Development and verification of multiplex fluorescence quantitative PCR (Taqman probe) for detection of telomerase activity / 中国生物制品学杂志

@#Objective To develop and verify a multiplex fluorescence quantitative PCR(Taqman probe) method for the detection of telomerase activity.Methods Specific reverse transcription primers,two pairs of quantitative primers and probes were designed for the CDS sequence of telomerase catalytic subunit telomerase reverse transcriptase(TERT).After optimization of the reverse transcription primers(specific reverse transcription primers and random primers) and quantitative primers(two pairs of quantitative primer probes used alone or in combination) in the reaction system,with the primer probe of internal reference gene GAPDH,multiplex fluorescence quantitative PCR was performed in a single tube.In addition,telomerase positive standard and negative standard were prepared with 293T and MRC-5 cells respectively,and the stability and precision of the method were verified.The telomerase activity in 19 normal mesenchymal cell samples and 32 breast cancer cell samples were detected by the developed method.Results The optimum reaction system was as follows:using cDNA synthesized with specific reverse transcription primers as the template,2 pairs of quantitative primer probes of TERT gene were mixed with internal reference gene GAPDH primer probes for multiplex fluorescence quantitative PCR reaction in a single tube.After optimization,the sensitivity and TERT fluorescence signal quantity of the system were greatly improved,and the ΔRn was enhanced by 3 times.The amplification curve of positive standard TERT gene was normal,and the ΔCt between TERT gene and GAPDH gene remained stable.The amplification curve of GAPDH gene in negative standard was normal,while there was no amplification curve of TERT gene.There was a little difference in ΔCt between TERT and GAPDH genes in the positive standard frozen and thawed for 3 and 5 times repeatedly and the positive standard without freezing and thawing,and the CVs of precision in intra-and inter-groups were all less than 1%.Telomerase activity was negative in 19 normal mesenchymal cell samples and positive in 32 breast cancer cell samples,and significant difference in Ct value of TERT gene between them was observed(t=4.236,P <0.001).Conclusion The developed multiplex fluorescence quantitative PCR(Taqman probe) method for the detection of telomerase activity has good stability and precision,which is expected to be used in early diagnosis and gene therapy of tumors.

Telomere Biology in Mood Disorders: An Updated, Comprehensive Review of the Literature

Major psychiatric disorders are linked to early mortality and patients afflicted with these ailments demonstrate an increased risk of developing physical diseases that are characteristically seen in the elderly. Psychiatric conditions like major depressive disorder, bipolar disorder and schizophrenia may be associated with accelerated cellular aging, indicated by shortened leukocyte telomere length (LTL), which could underlie this connection. Telomere shortening occurs with repeated cell division and is reflective of a cell’s mitotic history. It is also influenced by cumulative exposure to inflammation and oxidative stress as well as the availability of telomerase, the telomere-lengthening enzyme. Precariously short telomeres can cause cells to undergo senescence, apoptosis or genomic instability; shorter LTL correlates with compromised general health and foretells mortality. Important data specify that LTL may be reduced in principal psychiatric illnesses, possibly in proportion to exposure to the ailment. Telomerase, as measured in peripheral blood monocytes, has been less well characterized in psychiatric illnesses, but a role in mood disorder has been suggested by preclinical and clinical studies. In this manuscript, the most recent studies on LTL and telomerase activity in mood disorders are comprehensively reviewed, potential mediators are discussed, and future directions are suggested. An enhanced comprehension of cellular aging in psychiatric illnesses could lead to their re-conceptualizing as systemic ailments with manifestations both inside and outside the brain. At the same time this paradigm shift could identify new treatment targets, helpful in bringing about lasting cures to innumerable sufferers across the globe.

