromosome instability, which is closely related to cellular senescence (4). To avoid telomere shortening, transformed cells and germline cells appear to have certain compensatory mechanisms (2, 13). One such mechanism is mediated by the ribonucleoprotein enzyme telomerase, capable of synthesizing terminal TTAGGG telomeric repeats (14). Extension of telomere length by the enzyme compensates the shortening of the termini that occurs during chromosomal replication (14).
Usually telomerase activity is not undetectable (2, 3) or very low (15) in normal cells, except for continuously proliferating normal cells such as stem cells and epithelial cells (2, 1519). Human telomerase consists of two major components, human telomerase RNA component (hTERC) and human telomerase reverse transcriptase (hTERT) (2024). The template for telomeric DNA sequences, hTERC, is expressed even in normal cells negative for telomerase activity, and the expression level is not proportional to telomerase activity (25, 26). On the other hand, it has been repeatedly demonstrated that the expression level of hTERT parallels telomerase activity (21, 22). These findings indicate that hTERT expression is a rate-limiting determinant of telomerase activity in cells.
Normal human T cells have detectable telomerase activity (2729). Telomerase activity in peripheral blood T cells is profoundly enhanced by stimulation with mitogens such as PHA (3032) and phorbol myristate acetate (PMA)/ionomycin (28, 33), with anti-CD3 antibody (30) or a combination of anti-CD3 and anti-CD28 antibodies (28, 34) and with the natural antigen house dust mite (33). T cells are activated by antigenic stimulation through TCRs with CD3, which induces production of IL-2 and expression of its functional receptor (IL-2R). Stimulation with IL-2 promotes cell cycle progression, finally leading to cell division. Previous studies reported a close link between induction of telomerase activity and promotion of cell cycle progression in T cells (28, 31). Addition of IL-2 to PHA-activated T cells has been reported to facilitate telomerase activity (27, 29, 31). Collectively, IL-2 is implicated in the induction of telomerase activity in T cells during the immune response. The molecular mechanism of IL-2 stimulation of telomerase activity, however, remains to be elucidated.
In the present study, we investigated the molecular basis on expression of the hTERT gene in IL-2-stimulated T cells. Our results demonstrate that IL-2 induces expression of the hTERT gene through two distinct elements in the promoter region, implying that, in addition to promotion of cell growth signals, IL-2 signals for hTERT expression. IL-2-mediated augmentation of telomerase activity may serve to maintain telomere length to guard against attrition by repeated cell division for immune reactions.
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Methods
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Cells and cell culture
Peripheral blood lymphocytes (PBLs) were obtained from a consenting healthy adult by discontinuous density gradient sedimentation using Ficoll-Paque PLUS (Amersham Pharmacia-Biotech, Uppsala, Sweden). The cells were cultured in RPMI 1640 medium containing 20% FCS with PHA (Difco Laboratories, Detroit, MI, USA) at a concentration indicated by the protocol provided by the manufacturer. The cells were then washed twice with RPMI, cultured in RPMI containing 20% FCS without IL-2 (Ajinomoto, Yokohama, Japan) for 36 h and further cultured in the absence or presence of 1.0 nM IL-2 for 24 or 72 h. The IL-2-dependent human T cell line Kit 225 (35) was maintained in RPMI 1640 medium containing 10% FCS and 0.5 nM IL-2.
Real-time PCR
Total RNA was extracted from cell pellets using Isogen (Nippon Gene, Tokyo, Japan), and polyA RNA purification was carried out using PolyATtract (Promega, Madison, WI, USA) according to the protocols recommended by the manufacturer. Quantitative detection of mRNA for the telomerase component hTERT was performed with LightCycler (Roche Diagnostics, Mannheim, Germany) using the LightCycler Telo TAGGGhTERT Quantification Kit (Roche Diagnostics) using the procedure outlined in the kit manual. The assay was repeated three times, and the results were expressed as mean + SE.
Plasmids
The
