Basic Research
Copyright ©2006 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jul 7, 2006; 12(25): 4004-4008
Published online Jul 7, 2006. doi: 10.3748/wjg.v12.i25.4004
Inhibitory effects of antisense phosphorothioate oligodeoxynucleotides on pancreatic cancer cell Bxpc-3 telomerase activity and cell growth in vitro
Yun-Feng Wang, Ke-Jian Guo, Bei-Ting Huang, Yong Liu, Xiao-Yun Tang, Jian-Jun Zhang, Qiang Xia
Yun-Feng Wang, Department of General Surgery, The Center Hospital of Shanghai Yangpu District (Branch Hospital of The Affiliated Xinhua Hospital of Shanghai Jiaotong University), Shanghai 200127, China
Jian-Jun Zhang, Qiang Xia, Center of Liver Transplantation of The Affiliated Renji Hospital of Shanghai Jiaotong University, Shanghai 200127, China
Ke-Jian Guo, Yong Liu, Department of Biliary-Pancreatic Surgery, The First Affiliated Hospital of China Medical University, Shengyang 110001, Liaoning Province, China
Bei-Ting Huang, Department of Clinical Epidemiology, Staff Room of the First Affiliated Hospital of China Medical University, Shengyang 11001, Liaoning Province, China
Xiao-Yun Tang, The First Department of The Center of Experiment and Technology, The China Medical University, Shengyang 11001, Liaoning Province, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Dr. Yun-Feng Wang, Department of General Surgery, The Center Hospital of Shanghai Yangpu District (Branch Hospital of the Affiliated Xinhua Hospital of Shanghai Jiaotong University), Shanghai 200127, China.
Telephone: +86-13370291239 Fax: +86-21-68732693
Received: May 17, 2005
Revised: July 5, 2005
Accepted: July 15, 2005
Published online: July 7, 2006


AIM: To investigate the effect of telomerase hTERT gene antisense oligonucleotide (hTERT-ASO) on proliferation and telomerase activity of pancreatic cancer cell line Bxpc-3.

METHODS: MTT assay was used to detect the effect of different doses of hTERT-ASO on proliferation of Bxpc-3 cell for different times. To study the anti-tumor activity, the cells were divided into there groups: Control group (pancreatic cancer cell Bxpc-3); antisense oligonucleotide (hTERT-ASO) group; and nosense oligonucleotide group decorated with phosphorothioate. Telomerase activity was detected using TRAP-PCR-ELISA. Cell DNA distribution was examined using flow cytometry assay. Cell apoptosis was observed by transmission electron microscope in each group.

RESULTS: After treatment with 6 mmol/L hTERT-ASO, cell proliferation was inhibited in dose- and time-dependent manner. The telomerase activity decreased after treatment with hTERT-ASO for 72 h. Flow cytometry showed the cell number of G0/G1 phase increased from 2.7% to 14.7%, the cell number of S phase decreased from 72.7% to 51.0%, and a sub-G1 stage cell apoptosis peak appeared in front of G1 stage.

CONCLUSION: Telomerase antisense oligodeoxy-nucleotide can inhibit the proliferation of pancreatic cancer cell line Bxpc-3 and decrease the telomerase activity and increase cell apoptosis rate in vitro.

Key Words: Antisense oligodeoxynucleotide, hTERT, Telomerase, Telomerase reverse transcriptase


