To detect the expression of telomerase subunits (human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA) in gastric cancer and to examine the role that different telomerase subunits play in the gastric carcinogenesis, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect telomerase subunits messenger RNA in 24 samples of gastric cancer and corresponding non-cancerous tissue. The results showed that the positive rate of hTERT mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 25%, respectively. The former was significantly higher than the latter (chi2 = 26.4, P < 0.01). The positive rate of hTEP1 mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 91.7%, respectively and no significant difference was found between them (chi2 = 2.1, P > 0.05). The positive rates of hTR for gastric cancer and corresponding non-cancerous tissues were both 100% and no significant difference existed between them. It is concluded that in contrast to hTEP1 and hTR, the up-regulation of hTERT mRNA expression may play a more important role in the development of gastric cancer.
Carrier Proteins/biosynthesis ; Carrier Proteins/genetics ; RNA/biosynthesis ; RNA/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach Neoplasms/*metabolism ; Telomerase/*biosynthesis ; Telomerase/genetics

Total cDNA of human telomerase RNA(hTR) gene was cloned by means of RT-PCR and inverted into retroviral vector (pLNCX) to construct the mammalian cell expression plasmid. Then, by using lipofectin-mediated DNA transfection, the obtained expression plasmid was successfully transfected into human normal peripheral blood leukocyte. All data suggested that expression of transfected exogenous hTR gene can not reconstitute telomerase activity. Flow cytometry analysis and data from cell growth curve also indicated that expression of exogenous gene can not prolong the longevity of leukocyte, but rather inhibit the growth of leukocyte and induce its apoptosis. We conclude that expression of exogenous gene may block the coalition of telomerase RNA and its catalytic subunit(hTRT) and block the coalition of telomerase RNA template and telomere DNA, thus affecting telomerase activity and repressing cell proliferation.
Cells, Cultured ; Gene Expression ; Humans ; Leukocytes ; cytology ; enzymology ; metabolism ; RNA ; biosynthesis ; genetics ; Telomerase ; biosynthesis ; genetics ; Transfection

OBJECTIVETo study relation of the expression of hTERT mRNA and cyclin A, p53 protein, proliferation cell nuclear antigen (PCNA) in ameloblastoma (AB) and to investigate clinical biological characteristics of AB.

METHODSThe hTERT mRNA was detected by in situ hybridization, cyclin A, p53 protein and PCNA by SP method. Normal oral mucosa and odontogenic keratocyst (OKC) are comparition.

RESULTSThe positive ratio of hTERT mRNA was 1/7 cases in normal oral mucosa. The expression of cyclin A, p53 and PCNA in normal oral mucosa were similar, and the positive cells distributed in stratum basale. In OKC, the positive cells distributed in stratum basale and super-strrtum basale. And the positive ratio of hTERT, cyclin A, p53, PCNA in OKC was 14/16 cases, 4/32 cases, 11/25 cases, 5/9 cases, respectively. In AB, the positive ratio of hTERT mRNA, cyclin A, p53 protein and PCNA was 94.4% (51/54), 66.7% (40/60), 85.7% (42/49) and 78.1% (25/32), respectively. A strong correlation was found between hTERT mRNA with cyclin A, p53 protein and PCNA (r(s) = 0.914, 0.848, 0.804, P < 0.05).

CONCLUSIONSThe expression of hTETR mRNA, cyclin A, p53 and PCNA is different in different tissues and lesions, and correlates with cell differentiation and clinical biology behaviour. Telomerase activity is related to cumulation of p53 protein, related to cell proliferation and differentiation, regulated by cyclin A, and higher in S phase.

Ameloblastoma ; metabolism ; pathology ; Cyclin A ; biosynthesis ; Humans ; Jaw Neoplasms ; metabolism ; pathology ; Proliferating Cell Nuclear Antigen ; biosynthesis ; RNA, Messenger ; biosynthesis ; Telomerase ; biosynthesis ; genetics ; Tumor Suppressor Protein p53 ; biosynthesis

