Telomerase Reverse Transcriptase Expression Levels Research Articles

Elevated TERT Expression in TERT-Wildtype Adult Diffuse Gliomas: Histological Evaluation with a Novel TERT-Specific Antibody.

Telomerase reverse transcriptase (TERT) is important for the biology of diffuse gliomas. TERT promoter mutations are selectively observed among 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene- (IDH-) wildtype glioblastoma (GBM). However, TERT transcripts range widely in various cancers including gliomas, and TERT protein expression has been rarely investigated thus far. It would be thus critical to examine the expression level of TERT in tumors in addition to its mutational status, and sensitive and specific methods are urgently needed to examine TERT protein expression for the assessment of TERT biology in gliomas. Using our newly developed TERT-specific monoclonal antibody (TMab-6) applicable to human tissue, we found an unexpected increase in TERT expression in TERT-wildtype as well as TERT-mutated gliomas and in tumor vasculature. This is the first extensive analysis on the expression of TERT immunoreactivity in human glioma tissue, suggesting that TERT protein expression may be regulated by several mechanisms in addition to its promoter mutation.

Curcumin Affects Adipose Tissue-Derived Mesenchymal Stem Cell Aging Through TERT Gene Expression

Aging and losing cell survival is one of the main problems in cell therapy. Aging of Mesenchymal Stem Cells (MSCs) is associated with a rise in intracellular reactive oxygen species, decrease in telomerase reverse transcriptase (TERT) expression and finally eroded telomere ends. Given that the production of free radicals in the aging process is effective, the use of antioxidants can help in scavenging free radicals and prevent the aging of cells. The aim of this study is to evaluate the effects of curcumin on proliferation, aging and TERT expression of rat adipose tissue-derived stem cells (rADSC). rADSCs were isolated from inguinal rat adipose tissue and their viabilities were assessed by MTT after exposure to different concentrations of curcumin. Flow-cytometry was performed for investigating the cell surface markers. Adipogenic and osteogenic differentiation were carried out to evaluate the pluripotency of rADSCs. Telomerase expression and percentage of senescent cells were evaluated using real-time PCR and senescence-associated β-galactosidase activity, respectively. The results demonstrated significant proliferation of rADSCs after 48 h treatment with 1 and 5 µM curcumin. Additionally, these concentrations could significantly reduce the population doubling time and aging of rADSCs at different passages. The findings of SA-ß-gal staining showed that curcumin significantly decreased the number of senescent cells in the 5 and 7 cell passages. Moreover, expression levels of TERT increased in the presence of 1 and 5 µM curcumin than control group (P<0.001). As a conclusion, curcumin may be a good candidate to improve lifespan of rADSCs through promoting TERT gene expression.

Quantum Dots-siRNA Nanoplexes for Gene Silencing in Central Nervous System Tumor Cells.

RNA interfering (RNAi) using short interfering RNA (siRNA) is becoming a promising approach for cancer gene therapy. However, owing to the lack of safe and efficient carriers, the application of RNAi for clinical use is still very limited. In this study, we have developed cadmium sulphoselenide/Zinc sulfide quantum dots (CdSSe/ZnS QDs)-based nanocarriers for in vitro gene delivery. These CdSSe/ZnS QDs are functionalized with polyethyleneimine (PEI) to form stable nanoplex (QD-PEI) and subsequently they are used for siRNA loading which specially targets human telomerase reverse transcriptase (TERT). High gene transfection efficiency (>80%) was achieved on two glioblastoma cell lines, U87 and U251. The gene expression level (49.99 ± 10.23% for U87, 43.28 ± 9.66% for U251) and protein expression level (51.58 ± 7.88% for U87, 50.69 ± 7.59% for U251) of TERT is observed to decrease substantially after transfecting the tumor cells for 48 h. More importantly, the silencing of TERT gene expression significantly suppressed the proliferation of glioblastoma cells. No obvious cytotoxicity from these QD-PEI nanoplexes were observed over at 10 times of the transfected doses. Based on these results, we envision that QDs engineered here can be used as a safe and efficient gene nanocarrier for siRNA delivery and a promising tool for future cancer gene therapy applications.

