Effect of Lidamycin on Telomerase Activity in Human Hepatoma BEL-7402 Cells

Objective To 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. Methods Chromatin 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-13-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. Results Mitotic 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-13-gal activity and the changes of senescence-related protein expression. Telomerase activity markedly decreased （P〈0.01） in LDM-treated hepatoma BEL-7402 cells. Conclusion Mitotic 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.

Detection of telomerase activity by combination of telomeric repeat amplification protocol and electrochemiluminescence assay

A highly sensitive telomerase detection method that combines telomeric repeat amplification protocol （TRAP） and magnetic beads based electrochemiluminescence （ECL） assay has been developed. Briefly, telomerase recognizes biotinylated telomerase synthesis primer （B-TS） and synthesizes extension products, which then serve as the templates for PCR amplification using B-TS as the forward primer and tris-（2′2′-bipyridyl） ruthenium （TBR） labeled ACX （TBR-ACX） as the reversed primer. The amplified product is captured on streptavidin-coated paramagnetic beads and detected by ECL. Telomerase positive HeLa cells were used to validate the feasibility of the method. The experimental results showed down to 10 cancer cells can be detected easily. The method is a useful tool for telomerase activity analysis due to its sensitivity, rapidity, safety, high throughput, and low cost. It can be used for screening a large amount of clinical samples.

TELOMERASE ACTIVITY IN COLORECTAL CARCINOMA AND ITS CORRELATION WITH EXPRESSION OF C-MYC

Objective： To study the role of telomerase activity and c-myc in pathogenesis and progression of colorectal carcinoma, and to investigate the possible regulatory mechanism of telomerase activation. Methods： A modified telomeric repeat amplification protocol （TRAP） and immunohistochemical staining was used to detect telomerase activity and the expression of c-myc in tissue samples from colorectal carcinoma, paracarcinomatousl tissues, normal mucosa, and adenomatoid polyp. Results： The positive rates of telomerase activity and c-myc expression were 83.33% and 80.00% in colorectal carcinoma, 13.33% and 23.33% in paracarcinomatousl tissues, 13.33% and 20.00% in normal mucosa, and 10.00% and 45.00% in adenomatoid polyp respectively, they were significantly higher in colorectal carcinoma than in paracarcinomatousl tissues, normal mucosa, and adenomatoid polyp （P〈0.05）. The rates of telomerase activity and c-myc expression were much higher in colorectal carcinoma with lymph nodes metastases than that without lymph nodes metastases. The expression of c-myc was found being significantly higher in the telomerase positive colorectal carcinoma than in the telomerase negative group （P〈0.05）. Conclusion： The activation of telornerase and abnormal expression of c-myc might play an important role in the process of carcinogenesis and progression of colorectal carcinoma. The over-expression of c-myc may be related to telomerase activation and up-regulation in colorectal carcinoma.

DETECTION OF TELOMERASE ACTIVITY IN BRONCHOSCOPICBRUSHING CELLS OF LUNG CANCER PATIENTS

Objective: To investigate the diagnostic significance of the detection of telomerase activity in the brushing cells obtained from fiberobronchoscopy. Methods: The techniques of TRAP-PCR-ELISA and TRAP-silver staining were employed to detect telomerase activity in 42 patients (57 samples) with pulmonary diseases. Results: Telomerase activity in the lesion side of lung cancer patients (N=23) was significantly higher than that in the contralateral side of the same patient (P<0.05), and in patients with pneumonia (P<0.05). In 23 patients with lung cancer, 21 cases (91.3%) were showed positive in telomerase activity, while only 12 cases (52.3%) were positively diagnosed by cytological smear examination (P<0.05). In 6 cases with cytological dysplasia of exfoliated cells, 5 (83.3%) were found to be telomerase activity positive. Conclusion: Detection of telomerase activity in the brushing cells obtained from fiberobronchoscopy may be an effective and sensitive method in the diagnosis of pulmonary malignant diseases.

Inhibition of Cell Growth and Telomerase Activity in Osteosarcoma Cells by DN-hTERT

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.

