Aim: To characterize the coexpression of survivin, an inhibitor of apoptosis (IAF), and human telomerase reverse transcriptase (hTERT) in human testes with varying spermatogenic function. Methods: Transcript lev...Aim: To characterize the coexpression of survivin, an inhibitor of apoptosis (IAF), and human telomerase reverse transcriptase (hTERT) in human testes with varying spermatogenic function. Methods: Transcript levels of survivin mRNA and hTERT mRNA were determined in normal testes (n = 11) and testes with defective spermatogenesis (n = 28) using real-time reverse-transcription polymerase chain reaction (RT-PCR). The histological work-up was performed according to a modified Johnsen score. Results: Expressions of both survivin and hTERT were highest at median levels of 96.8 and 709 in normal spermatogenesis and dropped to 53.3 and 534 in testes with postmeiotic spermatogenic arrest (n = 10). In severe spermatogenic failure (n = 18), survivin expression was lacking in most specimens (n = 16), whereas at least low levels of testicular hTERT expression were largely detectable with a normalized expression of 73 in premeiotic spermatogenic arrest (n = 7) and 45 in patients with Sertoli cell-only syndrome (SCOS) (n = 3). Both survivin and hTERT expressions increased with a progressing Johnsen score (P for trend = 0.001). Conclusion: Although both survivin and hTERT are correlated with spermatogenic function, they show different expression patterns in testes of infertile patients. These findings substantiate results from studies in the rodent testis suggesting a predominant expression of survivin in meiotically dividing germ cells. (Asian J Andro12006 Jan; 8: 95-100)展开更多
BACKGROUND: Numerous current studies have suggested that human telomerase reverse transcriptase (hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the...BACKGROUND: Numerous current studies have suggested that human telomerase reverse transcriptase (hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE: To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 (AI325-35). DESIGN, TIME AND SETTING: The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS: AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Odk5 and mouse anti-p16 monoclonal antibodies (Lab Vision, USA), and mouse anti-hTERT monoclonal antibody (Epitomics, USA), were used in this study. METHODS: (1) Recombinant adenovirus vectors, encoding hTERT (Ad-hTERT) and green fluorescent protein (Ad-GFP), were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. (2) Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 pmol/L Aβ25-35 for 24 hours. Normal human embryonic cortical neurons treated with 10 pmol/Lβ25.35 for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35. MAIN OUTCOME MEASURES: Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol (TRAP) ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS: Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups (P 〈 0.01). Compared with the control group, cell activity was significantly decreased (P 〈 0.05), and cell apoptotic rate, Cdk5 and p16 expression were significantly increased (P 〈 0.01) in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection (P 〈 0.05). Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased (P 〈 0.01). CONCLUSION: Expression of hTERT in human embryonic cortical neurons can relieve Aβ25-35-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.展开更多
Aim: To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase (hTERT) antisense on tumor necrosis factor-α (TNF-α-induced apoptosis in prostate cancer cells (PC3). Meth...Aim: To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase (hTERT) antisense on tumor necrosis factor-α (TNF-α-induced apoptosis in prostate cancer cells (PC3). Methods: Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol (TRAP) and polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA was measured by reverse transcription PCR (RT-PCR) assay and gel-image system, hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by morphological method and determined by flow cytometry. Results: The telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide (S PS-ODN) and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF-α treatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF-α treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group. Conclusion: hTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF-α- induced apoptosis of PC3 cells.展开更多