FIXII phage clone containing the hTERT gene was kindly provided by R. Weinberg (Whitehead Institute for Biochemical Research, Cambridge, MA, USA) (22). Phage DNA was digested with EcoRI and SalI, and the 9 kb EcoRISalI fragment encompassing the 3.9 kb hTERT 5'-flanking sequence and hTERT gene was sub-cloned into pBluescriptII SK (Stratagene, La Jolla, CA, USA). To remove the hTERT coding region, the fragment was deleted from the 3'-end with ExoIII and mung bean nuclease using Exo Mung Bean Deletion Kit (Stratagene), and BglII linker was added at the 3'-end of the fragment. The resultant fragment (hTERT-L) containing 3927+51 [+1; one of transcription initiation site described in (36)] was cut with SacI and BglII, and sub-cloned into the SacIBglII sites of pGL3-Basic plasmid (Promega), generating pTERT-L. Another reporter plasmid pTERT-S harboring the 3'-deleted fragment containing 3927+8 (hTERT-S) was similarly constructed. Reconstitution of reporter plasmids by introduction of the 43-bp fragment (+9+51) in the sense and anti-sense orientations were named pTERT-S/FW43 and pTERT-S/RV43, respectively. Mutated fragments (+9+30, 31+51, +20+40, +9+11mut, +12+14mut, +15+17mut, +18+20mut, +40+42mut, +43+45mut, +46+48mut, +49+51mut and 43-bp-mutx2) derived from the +9 to +51 fragment and a 43 bp unrelated fragment (random43) were inserted at the BglII site of pTERT-S, generating pTERT-S/+9+30, pTERT-S/+31+51, pTERT-S/+20+40, pTERT-S/+9+11mut, pTERT-S/+12+14mut, pTERT-S/+15+17mut, pTERT-S/+18+20mut, pTERT-S/+40+42mut, pTERT-S/+43+45mut, pTERT-S/+46+48mut, pTERT-S/+49+51mut, pTERT-S/mutx2 and pTERT-S/random43, respectively. Nucleotide sequences of the DNA fragments of the promoter used for reporter plasmids are as follows:
- 43-bp:
- 5'-gatctGGCGCGAGTTTCAGGCAGCGCTGCGTCCTGCTGCGCACGTGGGa-3'
- 3'-aCCGCGCTCAAAGTCCGTCGCGACGCAGGACGACGCGTGCACCCtctag-5'
- +9+11mut:
- 5'-gatctATAGCGAGTTTCAGGCAGCGCTGCGTCCTGCTGCGCACGTGGGa-3'
- 3'-aTATCGCTCAAAGTCCGTCGCGACGCAGGACGACGCGTGCACCCtctag-5'
- +12+14mut:
- 5'-gatctGGCTTAAGTTTCAGGCAGCGCTGCGTCCTGCTGCGCACGTGGGa-3'
- 3'-aCCGAATTCAAAGTCCGTCGCGACGCAGGACGACGCGTGCACCCtctag-5'
- +15+17mut:
- 5'-gatctGGCGCGCTGTTCAGGCAGCGCTGCGTCCTGCTGCGCACGTGGGa-3'
- 3'-aCCGCGCGACAAGTCCGTCGCGACGCAGGACGACGCGTGCACCCtctag-5'
- +18+20mut:
- 5'-gatctGGCGCGAGTGCAAGGCAGCGCTGCGTCCTGCTGCGCACGTGGGa-3'
- 3'-aCCGCGCTCACGTTCCGTCGCGACGCAGGACGACGCGTGCACCCtctag-5'
- +40+42mut:
- 5'-gatctGGCGCGAGTTTCAGGCAGCGCTGCGTCCTGCCAAGCACGTGGGa-3'
- 3'-aCCGCGCTCAAAGTCCGTCGCGACGCAGGACGGTTCGTGCACCCtctag-5'
- +43+45mut:
- 5'-gatctGGCGCGAGTTTCAGGCAGCGCTGCGTCCTGCTGCAACCGTGGGa-3'
- 3'-aCCGCGCTCAAAGTCCGTCGCGACGCAGGACGACGTTGGCACCCtctag-5'
- +46+48mut:
- 5'-gatctGGCGCGAGTTTCAGGCAGCGCTGCGTCCTGCTGCGCAATCGGGa-3'
- 3'-aCCGCGCTCAAAGTCCGTCGCGACGCAGGACGACGCGTTAGCCCtctag-5'
- +49+51mut:
- 5'-gatctGGCGCGAGTTTCAGGCAGCGCTGCGTCCTGCTGCGCACGTATAa-3'
- 3'-aCCGCGCTCAAAGTCCGTCGCGACGCAGGACGACGCGTGCATATtctag-5'
- random43:
- 5'-gatctTGCAATCCGTAGCTACAGTCCGATCTACAGTCTAGTACCATTAa-3'
- 3'-aACGTTAGGCATCGATGTCAGGCTAGATGTCAGATCATGGTAATtctag-5'
- 43-bp-mutx2:
- 5'-gatctGGCTTAAGTTTCAGGCAGCGCTGCGTCCTGCTGCGCAATCGGGa-3'
- 3'-aCCGAATTCAAAGTCCGTCGCGACGCAGGACGACGCGTTAGCCCtctag-5'
- +9+30:
- 5'-gatctGGCGCGAGTTTCAGGCAGCGCTa-3'
- 3'-aCCGCGCTCAAAGTCCGTCGCGAtctag-5'
- +20+40:
- 5'-gatctCAGGCAGCGCTGCGTCCTGCTa-3'
- 3'-aGTCCGTCGCGACGCAGGACGAtctag-5'
- +31+51:
- 5'-gatctGCGTCCTGCTGCGCACGTGGGa-3'
- 3'-aCGCAGGACGACGCGTGCACCCtctag-5'
Underlines in +9+11mut, +12+14mut, +15+17mut, +18+20mut, +40+42mut, +43+45mut, +46+48mut, +49+51mut and 43-bp-mutx2 indicate mutant nucleotides and lower case letters show nucleotides for the BglII site for ligation.
Transfection assay
Reporter plasmids were introduced into asynchronously growing Kit 225 cells by the diethylaminoethyldextran method as described previously (37). After transfection, cells were cultured in the absence of IL-2 for 48 h to induce quiescence and then either stimulated with 1 nM IL-2 or left IL-2 starved for further 18 h. Cells were harvested and luciferase assays were performed using the Luciferase Assay System (Promega) according to the protocol recommended by the manufacturer. For kinetics study, the transfected cells were cultured in the absence of IL-2 for 48 h to induce quiescence and stimulated with 1 nM IL-2. The cells were harvested every 4 h after stimulation, and luciferase activities were determined. Luciferase activity was normalized relative to protein concentration. All assays were performed at least three times in duplicate, and the mean + SE values are presented.
Northern blot
PolyA RNA was purified from the same cell number of Kit 225 cells, transfected with either pTERT-S or pTERT-L, gel electrophoresed, transferred onto nylon membranes and hybridized as described previously (38). The probe used was a part of luciferase cDNA, the 664-bp NcoISphI fragment of pGL3-Basic (Promega). Human ribosomal phosphoprotein, large, P0 cDNA was used as a control. The membrane was exposed to an imaging plate (2040; Fujifilm, Tokyo, Japan), and analyzed with an image analyzer (BAS-1500; Fujifilm).
