OBJECTIVETo study the frequency of telomerase gene (TERC and TERT) mutation in Northern Chinese patients with acquired bone marrow failure syndromes (BMFS).

METHODSDNA extracted from blood samples of 90 patients with BMFS (including AA, MDS, and PNH) and 45 normal controls from 4 northern hospitals was collected. TERC and TERT mutation analysis was performed by PCR.

RESULTSTwo TERC mutations (n37 A-->G, and n66 G-->C) and two TERT mutations \[n1870 G-->T (E/*)\]; and \[n1780 G-->T (S/I)\] were identified in 90 BMFS patients. Among them, 3 mutations were reported for the first time. One patient with TERT mutation, however, was finally diagnosed as DKC instead of acquired AA, making the incidence of telomerase gene mutation in northern Chinese people with acquired BMFS 3.4%, similar to that of the western country people.

CONCLUSIONThe incidence of telomerase gene mutation in northern Chinese people with acquired bone marrow failure syndromes is 3.4%, similar to that of the western country people.

DNA Mutational Analysis ; Humans ; Mutation ; RNA ; genetics ; Syndrome ; Telomerase ; metabolism

Humans ; Male ; RNA ; analysis ; Telomerase ; genetics ; Testicular Neoplasms ; genetics

OBJECTIVETo investigate expression and significance of hTERT, telomerase associated-regulation protein (TRAP) and proliferating cell nuclear antigen (PCNA) in ovarian epithelial tumors.

METHODS106 specimens of ovarian epithelial tumors and their clinical history were collected, including 54 cases of malignancy, 33 borderline cases and 19 benign tumor cases. Immunohistochemical staining for hTERT, TRAP and PCNA were performed. Follow-up information was obtained for 45 of 87 cases (malignancy in 54 and borderline malignancy in 33).

RESULTSThe expression of hTERT was significantly different between benign (4/19) and borderline (90.9%, 30/33) cases, benign and malignant (94.4%, 51/54) cases (P < 0.001), as was the expression of TRAP between benign (4/15) and malignant (77.8%, 28/36) cases (P < 0.001). The expression of hTERT and TRAP was not higher in stage III, IV ovarian cancer patients than in stage I and II (P > 0.05, P > 0.3). The expression of PCNA between benign (6.9 +/- 5.9)% and borderline (26.4 +/- 17.8)% cases, benign and malignant (51.8 +/- 22.1)% cases, and borderline and malignant cases were different, and were statistically significant (P < 0.01, P < 0.001, P < 0.05). 33 cases of borderline malignancy are all survive. In 54 cases of malignancy, 35 of them have metastasis (64.8%), including 5 cases of lymph nodes metastasis. 4 of them died (7.4%).

CONCLUSIONSThe expression of hTERT and TRAP is associated with the malignant degree of ovarian cancer, but does not correlate with stage. The expression of TRAP resembles hTERT, which may be a new tumor-associated gene. Telomerase activity is positively associated with PCNA.

DNA-Binding Proteins ; Female ; Humans ; Immunohistochemistry ; Neoplasm Staging ; Ovarian Neoplasms ; enzymology ; pathology ; Proliferating Cell Nuclear Antigen ; analysis ; Telomerase ; analysis ; Transcription Factors ; analysis

This study was to investigate dynamics of biological properties of CD133(+) cells from human umbilical cord blood (UCB) during short-term culture containing the combination of hematopoietic growth factors and the feasibility of in vitro expansion of CD133(+) cells. The biology activities including analysis of cell cycle, immunophenotype, telomerase activity, expression of adhesion molecules and expansion potential of CD133(+) cells were monitored during ex-vivo expansion, and compared with those of CD34(+) cells. The results showed that the contents of CD133(+) and CD34(+) cells in fresh UCB were (1.05 +/- 0.73)% and (1.40 +/- 0.56)% respectively. About 79.62% of CD34(+) cells expressed CD133, and more than 97% of CD133(+) cells were CD133(+)CD34(+), markedly higher than that in CD34(+) fraction (P < 0.01). No significant differences were observed in content of cells expressing CD38, CD13, CD14, CD61 and glycophorin-A between the two fractions. Expansion of CD133(+), CD133(+)CD34(+) and CD34(+)CD38(-) cells at 10 days and those of CFU-mix, HPP-CFC and CD34(+)CD38(-) cells at 6 days from CD133(+) cells group were significantly higher than those from the CD34(+) cell group (P < 0.05). Analysis of immunophenotype showed that CD133(+)CD34(+) cells declined gradually while CD133(-)CD34(+) and CD133(-)CD34(-) cells increased during ex-vivo expansion; basal telomerase activities of fresh UCB CD133(+) and CD34(+) cells were low but significantly exceeded that of CD34(-) fraction (P < 0.05). At first week of expansion, telomerase activity was significantly upregulated, after two weeks, telomerase activity remarkably declined, and decreased to baseline or below the limits of detection in day 20. More than 90% of CD133(+) cells expressed CD49d and CD11a, and, more than 85% of the cells expressed CD54, about 50% of cells expressed CD62L. At the early stage of expansion, expression of CD49d was upregulated, expression of CD11a remaining no change, while as expression of CD54 and CD62L was downregulated. Expression of all adhesion molecules was decreased gradually with extend of culture. But expression of these adhesion molecules on CD34(+) subsets were not affected significantly during expansion. It is concluded that CD133(+) population may be a more primitive hematopoietic stem/progenitor cells (HSPC) than CD34(+) cells, CD133(+) cells have great expansion potential for ex-vivo expansion and is a suitable target cell for ex-vivo expansion of HSPC. Downregulation of adhesion molecules and telomerase activity may be one of the reasons for delayed engraftment of expanded products.
AC133 Antigen ; Antigens, CD ; Antigens, CD34 ; analysis ; Cell Cycle ; Cells, Cultured ; Fetal Blood ; cytology ; Glycoproteins ; analysis ; Hematopoietic Stem Cells ; physiology ; Humans ; Immunophenotyping ; Peptides ; analysis ; Telomerase ; metabolism