Effects of Tai Chi on telomerase activity and gerotranscendence in middle aged and elderly adults in Chinese society

Introduction:Telomeres are DNA protein structures at the end of chromosomes and are linked to the physical aging process.The improvement of quality of life is closely associated with aerobic exercise,and thedynamic effects of exerciseonphysiology and psychology are evident with aging.Tai Chi is popularly practiced in China.However,findings on the effects of Tai Chi on telomerase activity(TA)in peripheral blood mononuclear cells,and gerotranscendence(GT),as well as the association of TA and GT with Tai Chi,have been inconsistent.Purpose:This study aims to assess TA in peripheral blood mononuclear cells,GT,and the associations between them.The associations among these variables are determined during six months of Tai Chi intervention among Chinese middle aged and elderly adults.Methods:TA assessment was obtained by TE-ELISA(human telomeraseeenzyme linked immunosorbent assay),and GT was measured at the baseline level after six months of Tai Chi intervention.Results:TA increased significantly in the Tai Chi group from 23.75±3.78 u/mmol(preintervention)to 26.31±2.93 u/mmol(after 6 months)(p<0.05).Compared with the TA in the control group,the TA in the intervention group was statistically significant after six months(p<0.05).Compared with the GT in the control group,the GT in the intervention group improved significantly after six months(p<0.05).TA and GT had a positive correlation(r=0.325,p<0.01).Conclusion:Our data illustrated that Tai Chi had a protective effect on TA and might improve the GT in Chinese middle aged and elderly adults.The TA increased with the increasing GT in Chinese middle aged and elderly adults.

Resveratrol-induced augmentation of telomerase activity delays senescence of endothelial progenitor cells

Background Previous studies have shown that resveratrol increases endothelial progenitor cell （EPC） numbers and functional activity. Increased EPC numbers and activity are associated with the inhibition of EPC senescence. In this study, we investigated the effect of resveratrol on the senescence of EPCs, leading to potentiation of cellular function. Methods EPCs were isolated from human peripheral blood and identified immunocytochemically. EPCs were incubated with resveratrol （1, 10, and 50 pmol/L） or control for specified times. After in vitro cultivation, acidic 13-gatactosidase staining revealed the extent of senescence in the cells. To gain further insight into the underlying mechanism of the effect of resveratrol, we measured telomerase activity using a polymerase chain reaction （PCR）-enzyme-linked immunosorbent assay （ELISA） technique. Furthermore, we measured the expression of human telomerase reverse transcriptase （hTERT） and the phosphorylation of Akt by immunoblotting. Results Resveratrot dose-dependently inhibited the onset of EPC senescence in culture. Resveratrol also significantly increased telomerase activity. Interestingly, quantitative real-time PCR analysis demonstrated that resveratrol dose-dependently increased the expression of the catalytic subunit, hTERT, an effect that was significantly inhibited by pharmacological phosphatidylinositol 3-kinase （PI3-K） blockers （wortmannin）. The expression of hTERT is regulated by the PI3-K/Akt pathway; therefore, we examined the effect of resveratrol on Akt activity in EPCs. Immunoblotting analysis revealed that resveratrol led to dose-dependent phosphorylation and activation of Akt in EPCs. Conclusion Resveratrol delayed EPCs senescence in vitro, which may be dependent on telomerase activation.

Telomerase activity and expression of the telomerase catalytic subunit gene in non small cell lung cancer: correlation with decreased apoptosis and clinical prognosis