Abstract 2851: EGFR-TERT cooperation in the development of EGFR-TKI treatment induced pulmonary fibrosis

Abstract Background: Previous studies have shown that epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment in lung cancer can lead to the development of pulmonary fibrosis (PF). In other studies, it has been shown that reduced telomerase reverse transcriptase (TERT) function increases susceptibility to PF. In this work, we hypothesize that TERT is associated with the pathogenesis of EGFR-TKI induced PF via important signaling pathways. Methods: HCC827 cells (EGFR L858R) was treated with gefitinib and TERT expression level was detected following the treatment. To investigate the molecular changes following gefitinib treatment, we also performed a 84-TF qPCR- microarray and a qPCR microarray for 84-key secreted proteins of cytokines and chemokines. The level of interleukin-6(IL-6) was further measured with ELISA in cell medium following the gefitinib treatment. Fibrogenic markers were detected after co-culturing HCC827 with lung fibroblast IMR-90 cells. Results: TERT gene expression was significantly decreased over 90% after gefitinib treatment. Early growth response protein 1(EGR-1) and E2F transcription factor 1(E2F1) were significantly down-regulated as major TFs that are responsive to gefitinib treatment. Both EGR-1 and E2F1 have been shown to regulate TERT expression in previous studies. In addition, TFs including JUN and most REL members involved in the NF-κB pathway, were significantly up-regulated after gefitinib treatment, which are known to lead to the activation of the senescence-associated secretory phenotype (SASP) pathway. IL-6 mRNA as well as protein secretion were both significantly up-regulated after gefitinib treatment. After co-culturing HCC827 and IMR-90 for 7 days and treating with gefitinib, the growth of IMR-90 cells were increased compare to the control group. Fibrogenic markers, e.g. COL1A1 (Collagen, Type I, Alpha 1), α-SMA (alpha smooth muscle actin), FSP-1(Fibroblast-specific protein 1), etc. were all up-regulated. Conclusion: Our data suggest that EGFR inhibition leads to lung epithelial cell senescence via inhibiting TERT function and activating of the SASP pathway, which further activates fibroblasts and induces fribrogenesis. Continued studies are warranted to further understand the molecular signaling in this mechanism. Citation Format: Rongrong Wei, Wanqing Liu. EGFR-TERT cooperation in the development of EGFR-TKI treatment induced pulmonary fibrosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2851.

TERT alleviates irradiation-induced late rectal injury by reducing hypoxia-induced ROS levels through the activation of NF-κB and autophagy.

The hypoxic microenvironment which is present following irradiation has been proven to promote radiation-induced injury to normal tissues. Previous studies have demonstrated that telomerase reverse transcriptase (TERT) is regulated by hypoxia, and that it plays a protective role in the process of wound repair. However, its effects on radiation-induced injury remain unclear. In this study, we examined the effects of human TERT on irradiation-induced late rectal injury in fibroblasts under hypoxic conditions. We also performed in vivo experiments. The rectums of 5-week-old female C57BL/6N mice were irradiated locally with a single dose of 25 Gy. We then examined the fibrotic changes using hematoxylin and eosin staining, and Masson's staining. The expression of hypoxia inducible factor-1α (HIF-1α) and TERT was analyzed by immunohistochemistry. In in vitro experiments, apoptosis, reactive oxygen species (ROS) production and the autophagy level induced by exposure to hypoxia were assayed in fibroblasts. The association between TERT, nuclear factor-κB (NF-κB) and the autophagy level was examined by western blot analysis. The antioxidant effects of TERT were examined on the basis of the ratio of glutathione to glutathione disulfide (GSH/GSSG) and mitochondrial membrane potential. Rectal fibrosis was induced significantly at 12 weeks following irradiation. The HIF-1α and TERT expression levels increased in the fibrotic region. The TERT-overexpressing fibroblasts (transfected with an hTERT-expressing lentiviral vector) exhibited reduced apoptosis, reduced ROS production, a higher autophagy level, a higher GSH/GSSG ratio and stable mitochondrial membrane potential compared with the fibroblasts in which TERT had been silenced by siRNA. NF-κB was activated by TERT, and the inhibition of TERT reduced the autophagy level in the fibroblasts. These results demonstrate that TERT decreases cellular ROS production, while maintaining mitochondrial function and protecting the cells from hypoxia-induced apoptosis, which may thus attenuate the effects of irradiation-induced hypoxia on rectal injury following irradiation.

A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression.

