Telomerase-related advances in hepatocellular carcinoma:A bibliometric and visual analysis

BACKGROUND As a critical early event in hepatocellular carcinogenesis,telomerase activation might be a promising and critical biomarker for hepatocellular carcinoma(HCC)patients,and its function in the genesis and treatment of HCC has gained much attention over the past two decades.AIM To perform a bibliometric analysis to systematically assess the current state of research on HCC-related telomerase.METHODS The Web of Science Core Collection and PubMed were systematically searched to retrieve publications pertaining to HCC/telomerase limited to“articles”and“reviews”published in English.A total of 873 relevant publications related to HCC and telomerase were identified.We employed the Bibliometrix package in R to extract and analyze the fundamental information of the publications,such as the trends in the publications,citation counts,most prolific or influential writers,and most popular journals;to screen for keywords occurring at high frequency;and to draw collaboration and cluster analysis charts on the basis of coauthorship and co-occurrences.VOSviewer was utilized to compile and visualize the bibliometric data.RESULTS A surge of 51 publications on HCC/telomerase research occurred in 2016,the most productive year from 1996 to 2023,accompanied by the peak citation count recorded in 2016.Up to December 2023,35226 citations were made to all publications,an average of 46.6 citations to each paper.The United States received the most citations(n=13531),followed by China(n=7427)and Japan(n=5754).In terms of national cooperation,China presented the highest centrality,its strongest bonds being to the United States and Japan.Among the 20 academic institutions with the most publications,ten came from China and the rest of Asia,though the University of Paris Cité,Public Assistance-Hospitals of Paris,and the National Institute of Health and Medical Research(INSERM)were the most prolific.As for individual contributions,Hisatomi H,Kaneko S,and Ide T were the three most prolific authors.Kaneko S ranked first by H-index,G-index,and overall publication count,while Zucman-Rossi J ranked first in citation count.The five most popular journals were the World Journal of Gastroenterology,Hepatology,Journal of Hepatology,Oncotarget,and Oncogene,while Nature Genetics,Hepatology,and Nature Reviews Disease Primers had the most citations.We extracted 2293 keywords from the publications,120 of which appeared more than ten times.The most frequent were HCC,telomerase and human telomerase reverse transcriptase(hTERT).Keywords such as mutational landscape,TERT promoter mutations,landscape,risk,and prognosis were among the most common issues in this field in the last three years and may be topics for research in the coming years.CONCLUSION Our bibliometric analysis provides a comprehensive overview of HCC/telomerase research and insights into promising upcoming research.

Trichostatin A Induces Apoptosis by Inhibiting Telomerase Activity and Expression of Telomerase Reverse Transcriptase in HL-60 Cells

Aim To investigate the effects of trichostatin A （TSA） on telomerase activity and the expression of human telomerase reverse transcriptase （hTERT） during apoptosis in vitro and the mechanisms in HL-60 cells. Methods The proliferative activity of HL-60 cells was assessed by MTT assay. Cell apoptosis was analyzed by flow cytometry. Telomerase activity was examined by TRAP-ELISA. The expression of telomerase subunits was analyzed by RT-PCR. Results A time- and dose-dependent inhibition was detected in HL-60 cells treated with TSA. After treatment with 600 nmol· L^-1 TSA for 48 h, the apoptosis rate in HL-60 cells was 42. 6% and telomerase activity decreased 1.95 ± 0.25, 1.73 ± 0. 12, and 1.52 ± 0. 09 for 12, 24, and 48 h, respectively. The expression of hTERTmRNA decreased. No significant changes were observed in the expression of hTRmRNA and hTPI mRNA. Condusion TSA inhibits telomerase activity and induces apoptosis in HL-60 cells. The underlying mechanism may be related to the down-regulation of hTERT transcription.

Testicular expression of survivin and human telomerase reverse transcriptase(hTERT)associated with spermatogenic function in infertile patients

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）

Inhibition of telomerase with human telomerase reverse transcriptase antisense increases the sensitivity of tumor necrosis factor-α-induced apoptosis in prostate cancer cells

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.