BACKGROUNDIn humans telomerase is expressed in most cancers and immortal cell lines, and activation of telomerase may play important roles in tumorigenesis and immortalization. This study was to investigate the roles of telomerase activity (TA) and human telomerase RNA (hTR) in sebaceous carcinoma of the eyelid.

METHODSThe telomerase repeated amplification protocol (TRAP) was used to demonstrate telomerase activity in 12 cases of sebaceous carcinoma of the eyelid. In situ hybridization (ISH) was used to demonstrate the expression of hTR in 55 cases of paraffin-embedded sebaceous carcinoma of the eyelid, and the results were compared with the proliferative index determined by Mib-1 immuno-labeling, histological patterns and recurrence of the tumor.

RESULTSDifferent telomerase activity was shown in the 12 cases of sebaceous carcinoma of the eyelid. The positive expression of hTR was 85.5% (47/55) in tumor cells, but not in the adjacent tissues. The positive expression of hTR was correlated with the proliferative activity (as assessed by Mib-1 immunolabelling, r = 0.942, P < 0.001) and the differentiation of sebaceous carcinoma of the eyelid (chi(2) = 17.621, P < 0.001), but not significantly related to tumor recurrence. The level of hTR expression increased with the decrease of differentiation of sebaceous carcinoma of the eyelid.

CONCLUSIONSThe results suggest that the up-regulation of telomerase expression plays some roles in tarsal gland carcinogenesis, and the expression of hTR is a useful marker for malignant degree of sebaceous carcinoma of the eyelid.

Biomarkers, Tumor ; analysis ; Eyelid Neoplasms ; enzymology ; pathology ; Humans ; In Situ Hybridization ; Neoplasm Recurrence, Local ; enzymology ; RNA ; analysis ; Sebaceous Gland Neoplasms ; enzymology ; pathology ; Telomerase ; analysis

This study was aimed to investigate the growth inhibition effect of diimide G-quadruplex ligand on leukemia cells and to explore its molecular mechanisms. K562 leukemia cell lines were treated with various concentrations of the diimide G-quadruples ligand small molecule (0.1 - 10 micromol/L). Trypan blue exclusion assay was used to evaluate the proliferation inhibition. Cell apoptosis was observed using terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL). Telomerase activity was analyzed by telomere repeat amplification protocol. Gene expression was detected by microarray and confirmed by RT-PCR assay. The results showed that diimide small molecule inhibited the proliferation of K562 cells and induced apoptosis of these cells. After treating with diimide G-quadruplex ligand, telomerase activity of K562 cells was reduced and the transcriptional levels of some important genes were changed significantly. These genes were involved in cell apoptosis, cell signaling pathway and other key functions. In conclusion, the diimide G-quadruplex ligand is a small molecule that inhibits the proliferation and induces apoptosis in leukemia cells, and these functions may be related to telomerase inhibition and regulation of some important gene transcription.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; G-Quadruplexes ; Humans ; K562 Cells ; Leukemia ; genetics ; pathology ; Ligands ; Microarray Analysis ; Telomerase ; metabolism

To investigate the change of telomerase activity and human telomerase reverse transcriptase (hTERT) gene expression in HL-60 cells transfected with wild type p53 gene, wild type p53 gene was introduced into HL-60 cells by Lipofectin transfection. Apoptosis was analyzed by TUNEL assay. Telomerase activity and the level of hTERT mRNA were detected by telomeric repeat amplification protocol (TRAP)-ELISA and RT-PCR, respectively. The results showed that the apoptotic rate of HL-60-pN53cG cells was 8.3% and 21.0% respectively after cultured at 32.5 degrees C for 24 h and 72 h. The level of hTERT mRNA was decreased to 68.4% and 55.8% and telomerase activity to 27.3% and 8.9% of control value in HL-60-pN53cG cells at the same points. In conclusions, hTERT mRNA and telomerase activity were down-regulated in HL-60 cells transfected with p53 gene. This may be one of mechanisms of apoptosis induced by wild type p53 gene.
Apoptosis ; DNA-Binding Proteins ; Gene Expression ; Genes, p53 ; physiology ; HL-60 Cells ; Humans ; RNA, Messenger ; analysis ; Telomerase ; genetics ; metabolism