Telomerase is a ribonucleoprotein polymerase which adds telomeric sequences onto the ends of chromosomes to compensate for DNA end replication[1]. Telomerase plays an important role in the development of cellular immortality and oncogenesis[2]. It is different from reverse transcriptase DNA polymerase of commonly pure proteins. The activated telomerase takes the 3’ distal end of telomeres as the primer and its RNA component acts as the template[3]. In human, telomerase activity has been found in embryonic development, adult germ-line tissues, immortal cell[4,5] and most malignant tumors[6,7], while there is no evident expression in normal human tissue other than in germ cell, hemopoietic stem cells and cuticle basal cells[8,9]. Human telomerase is a ribonucleo-protein complex, composed of a catalytic reverse transcriptase subunit (hTERT), an RNA component (hTR) that serves as a template for the synthesis of telomeric repeats, and an associated protein subunit (TP1)[10-12]. Previous studies have shown that telomerase activity is found in 85%-90% of all human tumors, but not in their adjacent normal cells[13,14]. This makes telomerase a good target not only for cancer diagnosis, but also for the development of novel therapeutic agents[15,16]. Among them, only the expression of hTERT mRNA is correlated with telomerase[17], and mainly regulates the expression of human telomerase enzymatic activity[18-20]. hTERT is a useful marker for telomerase activation[21,22]. It is associated with the majority of human malignant cancers. Therefore, telomerase should be the target of anti-tumor drugs research.

In this study, we aimed to investigate the inhibitory effects of antisense phosphorothioate oligodeoxynucleotides on human pancreatic cancer cell line Bxpc-3 telomerase activity and cell growth, thereby exploring the potential value of telomerase hTERT gene as a target for antisense gene therapy in pancreatic cancer.

Cell culture

Human pancreatic carcinoma cell line (Bxpc-3) was obtained from Chinese National Cancer Institute, Chinese Academy of Medical Sciences, Beijing. Cells were cultured in RPMI 1640 medium supplemented with heat-inactivated fetal bovine serum, 2 mmol/L L-glutamine, 100 kU/L

penicillin and 100 mg/L streptomycin at 37°C in a humidified CO2 incubator containing 50 mL/L CO2 and 950 mL/L air. Cells were counted using 5 g/L trypan blue staining.

Oligodeoxynucleotides synthesis

Oligodeoxynucleotides synthesis was designed as described in GenBank, and they have not the same source searched with computer. Antisense oligodeoxynucleotides (ANS-ODN) with sequence 5’-CTCAGTTAGGGTTAGACA-3’, which can recognize the RNA template region of telomerase, and nosense oligodeoxynucleotides (NNS-ODN) with sequence 5’-CATTTCTTGCTCTCCACG-3’ were prepared on the 391 DNA synthesizer, and synthesized by Huamei Engineering Company of Beijing. The synthesized oligodeoxynucleotides were subjected to polyacrylamide gel electrophoresis and purified by running at 300 V for 1.5 h.

MTT assay

Cells were seeded at 2 × 106 cells /well in a 96-well plate, 100 μL per well in three wells. The ANS-ODN and NS-ODN were added to the cultured cells with the final concentrations of 1, 2, 4, 6 μmol/L, and further cultured for 24, 48, 72, 96 h, respectively. Four hours before the end of culture, 50 μL (1 g/L) 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma) was added, which was used to assay the activity of mitochondrial dehydrogenases. Four hours later, DMSO was added to solubilize the crystal product. The plate was then incubated at 37°C for another 12 h. The absorbance at 630 nm was measured with a model 550 microplate reader (Bio-Rad). The percent of cell growth inhibition was calculated as follows: (Ac-Ae)/Ac× 100%, where Ac is the absorbance value for the controls and Ae for the experimental cells.

Flow cytometry analysis of cell cycle and apoptosis

Approximately 2 × 106 cells were harvested and washed twice with PBS, fixed with 700 mL/L alcohol for 24 h, prepared as single-cell suspension and stored at 4°C, thawed rapidly at room temperature and centrifuged to collect the cells. The cells were resuspended in a solution containing 10 g/L RNase A (833.5 μkat/g, 50 mg/L) and 20 mg/L propidium iodide (PI) for 10 min. The cell cycle distribution and apoptosis were determined by the fluorescence of individual cells measured with flow cytometry[23]. All data were analyzed using Cell Quest software.