This study was purposed to investigate the inhibition of hTERT antisense oligodeoxynucleotide (ASODN) on the proliferation and telomerase activity in HL-60 cells and to explore the relativity between the telomerase activity and the expression of hTERT gene in HL-60 cells. After treated by hTERT ASODN the expression of hTERT was detected by RT-PCR, the morphological changes of HL-60 cells was observed with inverted microscopy, the cell proliferation was measured by MTT method, and the telomerase activity was determined with TRAP-ELISA and TRAP-PAGE. The results showed that after sealing hTERT gene with ASODN for 72 hours, the expression of hTERT gene was significantly inhibited, the cell growth was repressed and the ability of proliferation decreased, and the effect was specific in sequence and dependent in dose and time. OD(450-690) values were 2.648 +/- 0.42, 1.504 +/- 0.47, 1.223 +/- 0.39, 0.944 +/- 0.16 respectively, as the cells were treated with 0, 10, 20, 30 micromol/L ASODN for 72 hours. The difference was significant as compared 10, 20, 30 micromol/L groups with 0 micromol/L ASODN group respectively (P < 0.05), but the difference was no significant when compared 20 micromol/L SODN group (2.376 +/- 0.65) with untreated group (2.648 +/- 0.42) (P > 0.05). TRAP-PAGE detection revealed that comparing ASODN groups with SODN groups the telomerase image bands were decreased and least was found in groups of 30 +/- mol/L. It is concluded that the hTERT ASODN may inhibit the proliferation and down-regulate the telomerase activity in HL-60 cells by sealing the expression of hTERT gene.
Cell Proliferation ; drug effects ; HL-60 Cells ; Humans ; Oligonucleotides, Antisense ; biosynthesis ; genetics ; Telomerase ; biosynthesis ; genetics ; metabolism ; pharmacology ; Transfection

OBJECTIVETo investigate the expressions of telomerase reverse transcriptase (TRT) and vascular endothelial growth factor (VEGF) and their correlation in prostate cancer (PCa).

METHODSTRT and VEGF expressions were assayed in 30 cases of PCa and 30 cases of benign prostatic hyperplasia (BPH) by means of immunohistochemistry (SP) combined with computer assisted image analysis.

RESULTSThe expression of TRT was detected in 19 of the 30 cases of PCa and 5 of 30 cases of BPH, and that of VEGF in 23 of the 30 PCa and 14 of the 30 BPH patients. TRT and VEGF expressions were significantly higher in cancer tissues than in BPH (P < 0.05). A significant correlation was observed between TRT and VEGF expressions (r = 0.8333, P < 0.05).

CONCLUSIONThe expression of TRT or VEGF might be a malignant phenotype in PCa. The expression of TRT is significantly correlated with that of VEGF, but the mechanisms are yet to be further studied.

Animals ; Humans ; Immunohistochemistry ; Male ; Prostatic Hyperplasia ; metabolism ; pathology ; Prostatic Neoplasms ; metabolism ; pathology ; Rabbits ; Telomerase ; biosynthesis ; Vascular Endothelial Growth Factor A ; biosynthesis

Adenocarcinoma, Follicular ; genetics ; metabolism ; pathology ; Biomarkers, Tumor ; genetics ; Diagnosis, Differential ; Humans ; Matrix Metalloproteinase 1 ; biosynthesis ; genetics ; Telomerase ; biosynthesis ; genetics ; Thyroid Neoplasms ; genetics ; metabolism ; pathology ; ras Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate telomerase activity and expression of hTR and hTERT in human neuroepithelial tumors for exploring new strategy for clinical diagnosis and treatment.

METHODSTelomerase activity was detected by modified TRAP method and the expression of hTR and hTERT was measured by RT-PCR method in 65 human neuroepithelial tumors, respectively.

RESULTSThe positive rates of telomerase and hTERT were 61.54% and 70.77% respectively in human neuroepithelial tumors, and the positive rate and their level of expression were correlated with the degree of malignancy of tumors positively.

CONCLUSIONSTelomerase activity and hTERT are significantly correlated with the degree of malignancyin human neuroepithelial tumors. hTERT may play a key role in the regulation of telomerase activity.

DNA-Binding Proteins ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Humans ; Neoplasms, Neuroepithelial ; enzymology ; genetics ; Telomerase ; biosynthesis ; genetics ; metabolism

OBJECTIVETo observe the functional changes of dendritic cells (DCs) after infection by recombinant retrovirus carrying human telomerase reverse transcriptase (hTERT) gene fragment.