BackgroundTo facilitate indefinite proliferation, stem cells and most cancer cells require the activity of telomerase, which counteracts the successive shortening of telomeres caused by incomplete DNA replication at the very end of each chromosome. Human telomerase activity is often determined by the expression level of telomerase reverse transcriptase (TERT), the catalytic subunit of the ribonucleoprotein complex. The low expression level of TERT and the lack of adequate antibodies have made it difficult to study telomerase-related processes in human cells.ResultsTo overcome the low CRISPR-Cas9 editing efficiency at the TERT locus, we develop a two-step “pop-in/pop-out” strategy to enrich cells that underwent homologous recombination (HR). Using this technique, we fuse an N-terminal FLAG-SNAP-tag to TERT, which allows us to reliably detect TERT in western blots, immunopurify it for biochemical analysis, and determine its subcellular localization by fluorescence microscopy. TERT co-localizes detectably with only 5–7 % of the telomeres at a time in S-phase HeLa cells; no nucleolar localization is detected. Furthermore, we extend this approach to perform single base-pair modifications in the TERT promoter; reverting a recurrent cancer-associated TERT promoter mutation in a urothelial cancer cell line results in decreased telomerase activity, indicating the mutation is causal for telomerase reactivation.ConclusionsWe develop a two-step CRISPR-Cas9 genome editing strategy to introduce precise modifications at the endogenous TERT locus in human cell lines. This method provides a useful tool for studying telomerase biology, and suggests a general approach to edit loci with low targeting efficiency and to purify and visualize low abundance proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0791-1) contains supplementary material, which is available to authorized users.

The TERT promoter SNP rs2853669 decreases E2F1 transcription factor binding and increases mortality and recurrence risks in liver cancer.

A common single-nucleotide polymorphism in the telomerase reverse transcriptase (TERT) promoter, rs2853669 influences patient survival rates and the risk of developing cancer. Recently, several lines of evidence suggest that the rs2853669 suppresses TERT promoter mutation-mediated TERT expression levels and cancer mortality as well as recurrence rates. However, no reports are available on the impact of rs2853669 on TERT expression in hepatocellular carcinoma (HCC) and its association with patient survival. Here, we found that HCC-related overall and recurrence-free survival rates were not associated with TERT promoter mutation individually, but rs2853669 and the TERT promoter mutation in combination were associated with poor survival rates. TERT mRNA expression and telomere fluorescence levels were greater in patients with HCC who had both the combination. The combination caused TERT promoter methylation through regulating the binding of DNA methyltransferase 1 and histone deacetylase 1 to the TERT promoter in HCC cell lines. The TERT expression level was significantly higher in HCC tumor with a methylated promoter than in that with an unmethylated promoter. In conclusion, we demonstrate a substantial role for the rs2853669 in HCC with TERT promoter mutation, which suggests that the combination of the rs2853669 and the mutation indicate poor prognoses in liver cancer.

Telomerase reverse transcriptase promoter methylation is related to a risk of recurrence in hepatocellular carcinoma