Telomerase activity assay

Polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) was performed following the manufacturer’s instructions (Huamei Engineering Company of Beijing). Briefly, 2 × 106 cells were isolated, mixed with 50 μL lysis buffer and incubated on ice for 30 min. Supernatant was collected after centrifugation at 14 000 g for 20 min at 4°C. In an Eppendorf tube, 2 μL of the supernatant, 45 μL of reaction mixture and 30 μL of liquid olefin were added, and centrifuged for several seconds, placed in the water-bath at 25°C for 30 min, and then subjected to PCR amplification. The PCR conditions were as follows: primer elongation at 94°C for 2 min; telomerase inactivation at 94°C for 30 s; and 35 amplification cycles, each cycle consisted of denaturation at 48°C for 30 s, primer annealing at 72°C for 90 s, and extension at 72°C for 5 min. Then 50 μL of amplified product and 100 μL of hybridization reagent solution B were added and mixed; 25 μL of the mixture was distributed in the wells of a microtitering plate incubated at 37°C for 60 min. The mixture was washed at 37°C for 15 min using 100 μL of the hybridization reagent solution C, followed by addition of 25 μL of solution A and B in microtitering and incubation at 37°C for 15 min. Finally, 2 μL of stop reagent was added. The value in each well was read at the wave length of 450 nm. The negative control value was considered as 0.05. Sample’s value ≥ 2.1 times of the negative control was considered positive. Each sample was examined for more than twice. The final value was presented as mean ± SD, after analyzing with t test.

Cytologic morphology

Cytologic morphological changes were observed under the Olympus optical microscope and transmission electron microscope.

Statistical analysis

Results were expressed as follows. Statistical analyses were carried out with the software package SPSS11.0. The means of the groups were compared with repeated measures analysis of variance. P < 0.05 was considered statistically significant.

Growth arrest of Bxpc-3 cell in vivo

No significant difference in absorbance value of the control group and the NS-ODN group was observed (F = 0.013, P > 0.05), whereas a significant difference was observed compared to the Ans-hTERT group (F = 35.347, P < 0.01), thus indicating an obvious inhibition of cell growth caused by Ans-hTERT (Tables 1 and 2). This growth inhibitory effect was dose- and time-dependent (Figures 1 and 2).

Table 1 Absorbance values of the different groups (mean ± SD).
Groups24 h48 h72 h96 h
Control0.48 ± 0.100.60 ± 0.100.61 ± 0.030.65 ± 0.05
NS-ODN (6 μmol/L)0.50 ± 0.070.61 ± 0.190.60 ± 0.080.66 ± 0.19
ANS-ODN (1 μmol/L)0.60 ± 0.030.48 ± 0.060.37 ± 0.030.31 ± 0.05
ANS-ODN (2 μmol/L)0.56 ± 0.020.47 ± 0.090.34 ± 0.040.29 ± 0.03
ANS-ODN (4 μmol/L)0.51 ± 0.100.39 ± 0.080.33 ± 0.040.28 ± 0.03
ANS-ODN (6 μmol/L)0.43 ± 0.100.27 ± 0.020.26 ± 0.060.19 ± 0.00
Figure 1
Figure 1 The absorbency value was reduction, companied by the increase of ans-hTERT’s time and dose.
Table 2 Ratio of inhibition of Ans-hTERT group (mean ± SD).
ANS-ODN24 h48 h72 h96 h
1 μmol/L1.2 ± 6.419.6 ± 10.639.9 ± 4.852.0 ± 8.1
2 μmol/L2.5 ± 7.521.1 ± 15.443.9 ± 6.655.4 ± 4.6
4 μmol/L5.6 ± 9.734.6 ± 13.446.4 ± 6.157.3 ± 5.3
6 μmol/L11.4 ± 19.255.6 ± 3.657.9 ± 9.071.3 ± 0.4
Figure 2
Figure 2 The inhibition ratio was enhancement, companied by the increase of ans-hTERT’s dose and time.
Effect of antisense hTERT ODNs on induction of Bxpc-3 cells apoptosis

After incubation of the cells with 6 μmol/L ans-hTERT for 72 h, the G0/G1 stage cell had obvious difference (3.6 ± 1.6% vs 13.6±1.0%, t = 9.522, P = 0.01), and a sub-G1 stage cell apoptosis peak appeared in front of G1 stage.

