OBJECTIVETo explore the relationship of telomerase RNA component (hTERC) and the telomerase reverse transcriptase (hTERT) with telomerase activity in the marrow hemopoietic stem cells of children with aplastic anemia (AA).

METHODSFifty-two children with chronic AA, 13 children with acute AA and 21 normal controls were enrolled in the study. Telomerase activity and the expression of mRNA of hTERT and hTERC were detected by Telomeric Repeat Amplification Protocol (TRAP) with silver staining and real-time Q-PCR respectively.

RESULTSLevels of telomerase activity in both the chronic and acute AA groups were higher than in the control group (P<0.01). The AA groups had significantly higher expression of hTERT mRNA than the control group (P<0.01). The chronic AA group had higher expression of hTERT mRNA and telomerase activity than the acute AA group (P<0.05). There was no significant difference in the expression of hTERC mRNA among the three groups (P=0.812). There was a significant correlation between the expression of hTERT mRNA and telomerase activity (r=0.660, P<0.01).

CONCLUSIONSExpression of telomerase activity may be involved in the pathophysiology and development of AA, and hTERT plays a crucial role in expression of telomerase activity.

Adolescent ; Anemia, Aplastic ; enzymology ; Child ; Child, Preschool ; Female ; Hematopoietic Stem Cells ; enzymology ; Humans ; Infant ; Male ; RNA ; genetics ; RNA, Messenger ; analysis ; Telomerase ; genetics ; metabolism

OBJECTIVETo explore the feasibility and practical value of fluorescence in situ hybridization (FISH) detection of TERC gene amplification in cervical intraepithelial lesions (CIN) and squamous cell carcinoma (SCC).

METHODSTissue microarray was constructed to cover 150 cases of various cervical conditions, including 24 cases of normal cervical mucosa, 78 cases of CINs (CINI, 25 cases; CINII, 21 cases and CINIII, 32 cases) and 48 cases of SCC. FISH was used to detect TERC gene amplification.

RESULTSTERC gene amplification was detected in 8% (2/25) CINI, 47.6% (10/21) CINII, 71.9% (23/32) CINIII and 87.5% (42/48) SCC. There were significant differences among these groups (P < 0.05). The amplification rates of TERC gene in SCC, CINIII and CINII were significantly higher than those of normal cervical epithelium and CINI (P < 0.05). Significant differences were also observed among CINI and CINII, CINIII and SCC (P < 0.05), and between CINII and SCC (P < 0.05). There were no significant differences between normal cervical epithelium and CINI, CINII and CIN III, and between CINIII and SCC (P > 0.05). FISH detection of amplification of TERC gene in CINI and CINII-III demonstrated the following statistics: sensitivity of 62.3%, specificity of 92.0%, accuracy of 71.8%, positive and negative predictive values of 94.3% and 53.5%, respectively.

CONCLUSIONSFISH detection is a reliable method in detecting TERC gene amplification using paraffin tissue sections. When histological evaluation becomes difficult, TERC amplification detectable by FISH may offer a diagnostic distinction of CINI from CINII. Moreover, TERC amplification may be used as a biomarker in predicting CIN progression to invasive cancer.

Adenoma ; diagnosis ; genetics ; Adult ; Aged ; Biomarkers, Tumor ; analysis ; Carcinoma, Squamous Cell ; diagnosis ; genetics ; Cervical Intraepithelial Neoplasia ; diagnosis ; genetics ; Disease Progression ; Female ; Gene Amplification ; Humans ; In Situ Hybridization, Fluorescence ; Middle Aged ; RNA ; genetics ; Sensitivity and Specificity ; Telomerase ; genetics ; Uterine Cervical Neoplasms ; diagnosis ; genetics ; Young Adult

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

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

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

BACKGROUNDTelomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells. Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that plays an important role in telomerase activity. This study investigated the effect of the telomerase inhibition with an hTERT antisense oligodeoxynucleotide (ODN) in bladder cancer cells (T24) on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis.

METHODSAntisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR) assay and a gel-image system. hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by a morphological method and determined by flow cytometry.