Cell cycle analysis
Cells were harvested, fixed with 1x FACS Lysing Solution (Becton Dickinson, Franklin Lakes, NJ, USA) for 10 min at room temperature and washed twice with PBS containing 5% BSA. The cells were re-suspend in staining solution containing propidium iodide (50 µg ml1) and RNase (50 µg ml1) for 30 min at room temperature, and analyzed for their DNA content with a FACSCalibur (Becton Dickinson) with CellQuest and Modfit software (Becton Dickinson).
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Results
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IL-2-mediated induction of hTERT mRNA in normal PBLs
Stimulation of normal human PBLs with PHA has been reported to induce telomerase activity (3032), presumably resulting from expression of the hTERT gene, which codes for the rate-limiting component of the telomerase complex. To determine whether stimulation with IL-2 induces hTERT gene expression, we used real-time PCR to measure the hTERT mRNA level of PHA-stimulated normal PBLs after treatment with IL-2. PBLs were cultured with PHA for 68 h, washed to remove PHA and further cultured in the absence of IL-2 for 36 h (PHA stimulated in Fig. 1). These cells showed a profound increase in the level of hTERT mRNA, confirming previously reported results (32, 39). Further culture without IL-2 rapidly decreased the level of hTERT mRNA with time. On the other hand, IL-2 addition kept hTERT gene expression level high and IL-2-dependent enhancement increased with time of culture, 2.0- and 8.6-fold to IL-2-starved cells at 24 and 72 h after stimulation with IL-2, respectively (Fig. 1). These results clearly demonstrate that IL-2 is a pivotal molecule involved in the induction of hTERT mRNA in T cells.

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Fig. 1. Induction of hTERT gene expression in normal PBLs. Levels of hTERT mRNA were measured with real-time PCR. PBLs were isolated from heparin-treated blood (normal), stimulated with PHA for 68 h and cultured in RPMI 1640 medium containing 20% FCS without IL-2 for 36 h (PHA stimulated). The cells were further cultured in the absence (IL-2) or presence (+IL-2) of 1.0 nM IL-2 for 24 or 72 h. Data are mean + SE.
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Activation of the hTERT promoter by IL-2 stimulation
In the next step, we examined the molecular mechanism of IL-2-induced hTERT gene expression by luciferase reporter assay. Luciferase reporter pTERT-L plasmid carrying the hTERT promoter fragment (3927+51) (hTERT-L) was transfected into the IL-2-dependent human T cell line Kit 225. We used this cell line because its growth quiescence can be established by deprivation of IL-2 without significant apoptosis, and growth promotion can be re-induced by the addition of IL-2. After transfection, Kit 225 cells were depleted of IL-2 for 48 h to induce quiescence and then either stimulated with IL-2 or left IL-2 starved for another 18 h. Kit 225 cells depleted of IL-2 showed relatively low luciferase activities (Fig. 2A). In contrast, luciferase activities increased markedly upon stimulation with IL-2 compared with IL-2-starved cells. The results indicate that the hTERT promoter fragment (3927+51) contains an element that mediates IL-2-dependent expression.

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Fig. 2. Activation of the hTERT promoter by IL-2. (A) Reporter assay of the hTERT promoter. pTERT-L and pTERT-S were transfected into Kit 225 cells, and cells were cultured in the absence of IL-2 for 48 h and then either stimulated with 1 nM IL-2 or left IL-2 starved for further 18 h. Cells were harvested for luciferase assay. Luciferase activity was normalized by protein content. Data are mean + SE. (B) Nucleotide sequence of the 5'-flanking region of the hTERT gene. Numbers on the left indicate bases upstream or downstream of the transcription start site (position +1; arrow). The start codon ATG is underlined. A putative IL-2-responsive element is shaded. An E-box is indicated by a box.
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To identify the elements involved in IL-2-dependent expression, a mutant fragment with 43 bp sequences deleted from the 3'-end was generated (Fig. 2B). The deleted region encompassed the E-box located at +44+49, which has been reported to mediate transcriptional activation by c-Myc (36, 4043) and upstream stimulatory factors (USFs) (4447). The activity of the mutant fragment (3927+8, hTERT-S) in response to IL-2 was determined by transfection into Kit 225 cells. In contrast to the pTERT-L, pTERT-S showed little, if any, increase in luciferase activity in response to IL-2 (Fig. 2A). It should be noted that pTERT-S exhibited a higher basal activity than that of pTERT-L in the absence of IL-2. These results imply that the +9+51 hTERT promoter region containing the E-box element includes the putative IL-2-responsive element that presumably functions as an element primarily suppressing the promoter activity in resting Kit 225 cells.
Orientation and sequence specificity were investigated with the isolated 43 bp (+9+51) fragment. The fragment was inserted downstream of the hTERT-S fragment in pTERT-S in the sense (pTERT-S/FW43) or anti-sense (pTERT-S/RV43) orientation. These plasmids were transfected into Kit 225 cells and reporter gene expression was examined after IL-2 stimulation. Introduction of the 43 bp fragment could restore the ability of these cells to respond to IL-2; pTERT-S/FW43 exhibited a 12.5-fold increase in luciferase activity in response to IL-2 (Fig. 3A). The 43 bp fragment might repress the high basal promoter activity demonstrated by the hTERT-S fragment in the absence of IL-2 to a level equivalent to that of the wild-type hTERT-L fragment without IL-2. The effect was independent of orientation of the fragment. A random 43 bp sequence was similarly inserted in pTERT-S, generating pTERT-S/random43. The random 43 bp fragment did not lower the luciferase activity derived from the hTERT-S fragment in the absence of IL-2, resulting in no IL-2-dependent activation (Fig. 3A). To confirm that IL-2-mediated activation occurs at the transcription level, we performed northern blot analyses using Kit 225 cells transfected with pTERT-L and pTERT-S (Fig. 3B). IL-2 treatment of Kit 225 cells transfected with pTERT-L significantly induced expression of luciferase mRNA compared with the same transfectant without IL-2, while Kit 225 cells transfected with pTERT-S showed the high level of luciferase mRNA even without IL-2 treatment (Fig. 3B).