BACKGROUND/AIMS: Telomerase activity and telomerase reverse transcriptase (TERT) expression have been proposed as a marker for malignancy. However, little is known about those markers in intestinal metaplasia (IM). This study was performed to evaluate the usefulness of telomerase activity in gastric washing fluid and TERT expression in tissue as a marker for early diagnosis of stomach cancer. METHODS: Gastric washing fluid and biopsies were taken endoscopically. We examined the telomerase activity by telomeric repeat amplification protocol (TRAP) and the TERT expression by semiquantitative reverse transcription-polymerase chain reaction in 26, 21 and 15 cases of cancer, IM, and normal mucosa respectively. RESULTS: The telomerase activity was positive in 65% of cancer, 44% of incomplete IM, and 33% of complete IM. The TERT was expressed in 89% of cancer, 81% of IM, but not in normal mucosa. The TERT expression level was higher in cancer and incomplete IM than in complete IM and normal mucosa (p<0.05). CONCLUSIONS: Telomerase activity in gastric washing fluid and TERT expression in tissue may have limited usefulness as a marker for the early diagnosis of stomach cancer. However, the increased levels of TERT expression in IM and cancer suggest that TERT expression may be associated with carcinogenesis in stomach cancer.
DNA-Binding Proteins ; Gastric Lavage ; Gastric Mucosa/enzymology ; Humans ; Metaplasia ; Precancerous Conditions/diagnosis ; Stomach/enzymology ; Stomach Neoplasms/*diagnosis/enzymology ; Telomerase/*analysis ; Tumor Markers, Biological/*analysis

To explore the effect of antisense phosphorothioate oligodeoxynucleotide (ASODN) of human telomerase reverse transcriptase (hTERT) gene on telomerase activity in CEM cells, PCR enzyme-linked immunoassay was used to determine telomerase activity. The expression levels of hTERT mRNA and protein were assayed by RT-PCR and immunofluorescence assay using fluoresce isothiocyanate label respectively. The results showed that the expression levels of hTERT mRNA and protein in CEM cells decreased with time after hTERT ASODN treatment. There was no difference in hTERT mRNA and protein levels between control and sense oligodeoxynucleotide-treated cells. Telomerase activity decreased when CEM cells were treated with ASODN for 48 hours. Telomerase activity of CEM cells was significantly inhibited when treated with ASODN for 72 hours. There was no difference in telomerase activity levels between control and hTERT sense oligodeoxynucleotide-treated cells. These results suggested that hTERT ASODN inhibited telomerase activity of CEM cells.
Cell Division ; drug effects ; Cell Line ; DNA-Binding Proteins ; Flow Cytometry ; Humans ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; Telomerase ; analysis ; genetics ; metabolism

UNLABELLEDBACKGROUND AND OBJECTIVE Telomerase and CYFRA21-1 may be positively expressed in malignant pleural effusion, but the sensitivity and specificity of single tumor marker were low. The aim of this study is to investigate the diagnostic value of combining determination of telomerase activity and CYFRA21-1 levels in differentiating benign from malignant pleural effusion caused by lung cancer.

METHODS80 patients with malignant and 50 patients with benign pleural effusion were enrolled into this study. The telomerase activity in pleural effusion was tested by means of telomeric repeat amplification protocal-PCR-ELISA (TRAP-PCR-ELISA) and CYFRA21-1 levels were tested by the EIA method. All the results were analyzed by the statistical method.

RESULTSThe levels of telomerase and CYFRA21-1 in malignant pleural effusion was significantly higher than that in benign one (t = 17.252 and t = 13.951, P < 0.001). The sensitivity of telomerase activity testing for diagnosing malignant pleural effusion was 71.3%; the specificity was 86.0% and the overall accuracy was 76.9%. The sensitivity of CYFRA 21-1 testing was 60.0%, the specificity was 78.0% and the overall accuracy was 66.9%. The sensitivity of the combined testing was 90.0%, the specificity was 76.0% and the overall accuracy was 86.9%. The sensitivity and the overall accuracy of combined testing were higher than those of telomerase and CYFRA21-1 testing single (chi2 = 9.002 and chi2 = 19.201, P < 0.01; chi2 = 4.389 and chi2 = 14.647, P < 0.05).

CONCLUSIONThe combined testing oftelomerase with CYFRA21-1 can increase the sensitivity and overall accuracy of differential diagnosis of benign and malignant pleural effusion diagnosis.

Aged ; Antigens, Neoplasm ; analysis ; Diagnosis, Differential ; Female ; Humans ; Keratin-19 ; analysis ; Male ; Middle Aged ; Pleural Effusion ; diagnosis ; Pleural Effusion, Malignant ; diagnosis ; Telomerase ; metabolism