Aim: To evaluate the quantitative detection of human telomerase RNA (hTR) and human telomerase reverse tran-scriptase (hTERT) mRNA as diagnostic parameters in the workup of testicular tissue specimens from patients pr...Aim: To evaluate the quantitative detection of human telomerase RNA (hTR) and human telomerase reverse tran-scriptase (hTERT) mRNA as diagnostic parameters in the workup of testicular tissue specimens from patients presentingwith non-obstructive azoospermia. Methods: hTR and hTERT mRNA expression were quantified in 38 cryopre-served testicular tissue specimens by fluorescence real-time reverse transcription - polymerase chain reaction (RT-PCR)in a LightCycler(r). This was paralleled by conventional histological workup in all tissue specimens and additionalsemithin sectioning preparation in cases with maturation arrest (n = 12) and Sertoli-cell-only syndrome (n = 12). Re-sults; The average normalized hTERT expression (N_(hTERT)) was 131.9±48.0 copies (mean ± SD) in tissue speci-mens with full spermatogenesis, N_(hTERT) = 51.2 ±17.2 copies in those with maturation arrest and N_(hTERT) = 2.7 ± 2.4copies in those with Sertoli-cell-only syndrome (SCOS). The discriminant analysis showed that detection of N_(hTERT)(N_(hTR)) had a predictive value of 86.8% (55.3%) for correct classification in one of the three histological subgroups.Conclusion; Our results demonstrate that quantitative detection of hTERT mRNA expression in testicular tissue en-ables a molecular-diagnostic classification of gametogenesis. Quantitative detection of hTERT in testicular biopsies isthus well suited for supplementing the histopathological evaluation. (Asian J Androl 2001 Dec; 3: 263 - 270)展开更多
To investigate the effects of sodium selenite on telomerase activity and expression of hTERT mRNA in cadmium-transformed 16HBE cells. Methods Telomerase activity and expression of genes were measured after cultured ca...To investigate the effects of sodium selenite on telomerase activity and expression of hTERT mRNA in cadmium-transformed 16HBE cells. Methods Telomerase activity and expression of genes were measured after cultured cadmium-transformed 16HBE cells were exposed to sodium selenite at different doses (0.625, 1.25, 2.50, 5.00 pmol/L) for 24 hours. Results Selenium decreased telomerase activity in cadmium-transformed 16HBE cells. There existed an obvious dose-effect relationship between the selenium concentration and these changes. The expression of hTERT and c-myc mRNA also decreased but the expression of madl mRNA increased after exposure to selenium for 24 hours. No difference was found in expression of hTRF1 and hTRF2 mRNA after incubated with sodium selenite for 24 hours, compared with control group. Conclusion Selenium inhibits telomerase activity by decreasing hTERT and c-myc mRNA expression and increasing madl mRNA expression in cadmium-transformed 16HBE cells and selenium concentration is significantly correlated with these changes.展开更多
The effects of combined RNA interference(RNAi) of human telomerase RNA(hTR) and human telomerase reverse transcriptase(hTERT) genes on telomerase activity in a bladder cancer cell line(BIU-87 cells) were investigated ...The effects of combined RNA interference(RNAi) of human telomerase RNA(hTR) and human telomerase reverse transcriptase(hTERT) genes on telomerase activity in a bladder cancer cell line(BIU-87 cells) were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer(BTCC).Three TR-specific double-stranded small interfering RNAs(siRNAs) and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA.The phTR-siRNA,phTERT-siRNA,and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells.The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction,and a telomeric repeat amplification protocol was applied to detect telomerase activity.Growth inhibition of BIU-87 cells was measured by MTT assay.Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro.The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,and pRNAT-hTR-Ⅲ+hTERT-Ⅲ in BIU-87 cells.The inhibition efficiency of pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,pRNAT-hTERT-Ⅲ+pRNAT-hTR-Ⅲ was 67% for TERT mRNA,41% for TR mRNA,57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively.The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased,especially in the cells treated with combined RNAi-hTR and-hTERT.Gene chip analysis revealed that 21 genes were down-regulated(ATM,BAX,BCL2,BCL2L1,BIRC5,CD44,CTNNB1,E2F1,JUN,MCAM,MTA1,MYC,NFKB1,NFKBIA,NME4,PNN,PNN,SERPINE1,THBS1,TNFRSF1A,and UCC1).The results indicated that hTR-siRNA and hTERT-siRNA,especially their combination,siRNA hTR+hTERT,specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity.Molecular biological mechanism by which combined siRNA-TR and-TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation of the 21 genes.展开更多