Transplantation of human telomerase reverse transcriptase gene-transfected Schwann cells for repairing spinal cord injury

Transfection of the human telomerase reverse transcriptase（h TERT）gene has been shown to increase cell proliferation and enhance tissue repair.In the present study,h TERT was transfected into rat Schwann cells.A rat model of acute spinal cord injury was established by the modified free-falling method.Retrovirus PLXSN was injected at the site of spinal cord injury as a vector to mediate h TERT gene-transfected Schwann cells（1×10^（10）/L;10μL）or Schwann cells（1×10^（10）/L;10μL）without h TERT gene transfection.Between 1 and 4 weeks after model establishment,motor function of the lower limb improved in the h TERT-transfected group compared with the group with non-transfected Schwann cells.Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling and reverse transcription-polymerase chain reaction results revealed that the number of apoptotic cells,and gene expression of aquaporin 4/9 and matrix metalloproteinase 9/2decreased at the site of injury in both groups;however,the effect improved in the h TERT-transfected group compared with the Schwann cells without h TERT transfection group.Hematoxylin and eosin staining,PKH26 fluorescent labeling,and electrophysiological testing demonstrated that compared with the non-transfected group,spinal cord cavity and motor and sensory evoked potential latencies were reduced,while the number of PKH26-positive cells and the motor and sensory evoked potential amplitude increased at the site of injury in the h TERT-transfected group.These findings suggest that transplantation of h TERT gene-transfected Schwann cells repairs the structure and function of the injured spinal cord.

Unique case of oligoastrocytoma with recurrence and grade progression:Exhibiting differential expression of high mobility group-A1 and human telomerase reverse transcriptase

Mixed gliomas, primarily oligoastrocytomas, account for about 5%-10% of all gliomas. Distinguishing oligoastrocytoma based on histological features alone has limitations in predicting the exact biological behavior, necessitating ancillary markers for greater specificity. In this case report, human telomerase reverse transcriptase(hT ERT) and high mobility group-A1(HMGA1); markers of proliferation and stemness, have been quantitatively analyzed in formalin-fixed paraffin-embedded tissue samples of a 34 years old patient with oligoastrocytoma. Customized florescence-based immunohistochemistry protocol with enhanced sensitivity and specificity is used in the study. The patient presented with a history of generalized seizures and his magnetic resonance imaging scans revealed infiltrative ill-defined mass lesion with calcified foci within the left frontal white matter, suggestive of glioma. He was surgically treated at our center for four consecutive clinical events. Histopathologically, the tumor was identified as oligoastrocytoma-grade Ⅱ followed by two recurrence events and final progression to grade Ⅲ. Overall survival of the patient without adjuvant therapy was more than 9 years. Glial fibrillary acidic protein, p53, Ki-67, nuclear atypia index, pre-operative neutrophillymphocyte ratio, are the other parameters assessed. Findings suggest that hT ERT and HMGA1 are linked to tumor recurrence and progression. Established markers can assist in defining precise histopathological grade in conjuction with conventional markers in clinical setup.

Telomerase Activity and Telomerase Reverse Transcriptase Expression Induced by Selenium in Rat Hepatocytes

Objective To investigate the effects of sodium selenite on telomerase activity, apoptosis and expression of TERT, c-myc and p53 in rat hepatocytes. Methods Selenium at doses of 2.5, 5.0, and 10 μmol/kg was given to SD rats by garage. In rat hepatocytes, telomerase activity was measured by the telomeric repeat amplification protocol （TRAP）, apoptosis was detected by flow cytometry, and expressions of telomerase reverse transcriptase （TERT）, c-myc and p53 were analyzed by reverse transcription-polymerase chain reaction （RT-PCR）. c-Myc and P53 proteins were detected by immunochemistry. Results Selenium at doses of 2.5, 5.0, and 10 μmol/kg significantly increased hepatocellular telomerase activity and induced apoptosis in a dose-dependent manner. Although selenium at doses of 2.5, 5.0, and 10 μmol/kg displayed no obvious enhancing effect on the TERT mRNA expression in rat hepatocytes （P〉0.05）, it significantly increased the c-myc mRNA and p53 mRNA expression at the dose of 10 μmol/kg （P〈0.05）. Selenium at doses of 5.0 and 10 μmol/kg obviously increased the content of P53 protein in rat hepatocytes, but only at the dose of 10 μmol/kg, it significantly promoted the value of c-Myc protein in them. Conclusion Selenium can slightly increase telomerase activity and TERT expression, and significantly induce apoptosis and over-expression of c-myc and p53 at relatively high doses. The beneficial effects of selenium on senescence and aging may be mediated by telomerase activation and expression of TERT, c-myc, and p53 in rat hepatocytes.