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Fig. 3. IL-2-responsive enhancer of the hTERT promoter. (A) Responsiveness of the 43 bp region to IL-2. The 43 bp fragment was introduced into reporter plasmids with cognate promoter, generating pTERT-S/FW43 and pTERT-S/RV43. A random 43 bp sequence was also put to the same reporter plasmid (pTERT-S/random43). The plasmids, pTERT-S, pTERT-L and pTERT-S/random43 were used as controls. These plasmids were transfected into Kit 225 cells and luciferase assay was performed as described in Fig. 2(A). Data are mean + SE. (B) Northern blot analysis of IL-2-mediated promoter activation. Kit 225 cells transfected with pTERT-S or pTERT-L were used as sources for RNA extraction. mRNA was purified from the same numbers of cells transfected with pTERT-S (lanes 3 and 4), pTERT-L (lanes 5 and 6) or mock transfection (lanes 1 and 2). Cells were cultured without (lanes 1, 3 and 5) and with (lanes 2, 4 and 6) IL-2 (Genzyme) for 18 h. mRNA derived from the hTERT promoter was detected by northern blot hybridization with the luciferase gene-specific probe. As a cellular RNA-loading control, mRNA for the human ribosomal phosphoprotein, large, P0 (RPLP0) gene was monitored. mRNAs loaded were 2.58 µg at lane 1, 5.84 µg at lane 2, 2.19 µg at lane 3, 5.46 µg at lane 4, 2.26 µg at lane 5 and 5.12 µg at lane 6. Arrows a and b indicate specific bands of luciferase mRNA with a size of 2.9 kb and RPLP0 mRNA with a size of 1.3 kb, respectively.
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Involvement of two distinct sequences in IL-2-dependent activation
To gain further insights into the molecular mechanism of IL-2-dependent activation of the hTERT promoter, we designed further studies to identify the elements responsible for IL-2-dependent activation. For this purpose, three sub-fragments (+9+30, +20+40 and +31+51) of the 43 bp fragment, which partly overlap, were inserted downstream of the hTERT-S fragment in pTERT-S, generating pTERT-S/+930, pTERT-S/+20+40 and pTERT-S/+31+51, respectively. These reporter plasmids were transfected into Kit 225 cells and the luciferase activities were determined to monitor their abilities to mediate IL-2-dependent activation. pTERT-S/+9+30 and pTERT-S/+31+51 were reproducibly activated in response to IL-2 with 6.4- and 4.7-fold activation, respectively (Fig. 4A). The magnitude of the activation mediated by these sub-fragments was about a half of that induced by wild-type 43 bp fragment. Again, note that the addition of each of the sub-fragments significantly, although less than that of the 43 bp fragment, lowered the promoter activity in the absence of IL-2, thus making the promoter responsive to IL-2. Introduction of the +20+40 sub-region, pTERT-S/+20+40, did not show any significant activation upon IL-2 stimulation; the promoter activity remained high in the absence and presence of IL-2. The results demonstrate that at least two distinct sequences in the 43 bp fragment are independently involved in IL-2-induced activation of the hTERT gene promoter.

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Fig. 4. Involvement of two distinct regions in IL-2-dependent activation of the hTERT promoter. (A) Activation of two distinct regions in response to IL-2. pTERT-S, pTERT-S/FW43, pTERT-S/+9+30, pTERT-S/+20+40 and pTERT-S/+31+51 were transfected into Kit 225 cells and luciferase assay was performed as described in Fig. 2(A). Data are mean + SE. (B) Analysis of IL-2-responsive elements with mutant fragments. Reporter plasmids containing mutant fragments (+9+11mut, +12+14mut, +15+17mut, +18+20mut, +40+42mut, +43+45mut, +46+48mut, +49+51mut and 43-bp-mutx2) were transfected into Kit 225 cells and luciferase assay was performed as described in Fig. 2(A). Data are mean + SE.
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The putative functional sub-regions were further examined for their sequences and functional properties by the introduction of substitution mutation in three consecutive nucleotides. The eight mutant fragments were separately linked to pTERT-S for luciferase assays. All mutants except for a mutant at +40+42 showed distinct but low promoter activities in response to IL-2. The fold activation of these mutants was from 2.1 to 4.8, while the 43 bp fragment exhibited 7.0-fold activation (Fig. 4B). The mutant at +40+42 exerted full activation, in a similar fashion to the wild-type 43 bp fragment. Simultaneous introduction of three consecutive nucleotide substitutions at +12+14 and +46+48 (pTERT-S/mutx2) completely abolished the responsiveness to IL-2. These results are compatible with those obtained by experiments with the isolated sub-region fragments, confirming that two distinct sub-regions (+9+30 and +31+51) are responsible for IL-2-mediated activation of the hTERT promoter.