Determining Osteogenic Differentiation Efficacy of Pluripotent Stem Cells by Telomerase Activity

BACKGROUND: Bone tissue engineering based on pluripotent stem cells (PSCs) is a new approach to deal with bone defects. Protocols have been developed to generate osteoblasts from PSCs. However, the low efficiency of this process is still an important issue that needs to be resolved. Many studies have aimed to improve efficiency, but developing accurate methods to determine efficacy is also critical. Studies using pluripotency to estimate efficacy are rare. Telomerase is highly associated with pluripotency. METHODS: We have described a quantitative method to measure telomerase activity, telomeric repeat elongation assay based on quartz crystal microbalance (QCM). To investigate whether this method could be used to determine the efficiency of in vitro osteogenic differentiation based on pluripotency, we measured the pluripotency pattern of cultures through stemness gene expression, proliferation ability and telomerase activity, measured by QCM. RESULTS: We showed that the pluripotency pattern determined by QCM was similar to the patterns of proliferation ability and gene expression, which showed a slight upregulation at the late stages, within the context of the general downregulation tendency during differentiation. Additionally, a comprehensive gene expression pattern covering nearly every stage of differentiation was identified. CONCLUSION: Therefore, this assay may be powerful tools for determining the efficiency of differentiation systems based on pluripotency. In this study, we not only introduce a new method for determining efficiency based on pluripotency, but also provide more information about the characteristics of osteogenic differentiation which help facilitate future development of more efficient protocols.

Effects of estradiol on naphthalene-induced telomerase activity and apoptosis of LEC in ovariectomized female rats / 眼科新进展

Objective To explore the effects of estradiol on the telomerase activity and apoptosis of lens epithelial cells and its mechanisms.Methods Fifty adult female Sprague-Dawley rats were randomly divided into five groups (n =10):ovariectomized group,estradiol group 1,estradiol group 2,estradiol group 3 and sham group.All rats were prepared for ovariectomized models except that of the sham group.After 2 weeks of the operation,rats in the 5 groups were given naphthalene solution through a stomach tube,and meanwhile,estradiol group 1,estradiol group 2,estradiol group 3 received estradiol valerate with the dose of 0.21 mg · kg-1 · d-1,0.42 mg · kg-1 · d-1 and 0.84 mg · kg-1 · d-1,accordingly,while rats in the ovariectomized group and sham group were given 9 g · L-1 Nacl by stomach perfusion with a dose of 0.42 mg · kg-1 ·d-1.Then,the opacity of the lens in each group was examined by a slit lamp microscope every week.After 12 weeks of naphthalene solution administration,all rats were sacririced and the serum estradiol concentration was determined by radioimmunoassay.Next,the lenses were taken out,and TERT mRNA expression of lens epithelial cells (LEC) was measured by RT-qPCR,and finally,the apoptotic rate was detected by TUNEL method.Results The opacity of lens in the ovariectomized group was different from that in the estradiol group 1,estradiol group 2,estradiol group 3 and sham group,with statistical significances (all P ＜ 0.05),and the opacity of lens in the estradiol group 1,2,3 and sham group were mild and occurred later.The serum estradiol concentration in the ovariectomized group (8.19 ± 1.45)ng · L-1 was significantly lower than that of estradiol groupl,2,3 and sham group,and there were significant differences (all P ＜ 0.05).Thc relative expression of TERT mRNA in LEC in the ovariectomized group (0.371 2-±0.056 4) was significantly lower than that in estradiol group 1,2,3,but the apoptotic rate of LEC (0.602 1 ±0.010 8) was obviously higher than that of estradiol group 1,2,3 in a dose-dependent manner,and there were significant differences (all P ＜ 0.05).Pearson correlation analysis showed the relative expression of LEC TERT mRNA in rats was negatively correlated with the apoptosis rate(r =-0.859,P ＜ 0.05).Conclusion Estradiol can up-regulate TERT mRNA expression and enhance telomerase activity of LEC in naphthalene-induced ovariectomized female rats in a dose-dependent manner.Estradiol can inhibit the LEC apoptosis in naphthalene-induced ovariectomized female rats,and the mechanism may be related to the increase of telomerase activity in the LEC.

Vinorelbine induces apotosis and decreases telomerase activity in human epithelial ovarian cancer cells line SKOV 3 / 基础医学与临床

Objective To investigate the effect of different concentrations of vinorelbine on apoptosis ,telomerase ac-tivity and expression of human telomerase-reverse transcriptase gene ( hTERT ) in human epithelial ovarian cancer cells SKOV3.Methods Ovarian cancer cells SKOV 3 were treated with vinorelbine under different concentrations . The cell proliferation was measured by cell counting kit-8 ( CCK-8) assay , and the cell apoptosis was detected by flow cytometry.The telomerase activity of SKOV3 cells was determined by TRAP-PAGE-silver staining;The mRNA expression of hTERT was performed by RT-PCR assay .Results Vinorelbine could significantly inhibited the prolifer-ation of SKOV3 cells,induce cell apoptosis (P<0.01),reduce the telomerase activity and expression of hTERT mRNA (P<0.01), in dependent of a concentration-time manner.Conclusions The detection of telomerase activity and the mRNA expression of hTERT might be vital for predicting the prognosis of patients with epithelial ovarian cancer .

