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

OBJECTIVETo investigate the effect of RNA interference by targeting human telomerase reverse transcriptase (hTERT) mRNA in the larynx cancer cell line, Hep-2.

METHODSThe primary structures of hTERT cDNA were found in GenBank. Then the structure analysis were done according to RNAi strategy which determined the specific base sequences to design shRNA plasmid. Two types of plasmid, pshRNA1 and pshRNA2, involved in fluorescein gene were synthesized based on the specific base sequences. Control pshRNA3, a random sequence, and control pshRNA4, without additional specific sequence were also constructed. Cells were treated daily with pshRNA1-4 or normal culture medium respectively. The pshRNA1-3 was identified by electrophoresis. After administration of pshRNA1-4, fluorescence expression was detected by confocal microscopy, the expression of hTERT of the transfected cells was determined by Western blotting, telomerase activity was measured by TRAP-PCR ELISA, cell viability was determined by MTT assay, morphological changes and apoptosis were examined by inverted microscope and TUNEL respectively.

RESULTSThere was a 400 bp balteum in pshRNA1-3 after cut by SalI, which was identical with the size of the objective gene. Many cells presented green fluorescence after being treated by pshRNA1-4, but there are much more dead green fluorescent cells in the pshRNA1 and pshRNA2 group. hTERT protein and telomerase activity was significantly decreased after treated by pshRNA1 or pshRNA2. It was observed that treatment with pshRNA1 or pshRNA2 in the presence of a valid transfection reagent could reduce cell viability of Hep-2 cells within 96 h (P < 0.01). Under the same culture conditions, cells grew more sparsely and the number of apoptotic cell increased significantly.

CONCLUSIONSshRNA plasmid directed against human telomerase reverse transcriptase can effectively transfect Hep-2 cells. shRNA targeted hTERT gene can significantly inhibit the growth and proliferation of Hep-2 cells, which results in apoptotic cell death. RNA interference may be a promising strategy for the treatment of laryngeal cancer.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Plasmids ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; Telomerase ; biosynthesis ; genetics ; Transfection

Adult ; Biomarkers, Tumor ; biosynthesis ; Carcinoma, Hepatocellular ; diagnosis ; enzymology ; DNA-Binding Proteins ; Female ; Humans ; Liver Neoplasms ; diagnosis ; enzymology ; Male ; Prognosis ; Telomerase ; biosynthesis ; genetics