Supported by the Korea Healthcare Technology Research and Development Project of the Ministry of Health and Welfare of the Republic of Korea (HI12C1437). Potential conflict of interest: Nothing to report. To the Editor: We read with great interest the article published in this journal by Nault et al.,1 who observed 0% frequency of somatic mutations in the telomerase reverse transcriptase (TERT) promoter in cirrhotic livers, 6% in low‐grade dysplastic nodules, 19% in high‐grade dysplastic nodules, and 61% in early hepatocellular carcinoma (HCC). These data indicate that TERT promoter mutations are early prognostic markers for HCC, based on their increased frequency in liver carcinogenesis; such an effect was not observed in 10 cancer genes (CTNNB1, TP53, ARID1A, ARID2, NFE2L2, AXIN1, PIK3CA, KEAP1, RPS6KA3, and CDKN2A).1 Nault et al.1 demonstrated that TERT promoter mutations significantly contribute to increased TERT expression in liver carcinogenesis; here, we suggest an additional mechanism regulating TERT expression. Promoter methylation of TERT is observed in various human tumor tissues and transformed cell lines,2 and its hypermethylation is positively associated with TERT expression and poor prognosis in childhood brain tumors.3 Our study of 78 Korean HCC patients reinforces the possibility that TERT promoter methylation is involved in liver carcinogenesis. First, TERT messenger RNA expression was significantly higher in HCC tumor tissues possessing a methylated TERT promoter than in those possessing an unmethylated promoter (Mann‐Whitney test, P < 0.001; Fig. 1A). Second, TERT promoter methylation was associated with poor recurrence‐free survival in HCC patients (log‐rank test, P = 0.026; Fig. 1B) and a significant risk of recurrence, as evidenced by a hazard ratio of 4.612 (95% confidence interval = 1.048‐20.3, P = 0.0432). Because TERT promoter methylation was not associated with somatic mutations at the promoter in HCC tumors (chi‐squared test, P = 0.154), these two factors independently regulate TERT expression. Taken together, these results are consistent with those of previous studies,2 indicating that TERT promoter methylation is positively associated with TERT expression.Figure 1: Methylation at the TERT promoter correlates with high TERT expression and poor recurrence‐free survival rate. (A) The TERT expression level in HCC tumor tissues possessing unmethylated or methylated TERT promoters. Methylation status was checked by methylation‐specific polymerase chain reaction using primer pairs specific for unmethylated or methylated TERT promoters (the region is from 270 bp upstream of the ATG start site to 31 bp upstream of the ATG start site2). Boxes show median, 25th percentile, and 75th percentile, while whiskers show 5th and 95th percentiles. (B) Recurrence‐free survival rates of HCC patients. Recurrence‐free survival rates were estimated using the Kaplan‐Meier method, and the differences in survival rates were compared using the log‐rank test.Findings of this study and that by Nault et al.1 demonstrate that increased TERT expression contributes substantially to HCC progression, suggesting the potential of therapeutic strategies aimed at suppressing TERT expression. Acknowledgment The biospecimens for this study were provided by Seoul St. Mary's Hospital of the Catholic University of Korea and the Korea University Guro Hospital of National Biobank, a member of the National Biobank of Korea.

Promoter hypermethylation of TERT is associated with hepatocellular carcinoma in the Han Chinese population

Upstream of the transcription start site (UTSS), hypermethylation of the telomerase reverse transcriptase (TERT) gene has been shown to be associated with tumour progression and a poor prognosis in paediatric brain tumours (Castelo-Branco 2013). It has been inferred that the UTSS region of TERT is a potentially accessible biomarker for various cancers. In this study, we aimed to explore the role of TERT in hepatocellular carcinoma (HCC) and to investigate whether the UTSS region of the TERT promoter shows the same methylation pattern in HCC. We analysed the results of a methylation assay for TERT, including the UTSS region, from 125 paired HCC samples using Mass Array EpiTyper (Sequenom, San Diego, CA, USA). To determine the relationship between TERT promoter methylation status and the TERT expression level, we analysed a validation group of 12 paired HCC samples and acquired the FPKM values for the TERT gene. Our results showed aberrant methylation of the UTSS region of the TERT promoter in HCC (mean=15.1) compared with the adjacent normal tissues (mean=6.1, P<0.00001). Furthermore, a nearly 56-fold increase in TERT expression from the hypermethylated promoter was found in HCC (P<0.05), indicating a positive relationship between TERT methylation and expression. As hypermethylation was positively correlated with high expression of TERT in HCC, TERT is likely to be involved in the aetiology of HCC. Our findings indicate that future studies on TERT might be fruitful.

Nuclear translocation of telomerase reverse transcriptase is a critical process in lymphatic metastasis of nasopharyngeal carcinoma.

Telomerase reverse transcriptase (TERT) is the predominant functional unit of telomerase and maintains the telomere length and the stability of chromosomes. Recently, TERT has been shown to be a critical factor in a number of other biological processes, including cell proliferation and cancer metastasis. In addition, although numerous studies have been conducted, the subcellular localization of the TERT protein and the association of such with cancer metastasis remains unclear. To investigate the involvement of TERT in in vivo metastasis, quantum dots-based immunofluorescence and western blot analysis were conducted to detect changes in the subcellular localization of TERT in human nasopharyngeal carcinoma (NPC) tissues and metastatic lymph nodes. To further investigate, metastatic and non-metastatic models of NPC were generated using 5–8F (high metastasis capability) and 6–10B (low metastasis capability) cell lines, respectively. It was found that TERT protein was overexpressed in NPC tissue samples and metastatic lymph nodes and TERT was predominantly located in the cytoplasm of primary NPC tissues, while TERT was predominantly located in the nucleus of the metastatic lymph nodes. The ratio of cytoplasmic TERT/nuclear TERT for the primary tumor of the 6–10B cell line was almost six-fold higher than that of the metastatic lymph nodes of the 5–8F cell line. TERT translocation from the cytoplasm to nucleus may present a critical step in the lymphatic metastasis of NPC. Thus, TERT translocation may be more useful than TERT expression level and telomerase activity for predicting the metastasis of NPC.