RESULTSAS PS-ODN significantly inhibited telomerase activity and decreased the levels of hTERT mRNA which preceded the decline in the telomerase activity. AS PS-ODN significantly reduced the percentage of positive cells expressing hTERT protein following the decline of hTERT mRNA levels. There was no difference seen in the telomerase activity, hTERT mRNA expression or the protein levels between the sense phosphorothioate oligodeoxynucleotide (SPS-ODN) and the control group. AS PS-ODN treatment significantly decreased the cell viability and enhanced the apoptotic rate of T24 cells in response to TNF-alpha while there was no difference in cell viability and apoptotic rate between the S PS-ODN and the control group.

CONCLUSIONSAS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression. Treatment with AS PS-ODN may be a potential and most promising strategy for bladder cancer with telomerase activity.

Apoptosis ; Cell Line, Tumor ; Flow Cytometry ; Humans ; Oligonucleotides, Antisense ; therapeutic use ; RNA, Messenger ; analysis ; Telomerase ; analysis ; antagonists & inhibitors ; genetics ; Thionucleotides ; therapeutic use ; Tumor Necrosis Factor-alpha ; physiology ; Urinary Bladder Neoplasms ; enzymology ; pathology ; therapy

OBJECTIVETo investigate the effect of lidamycin (LDM) on telomerase activity in human hepatoma BEL-7402 cells under the condition of LDM inducing mitotic cell death and senescence.

METHODSChromatin condensation was detected by co-staining with Hoechst 33342 and PI. Cell multinucleation was observed by Giemsa staining and genomic DNA was separated by agarose gel electrophoresis. Fluorescent intensity of Rho123 was determined for mitochondrial membrane potential. MTT assay and SA-beta-gal staining were employed to analyze the senescence-like phenotype. The expression of proteins was analyzed by Western blot. Telomerase activity was assayed by telomerase PCR-ELISA.

RESULTSMitotic cell death occurred in LDM-treated cells characterized by unique and atypical chromatin condensation, multinucleation and increased mitochondrial membrane potential. However, no apoptotic bodies or DNA ladders were found. In addition, apoptosis-related proteins remained nearly unaltered. Senescence-like phenotype was identified by increased and elongated size of cells, growth retardation, enhanced SA-beta-gal activity and the changes of senescence-related protein expression. Telomerase activity markedly decreased (P<0.01) in LDM-treated hepatoma BEL-7402 cells.

CONCLUSIONMitotic cell death and senescence could be triggered simultaneously or sequentially after exposure of hepatoma BEL-7402 cells to LDM. The decrease in telomerase activity may play a key role in the defective mitosis and aging morphology. Further investigation of detailed mechanism is needed.

Aminoglycosides ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Azure Stains ; Benzimidazoles ; Carcinoma, Hepatocellular ; enzymology ; pathology ; Cell Nucleus ; drug effects ; metabolism ; Cellular Senescence ; drug effects ; Chromatin ; metabolism ; DNA, Neoplasm ; analysis ; Dose-Response Relationship, Drug ; Enediynes ; pharmacology ; Genome, Human ; genetics ; Humans ; Liver Neoplasms ; enzymology ; pathology ; Membrane Potential, Mitochondrial ; drug effects ; Mitosis ; drug effects ; Phenotype ; Propidium ; Telomerase ; metabolism ; Time Factors ; beta-Galactosidase ; metabolism

Telomere length maintenance is regarded as a fundamental step in tumorigenesis, as most human brain tumors, including meningiomas, stabilize the ends of their chromosomes using telomerase. This investigation represents an introduction to telomerase expression in canine and feline meningiomas. Twenty-five archived cases (14 dogs and 11 cats) were immunohistochemically tested for human-telomerase reverse transcriptase (h-TERT), scored, and quantified; furthermore, mitoses were counted on sections stained with a modified toluidine blue. The h-TERT antibody immunolabelled the nucleus and nucleolus of meningeal neoplastic cells, with an intensity ranging from mild to strong and a speckled distribution; a significantly higher expression in cats was noted, while no significant association between h-TERT immunolabelling and sex or histotype was evident in dogs or cats. The telomerase enzyme represents a fundamental parameter of potential malignant transformation, which may occur independently of the signal to proliferate, thereby supplying the cells with unlimited growth capabilities. Telomerase expression could be a prognostic indicator independent of the kinetic parameters, although this should be evaluated using a larger dataset with available clinical information.
Animals ; Brain Neoplasms/enzymology/pathology/*veterinary ; Cat Diseases/*enzymology/pathology ; Cats ; Dog Diseases/*enzymology/pathology ; Dogs ; Female ; Immunohistochemistry/veterinary ; Male ; Meningioma/enzymology/pathology/*veterinary ; Mitosis/physiology ; Regression Analysis ; Telomerase/*biosynthesis