METHODSInterleukin-12 (IL-12) levels in DC culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). The abilities of DCs infected with recombinant retrovirus carrying hTERT gene (hTERT-DCs) and non-infected DCs (N-DCs) to stimulate allogeneic lymphocyte proliferation were evaluated with mixed leukocytes reaction (MLR), and the surface markers of DCs including CD80, CD83, CD86 and HLA-DR were detected by flow cytometry. Cytotoxic T lymphocyte (CTL) assay was performed with CytoTox 96 non-radioactive cytoxicity assay.

RESULTSCompared with N-DCs, hTERT-DCs showed no significant changes in IL-12 secretion and capacity to stimulate allogeneic lymphocytes reaction, but had significantly lower CD83 expression. Specific CTLs induced by hTERT-DCs resulted in higher cytotoxicity against telomerase-positive target cells than that against the negative target cells.

CONCLUSIONInfection with the recombinant retrovirus carrying hTERT fragment may jeopardize the maturation of DCs, which, however, still retain their capacity to activate and stimulate lymphocyte proliferation and to prime autologous T lymphocytes to generate specific CTL against hTERT.

Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; virology ; Genetic Vectors ; Humans ; Interleukin-12 ; biosynthesis ; Recombination, Genetic ; Retroviridae ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Telomerase ; biosynthesis ; genetics

This study was aimed to investigate the activation of P38MAPK/STAT3 and expression of telomerase reverse transcriptase during sodium nitroprusside (SNP) inducing apoptosis of human leukemia cell line K562 and to explore the molecular mechanisms of SNP-inducing apoptosis in K562 cells. The K562 cell were treated with different concentrations of SNP and were cultured for different time. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and Annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to quantitate the in situ cell apoptosis. The expressions of phosphorylated p38MAPK or STAT3 were analysed by flow cytometry, while the expression of hTERT mRNA in transcriptional level was measured by fluorescence quantitative RT-PCR. The results showed that SNP inhibited K562 cell growth. The K562 cell apoptosis was confirmed by typical cell morphology and DNA fragment, peak of sub-G1 phase, TUNEL and Annexin-V/PI labeling. A majority of K562 cells were arrested in G0/G1 phase. After treatment with SNP at 0.5-3.0 mmol/L, the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 increased first and decreased afterwards. Incubation of K562 cell with SNP (2 mmol/L) could increase the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 at 12 hours and 24 hours respectively, and down-regulated at 72 hours and 48 hours. SNP could decrease the expression of hTERT-mRNA in time-and dose-dependent manner. It is concluded that SNP can significantly induce K562 cells apoptosis, its mechanism may be related to the activation of P38MAPK and suppression of phosphorylated-STAT3 and hTRET-mRNA.
Apoptosis ; drug effects ; Humans ; K562 Cells ; Nitroprusside ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; STAT3 Transcription Factor ; genetics ; metabolism ; Telomerase ; biosynthesis ; genetics ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

The gene for LPTS is originally cloned as a human liver-related putative tumor suppressor (LPTS) gene that encodes a full length protein of 328 amino acids (LPTS-L). LPTS-L is also identified as a telomerase inhibitor to regulate telomere length in the cells. To facilitate the functional and structural studies of LPTS-L protein, the cDNA for LPTS-L was cloned into the expression vector pET-24 in frame to generate a recombinant plasmid pET-24-LPTS. The LPTS-L protein was expressed in E. coli BL21 solublely, and purified by Ni Sepharose affinity chromatography which, however, is not fit for large scale protein purification. The gene of LITS-L was then PCR amplified to remove the 6 x His tag, and cloned into pET-24a. The non-fusion protein of LPTS-L was expressed in E. coli B21, and purified by phosphocellulose P11 chromatography. The purity of LPTS-L protein was about 55% after that procedure,and arrived at 80% after second purification by Sephadex G-100 chromatography. Western Blotting analysis showed that the band reflects the specific binding of anti-LPTS antiserum against the purified LPTS-L protein. The TRAP assay was performed to detect the telomerase inhibitory activity of LPTS-L protein in vitro. It was observed that the purified LPTS-L inhibited the activity of telomerase greatly, similarly with that of LPTS-L protein purified by Ni Sepharose 4B. Our results suggest that phosphocellulose P11 plus Sephadex G-100 chromatography could substitute for Ni Sepharose 4B affinity chromatography for preparation of purified LPTS-L protein. Through this study, a technique for preparation of LPTS-L protein in a large scale is established.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Recombination, Genetic ; Telomerase ; antagonists & inhibitors ; Tumor Suppressor Proteins ; biosynthesis ; genetics