Neuroprotective effects of human telomerase reverse transcriptase on beta-amyloid fragment 25-35-treated human embryonic cortical neurons

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.

Quantification of human telomerase RNA(hTR)and human telomerase reverse transcriptase(hTERT)mRNA in testicular tissue of infertile patients

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.

RADIATION- INDUCED PROGRESSIVE DECREASING IN THE EXPRESSION OF REVERSE TRANSCRIPTASE GENE OF hEST2 AND TELOMERASE ACTIVITY

Objectives. In order to identify the relationship between telomerase and the biological effect of radiation injury,and investigate the role of human telomerase catalytic subunit gene (hEST2) reverse transcriptase(RT) segment in the expression of telomerase activity. Methods. Tumor HeLa cells, KB cells and A431 cells were employed to measure the change in telomerase activity after 60Co- ray irradiation at RNA level and protein level. Quantitative PCR and Northern blotting were used to determine the expression of hEST2 RT segment that encodes seven motifs of the human telomeres, a PCR- based telomeric repeat amplification protocol (TRAP)was used to assay telomerase activity after exposure to radiation. Results. Both of telomerase activity and the expression hEST2 RT segment were decreased with increasing dosage of radiation. In addition, testing the expression of motifs domain is similar to the measurement of telomerase activity. Conclusion. The detection of the hEST2 RT segment by Northern blotting and quantitative PCR are new methods for testing telomerase activity. Furthermore, radiation can cause a dose- dependent decrease in telomerase activity. The effect of radiation on telomerase is one possible reason for the death of cancer cells after irradiation.

Expression of Telomerase Reverse Transcriptase during the Malignant Transformation of Cadmium-Induced Cells

The objective of the present study was to investigate human telomerase reverse transcriptase (hTERT) mRNA and protein expressions during the cadmium chloride-induced malignant transformation of human bronchial epithelial (16HBE) cells. Fluorescence quantitative PCR (FQ-PCR) and Western blot analyses were performed to detect the hTERT mRNA and protein expressions in normal 16HBE cells, cadmium chloride-transformed 16HBE cells, and tumorigenic cells from nude mice inoculated with cadmium chloride-transformed 16HBE cells. Under the inner standard of GAPDH, the hTERT mRNA expression was significantly higher at different stages of malignant transformation (cadmium chloride-transformed 16HBE cells at passages 15 and 35 and tumorigenic cells from nude mice) than in normal 16HBE cells, and increased with the development of malignancy (P < 0.01). In addition, hTERT protein expression increased with the development of malignancy. These findings demonstrate that hTERT expression is related to cadmium chlorideinduced malignant transformation. Cadmium chloride-induced malignant transformation is involved in changes in the hTERT activity, and might be an early event in cadmium chloride-induced malignant transformation.

Study on neoplasia of hydatidiform mole by detecting telomerase reverse transcriptase mRNA expression in peripheral blood mononuclear cells