Dysregulation of Telomere Lengths and Telomerase Activity in Myelodysplastic Syndrome

BACKGROUND: Telomere shortening is thought to be involved in the pathophysiology of myeloid malignancies, but telomere lengths (TL) during interphase and metaphase in hematopoietic malignancies have not been analyzed. We aimed to assess the TLs of interphase and metaphase cells of MDS and telomerase activity (TA) and to find out prognostic significances of TL and TA. METHODS: The prognostic significance of TA by quantitative PCR and TL by quantitative fluorescence in situ hybridization (QFISH) of interphase nuclei and metaphase chromosome arms of bone marrow cells from patients with MDS were evaluated. RESULTS: MDS patients had shorter interphase TL than normal healthy donors (P<0.001). Average interphase and metaphase TL were inversely correlated (P=0.013, p arm; P=0.029, q arm), but there was no statistically significant correlation between TA and TL (P=0.258). The progression free survival was significantly shorter in patients with high TA, but the overall survival was not different according to average TA or interphase TL groups. Multivariable Cox analysis showed that old age, higher International Prognostic Scoring System (IPSS) subtypes, transformation to AML, no history of hematopoietic stem cell transplantation and short average interphase TL (<433 TL) as independent prognostic factors for poorer survival (P=0.003, 0.001, 0.005, 0.005, and 0.013, respectively). CONCLUSIONS: The lack of correlation between age and TL, TA, and TL, and the inverse relationship between TL and TA in MDS patients reflect the dysregulation of telomere status and proliferation. As a prognostic marker for leukemia progression, TA may be considered, and since interphase TL has the advantage of automated measurement by QFISH, it may be used as a prognostic marker for survival in MDS.

Effect of estradiol and bisphenol A on human hepatoblastoma cell viability and telomerase activity

Sex hormones from environmental and physiological sources might play a major role in the pathogenesis of hepatoblastoma in children. This study investigated the effects of estradiol and bisphenol A on the proliferation and telomerase activity of human hepatoblastoma HepG2 cells. The cells were divided into 6 treatment groups: control, bisphenol A, estradiol, anti-estrogen ICI 182,780 (hereinafter ICI), bisphenol A+ICI, and estradiol+ICI. Cell proliferation was measured based on average absorbance using the Cell Counting-8 assay. The cell cycle distribution and apoptotic index were determined by flow cytometry. Telomerase activity was detected by polymerase chain reaction and a telomeric repeat amplification protocol assay. A higher cell density was observed in bisphenol A (P<0.01) and estradiol (P<0.05) groups compared with the control group. Cell numbers in S and G2/M phases after treatment for 48 h were higher (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h (P<0.05) were higher in these groups than in the control group. The cell density was also higher in bisphenol A+ICI (P<0.01) and estradiol+ICI (P<0.05) groups compared with the ICI group. Furthermore, cell numbers were increased in S and G2/M phases (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h were higher (P<0.05) in these groups than in the ICI group. Therefore, bisphenol A and estradiol promote HepG2 cell proliferation in vitro by inhibition of apoptosis and stimulation of telomerase activity via an estrogen receptor-dependent pathway.

Modeling pulmonary fibrosis by abnormal expression of telomerase/apoptosis/collagen V in experimental usual interstitial pneumonia