Effect of antisense hTERT ODNs on telomerase activity in Bxpc-3 cells

After incubation of the cells with 6 μmol/L ans-hTERT for 72 h, a significant increased telomerase activity of ans-hTERT group was observed (1.209 ± 0.308 vs 0.447 ± 0.087, t = 4.128, P < 0.05) compared to the control group. The ratio of inhibition of the telomerase activity was 63% (Ratio of inhibition = 1 - ans-hTERT group/control group × 100%).

Change in cytologic morphology

After incubation of the cells with 6 μmol/L ans- hTERT for 72 h, the chromatin in the nucleolus was congregated, distributed in the karyotheca and appeared as half-noon type. The villus on the surface of karyotheca died away and came into being the big and small varied apophysis of vesicular shape. The cell appeared undergone apoptosis (Figure 3A and B).

Figure 3
Figure 3 Chromatin of nucleolus congregated and distributed inside karyotheca, appearing half-moon type. A: Bxpc-3 cell control group; B: ans-hTERT group.

Telomeres form the ends of eukaryotic chromosomes consisting of an array of tandem repeats of hexanucleotide 5’-TTAGGG-3’. Telomeres protect the chromosomes from DNA degradation, end-to-end fushions, rearrangements and maintain nuclear structure[24]. Human telomerase is a ribonucleoprotein complex, composed of a catalytic reverse transcriptase subunit (hTERT), an RNA component (hTR) that serves as a template for the synthesis of telomeric repeats, and an associated protein subunit (TP1)[10,12,25]. It adds telomeric repeats to the 3’ end of telomeric DNA. This telomere stabilization by telomerase can lead to unlimited cell proliferation. Therefore, inhibition of telomerase activity will be a new therapeutic modality for cancer[26-32]. In most researches, the target gene was hTR, which is the replication template for telomere, but not the regulatory region of hTERT, the most important region which regulates the activity of tolemerase. Studies have reported that anti-hTR complementary to the template region of hTR is sufficient to inhibit P3 cell telomerase activity and cell proliferation in vitro[3,33]. But the expression level of hTR has no relation with the tumor’s development. Most current studies have proposed that reactivation of telomerse is a critical step in tumorigenesis[34-38]. There is a close correlation between hTERT and telomerase activity. Several researchers, using ISH techniques, have demonstrated high levels of hTERT expression in malignant tissues but not in non-malignant tissues. Du et al[39] showed that the telomerase activity was correlated with the cell growth. Our findings also revealed a good correlation between the inhibition of telomerase activity and reduction in cell growth. However, in our study, the cell growth inhibition was mainly the result of cell cycle arrest, not the increased cell apoptosis rate.

In conclusion, growth of pancreatic cancer cell Bxpc-3 is inhibited by ans-hTERT oligodeoxynucleotides in a dose- and time-dependent manner, which is associated with the cell cycle accumulation in G0/G1 phase. The cell ratio of G0/G1 phase increased from 2.70% to 14.69%, the cell ratio of S phase decreased from 72.7% to 51.0% and a sub-G1 stage cell apoptosis peak appeared in front of G1 stage. Our results showed that ans-hTERT oligodeoxynucleotides can effectively inhibit hTERT gene expression, decrease telomerase activity, and trigger apoptosis. It is likely to raise the possibility that antitumor effect of ans-hTERT oligodeoxynucleotides occurs through following two pathways: (1) a short-term effect on apoptosis is induced rapidly by as-hTERT[40]; and (2) a long-term effect on telomerase activity is inhibited, and cell death is caused when telomere length is critically shortened by telomeric DNA. Apoptosis induction may be one of the potential mechanisms of ans-hTERT oligodeoxynucleotides-mediated inhibition for tumor cell growth. Continuous ans-hTERT oligodeoxynucleotides treatment might shorten the telomere to a size that leads to cell senescence. The treatment with ans-hTERT oligodeoxynucleotides may be a potential strategy for cancer with telomerase activity.