To investigate the level of telomerase activation (TA) and telomerase catalytic subunit (hEST 2) gene mRNA in patients with non small cell lung cancer, and determine whether they are associated with tumor cell apoptosis, stage, and clinical outcome Methods Primary tumor specimens from 58 patients untreated with chemotherapy and 10 cases of histologically benign and adjacent lung tissue were analyzed TA and hEST 2 were measured by means of a modified telomerase repeat amplification protocol (TRAP) assay and in situ hybridization (ISH), respectively Terminal deoxynucleotidyl transferase (TdT) mediated biotin dUTP nick end labeling (TUNEL) was used to evaluate apoptotic cells Reverse transcription polymerase chain reaction (RT PCR) was used to detect bcl 2 mRNA expression Results TA and hEST 2 were detected in 45 (77 6%) and 43 (74 1%) of 58 tumor specimens, respectively, and not detected in specimens of adjacent and benign lung tissue One case expressed hEST 2 as a weak positive Statistically significant positive association was found between the level of TA and hEST 2( r =0 85, P =0 001) TA and hEST 2 were associated with tumor stage, but not associated with tumor grade, gender and patient age Positive rate of bcl 2 mRNA was 38 (65 5%) of 58 tumor specimens The mean apoptotic index in the bcl 2 positive group (9 5±1 3) was lower than that in the bcl 2 negative one (19 8±2 1, P <0.05), suggesting that apoptotic index may be inversely associated with bcl 2 expression ( r =-0 48, P =0 041) Bcl 2 expression in the TA and hEST 2 positive group (92 1% and 89 4%) was higher than that in the negative one (50 0% and 45 0%, P =0 043 and P =0 032, respectively) The apoptotic index was lower in the TA or hEST 2 positive group (8 2±1 4, 10 7±1 1) than in the negative one (20 5±1 6, 24 2±2 1, P <0 05) A statistically significant inverse association was found between TA or hEST 2 and apoptotic index ( r =-0 45, P =0 02 and r = -0 51, P =0 001, respectively) Positive correlation was also detected between TA or hEST 2 and bcl 2 expression ( r =0 86, P =0 01 and r =0 73, P =0 024, respectively) The level of hEST 2 mRNA and apoptotic index were associated with clinical outcome in a multivariate cox regression analysis Conclusions High TA and hEST 2 were frequently detected in primary non small cell lung cancer untreated with chemotherapy, having high bcl 2 expression and a low tumor cell apoptotic rate This suggests that both TA and hEST 2 are correlated with the deregulation of apoptosis hEST 2 and apoptotic index have prognostic significance in patients with non small cell lung cancer

Comparative Analysis of Telomerase Activity in CD117^＋CD34^＋ Cardiac Telocytes with Bone Mesenchymal Stem Cells, Cardiac Fibroblasts and Cardiomyocytes

Background： This study characterized the cardiac telocyte （TC） population both in vivo and in vitro, and investigated its telomerase activity related to mitosis. Methods： Using transmission electron microscopy and a phase contrast microscope, the typical morphological features of cardiac TCs were observed; by targeting the cell surface proteins CD1 17 and CD34, CD 117^＋CD34^＋ cardiac TCs wcrc sorted via flow cytomctry and validated by immunofluorescence based on the primary cell culture. Then the optimized basal nutrient medium for selected population was examined with the cell counting kit 8. Under this conditioned medium, the process of cell division was captured, and the telomerase activity of CD117^＋ CD34^＋ cardiac TCs was detected in comparison with bone mesenchymal stem cells （BMSCs）, cardiac fibroblasts （CFBs）, cardiomyocytes （CMs）. Results： Cardiac TCs projected characteristic telopodes with thin segments （podomers） in alternation with dilation （podoms）. In addition, 64% of the primary cultured cardiac TCs were composed of CD117^＋CD34^＋ cardiac TCs： which was verified by immunofluorescence. In a live cell imaging system, CD117^＋CD34^＋ cardiac TCs were observed to enter into cell division in a short time. followed by an significant invagination forming across the middle of the cell body. Using a real-time quantitative telomeric-repeat amplification assay, the telomerase concentration in CD117^＋CD34^＋ cardiac TCs was obviously lower than in BMSCs and CFBs, and significantly higher than in CMs. Conclusions： Cardiac TCs represent a unique cell population and CD11 7^＋CD34^＋ cardiac TCs have relative low telomerase activity that differs from BMSCs, CFBs and CMs and thus they might play an important role in maintaining cardiac homeostasis.