Limitations on tissue proliferation capacity determined by telomerase/apoptosis balance have been implicated in pathogenesis of idiopathic pulmonary fibrosis. In addition, collagen V shows promise as an inductor of apoptosis. We evaluated the quantitative relationship between the telomerase/apoptosis index, collagen V synthesis, and epithelial/fibroblast replication in mice exposed to butylated hydroxytoluene (BHT) at high oxygen concentration. Two groups of mice were analyzed: 20 mice received BHT, and 10 control mice received corn oil. Telomerase expression, apoptosis, collagen I, III, and V fibers, and hydroxyproline were evaluated by immunohistochemistry, in situ detection of apoptosis, electron microscopy, immunofluorescence, and histomorphometry. Electron microscopy confirmed the presence of increased alveolar epithelial cells type 1 (AEC1) in apoptosis. Immunostaining showed increased nuclear expression of telomerase in AEC type 2 (AEC2) between normal and chronic scarring areas of usual interstitial pneumonia (UIP). Control lungs and normal areas from UIP lungs showed weak green birefringence of type I and III collagens in the alveolar wall and type V collagen in the basement membrane of alveolar capillaries. The increase in collagen V was greater than collagens I and III in scarring areas of UIP. A significant direct association was found between collagen V and AEC2 apoptosis. We concluded that telomerase, collagen V fiber density, and apoptosis evaluation in experimental UIP offers the potential to control reepithelization of alveolar septa and fibroblast proliferation. Strategies aimed at preventing high rates of collagen V synthesis, or local responses to high rates of cell apoptosis, may have a significant impact in pulmonary fibrosis.

Telomerasa y telómero: su estructura y dinámica en salud y enfermedad / Telomerase and telomere: their structure and dynamics in health and disease

La telomerasa es la enzima responsable del mantenimiento de la longitud de los telómeros mediante la adición de secuencias repetitivas ricas en guanina, y su actividad se observa principalmente en gametos, células madre y células tumorales. En las células somáticas humanas el potencial de proliferación es limitado, alcanzando la senescencia luego de 50-70 divisiones celulares, debido a que la ADN polimerasa no es capaz de copiar el ADN en los extremos de los cromosomas. Por el contrario, en la mayoría de las células tumorales el potencial de replicación es ilimitado debido al mantenimiento de la longitud telomérica dado por la telomerasa. Los telómeros tienen proteínas adicionales que regulan la unión de la telomerasa. De la misma manera la telomerasa también se asocia con un complejo de proteínas que regulan su actividad. Este trabajo se centra en la estructura y función del complejo telómero/telomerasa y a cómo las alteraciones en su comportamiento conducen al desarrollo de diversas enfermedades, principalmente cáncer. El desarrollo de inhibidores del sistema telómero / telomerasa podría ser un blanco con posibilidades prometedoras.

Telomerase is the enzyme responsible for the maintenance of telomere length by adding guanine-rich repetitive sequences. Its activity can be seen in gametes, stem cells and tumor cells. In human somatic cells the proliferative potential is limited, reaching senescence after 50-70 cell divisions, because the DNA polymerase is not able to copy the DNA at the ends of chromosomes. By contrast, in most tumor cells the replicative potential is unlimited due to the maintenance of the telomeric length given by telomerase. Telomeres have additional proteins that regulate the binding of telomerase, likewise telomerase associates, with a protein complex that regulates its activity. This work focuses on the structure and function of the telomere/telomerase complex and how changes in its behavior lead to the development of different diseases, mainly cancer. Development of inhibitors of the telomere/telomerase complex could be a target with promising possibilities.

Combined SYBR Green Real-Time with Telomeric Repeat Amplification Protocol (RQ-TRAP) to Detect Telomerase Activity / 天津医药

Objective To establish methodology to detect telomerase activity based on real-time quantitative PCR technique combined with telomeric repeat amplification protocol (TRAP). Methods RQ-TRAP system was developed by combining real-time quantitative PCR technique with conventional TRAP method. Telomerase activity was assessed and compared by RQ-TRAP assay and TRAP connected with enzyme-linked immunosorbent assay (TRAP-ELISA) respectively in 12 kinds of cells. Results The RQ-TRAP method was both accurate and specified in measuring telomerase activity in a series dilution of protein extracts from 293T cells. The sensitivity of this method was 8 cells and the amplification efficiency was 98.92%. Telomerase activity was not detected in negative control group. Statistical analysis revealed a strong correlation between the two assays (r2=0.762 5). Conclusion The feasibility of RQ-TRAP was proved in this article. Compared with TRAP-ELISA, RQ-TRAP has many advantages. Apart from sample extraction and real-time PCR cycling, no other extra time-consuming steps are needed for telomerase quantification;RQ-TRAP is less costly and more rapid and reliable than TRAP-ELISA for quantification of telomerase activity and it also support high throughput.

Stromal Stem Cells from Parathyroid Glands of Patients with Secondary Hyperparathyroidism Demonstrate Higher Telomerase Activity and Osteogenic Differentiation Ability than Normal Bone Marrow Derived Stromal Stem Cells.