S- Editor Pan BR L- Editor Kumar M E- Editor Bi L

1.  Jiang YA, Luo HS, Zhang YY, Fan LF, Jiang CQ, Chen WJ. Telomerase activity and cell apoptosis in colon cancer cell by human telomerase reverse transcriptase gene antisense oligodeoxynucleotide. World J Gastroenterol. 2003;9:1981-1984.  [PubMed]  [DOI]
2.  Dalerba P, Guiducci C, Poliani PL, Cifola I, Parenza M, Frattini M, Gallino G, Carnevali I, Di Giulio I, Andreola S. Reconstitution of human telomerase reverse transcriptase expression rescues colorectal carcinoma cells from in vitro senescence: evidence against immortality as a constitutive trait of tumor cells. Cancer Res. 2005;65:2321-2329.  [PubMed]  [DOI]
3.  Zhou JH, Zhang HM, Chen Q, Han DD, Pei F, Zhang LS, Yang DT. Relationship between telomerase activity and its subunit expression and inhibitory effect of antisense hTR on pancreatic carcinoma. World J Gastroenterol. 2003;9:1808-1814.  [PubMed]  [DOI]
4.  Shen ZY, Xu LY, Li EM, Cai WJ, Chen MH, Shen J, Zeng Y. Telomere and telomerase in the initial stage of immortalization of esophageal epithelial cell. World J Gastroenterol. 2002;8:357-362.  [PubMed]  [DOI]
5.  Bailey SM, Murnane JP. Telomeres, chromosome instability and cancer. Nucleic Acids Res. 2006;34:2408-2417.  [PubMed]  [DOI]
6.  Endoh T, Tsuji N, Asanuma K, Yagihashi A, Watanabe N. Survivin enhances telomerase activity via up-regulation of specificity protein 1- and c-Myc-mediated human telomerase reverse transcriptase gene transcription. Exp Cell Res. 2005;305:300-311.  [PubMed]  [DOI]
7.  Sanders RP, Drissi R, Billups CA, Daw NC, Valentine MB, Dome JS. Telomerase expression predicts unfavorable outcome in osteosarcoma. J Clin Oncol. 2004;22:3790-3797.  [PubMed]  [DOI]
8.  Ohuchida K, Mizumoto K, Ogura Y, Ishikawa N, Nagai E, Yamaguchi K, Tanaka M. Quantitative assessment of telomerase activity and human telomerase reverse transcriptase messenger RNA levels in pancreatic juice samples for the diagnosis of pancreatic cancer. Clin Cancer Res. 2005;11:2285-2292.  [PubMed]  [DOI]
9.  Sumi M, Tauchi T, Sashida G, Nakajima A, Gotoh A, Shin-Ya K, Ohyashiki JH, Ohyashiki K. A G-quadruplex-interactive agent, telomestatin (SOT-095), induces telomere shortening with apoptosis and enhances chemosensitivity in acute myeloid leukemia. Int J Oncol. 2004;24:1481-1487.  [PubMed]  [DOI]
10.  Snow BE, Erdmann N, Cruickshank J, Goldman H, Gill RM, Robinson MO, Harrington L. Functional conservation of the telomerase protein Est1p in humans. Curr Biol. 2003;13:698-704.  [PubMed]  [DOI]
11.  Vulliamy TJ, Walne A, Baskaradas A, Mason PJ, Marrone A, Dokal I. Mutations in the reverse transcriptase component of telomerase (TERT) in patients with bone marrow failure. Blood Cells Mol Dis. 2005;34:257-263.  [PubMed]  [DOI]
12.  Liu Y, Snow BE, Kickhoefer VA, Erdmann N, Zhou W, Wakeham A, Gomez M, Rome LH, Harrington L. Vault poly(ADP-ribose) polymerase is associated with mammalian telomerase and is dispensable for telomerase function and vault structure in vivo. Mol Cell Biol. 2004;24:5314-5323.  [PubMed]  [DOI]
13.  Terasaki T, Kyo S, Takakura M, Maida Y, Tsuchiya H, Tomita K, Inoue M. Analysis of telomerase activity and telomere length in bone and soft tissue tumors. Oncol Rep. 2004;11:1307-1311.  [PubMed]  [DOI]