To construct a mutant pEGFP- hTERTexpression vector, to observe its steady expression intransfected human bladder carcinoma cell line T24 and its role in molecular regulatory mechanisms of telomerase, and to provide a...To construct a mutant pEGFP- hTERTexpression vector, to observe its steady expression intransfected human bladder carcinoma cell line T24 and its role in molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer. Methods: PCR amplification was performed by using primers basedon the known gene sequence of hTERT. PCR productionwas cloned into plasmid pGEMT-T easy and the sequenceof mutant hTERT gene was analyzed. A recombinantmutant hTERT vector (pEGFP-hTERT) was constructed at the EcoR I and Sal I sites of the pEGFP-C1 vector. Aftertransfecting the fusion gene into bladder carcinoma cell line T24 by calcium phosphate-DNA coprecipitation, the steady expression of GFP-hTERT fusion protein was tested by fluorescent light microscopy. The proliferation changes ofbladder carcinoma cell line T24 were detected by lightmicroscopy and senescence correlated b-galactosidase staining. Results: Identification of pEGFP-hTERT byenzyme digestion showed that mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. The steady expression of GFP-hTERT fusion protein was localized in the nucleus of transfected cells. Expression of senescence-associated b-galactosidase in transfected cells gradually increased with extended cultured time and cellgrowth was suppressed. Conclusion: The mutant-type hTERT gene suppresses the proliferation of bladder carcinoma cell line T24 by competitive effect on telomerase activity. This suggests that hTERT gene might be a suitable gene target for bladder cancer therapy.展开更多
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-I...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.展开更多
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 telom...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 suhunits 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 (X^2 = 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 (X^2 =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.展开更多
BACKGROUND:The human telomerase reverse transcriptase(hTERT) gene encodes the catalytic subunit of telomerase,which mediates pleiotropic effects,including the regulation of senescence and proliferation and plays an im...BACKGROUND:The human telomerase reverse transcriptase(hTERT) gene encodes the catalytic subunit of telomerase,which mediates pleiotropic effects,including the regulation of senescence and proliferation and plays an important role in carcinogenesis.This study attempts to clarify the genetic predisposition to hepatocellular carcinoma(HCC),focusing on the hTERT gene rs2736098 polymorphism.METHOD:Four hundred patients with HCC and 400 noncancer controls were genotyped to elucidate the potential association between hTERT rs2736098 polymorphism and HCC risks.RESULTS:Compared with the controls,the patients with HCC had a lower frequency of G/G genotype(33.3% vs 44.3%,P=0.001) and a higher frequency of G/A(51.5% vs 39.5%,P=0.001).Allele genotypic frequencies in the patients differed from those of the controls(P=0.040).The data of this study rs2736098[A] allele contributed significantly to HCC risk in female patients(OR=1.78,95% CI,1.17-2.72,P=0.007),patients with HCV infection(OR=2.89,95% CI,1.08-7.70,P=0.031),non-drinker patients(OR=1.32,95% CI,1.06-1.65,P=0.015),and patients not affected by HBV(OR=1.77,95% CI,1.30-2.40,P【0.001).CONCLUSIONS:rs2736098[A] may be an independent hereditary parameter in HCC,but some risk factors would cover up the association by more powerful hepatocarcinogenesis.These results are important guidance for further studies in detecting HCC-associated single nucleotide polymorphisms.展开更多
Objective To determine the expression of human telomerase reverse transcriptase (hTERT) and P53 in thyroid carcinoma and its relationship with development and prognosis of the carcinoma. Methods Totally 90 cases of th...Objective To determine the expression of human telomerase reverse transcriptase (hTERT) and P53 in thyroid carcinoma and its relationship with development and prognosis of the carcinoma. Methods Totally 90 cases of thyroid specimens (60 thyroid carcinomas,10 thyroid adenomas,10 goitres and 10 normal thyroid tissues) were studied by SP immunohistochemical method. Results Positive immunoreactivity of hTERT and P53 was higher in thyroid carcinoma (P<0.05). The positive rates of hTERT and P53 were higher in undifferentiated carcinomas,carcinomas with lymph nodes metastasis or at stage Ⅲ+Ⅳ than in well-differentiated carcinomas,carcinomas without lymph nodes metastasis or at stage Ⅰ+Ⅱ (P<0.05). The expression of hTERT was significantly related with that of P53 (P<0.05). Conclusion Over-expressed hTERT and P53 may be related to the carcinogenesis and progression of thyroid carcinoma and hTERT expression is related to P53 protein. Examination of expression of hTERT and P53 proteins may be helpful to judge the thyroid cancer’s behavior and prognosis.展开更多