Aims: The aim of this study was to isolate and extensively characterize parathyroid gland stem cells (PT-SCs) from secondary hyperparathyroidism cases. For this purpose, proliferation capacity, phenotypic properties, differentiation characteristics and gene expression profiles were analyzed and compared with mesenchymal stem cells from bone marrow (BM-MSCs) of the human. Methods: Stem cells isolated from PT and BM were analyzed by flow cytometry, RT-PCR, Real Time-PCR, and immunocytochemistry. Both cell lines were directionally differentiated towards adipogenic, osteogenic and neurogenic cell lineages. Results: The isolated hPT-SCs share similar characteristics of hBM-MSCs by immunophenotypic, histological and molecular analyses. Both cells were shown to differentiate successfully into adipogenic and osteogenic cell lines. Embryonic stem cell markers Pou5F1, Zpf42, FoxD3, Sox2 and Nanog were also expressed beside 5 fold higher telomerase activity in hPT-SCs that could indicate the regenerative ability of the human parathyroid gland. The osteogenic cell markers were expressed by hPT-SCs, which transformed efficiently into osteogenic cell lines, both at the level of genes (BMP2, BMP4, BGLAP, Coll11a1, Runx2, Sparc) and of proteins (BMP2, BMP4, Osteocalcin, Osteonectin, Osteopontin). Higher alkaline phosphatase (ALP) activity indicating osteogenic differentiation was determined in hPT-SCs from secondary hyperparathyroidism patients. Conclusion: PT-SCs might responsible for the calcified parathyroid glands and other ectopic calcifications including the vascular ones, observed in the secondary hyperparathyroidism cases, beside parathyroid hormone-dependent hypercalcemia leading diffusion of calcium phosphate precipitation in tissues.

Expression of telomerase & its significance in the diagnosis of pancreatic cancer.

Pancreatic cancer has one of the worst prognoses among all types of cancers. The survival rate is less than 5 per cent; this is due to difficulty in diagnosing at an early stage. Despite the improvements in diagnostic imaging techniques such as computed tomography, magnetic resonance imaging, etc., the early diagnosis of pancreatic cancer is still difficult. Alternative methods of diagnosing pancreatic cancer at an early stage are presently been explored. The detection of telomerase activity has been proposed to be a useful tool in the diagnosis of pancreatic cancer. Telomerase is made up of three major parts namely, human telomerase reverse transcriptase, human telomerase and telomerase -associated protein. Several researchers have shown telomerase activity in tissues and fluids of patients with pancreatic and other types of cancers. About 95 per cent telomerase activity has been detected in pancreatic adenocarcinoma. Since telomerase activity is present in a vast majority of human cancers, it might have a role in the diagnosis and treatment of cancer.

Telomerase Activity and the Risk of Lung Cancer

Telomerase play a key role in the maintenance of telomere length and chromosome integrity. We have evaluated the association between telomerase activity and the risk of lung cancer in peripheral blood. Telomerase activity in peripheral blood mononuclear cells was measured by a PCR-designed telomeric repeat amplification protocol in 63 lung cancer patients and 190 healthy controls that were matched for age, gender, and smoking status. Telomerase activity was significantly lower in the lung cancer patients than in controls (mean +/- standard deviation; 1.32 +/- 1.65 vs 2.60 +/- 3.09, P < 1 x 10(-4)). When telomerase activity was categorized into quartiles based on telomerase activity in the controls, the risk of lung cancer increased as telomerase activity reduced (Ptrend = 1 x 10(-4)). Moreover, when the subjects were categorized based on the median value of telomerase activity, subjects with low telomerase activity were at a significantly increased risk of lung cancer compared to subjects with high telomerase activity (adjusted odds ratio = 3.05, 95% confidence interval = 1.60-5.82, P = 7 x 10-4). These findings suggest that telomerase activity may affect telomere maintenance, thereby contributing to susceptibility to lung cancer.