By setting up a real-time fluorescent quantitative RT-PCR assay to detect human telomerase reverse transcriptase （hTERT） mRNA in hydatidiform mole in peripheral blood mononuclear cells, to analyze the correlation between the expression level of hTERT mRNA and the prognosis of hydatidiform mole, and to evaluate the clinic value of quantitative determination of hTERT mRNA in the diagnosis of hydatidiform mole. Methods： A real-time fluorescent quantitative RT-PCR （FQ RT-PCR） assay based on TaqMan fluorescence methodology and the Light-Cycler system was used to quantify the full range of hTERT mRNA copy numbers in 30 samples of hydatidiform mole and the neoplasia of hydatidiform mole. The normalized hTERT （NhTERT） was standardized by quantifying the number of GAPDH transcripts as internal control and expressed as 100x （hTERT/GAPDH） ratio. Based on the prognosis of the hydatidiform mole, the patients were divided into two groups： the experimental group and the control group, to compare the telomerase reverse transcriptase mRNA expression in peripheral blood mononuclear cells. Results： hTERT mRNA was both expressed in the peripheral blood mononuclear cells and pathological tissues in the mole experimental group and the control group. In the mole experimental group, the values were 6.31±0.32 and 6.24±0.44, respectively, and there was no significant difference between them （P〉0.05）. But in the control group the values were 1.21±0.65 and 1.40±0.61, respectively, and there was no significant difference between them （P〉0.05） The values in experimental group was significantly higher than those in the control group （P〈0.01）. Conclusion： Quantitative determination of hTERT mRNA by FQ RT-PCR is a rapid and sensitive method, hTERT in peripheral blood mononuclear cells may have potential use as a biomarker for the early detection of the prognosis of the hydatidiform mole.

Inhibition of Telomerase with hTERT Antisense Increases Susceptibility of Leukemic Cells to CDDP-induced Apoptosis

Objective: To investigated the e?ect of inhibition of telomerase with hTERT antisense on leukemic cells (HL-60 and K562) to CDDP-induced apoptosis. Methods: Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and puri?ed. Telomerase activity was detected by Telomerase PCR ELASA kit and cell apoptosis was observed by morphological method and determined by ?owcytometry. Results: AS PS-ODN could signi?cantly inhibit telomerase activity by down regulat- ing the hTERT expression, and increase the susceptibility of leukemic cells to CDDP-induced apoptosis. Conclusion: Inhibition of telomerase with hTERT antisense can increases the susceptibility of leukemic cells to CDDP-induced apoptosis.

Inhibitory Effects of Selenium on Telomerase Activity and hTERT Expression in Cadmium-transformed 16HBE Cells

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.

Telomerase and hTERT: Can they serve as markers for gastric cancer diagnosis?

AIM: To investigate telomerase activity and human telomerase reverse transcriptase (hTERT) expression in normal human gastric mucosal epithelial cells (nhGMECs) and fibroblasts (nhGMFs).

Telomeres,telomerase and colorectal cancer

Colorectal cancer(CRC)is the third most common cancer worldwide and,despite improved treatments,is still an important cause of cancer-related deaths.CRC encompasses a complex of diseases arising from a multistep process of genetic and epigenetic events.Besides heterogeneity in the molecular and biological features of CRC,chromosomal instability is a hallmark of cancer and cancer cells may also circumvent replicative senescence and acquire the ability to sustain unlimited proliferation.Telomere/telomerase interplay is an important mechanism involved in both genomic stability and cellular replicative potential,and its dysfunction plays a key role in the oncogenetic process.The erosion of telomeres,mainly because of cell proliferation,may be accelerated by specific alterations in the genes involved in CRC,such as APC and MSH2.Although there is general agreement that the shortening of telomeres plays a role in the early steps of CRC carcinogenesis by promoting chromosomal instability,the prognostic role of telomere length in CRC is still under debate.The activation of telomerase reverse transcriptase(TERT),the catalytic component of the telomerase complex,allows cancer cells to grow indefinitely by maintaining the length of the telomeres,thus favouring tumour formation/progression.Several studies indicate that TERT increases with disease progression,and most studies suggest that telomerase is a useful prognostic factor.Plasma TERT mRNA may also be a promising marker for the minimally invasive monitoring of disease progression and response to therapy.