文摘Aim: To characterize the coexpression of survivin, an inhibitor of apoptosis (IAF), and human telomerase reverse transcriptase (hTERT) in human testes with varying spermatogenic function. Methods: Transcript levels of survivin mRNA and hTERT mRNA were determined in normal testes (n = 11) and testes with defective spermatogenesis (n = 28) using real-time reverse-transcription polymerase chain reaction (RT-PCR). The histological work-up was performed according to a modified Johnsen score. Results: Expressions of both survivin and hTERT were highest at median levels of 96.8 and 709 in normal spermatogenesis and dropped to 53.3 and 534 in testes with postmeiotic spermatogenic arrest (n = 10). In severe spermatogenic failure (n = 18), survivin expression was lacking in most specimens (n = 16), whereas at least low levels of testicular hTERT expression were largely detectable with a normalized expression of 73 in premeiotic spermatogenic arrest (n = 7) and 45 in patients with Sertoli cell-only syndrome (SCOS) (n = 3). Both survivin and hTERT expressions increased with a progressing Johnsen score (P for trend = 0.001). Conclusion: Although both survivin and hTERT are correlated with spermatogenic function, they show different expression patterns in testes of infertile patients. These findings substantiate results from studies in the rodent testis suggesting a predominant expression of survivin in meiotically dividing germ cells. (Asian J Andro12006 Jan; 8: 95-100)
基金the National Key Basic Research Program of China,No. 2006cb500700the National Natural Science Foundation of China,No.30470904the Natural Science and Technology Foundation of Guangdong Province,No. 04009356, 2008B030301320
文摘BACKGROUND: Numerous current studies have suggested that human telomerase reverse transcriptase (hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE: To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 (AI325-35). DESIGN, TIME AND SETTING: The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS: AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Odk5 and mouse anti-p16 monoclonal antibodies (Lab Vision, USA), and mouse anti-hTERT monoclonal antibody (Epitomics, USA), were used in this study. METHODS: (1) Recombinant adenovirus vectors, encoding hTERT (Ad-hTERT) and green fluorescent protein (Ad-GFP), were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. (2) Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 pmol/L Aβ25-35 for 24 hours. Normal human embryonic cortical neurons treated with 10 pmol/Lβ25.35 for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35. MAIN OUTCOME MEASURES: Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol (TRAP) ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS: Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups (P 〈 0.01). Compared with the control group, cell activity was significantly decreased (P 〈 0.05), and cell apoptotic rate, Cdk5 and p16 expression were significantly increased (P 〈 0.01) in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection (P 〈 0.05). Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased (P 〈 0.01). CONCLUSION: Expression of hTERT in human embryonic cortical neurons can relieve Aβ25-35-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.
文摘Aim: To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase (hTERT) antisense on tumor necrosis factor-α (TNF-α-induced apoptosis in prostate cancer cells (PC3). Methods: Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol (TRAP) and polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA was measured by reverse transcription PCR (RT-PCR) assay and gel-image system, hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by morphological method and determined by flow cytometry. Results: The telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide (S PS-ODN) and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF-α treatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF-α treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group. Conclusion: hTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF-α- induced apoptosis of PC3 cells.