EFFECT OF wt-P53 PROTEIN ON TELOMERASE ACTIVITY IN KELOID FIBROBLASTS / 中国修复重建外科杂志

Objective To evaluated the role of wt-P53 protein in telomerase regulation in keloid fibroblasts(KFBs). Methods The fibroblasts were derived from human keloid tissue which was proved by pathological diagnosis. KFBs were divided into 2 groups, the transfection group and the untransfection group. wt-p53 gene was transfected into the fibroblasts by adenovirus vectors in the transfection group. The KFBs untransfected with wt-p53 gene served as control (untransfection group). After 48 hours of transfection, the expression of wt-P53 protein was analyzed by both Western blotting and immunofluorescence method, respectively. The telomerase activity was evaluated by TRAP-ELISA after 1-7 days of transfection.Results All the KFBs from 2 groups expressed wt-P53 protein. But the expression level of wt-P53 protein in the transfection group was significantly higher than that in the untransfection group. At the same time of high expression of wt-P53 protein, the telomerase activity of KFBs in transfection group was significantly lower than that in the untransfection group( P＜0.05). Conclusion High level expression of wt-P53 protein can transiently inhibit the telomerase activity of KFBs.

The value of CT scan and tumor markers in sputum for early diagnosis of peripheral lung cancer / 重庆医科大学学报

Objective:To investigate the diagnostic value of telomerase activity and p16 gene methylation from exfoliated cells of sputum in 55 cases of solitary nodules and of being suspected of early peripheral lung cancer(T1N0M0).Methods:The sputum specimens from 34 cases of cancer nodes and 21 cases of benign lung lesion were detected for telomerase activity and p16 gene methylation by methylation analysis.Results:The qualitative diagnosis accuracy of CT scan was 61.8%(34/55) for peripheral lung cancer.Telomerase activity was positive in 29 cases:Sensitivity was 79.4%;Specificity was 90.5%;and accuracy was 83.6%.p16 gene methylation was in 11 cases:Sensitivity was 32.4%;specificity was 100%,and veracity was 58.2%.The sensitivity was increased to 86.1% by the combination of telomerase activity and p16 gene methylatiion.Conclusion:The results suggest that combining CT scan with telomerase activity and p16 gene methylation detection in sputum for patients with lung cancer may enhance the diagnostic value of conventional cytology.It is a promising approach to early diagnosis of lung cancer and massive screening in terms of its rapidity,economy and simplicity.

Telomerase activity could be used as a marker for neoplastic transformation in gastric adenocarcinoma: but it does not have a prognostic significance

Telomerase activity is responsible for telomere maintenance and is believed to be crucial in most immortal cells and cancer cells; however, its clinicopathological significance in gastric cancer remains to be clarified. The aim of the present study was to assess whether malignant progression of gastric adenocarcinoma correlates with telomerase activity. We also investigated the correlation between telomerase activity and histopathological findings. We examined telomerase activity in tumor specimens and adjacent normal tissues from 43 patients with gastric adenocarcinoma. Telomerase activity was measured quantitatively by the TRAPEZE Gel Based Telomerase Detection Kit. Approximately 98% of the tumor tissues were telomerase positive, but telomerase activity was detected not only in tumor tissues but also in normal gastric mucosa. Although telomerase activity was found to be higher in tumor samples than normal tissue for each subject, we could not find a general cut-off level for telomerase activity in gastric adenocarcinoma. In addition, telomerase activity was not correlated with tumor invasion, lymph node involvement and histological stage. Our results support the idea that telomerase reactivation is a common event in gastric adenocarcinoma and it is not related to histopathological parameters. Since it is difficult to set a cut-off level for this type of cancer, we suggest that the prognostic utility of telomerase assay has not yet reached the clinic in terms of predicting outcome for patients with gastric adenocarcinoma. For the assessment of gastric carcinoma, telomerase activity should be evaluated in both tumor and normal tissues, because normal gastric mucosa samples show appreciable telomerase activity.

Inhibition of Cell Growth and Telomerase Activity in Osteosarcoma Cells by DN-hTERT / 华中科技大学学报（医学）（英德文版）

In order to study the effects of dominant negative human telomerase reverse transcriptase (DN-hTERT) on cell growth and telomerase activity in osteosarcoma cell line MG63, MG63 cells were transfected with DN-hTERT-IRES2-EGFP9 (DN) or IRES2-EGF (I, blank vector) with lipofectamine 2000. The stably transfected cells were selected with G-418. Cell growth properties were examined under a fluorescence microscope. The hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Telomerase activities were measured by TRAP-ELISE. The tumorigenicity was studied with tumor xenografts by subcutaneous injection of cancer cells into nude mice. The results showed that cell growth was suppressed in MG63 cells transfected with DN-hTERT. The hTERT mRNA was increased in N-hTERT transfected-MG63 cells (MG63/DN). The telomerase activity was 2.45±0.11 in MG63/DN cells, while 3.40±0.12 in the cells transfected with blank vector (MG63/I), (P＜0.05); DN-hTERT-expressing clones did not form tumors in 2 weeks, but the ratio of tumorigenesis was 30 % in nude mice bearing MG63/I (P＜0.01). It was concluded that DN-hTERT could specifically inhibit the cell growth and telomerase activity in MG63 cells.

