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

Telomerase reverse transcriptase (TERT) is the protein component of telomerase and combined with an RNA molecule, telomerase RNA component, forms the telomerase enzyme responsible for telomere elongation. Telomerase is essential for maintaining telomere length from replicative attrition and thus contributes to the preservation of genome integrity. Although diverse mouse models have been developed and studied to prove the physiological roles of telomerase as a telomere-elongating enzyme, recent studies have revealed non-canonical TERT activities beyond telomeres. To gain insights into the physiological impact of extra-telomeric roles, this review revisits the strategies and phenotypes of telomerase mouse models in terms of the extra-telomeric functions of telomerase.
Animals ; Mice ; Mice, Knockout ; Telomerase/genetics/*metabolism ; Telomere/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

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

The purpose of this study was to construct a recombinant conditionally replicating adenovirus (CRAd) expressing programmed cell death 5 (pdcd5). Pdcd5 gene was inserted in the E3 region of SG600-a CRAd in which the key genes for virus replication E1a and E1b were controlled under the human telomerase reverse transcriptase promoter (hTERTp) and the hypoxia response element (HRE) respectively, and with a deletion of 24 nucleotides within CR2 region of E1a. The insertion and orientation of all recombined plasmids were confirmed by restriction enzyme digestion and polymerase chain reaction (PCR). The infection efficiencies of a recombined virus carrying enhanced green fluorescent protein (EGFP) in leukemic cell lines were observed by using fluorescence microscope. The relative pdcd5 expression levels of K562 after being infected with SG611-pdcd5 were detected by real-time quantitative PCR. The results showed that the construction of SG611-pdcd5 was completed and confirmed. Pdcd5, hTERTp, HRE, skeleton and fiber11 of recombinant adenovirus SG611-pdcd5 were successfully amplified. The infection efficiencies of SG611-EGFP were all above 70% in both leukemic K562 and MEG-01 cell lines. SG611-pdcd5 expressed pdcd5 with high efficiency in leukemic cells as compared with Ad-pdcd5 or SG611 (p < 0.001). The expression level of pdcd5 increased gradually along with the increase of MOI. It is concluded that the triple-regulated adenovirus of SG611-pdcd5 containing the pro-apopro-tic gene pdcd5 has been successfully established with high pdcd5 expression level in leukemic cells, indicating that the recombinant adenovirus, SG611-pdcd5, promises further development of targeted tumor gene therapy.
Adenoviridae ; genetics ; Apoptosis Regulatory Proteins ; genetics ; Genetic Therapy ; methods ; Neoplasm Proteins ; genetics ; Oncolytic Viruses ; genetics ; Promoter Regions, Genetic ; Telomerase ; genetics

OBJECTIVETo investigate the variety of proliferating ability of umbilical endothelia (UE) transfected by plasmid pBABE-HYGR-hTERT.

METHODSUE was identified from two aspects: morphology and CD34 labeling technique. The plasmid was obtained and identified by alkali splitting and gel electrophoresis. Liposomes were used to transfect UE. RT-PCR based telomeric repeat amplification protocol (TRAP) assay was used to measure the telomerase activity of endothelia.

RESULTSUE arranged as "cobblestone" and were positive of CD34 labeling. Endothelia transfected by pBABE-HYGR-hTERT(HC) had an raised absorbance of 0.889. The shape of growth curve of HC was similar to UE. But the absorbance of MTT test and the amount of HC were prior to UE at every measuring time and the amount of HC increased four times within 8 days (P < 0.05).

CONCLUSIONThe transfection of pBABE-HYGRO-hTERT had greatly improved the proliferating abilities and activated the telomerase of UE.

Catalytic Domain ; genetics ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Humans ; Telomerase ; genetics ; Transfection ; Umbilical Veins ; cytology

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

OBJECTIVEHuman telomerase reverse transcriptase (hTERT) is a rate-limiting enzyme which dictates the activity of human telomerase and thus decides the life span of cells. The aim of this study was to explore the expression of hTERT in bone marrow from children with beta-thalassemia major and the relationship between the expression of hTERT and hemoglobin levels.

METHODSMultiple allele specific polymerase chain reaction (MASPCR) was used for targeted DNA amplification and gene mutation analysis of beta-thalassemia. hTERT mRNA expression in bone marrow was examined using real-time reverse transcription polymerase chain reaction (RT-PCR) analysis in 29 children with beta-thalassemia major, in 10 children with agranulocytosis and in K562 cell line. The hemoglobin levels in peripheral blood were measured. The relationship between hTERT expression and hemoglobin levels was evaluated by the Spearman test in the beta-thalassemia major group.

RESULTShTERT mRNA expression significantly increased in bone marrow from children with beta-thalassemia major compared with that from children with agranulocytosis (0.2928+/- 0.0838 vs 0.0993+/- 0.0336; P<0.01), but was significantly lower than that in K562 cell line (0.8291+/- 0.0908) (P<0.01). A significantly inverse correlation was found between hTERT mRNA expression and hemoglobin levels (r=-0.841, P<0.01).

CONCLUSIONSA low hemoglobin concentration might contribute to the up-regulation of marrow hTERT expression in children with beta-thalassemia major.

Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Telomerase ; genetics ; beta-Thalassemia ; genetics

OBJECTIVETo investigate the effect of hTERT single nucleotide polymorphisms on the development and metastasis of hepatocellular carcinoma.

METHODSA total of 290 male patients were divided into hepatitis-induced primary hepatocellular carcinoma (HCC) group (n%162), metastatic HCC group (n%22), and control group (n%106). hTERT gene was amplified and hTERT single nucleotide polymorphisms (SNPs) were tested in these subjects.

RESULTSSignificant differences were found in rs2853690 and rs10069690 distribution, but the difference in rs6554743 remained uncertain. The C and T alleles of rs10069690 and rs6554743 showed significant differences between the 3 groups; the carriers of non-T allele of rs10069690 had higher frequencies in both primary and metastatic HCC groups.

CONCLUSIONSome of the polymorphisms of hTERT may increase the risks of development and metastasis of hepatocellular carcinoma.

Carcinoma, Hepatocellular ; genetics ; pathology ; Humans ; Liver Neoplasms ; genetics ; pathology ; Male ; Neoplasm Metastasis ; genetics ; Polymorphism, Single Nucleotide ; Risk Factors ; Telomerase ; genetics

OBJECTIVETo construct a bait plasmid containing human telomerase RNA with multiple point mutations in a yeast three-hybrid system and evaluate the toxicity of the recombinant bait plasmid.

METHODSThe primers were designed according to the hTR sequence and the target mutation sites for inducing T-->A mutations at the 41st, the 80th and 102nd nucleotides of the hTR gene using the overlapping extension PCR (OE-PCR) method. The mutant was cloned into PMD18T vector, confirmed by sequencing, sub-cloned into the bait plasmid PRH3' and identified with PCR and restriction enzyme digestion. The recombinant bait plasmid was then transformed into yeast L40 ura3/pHyblex/ZeoMS2 for toxicity test.

RESULTSSequence analysis demonstrated successful introduction of point mutations at the target sites without causing random mutation. The recombined bait plasmid constructed showed no obvious toxicity against the host yeast cells.

CONCLUSIONSThe recombinant plasmid containing the human telomerase RNA mutant (PRH3'-hTRm) has been successfully constructed and can be used as the bait plasmid in yeast three-hybrid system.

Base Sequence ; Humans ; Plasmids ; genetics ; Point Mutation ; Polymerase Chain Reaction ; RNA ; genetics ; Telomerase ; genetics ; Two-Hybrid System Techniques