14.  Deschatrette J, Ng KH, Gouthière L, Maigné J, Guerroui S, Wolfrom C. Telomere dynamics determine episodes of anticancer drug resistance in rat hepatoma cells. Anticancer Drugs. 2004;15:671-678.  [PubMed]  [DOI]
15.  Pendino F, Tarkanyi I, Dudognon C, Hillion J, Lanotte M, Aradi J, Ségal-Bendirdjian E. Telomeres and telomerase: Pharmacological targets for new anticancer strategies. Curr Cancer Drug Targets. 2006;6:147-180.  [PubMed]  [DOI]
16.  Zou SQ, Qu ZL, Li ZF, Wang X. Hepatitis B virus X gene induces human telomerase reverse transcriptase mRNA expression in cultured normal human cholangiocytes. World J Gastroenterol. 2004;10:2259-2262.  [PubMed]  [DOI]
17.  Oikonomou P, Mademtzis I, Messinis I, Tsezou A. Quantitative determination of human telomerase reverse transcriptase messenger RNA expression in premalignant cervical lesions and correlation with human papillomavirus load. Hum Pathol. 2006;37:135-142.  [PubMed]  [DOI]
18.  Li C, Wu MY, Liang YR, Wu XY. Correlation between expression of human telomerase subunits and telomerase activity in esophageal squamous cell carcinoma. World J Gastroenterol. 2003;9:2395-2399.  [PubMed]  [DOI]
19.  Wang L, Wei Q, Wang LE, Aldape KD, Cao Y, Okcu MF, Hess KR, El-Zein R, Gilbert MR, Woo SY. Survival prediction in patients with glioblastoma multiforme by human telomerase genetic variation. J Clin Oncol. 2006;24:1627-1632.  [PubMed]  [DOI]
20.  Chang JT, Lu YC, Chen YJ, Tseng CP, Chen YL, Fang CW, Cheng AJ. hTERT phosphorylation by PKC is essential for telomerase holoprotein integrity and enzyme activity in head neck cancer cells. Br J Cancer. 2006;94:870-878.  [PubMed]  [DOI]
21.  Mavrommatis J, Mylona E, Gakiopoulou H, Stravodimos C, Zervas A, Giannopoulos A, Nakopoulou L. Nuclear hTERT immunohistochemical expression is associated with survival of patients with urothelial bladder cancer. Anticancer Res. 2005;25:3109-3116.  [PubMed]  [DOI]
22.  Luzar B, Poljak M, Gale N. Telomerase catalytic subunit in laryngeal carcinogenesis--an immunohistochemical study. Mod Pathol. 2005;18:406-411.  [PubMed]  [DOI]
23.  el-Awady MK, Tabll AA, Redwan el-RM, Youssef S, Omran MH, Thakeb F, el-Demellawy M. Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes: binding and entry. World J Gastroenterol. 2005;11:5203-5208.  [PubMed]  [DOI]
24.  Hsu CP, Lee LW, Shai SE, Chen CY. Clinical significance of telomerase and its associate genes expression in the maintenance of telomere length in squamous cell carcinoma of the esophagus. World J Gastroenterol. 2005;11:6941-6947.  [PubMed]  [DOI]
25.  Ting NS, Yu Y, Pohorelic B, Lees-Miller SP, Beattie TL. Human Ku70/80 interacts directly with hTR, the RNA component of human telomerase. Nucleic Acids Res. 2005;33:2090-2098.  [PubMed]  [DOI]
26.  Lankat-Buttgereit B, Hörsch D, Barth P, Arnold R, Blöcker S, Göke R. Effects of the tyrosine kinase inhibitor imatinib on neuroendocrine tumor cell growth. Digestion. 2005;71:131-140.  [PubMed]  [DOI]