Combination of telomerase antisense oligonucleotides simultaneously targeting hTR and hTERT produces synergism of inhibition of telomerase activity and growth in human colon cancer cell line

AIM: To investigate synergism of inhibition of telomerase activity and proliferation of human colon cancer cells by combination of telomerase antisense oligonucleotides (ASODNs) simultaneously targeting human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in vitro. METHODS: ASODN of hTR and ASODN of hTERT were transfected into human colon cancer SW480 cells by liposomal transfection reagents. Telomerase activity of SW480 cells was examined using telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (PCR-ELISA). Proliferation activity of SW480 cells was tested by methyl thiazolyl tetrazolium assay. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: The telomerase activity and cell survival rate in SW480 cells transfected with 0.2 μmol/L of ASODN of hTR or ASODN of hTERT for 24-72 h were significantly decreased in a time-dependent manner compared with those after treatment with sense oligonucleotides and untreated (telomerase activity: 24 h, 73%, 74% vs99%, 98%; 48 h, 61%, 55% vs98%, 99%; 72 h, 41%, 37% vs 99%, 97%; P<0.01; cell survival rate: 24 h, 88%, 86% vs594%, 98%; 48 h, 49%, 47% vs94%, 97%; 72 h, 44%, 42% vs92%, 96%; P<0.01). Moreover, the telomerase activity and the cell survival rate in SW480 cells treated by the combination of telomerase anti-hTR and anti-hTERT were more significantly suppressed than single anti-hTR or anti-hTERT (telomerase activity: 24 h, 59% vs 73%, 74%; 48 h, 43% vs61%, 55%; 72 h, 18% vs41%, 37%; P<0.01; cell survival rate: 24 h, 64% vs88%, 86%; 48 h, 37% vs49%, 47%; 72 h, 25% vs44%, 42%; P<0.01). Meanwhile, the apoptosis rates in the combination group were markedly increased compared with those in the single group (24 h, 18.0% vs7.2%, 7.4%; 48 h, 23.0% vs13.0%, 14.0%; 72 h, 28.6% vs 13.2%, 13.75; P<0.01). Cells in combination group were arrested at G0/G1 phase. CONCLUSION: Telomerase anti-hRT and anti-hTERT suppress telomerase activity, and inhibit growth of human colon cancer cells probably via induction of apoptosis and retardation of cell cycle. Additionally, combined use of telomerase ASODNs targeting both hTR and hTERT yields synergistic action selective for human colon cancer.

Effects of Cadmium on Telomerase Activity,Expressions of TERT,C-myc and P53,and Apoptosis of Rat Hepatocytes

This study investigated the effect of cadmium on the telomerase activity,the expression of TERT,c-myc and p53 and the apoptosis of rat hepatocytes.The rats were administrated 5,10 and 20 μmol/kg cadmium chloride intraperitoneally and sacrificed 48 h after the initial treatment.The telomerase activity of the rat hepatocytes was measured by the telomeric repeat amplification protocol (TRAP),and apoptosis was detected by flow cytometry.The mRNA expressions of TERT,c-myc and p53 were measured by reverse transcription-polymerase chain reaction (RT-PCR).C-myc and P53 proteins were determined by immunochemistry.The results showed that cadmium chloride increased the hepatocellular telomerase activity in a dose-dependant manner and induced the apoptosis of hepatocytes significantly.The value of relative coefficient between the telomerase activity and the apoptosis rate was 0.9398.RT-PCR revealed that specific bands corresponding to the TERT mRNA,c-myc mRNA,and p53 mRNA were displayed at 185,342 and 538 bp respectively.Cadmium chloride could substantially increase the mRNA expressions of TERT,c-myc and p53 in rat hepatocytes,as compared with control.Moreover,cadmium chloride at the doses of 5,10 and 20 μmol/kg could increase the content of P53 protein in rat hepatocytes obviously,but only that at the doses of 10 and 20 μmol/kg substantially promoted the c-myc protein level in rat hepatocytes.Our study herein suggested that cadmium may contribute to the carcinogenesis by activating telomerase,and overexpressing the mRNAs of TERT,c-myc and p53,and causing apoptosis of normal cells.

Effects of Combined siRNA-TR and-TERT on Telomerase Activity and Growth of Bladder Transitional Cell Cancer BIU-87 Cells

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.