Expression of PCNA in Gastric Cancer and Its Relationship with Telomerase Activity of Peritoneal Washings and Peritoneal Dissemination / 中国普外基础与临床杂志

Objective To investigate the expression of PCNA in gastric cancer and its relationship with telomerase activity of peritoneal washings and peritoneal dissemination, and to compare the efficacy of telomerase activity and cytology of peritoneal washings for prediction of peritoneal metastasis of gastric cancer. Methods Telomeric repeated amplification protocol (TRAP)-enzyme-linked immunosorbent assay (ELISA) was performed to measure the telomerase activity of peritoneal washings collected from 60 patients with gastric cancer. Exfoliate cytologic analysis of the corresponding samples was used for comparison.Expression of PCNA was measured with immunohistochemical staining.Their relationship with clinicopathologic features were evaluated. Results The positive rate of telomerase activity in peritoneal washing collected from patients with gastric cancer was 41.7%,which well related to serosal invasion, histology types, depth of infiltration and peritoneal metastasis of gastric cancer. The positive rate of telomerase activity increased with the increased depth of infiltration and serosal involvement areas (P

Inhibition of telomerase activity on breast cancer cells by dominant negative human telomerase reverase transcriptase gene / 医学研究生学报

Objective:To explore inhibition of telomerase activity and malignant phenotype on breast cancer cells MCF7 by Dominant Negative human telomerase reverase transcriptase gene(DN-hTERT).Methods:DN-hTERT eukaryotic expression vector DN-hTERT-IRES2-EGFP and empty vector(I) IRES2-EGFP were transfected into MCF7 by lipofectamine2000,after being selected by G418,positive clones were obtained;Tthe transfected cells growth was observed with inverted fluorescence microscope.The expression levels of hTERT mRNA of transfected cells were determined by reverse transcriptase polymerase chain reaction(RT-PCR).Telomerase activity of transfected cells was measured by TRAP-ELISE.Results:The proliferation of MCF7/DN cells were inhibited by DN-hTERT transtection;The expression levels of hTERT mRNA increased in MCF7/DN.Telomeric length was shorter in MCF7/DN than that in MCF7/I.The telomerase activity measured by TRAP-ELISE was 2.36?0.12 in MCF7/DN and was 3.32?0.14 in vacant vector MCF7/I.The telomerase activity in MCF7/DN was significantly lower than in vacant vector transfected MCF7/I(P

Telomerase Activity and Expression of hTR and TERT in Human Soft Tissue Sarcomas / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Sarcomas have rarely been analyzed for telomerase, which is an RNA-dependent DNA polymerase to maintain telomeres and prevent telomere shortening. This study was undertaken to determine telomerase activity and the expression of the telomerase subunits human telomerase RNA (hTR) and telomerase reverse transcriptase (TERT) in soft tissue sarcomas. MATERIALS AND METHODS: Twenty three sarcomas were analyzed for the telomerase activity by a radioactive PCR-based TRAP assay. All of the samples were further investigated for the expression of hTR by in situ hybridization and for TERT and p53 by immunohistochemistry. RESULTS: Telomerase activity was detected in four (17%) samples. Expression of hTR was demonstrated in 11 (48%) cases, whereas TERT was expressed in 20 (87%).Of the four telomerase-positive tumors, three were positive for both hTR and TERT, and one was positive only for TERT. p53 overexpression was observed in nine (39%) tumors. The frequency of p53 expression increased as the tumor grade advanced (p= .064). CONCLUSION: These data indicate that the reactivation of telomerase is an uncommon event in human soft tissue sarcomas. The high frequency of the expression of hTR and TERT in these tumors suggests that telomerase activity may be regulated at the transcriptional level and an additional event leading to telomerase activation exist.