文摘Aim: To evaluate the quantitative detection of human telomerase RNA (hTR) and human telomerase reverse tran-scriptase (hTERT) mRNA as diagnostic parameters in the workup of testicular tissue specimens from patients presentingwith non-obstructive azoospermia. Methods: hTR and hTERT mRNA expression were quantified in 38 cryopre-served testicular tissue specimens by fluorescence real-time reverse transcription - polymerase chain reaction (RT-PCR)in a LightCycler(r). This was paralleled by conventional histological workup in all tissue specimens and additionalsemithin sectioning preparation in cases with maturation arrest (n = 12) and Sertoli-cell-only syndrome (n = 12). Re-sults; The average normalized hTERT expression (N_(hTERT)) was 131.9±48.0 copies (mean ± SD) in tissue speci-mens with full spermatogenesis, N_(hTERT) = 51.2 ±17.2 copies in those with maturation arrest and N_(hTERT) = 2.7 ± 2.4copies in those with Sertoli-cell-only syndrome (SCOS). The discriminant analysis showed that detection of N_(hTERT)(N_(hTR)) had a predictive value of 86.8% (55.3%) for correct classification in one of the three histological subgroups.Conclusion; Our results demonstrate that quantitative detection of hTERT mRNA expression in testicular tissue en-ables a molecular-diagnostic classification of gametogenesis. Quantitative detection of hTERT in testicular biopsies isthus well suited for supplementing the histopathological evaluation. (Asian J Androl 2001 Dec; 3: 263 - 270)
基金Supported by Scientific Foundation of Guangdong Pharmaceutical University, No. 2006GGW01National Natural ScienceFoundation of China,No. 30271110.
文摘To investigate the effects of sodium selenite on telomerase activity and expression of hTERT mRNA in cadmium-transformed 16HBE cells. Methods Telomerase activity and expression of genes were measured after cultured cadmium-transformed 16HBE cells were exposed to sodium selenite at different doses (0.625, 1.25, 2.50, 5.00 pmol/L) for 24 hours. Results Selenium decreased telomerase activity in cadmium-transformed 16HBE cells. There existed an obvious dose-effect relationship between the selenium concentration and these changes. The expression of hTERT and c-myc mRNA also decreased but the expression of madl mRNA increased after exposure to selenium for 24 hours. No difference was found in expression of hTRF1 and hTRF2 mRNA after incubated with sodium selenite for 24 hours, compared with control group. Conclusion Selenium inhibits telomerase activity by decreasing hTERT and c-myc mRNA expression and increasing madl mRNA expression in cadmium-transformed 16HBE cells and selenium concentration is significantly correlated with these changes.
文摘The effects of combined RNA interference(RNAi) of human telomerase RNA(hTR) and human telomerase reverse transcriptase(hTERT) genes on telomerase activity in a bladder cancer cell line(BIU-87 cells) were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer(BTCC).Three TR-specific double-stranded small interfering RNAs(siRNAs) and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA.The phTR-siRNA,phTERT-siRNA,and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells.The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction,and a telomeric repeat amplification protocol was applied to detect telomerase activity.Growth inhibition of BIU-87 cells was measured by MTT assay.Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro.The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,and pRNAT-hTR-Ⅲ+hTERT-Ⅲ in BIU-87 cells.The inhibition efficiency of pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,pRNAT-hTERT-Ⅲ+pRNAT-hTR-Ⅲ was 67% for TERT mRNA,41% for TR mRNA,57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively.The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased,especially in the cells treated with combined RNAi-hTR and-hTERT.Gene chip analysis revealed that 21 genes were down-regulated(ATM,BAX,BCL2,BCL2L1,BIRC5,CD44,CTNNB1,E2F1,JUN,MCAM,MTA1,MYC,NFKB1,NFKBIA,NME4,PNN,PNN,SERPINE1,THBS1,TNFRSF1A,and UCC1).The results indicated that hTR-siRNA and hTERT-siRNA,especially their combination,siRNA hTR+hTERT,specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity.Molecular biological mechanism by which combined siRNA-TR and-TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation of the 21 genes.