Telomerase adds telomeric repeats to the ends of telomeres to compensate for their progressive loss. A favorable prognosis is associated with low or no telomerase in some tumors. The authors investigated whether telomerase activity is associated with survival of patients with brain tumors. Sixty-two consecutive patients with brain tumors underwent surgery, and their surgical specimens were investigated. The patients were pathologically categorized as group I (aggressive group) and group II (non-aggressive group). Telomerase activity was examined by the telomeric repeat amplification protocol (TRAP) assay. The median time was calculated in association with overall survival and progression-free survival in each group. The significant difference was noted in telomerase activity between high-grade gliomas and lowgrade gliomas (p=0.022). Telomerase activity was significantly associated with the median overall survival and progression-free survival in all tumors of the aggressive group. On the other hand, the median overall survival in the non-aggressive group was not dependent on telomerase activity, while the median progression-free survival was. Our data suggests that telomerase is an important prognostic indicator of survival in patients with brain tumors.
Adolescent ; Adult ; Aged ; Brain Neoplasms/enzymology/genetics/*pathology ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay/methods ; Female ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction/methods ; Prognosis ; Research Support, Non-U.S. Gov't ; Survival Analysis ; Telomerase/genetics/*metabolism

BACKGROUNDTo investigate the relationship among human papillomavirus (HPV)16 infection and the expression of telomerase catalytic protein subunit (hTERT), tumor suppressor gene p21waf1, proliferation antigen Ki67 in cervical intraepithelial neoplasias (CIN) and squamous cell carcinomas (SCC) of cervix uteri and their significance.

METHODSTissue microarray combined with in situ hybridization (ISH) and immunohistochemical staining (EliVision plus method) was used to detect the expression of HPV16 RNA, hTERT, Ki67 and p21waf1 proteins in the cervix uteri specimens from 130 subjects, including normal cervical tissue (n=26), CIN (n=46) and SCC (n=58).

RESULTS(1) The positive rate of HPV16 hybridization signals and expression of hTERT, Ki67 in CINII-III, in situ carcinoma and invasive SCC were all significantly higher than in normal cervical tissue (P < 0.05 for all), and was also higher in SCC than in CIN (P < 0.05 for all). There was a significant difference among CIN I, II and III (P < 0.05 for all). (2) The positive expression of p21waf1 protein only in SCC was significantly lower than in normal cervical tissue (P < 0.05), but there was no significant differences among other groups (all P > 0.05). (3) The positive rate of HPV16 and the expression of Ki67 showed respectively positive being correlated with the expression of hTERT (P < 0.05, r=0.339; P < 0.05, r=0.398); HPV16, hTERT and Ki67 showed respectively negative correlation with the expression of p21waf1 (P < 0.05, r=-0.337; P < 0.05, r=-0.248; P < 0.05, r=-0.446); There was no significant relation between HPV16 and Ki67 (P > 0.05).

CONCLUSIONThe results suggest that in tissues of CIN and SCC changes in hTERT, p21waf1 and Ki67 expression may be associated with HPV16 infection and they interact with each other, which can influent the progression of CIN and carcinogenesis of SCC. These biomarkers may be analyzed comprehensively to reveal the detailed mechanism by which HPV16 participate in malignant transformation and to provide some informations on the diagnosis of patients with high risk for malignant progression. Tissue microarray is an efficient platform for high-throughput analysis of genes and their expression products in investigations.

Carcinoma, Squamous Cell ; genetics ; metabolism ; virology ; Cervical Intraepithelial Neoplasia ; genetics ; metabolism ; virology ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; Host-Pathogen Interactions ; Human papillomavirus 16 ; physiology ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Ki-67 Antigen ; biosynthesis ; genetics ; Middle Aged ; Papillomavirus Infections ; genetics ; metabolism ; virology ; Peptide Fragments ; biosynthesis ; genetics ; Telomerase ; biosynthesis ; genetics ; Tissue Array Analysis ; Uterine Cervical Neoplasms ; genetics ; metabolism ; virology