Kinetics of hTERT promoter activation by stimulation with IL-2
A link between induction of telomerase activity and entry into the cell cycle in T cells by stimulation with mitogens has been reported (28, 31). These observations prompted us to examine coordination of hTERT gene expression with cell cycle progression. To this end, the kinetics of activation of the hTERT promoter following stimulation of resting Kit 225 cells with IL-2 was studied. Cells transfected with pTERT-S and pTERT-L were depleted of IL-2 for 48 h to induce quiescence, and then stimulated with IL-2 up to 24 h. Cells were harvested every 4 h after IL-2 stimulation and luciferase activities were determined. The promoter activity of pTERT-L was up-regulated from 8 h after the addition of IL-2 and reached 13.0-fold activation at 24 h (Fig. 5A). The point at which the hTERT promoter began to be activated corresponded to the G1/S boundary of the cell cycle of Kit 225 cells stimulated with IL-2, as determined by FACS analysis (Fig. 5D). The transcription factor E2F is known to transcriptionally activate a panel of genes involved in cell cycle progression at the G1/S boundary. The kinetics of the IL-2-mediated activation of the hTERT promoter paralleled that of E2F-mediated transcriptional activation (Fig. 5C). The basal promoter activity of pTERT-S was markedly higher than that of hTERT-L (see Fig. 2A), and this high activity was maintained for at least 8 h after the addition of IL-2 (Fig. 5A). Interestingly, a non-functional mutant of the E2F binding site showed a high level of transcriptional activity like the pTERT-S (Fig. 5C).

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Fig. 5. Kinetics of IL-2-dependent activation of the hTERT promoter. (A) Time course of promoter activation of pTERT-L and pTERT-S. Reporter plasmids pTERT-L and pTERT-S were transfected into Kit 225 cells. The cells were cultured in the absence of IL-2 for 48 h and then stimulated with 1 nM IL-2. Cells were harvested every 4 h after stimulation with IL-2, and luciferase assay was performed as described in Fig. 2(A). Data are mean + SE. (B) Time course of promoter activation in pTERT-S/FW43, pTERT-S/+12+14mut, pTERT-S/+46+48mut and pTERT-S/mutx2. These reporter plasmids were transfected into Kit 225 cells. Luciferase assay was performed as described in (A). Data are mean + SE. (C) Time course of promoter activation in E2WTx4 and E2mutx4. E2WTx4 and E2mutx4 were transfected into Kit 225 cells. Luciferase was performed as described in (A). Data are mean + SE. (D) Cell cycle progression of Kit 225 cells upon IL-2 stimulation. Cells were cultured in the absence of IL-2 for 48 h and stimulated with 1 nM IL-2. After collection at every 4 h, the cells were stained with propidium iodide, and DNA content was analyzed by flow cytometry.
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Since the 43 bp fragment has two putative IL-2-responsive elements, we further examined the kinetics of activation of each element using pTERT-S/+12+14mut, pTERT-S/+46+48mut and pTERT-S/mutx2. The pTERT-S/+12+14mut and pTERT-S/+46+48mut following IL-2 stimulation activated both mutant promoters 8 h later and induced a 2.2- and 3.1-fold activation at 24 h, respectively. The basal activities of the pTERT-S/+12+14mut and pTERT-S/+46+48mut were intermediate between the pTERT-L and pTERT-S. The kinetics of pTERT-S/FW43 was similar to those of pTERT-L, while the kinetics profile of pTERT-S/mutx2 resembled that of hTERT-S fragment (Fig. 5B). These results indicate that the two elements responsible for the IL-2-mediated activation are induced independently with similar kinetics.
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Discussion
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The major finding of the present study was that IL-2 induces signals for hTERT gene expression by activation of the hTERT promoter through two distinct promoter elements, the +9+30 and +31+51 sub-regions. Our results indicate that the promoter is primarily under repression and IL-2 signals for de-repression of the promoter. This mechanism may reflect a strict control of unfavorable expression of telomerase in T cells.
PBLs stimulated with mitogenic reagents such as PHA, PMA/ionomycin and anti-CD3/CD28 antibodies have been reported to up-regulate telomerase activity (28, 3032, 34). These reagents are known to induce production of IL-2, which promotes cell cycle progression and cell growth of T cells through interaction of functional IL-2R, which is also induced by stimulation with the reagents. Our semi-quantitative measurement of hTERT mRNA level indicates that IL-2 stimulation of PHA-stimulated PBLs further up-regulates the expression of the hTERT gene. This result is consistent with the report examined by one-point reverse transcriptionPCR (39, 48). Thus, induction of hTERT gene expression by PHA seems likely to be mediated mainly by IL-2 produced by stimulation with PHA.
Experiments using endogenous and exogenous hTERT gene promoters suggest that the effect of IL-2 is mainly mediated through activation of the hTERT promoter, demonstrating that IL-2 signals for cell cycle promotion that is associated with the induction of telomerase activity to maintain adequate proliferation capacity for proper immune response.
A series of reporter assays using the hTERT promoter revealed that the 43 bp sequence (+9+51) is responsible for IL-2-dependent activation. Further examination identified two elements (+9+30 and +31+51) in the 43 bp sequence, which confer IL-2 responsiveness in a similar but independent manner, and contribute additively to each other in gene expression when present together. Sequence comparison showed no significant homology between the two elements. The 43 bp sequence contains a couple of CpG dinucleotide sequences, which are thought to be possible methylation sites (49). Methylation in promoter sequences is thought to be one of the mechanisms responsible for suppression of expression of genes. At present, the correlation between hTERT gene expression and methylation status of specific sites of the hTERT promoter seems to be dependent on cells (50).