DETECTION OF TELOMERASE ACTIVITY IN BREAST CARCINOMA

Objective: To investigate the significance of telomerase activity in breast carcinoma with its respect to axillary lymph node status Methods: Telomerase activity was analyzed in 88 breast carcinomas and 16 benign breast lesions, using polymerase chain reaction (PCR) based telomeric repeat amplification protocol (TRAP) assay Results: Telomerase activity was detected in 75 (85%) of 88 breast carcinomas (including three breast carcinomas in situ which were all positive for telomerase activity), whereas in benign breast lesions analyzed only 2(12 5%) of 16 cases were positive for telomerase activity The difference between the two groups was statistically significant ( P <0 001) Besides, telomerase activity was expressed significantly higher in node positive breast carcinoma (93%) than in node negative ones (77%) ( P <0 05) Conclusion: Our results suggest that telomerase activation plays an important role during breast carcinoma development It is possible that this enzyme may serve as an early indication of breast carcinoma

Effects of arotinoid acid on induction of apoptosis and differentiation and telomerase activity and cell cycle in the HL 60 cell line

Objective To investigate the effects of arotinoid acid (Ro13 7410) on the morphological and functional alterations of leukemia HL 60 cell line and compared with those of RA Methods Differentiation of HL 60 cells was assessed by morphology and by NBT reduction Trypan blue exclusion was used to determine viability Apoptosis was assessed by changes in cell morphology and by measurement of fragmented DNA using the PCD assay kit Telomerase PCR ELISA kit tested telomerase activity The cell cycle was analyzed by flow cytometry Results Incubation of the HL 60 cells with 10 -6 10 -8 ?mol/L Ro13 7410 resulted in suppression of cell growth Apoptotic cells were detected following exposure to 10 -6 ?mol/LRo13 7410 for 3 hours by measurement of the “in situ” enzymatic labeling of DNA breaks with biotinylated dUTP Ultrastructural examination of Ro13 7410 treated samples showed cells with chromatin compaction and cytoplasm condensation and the presence of “apoptotic bodies” Cells induced into apoptosis were accompanied by Department of Hematology, the First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China (Liu XS, Lou LS, Zeng SR and Tang ZH) Department of Clinical Biochemistry, Chongqing University of Medical Sciences, Chongqing 400046, China (Jiang JK, Zhang Y, Xu XG, Liu BZ, He YJ and Kang GF) increase of intracellular free Ca 2+ Percentage of HL 60 cells reduced NBT following incubation with Ro13 7410 was lower than with all trans retinoic acid (RA) (27% vas 85%) Telomerase PCR ELISA assay showed that HL 60 cells cultured in the absence of inducing agents had significant telomerase activity Telomerase activity declined gradually after 10 -6 ?mol/L Ro13 7410 treatment, and changes becoming evident at 1 day The inhibition of telomerase activity at day 5 of treatment with Ro13 7410 was less effective than with RA DNA flow cytofluorimetric analysis revealed that Ro13 7410 caused partial cell arrest in the G 2/M phase after a 2 day treatment and the percentage of cells arrested in the G 2/M phase increased after 4 days treatment With RA treated cells, a reduction in the percentage of cells in the G 2/M phase was observed after 2 day of treatment Conclusion Our study shows that Ro13 7410 suppresses HL 60 cells growth mainly via the induction of apoptosis and is less effective than RA in induction differentiation Ro13 7410 dramatically inhibits telomerase activity during the course of induction and results in G 2/M arrest within 2 days These findings suggest that Ro13 7410 is worthy of further study for its effects on leukemic cells

MicroRNA-mediated NBS1 Gene Silence and Its Effects on Telomerase Activation in Hela Cells

Objective： To research the silence of NBS1 after transfection microRNA expressing eukaryotic recombinants and the changes of telomerase activation in telomerase-positive cell line Hela. Methods： According to the sequence of NBS1 mRNA, the NBS1 pre-microRNA was designed and synthesized, then cloned into the GFP reporter pcDNA6.2-GW/EmGFP-miR vector and transfected into Hela cells. The integrity of the insert fragment was verified through colony PCR and sequencing analysis. The NBS1 gene expression of NBS1 microRNA recombinants was detected by Real-Time PCR and western blot. Telomerase activity in Hela cells was assayed by TRAP-PCR-EB. Results： Sequences of insert fragment in microRNA expressing recombinants were correct. The NBS1 gene expression was decreased, and the telomerase activation of Hela cell reduced. Conclusion： NBS1 microRNA inhibits NBS1 gene expression, and depresses telomerase activation of Hela cells. This confirms that there is relevance between NBS 1 gene and telomerase activity.