27.  Jacob D, Schumacher G, Bahra M, Davis J, Zhu HB, Zhang LD, Teraishi F, Neuhaus P, Fang BL. Fiber-modified adenoviral vector expressing the tumor necrosis factor-related apoptosis-inducing ligand gene from the human telomerase reverse transcriptase promoter induces apoptosis in human hepatocellular carcinoma cells. World J Gastroenterol. 2005;11:2552-2556.  [PubMed]  [DOI]
28.  Katz MH, Spivack DE, Takimoto S, Fang B, Burton DW, Moossa AR, Hoffman RM, Bouvet M. Gene therapy of pancreatic cancer with green fluorescent protein and tumor necrosis factor-related apoptosis-inducing ligand fusion gene expression driven by a human telomerase reverse transcriptase promoter. Ann Surg Oncol. 2003;10:762-772.  [PubMed]  [DOI]
29.  Biroccio A, Leonetti C. Telomerase as a new target for the treatment of hormone-refractory prostate cancer. Endocr Relat Cancer. 2004;11:407-421.  [PubMed]  [DOI]
30.  Wong SC, Yu H, Moochhala SM, So JB. Antisense telomerase induced cell growth inhibition, cell cycle arrest and telomerase activity down-regulation in gastric and colon cancer cells. Anticancer Res. 2003;23:465-469.  [PubMed]  [DOI]
31.  Jacob D, Davis J, Schumacher G, Bahra M, Neuhaus P, Fang B. [Adenoviral vector expressing the TRAIL gene driven by the hTERT promoter]. Z Gastroenterol. 2004;42:1363-1370.  [PubMed]  [DOI]
32.  Folini M, Zaffaroni N. Targeting telomerase by antisense-based approaches: perspectives for new anti-cancer therapies. Curr Pharm Des. 2005;11:1105-1117.  [PubMed]  [DOI]
33.  Zhao JM, Li MY, Yang Z, Li Z, Zhang Y. [Cleavage of telomerase RNA component by two DNAzymes and their effects on expression of two apoptosis-related genes in human mammary cancer cells]. Di Yi Jun Yi Da Xue Xue Bao. 2005;25:638-642.  [PubMed]  [DOI]
34.  Ye J, Wu YL, Zhang S, Chen Z, Guo LX, Zhou RY, Xie H. Inhibitory effect of human telomerase antisense oligodeoxyribonucleotides on the growth of gastric cancer cell lines in variant tumor pathological subtype. World J Gastroenterol. 2005;11:2230-2237.  [PubMed]  [DOI]
35.  Hathcock KS, Jeffrey Chiang Y, Hodes RJ. In vivo regulation of telomerase activity and telomere length. Immunol Rev. 2005;205:104-113.  [PubMed]  [DOI]
36.  Hayashidani Y, Hiyama E, Murakami Y, Sueda T. Attenuation of telomerase activity by hammerhead ribozymes targeting human telomerase RNA and telomerase reverse transcriptase in pancreatic carcinoma cells. Hiroshima J Med Sci. 2005;54:21-27.  [PubMed]  [DOI]
37.  Jacob D, Davis JJ, Zhang L, Zhu H, Teraishi F, Fang B. Suppression of pancreatic tumor growth in the liver by systemic administration of the TRAIL gene driven by the hTERT promoter. Cancer Gene Ther. 2005;12:109-115.  [PubMed]  [DOI]
38.  Gulmann C, Lantuejoul S, Grace A, Leader M, Patchett S, Kay E. Telomerase activity in proximal and distal gastric neoplastic and preneoplastic lesions using immunohistochemical detection of hTERT. Dig Liver Dis. 2005;37:439-445.  [PubMed]  [DOI]
39.  Tian FJ, Wang ZY, Ma JY, Zhao YX, Lu W. [Inhibitory effect of hTERT dsRNA on telomerase activity in lung carcinoma cell line A549]. Ai Zheng. 2005;24:257-261.  [PubMed]  [DOI]
40.  Du QY, Wang XB, Chen XJ, Zheng W, Wang SQ. Antitumor mechanism of antisense cantide targeting human telomerase reverse transcriptase. World J Gastroenterol. 2003;9:2030-2035.  [PubMed]  [DOI]