In this context, it may be intriguing to note that the two elements function as repressors of the hTERT promoter in the absence of IL-2, and signals from IL-2R release the repression, thus making the hTERT promoter responsive to stimulation with IL-2. This may be a mechanism to secure cells from incidental expression of the hTERT gene. Once IL-2 is present, the gene is profoundly expressed to secure cells from senescence. In addition, treatment of T cells with tricostatin A has been reported to induce hTERT mRNA (51), indicating that histone deacetylation is involved in the repression of the hTERT gene expression (42, 52, 53). The mode of regulation mediated by the elements in the hTERT promoter is reminiscent of regulation of E2F-mediated expression in which E2F complexed with pRB family proteins actively suppresses target promoter activity and the addition of growth factors negates the suppression via phosphorylation of pRB family proteins by cyclin-dependent kinases, resulting in activation of the target genes (54). The similarity between the hTERT promoter and E2F-responsive promoter may raise the possibility that E2F is involved in the regulation of hTERT promoter activity.
One element (+31+51) contains an E-box (+44+49) and Myc/Max or Mad1/Max has been reported to bind to the E-box in human cell lines (43, 45, 5557). Our experiments of electrophoretic mobility shift assay showed that complex formation with the +31+51 fragment was not competed out by the consensus oligonucleotide sequence for Myc/Max and Mad1/Max, and that the addition of anti-c-Myc, anti-Mad1 or anti-Mnt1 antibodies had no or little effect on the complex formation (data not shown). Previous study has reported that USFs bind to the E-box (4547) and is involved in the regulation of hTERT promoter activity in lymphocytes (44). Our preliminary experiments similarly illustrated that anti-USF1 and anti-USF2 antibodies induced super-shift of complexes formed with the +31+51 DNA fragment and the Kit 225 nuclear extract (T. Hara, unpublished data). Another functional element, named MT-box, has been found at +46+54 and implicated in the hTERT promoter activity (56, 57). Since the 43 bp fragment does not encompass the entire MT-box, it may be possible that the MT-box is not implicated in IL-2-dependent activation. The +9+30 fragment also formed a complex with factors in the lysates from Kit 225 cells. The complex formation with the +9+30 fragment was not abolished by the addition of consensus oligonucleotides for E2F, activator protein-1-1, nuclear factor-
B, Ets/PEA3, Stat5 and Sp-1 (data not shown).
We did not observe appreciable differences between in both the fragment complexes with resting cell lysates and IL-2-stimulated cell lysates. The reason is not known at present and further experiments will be needed to clarify the factors and mechanism involved in the IL-2-mediated activation of the hTERT promoter.
The kinetics study of activation of the hTERT promoter following stimulation with IL-2 indicates that the promoter is activated at the G1/S boundary of the cell cycle. In addition, the mutants with mutation at +12+14 and +46+48, which are independently responsible for IL-2 activation, show kinetics similar to that of the +9+51 fragment. The coincidence presumably indicates that activation of hTERT gene expression by IL-2 occurs concomitantly with cell cycle progression. This notion is consistent with the previous observation that entry into the cell cycle is important for induction of telomerase activity in PBLs after stimulation with mitogens (28, 31). In this regard, expression of pRB has been reported to suppress telomerase activity in cancer cell lines, which is overcome by over-expression of E2F1 (58). Our examination of co-transfection of E2F1 expression vector along with pTERT-L, however, showed that E2F1 has scarcely any effect on hTERT promoter activity under the same conditions that allowed E2F1 activation of a typical E2F target gene promoter (our unpublished data). Other transcriptional mechanisms, which coincide with the activation of E2F, may be involved in the regulation of hTERT gene expression.
A variety of genes are known to be activated or repressed in order for the cell cycle to progress. Since activation of the hTERT promoter occurs at the G1/S boundary, activation of the hTERT promoter by IL-2 does not seem to be directly mediated through signals originating from IL-2R and the cascade of events is expected to occur before activation of the hTERT promoter. Considering that the promoter is under repression, which is presumably sustained by binding factors to the promoter elements, these events consequently relieve the repression. Related to this, the two distinct sub-regions may be expected to bind cellular factors to repress hTERT promoter function in resting cells.
In conclusion, association of telomerase activation with cell cycle progress seems reasonable to prevent T cells from senescence induced by vigorous cell proliferation during immune reactions.
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Acknowledgements
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This study was supported in part by Grants-in-Aid for Scientific Research and Cancer Research from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation. We are indebted to R. Weinberg for providing the
FIXII phage clone containing the hTERT gene and J. Hamuro for IL-2.
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Abbreviations
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hTERC | human telomerase RNA component |
hTERT | human telomerase reverse transcriptase |
PBLs | peripheral blood lymphocytes |
PMA | phorbol myristate acetate |
USF | upstream stimulatory factor |
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Notes
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Transmitting editor: K. Sugamura
Received 19 April 2004,
accepted 29 November 2004.