基金This work was supported by the grants from the National Natural Science Foundation of China (No. 39870783) and the National 973 Project of China (No.G2000057001)
文摘To construct a mutant pEGFP- hTERTexpression vector, to observe its steady expression intransfected human bladder carcinoma cell line T24 and its role in molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer. Methods: PCR amplification was performed by using primers basedon the known gene sequence of hTERT. PCR productionwas cloned into plasmid pGEMT-T easy and the sequenceof mutant hTERT gene was analyzed. A recombinantmutant hTERT vector (pEGFP-hTERT) was constructed at the EcoR I and Sal I sites of the pEGFP-C1 vector. Aftertransfecting the fusion gene into bladder carcinoma cell line T24 by calcium phosphate-DNA coprecipitation, the steady expression of GFP-hTERT fusion protein was tested by fluorescent light microscopy. The proliferation changes ofbladder carcinoma cell line T24 were detected by lightmicroscopy and senescence correlated b-galactosidase staining. Results: Identification of pEGFP-hTERT byenzyme digestion showed that mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. The steady expression of GFP-hTERT fusion protein was localized in the nucleus of transfected cells. Expression of senescence-associated b-galactosidase in transfected cells gradually increased with extended cultured time and cellgrowth was suppressed. Conclusion: The mutant-type hTERT gene suppresses the proliferation of bladder carcinoma cell line T24 by competitive effect on telomerase activity. This suggests that hTERT gene might be a suitable gene target for bladder cancer therapy.
文摘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.
文摘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 suhunits 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 (X^2 = 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 (X^2 =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.
基金supported by a grant from the Natural Science Funds of Nankai University (06-h83)
文摘BACKGROUND:The human telomerase reverse transcriptase(hTERT) gene encodes the catalytic subunit of telomerase,which mediates pleiotropic effects,including the regulation of senescence and proliferation and plays an important role in carcinogenesis.This study attempts to clarify the genetic predisposition to hepatocellular carcinoma(HCC),focusing on the hTERT gene rs2736098 polymorphism.METHOD:Four hundred patients with HCC and 400 noncancer controls were genotyped to elucidate the potential association between hTERT rs2736098 polymorphism and HCC risks.RESULTS:Compared with the controls,the patients with HCC had a lower frequency of G/G genotype(33.3% vs 44.3%,P=0.001) and a higher frequency of G/A(51.5% vs 39.5%,P=0.001).Allele genotypic frequencies in the patients differed from those of the controls(P=0.040).The data of this study rs2736098[A] allele contributed significantly to HCC risk in female patients(OR=1.78,95% CI,1.17-2.72,P=0.007),patients with HCV infection(OR=2.89,95% CI,1.08-7.70,P=0.031),non-drinker patients(OR=1.32,95% CI,1.06-1.65,P=0.015),and patients not affected by HBV(OR=1.77,95% CI,1.30-2.40,P【0.001).CONCLUSIONS:rs2736098[A] may be an independent hereditary parameter in HCC,but some risk factors would cover up the association by more powerful hepatocarcinogenesis.These results are important guidance for further studies in detecting HCC-associated single nucleotide polymorphisms.
文摘Objective To determine the expression of human telomerase reverse transcriptase (hTERT) and P53 in thyroid carcinoma and its relationship with development and prognosis of the carcinoma. Methods Totally 90 cases of thyroid specimens (60 thyroid carcinomas,10 thyroid adenomas,10 goitres and 10 normal thyroid tissues) were studied by SP immunohistochemical method. Results Positive immunoreactivity of hTERT and P53 was higher in thyroid carcinoma (P<0.05). The positive rates of hTERT and P53 were higher in undifferentiated carcinomas,carcinomas with lymph nodes metastasis or at stage Ⅲ+Ⅳ than in well-differentiated carcinomas,carcinomas without lymph nodes metastasis or at stage Ⅰ+Ⅱ (P<0.05). The expression of hTERT was significantly related with that of P53 (P<0.05). Conclusion Over-expressed hTERT and P53 may be related to the carcinogenesis and progression of thyroid carcinoma and hTERT expression is related to P53 protein. Examination of expression of hTERT and P53 proteins may be helpful to judge the thyroid cancer’s behavior and prognosis.