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References
|
---|
- Zhang, J., MacLennan, I. C., Liu, Y. J. and Lane, P. J. 1988. Is rapid proliferation in B centroblasts linked to somatic mutation in memory B cell clones? Immunol. Lett. 18:297.[CrossRef][ISI][Medline]
- Kim, N. W., Piatyszek, M. A., Prowse, K. R. et al. 1994. Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011.[ISI][Medline]
- Harley, C. B., Futcher, A. B. and Greider, C. W. 1990. Telomeres shorten during ageing of human fibroblasts. Nature 345:458.[CrossRef][ISI][Medline]
- Allsopp, R. C., Vaziri, H., Patterson, C. et al. 1992. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl Acad. Sci. USA 89:10114.[Abstract/Free Full Text]
- Allsopp, R. C., Chang, E., Kashefi-Aazam, M. et al. 1995. Telomere shortening is associated with cell division in vitro and in vivo. Exp. Cell Res. 220:194.[CrossRef][ISI][Medline]
- Blackburn, E. H. 1991. Structure and function of telomeres. Nature 350:569.[CrossRef][ISI][Medline]
- Counter, C. M., Avilion, A. A., LeFeuvre, C. E. et al. 1992. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 11:1921.[Abstract]
- Zakian, V. A. 1995. Telomeres: beginning to understand the end. Science 270:1601.[Abstract]
- Allshire, R. C., Dempster, M. and Hastie, N. D. 1989. Human telomeres contain at least three types of G-rich repeat distributed non-randomly. Nucleic Acids Res. 17:4611.[Abstract]
- de Lange, T., Shiue, L., Myers, R. M. et al. 1990. Structure and variability of human chromosome ends. Mol. Cell. Biol. 10:518.[ISI][Medline]
- Watson, J. D. 1972. Origin of concatemeric T7 DNA. Nat. New Biol. 239:197.[ISI][Medline]
- Olovnikov, A. M. 1973. A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J. Theor. Biol. 41:181.[ISI][Medline]
- Counter, C. M., Hirte, H. W., Bacchetti, S. and Harley, C. B. 1994. Telomerase activity in human ovarian carcinoma. Proc. Natl Acad. Sci. USA 91:2900.[Abstract/Free Full Text]
- Blackburn, E. H. 1992. Telomerases. Annu. Rev. Biochem. 61:113.[CrossRef][ISI][Medline]
- Masutomi, K., Yu, E. Y., Khurts, S. et al. 2003. Telomerase maintains telomere structure in normal human cells. Cell 114:241.[ISI][Medline]
- Broccoli, D., Young, J. W. and de Lange, T. 1995. Telomerase activity in normal and malignant hematopoietic cells. Proc. Natl Acad. Sci. USA 92:9082.[Abstract/Free Full Text]
- Harle-Bachor, C. and Boukamp, P. 1996. Telomerase activity in the regenerative basal layer of the epidermis in human skin and in immortal and carcinoma-derived skin keratinocytes. Proc. Natl Acad. Sci. USA 93:6476.[Abstract/Free Full Text]
- Yasumoto, S., Kunimura, C., Kikuchi, K. et al. 1996. Telomerase activity in normal human epithelial cells. Oncogene 13:433.[ISI][Medline]
- Kyo, S., Takakura, M., Kohama, T. and Inoue, M. 1997. Telomerase activity in human endometrium. Cancer Res. 57:610.[Abstract]
- Feng, J., Funk, W. D., Wang, S. S. et al. 1995. The RNA component of human telomerase. Science 269:1236.[ISI][Medline]
- Nakamura, T. M., Morin, G. B., Chapman, K. B. et al. 1997. Telomerase catalytic subunit homologs from fission yeast and human. Science 277:955.[Abstract/Free Full Text]
- Meyerson, M., Counter, C. M., Eaton, E. N. et al. 1997. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 90:785.[ISI][Medline]
- Harrington, L., Zhou, W., McPhail, T. et al. 1997. Human telomerase contains evolutionarily conserved catalytic and structural subunits. Genes Dev. 11:3109.[Abstract/Free Full Text]
- Kilian, A., Bowtell, D. D., Abud, H. E. et al. 1997. Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types. Hum. Mol. Genet. 6:2011.[Abstract/Free Full Text]
- Avilion, A. A., Piatyszek, M. A., Gupta, J. et al. 1996. Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues. Cancer Res. 56:645.[Abstract]
- Weng, N., Levine, B. L., June, C. H. and Hodes, R. J. 1997. Regulation of telomerase RNA template expression in human T lymphocyte development and activation. J. Immunol. 158:3215.[Abstract]
- Hiyama, K., Hirai, Y., Kyoizumi, S. et al. 1995. Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J. Immunol. 155:3711.[Abstract]
- Weng, N. P., Levine, B. L., June, C. H. and Hodes, R. J. 1996. Regulated expression of telomerase activity in human T lymphocyte development and activation. J. Exp. Med. 183:2471.[Abstract/Free Full Text]
- Yamada, O., Motoji, T. and Mizoguchi, H. 1996. Up-regulation of telomerase activity in human lymphocytes. Biochim. Biophys. Acta 1314:260.[ISI][Medline]
- Igarashi, H. and Sakaguchi, N. 1996. Telomerase activity is induced by the stimulation to antigen receptor in human peripheral lymphocytes. Biochem. Biophys. Res. Commun. 219:649.[CrossRef][ISI][Medline]
- Buchkovich, K. J. and Greider, C. W. 1996. Telomerase regulation during entry into the cell cycle in normal human T cells. Mol. Biol. Cell 7:1443.[Abstract]
- Kosciolek, B. A. and Rowley, P. T. 1999. Telomere-related components are coordinately synthesized during human T-lymphocyte activation. Leuk. Res. 23:1097.[CrossRef][ISI][Medline]
- Haruta, Y., Hiyama, K., Ishioka, S. et al. 1999. Activation of telomerase is induced by a natural antigen in allergen-specific memory T lymphocytes in bronchial asthma. Biochem. Biophys. Res. Commun. 259:617.[CrossRef][ISI][Medline]
- Bodnar, A. G., Kim, N. W., Effros, R. B. and Chiu, C. P. 1996. Mechanism of telomerase induction during T cell activation. Exp. Cell Res. 228:58.[CrossRef][ISI][Medline]
- Hori, T., Uchiyama, T., Tsudo, M. et al. 1987. Establishment of an interleukin 2-dependent human T cell line from a patient with T cell chronic lymphocytic leukemia who is not infected with human T cell leukemia/lymphoma virus. Blood 70:1069.[Abstract]
- Takakura, M., Kyo, S., Kanaya, T. et al. 1999. Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells. Cancer Res. 59:551.[Abstract/Free Full Text]
- Ohtani, K., Nakamura, M., Saito, S. et al. 1987. Identification of two distinct elements in the long terminal repeat of HTLV-I responsible for maximum gene expression. EMBO J. 6:389.[Abstract]
- Johnson, D. G., Schwarz, J. K., Cress, W. D. and Nevins, J. R. 1993. Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature 365:349.[CrossRef][ISI][Medline]
- Sinha-Datta, U., Horikawa, I., Michishita, E. et al. 2004. Transcriptional activation of hTERT through the NF-
B pathway in HTLV-I-transformed cells. Blood 104:2523.[Abstract/Free Full Text]
- Wu, K. J., Grandori, C., Amacker, M. et al. 1999. Direct activation of TERT transcription by c-MYC. Nat. Genet. 21:220.[CrossRef][ISI][Medline]
- Greenberg, R. A., O'Hagan, R. C., Deng, H. et al. 1999. Telomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation. Oncogene 18:1219.[CrossRef][ISI][Medline]
- Oh, S., Song, Y. H., Kim, U. J., Yim, J. and Kim, T. K. 1999. In vivo and in vitro analyses of Myc for differential promoter activities of the human telomerase (hTERT) gene in normal and tumor cells. Biochem. Biophys. Res. Commun. 263:361.[CrossRef][ISI][Medline]
- Gunes, C., Lichtsteiner, S., Vasserot, A. P. and Englert, C. 2000. Expression of the hTERT gene is regulated at the level of transcriptional initiation and repressed by Mad1. Cancer Res. 60:2116.[Abstract/Free Full Text]
- Yago, M., Ohki, R., Hatakeyama, S., Fujita, T. and Ishikawa, F. 2002. Variant forms of upstream stimulatory factors (USFs) control the promoter activity of hTERT, the human gene encoding the catalytic subunit of telomerase. FEBS Lett. 520:40.[CrossRef][ISI][Medline]
- Horikawa, I., Cable, P. L., Mazur, S. J., Appella, E., Afshari, C. A. and Barrett, J. C. 2002. Downstream E-box-mediated regulation of the human telomerase reverse transcriptase (hTERT) gene transcription: evidence for an endogenous mechanism of transcriptional repression. Mol. Biol. Cell 13:2585.[Abstract/Free Full Text]
- McMurray, H. R. and McCance, D. J. 2003. Human papillomavirus type 16 E6 activates TERT gene transcription through induction of c-Myc and release of USF-mediated repression. J. Virol. 77:9852.[Abstract/Free Full Text]
- Goueli, B. S. and Janknecht, R. 2003. Regulation of telomerase reverse transcriptase gene activity by upstream stimulatory factor. Oncogene 22:8042.[CrossRef][ISI][Medline]
- Xu, D., Erickson, S., Szeps, M. et al. 2000. Interferon alpha down-regulates telomerase reverse transcriptase and telomerase activity in human malignant and nonmalignant hematopoietic cells. Blood 96:4313.[Abstract/Free Full Text]
- Poole, J. C., Andrews, L. G. and Tollefsbol, T. O. 2001. Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT). Gene 269:1.[CrossRef][ISI][Medline]
- Devereux, T. R., Horikawa, I., Anna, C. H., Annab, L. A., Afshari, C. A. and Barrett, J. C. 1999. DNA methylation analysis of the promoter region of the human telomerase reverse transcriptase (hTERT) gene. Cancer Res. 59:6087.[Abstract/Free Full Text]
- Xu, D., Popov, N., Hou, M. et al. 2001. Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells. Proc. Natl Acad. Sci. USA 98:3826.[Abstract/Free Full Text]
- Cong, Y. S. and Bacchetti, S. 2000. Histone deacetylation is involved in the transcriptional repression of hTERT in normal human cells. J. Biol. Chem. 275:35665.[Abstract/Free Full Text]
- Takakura, M., Kyo, S., Sowa, Y. et al. 2001. Telomerase activation by histone deacetylase inhibitor in normal cells. Nucleic Acids Res. 29:3006.[Abstract/Free Full Text]
- Harbour, J. W. and Dean, D. C. 2000. The Rb/E2F pathway: expanding roles and emerging paradigms. Genes Dev. 14:2393.[Free Full Text]
- Oh, S., Song, Y. H., Yim, J. and Kim, T. K. 2000. Identification of Mad as a repressor of the human telomerase (hTERT) gene. Oncogene 19:1485.[CrossRef][ISI][Medline]
- Tzukerman, M., Shachaf, C., Ravel, Y. et al. 2000. Identification of a novel transcription factor binding element involved in the regulation by differentiation of the human telomerase (hTERT) promoter. Mol. Biol. Cell 11:4381.[Abstract/Free Full Text]
- Braunstein, I., Cohen-Barak, O., Shachaf, C. et al. 2001. Human telomerase reverse transcriptase promoter regulation in normal and malignant human ovarian epithelial cells. Cancer Res. 61:5529.[Abstract/Free Full Text]
- Crowe, D. L. and Nguyen, D. C. 2001. Rb and E2F-1 regulate telomerase activity in human cancer cells. Biochim. Biophys. Acta 1518:1.[ISI][Medline]