Combinatorial molecular marker assays of WT1,survivin, and TERT at initial diagnosis of adult acute myeloid leukemia

High levels of expression of Wilms' tumor gene 1 (WT1), survivin, or telomerase reverse transcriptase (TERT) genes are introduced as leukemia-associated targets predicting clinical outcome. We prospectively investigated the leukemia-associated gene transcripts by real-time quantitative polymerase chain reaction from 151 adult patients with AML associated with the patients' clinical characteristics. The maximum levels of each gene in bone marrow were 64.4-, 8.1-, and 3.9-fold higher than those in the normal control, respectively. In contrast to the WT1 and TERT levels, survivin showed comparatively higher expression in the unfavorable cytogenetic group of patients. We found a significant difference in survivin levels between the CR and non-CR groups (P = 0.0237). TERT expression levels were higher in patients who had a greater number of peripheral blood leukemic blasts at diagnosis (P = 0.0191). Non-MRC subtypes and patients without specific mutations were the most powerful predictive factors for a better CR rate, by multivariate analyses. The lower levels of both WT1 and survivin co-expression (P = 0.0129) and both survivin + TERT co-expression (P = 0.0115) were significant factors for better OS. Besides lower initial levels of serum ferritin (P = 0.0401), lower levels of WT1 (P = 0.0438) and survivin (P = 0.0401), lower levels of both WT1 and survivin co-expression (P = 0.0031), and the three-gene combination of lower WT1 + survivin + TERT (P = 0.0454) were powerful predictive factors for better EFS. As our findings were based on a single disease entity, that is, adult AML, they suggest that the expression of these genes may be critical for the immunobiology of AML to influence the clinical outcome in various ways.

Upregulating mutations in the TERT promoter commonly occur in adult malignant gliomas and are strongly associated with total 1p19q loss

Telomere lengthening is one of the key events in most cancers, and depends largely on telomerase activation. Telomerase activation is a well-known phenomenon in gliomas; however, its mechanism remains obscure. In this study, we investigated the presence of mutations in the promoter of the telomerase reverse transcriptase (TERT) gene in a series of 546 gliomas. We found a high incidence of mutually exclusive mutations located at two hot spots, C228T and C250T, in all subtypes of gliomas (55 %). The frequency of mutation was particularly high among primary glioblastomas (70 %) and pure oligodendroglial tumors (74 %), while relatively low in diffuse astrocytomas and anaplastic astrocytomas (19 and 25 %, respectively). The expression level of TERT in tumors carrying those mutations was on average 6.1 times higher than that of wild-type tumors, indicating that the mutated promoter leads to upregulation of TERT. TERT promoter mutations were observed in almost all tumors harboring concurrent total 1p19q loss and IDH1/2 mutations (98 %). Otherwise TERT promoter mutations were mostly observed among IDH wild-type tumors. Most EGFR amplifications (92 %) were also associated with TERT promoter mutations. Our data indicate that mutation of the TERT promoter is one of the major mechanisms of telomerase activation in gliomas. The unique pattern of TERT promoter mutations in relation to other genetic alterations suggests that they play distinct roles in the pathogenesis of oligodendroglial and astrocytic tumors. Our results shed a new light on the role of telomerase activation in the development of adult gliomas.

SAT0123 Reduction Of T-Cell Clonal Expansion in Rheumatoid Arthritis Patients After 12 Months of Abatacept Treatment.

BackgroundThe T-cell compartment of patients with rheumatoid arthritis (RA) is characterized by profound changes, including an acceleration of immune-senescence, reduced diversity of the T-cell repertoire, and expansion of clonal populations....

Abstract 3820: Wnt signaling and telomerase activation in hepatoblastoma.

Abstract Purpose: The abnormal Wnt/β-catenin signaling plays a key role in hepatoblastoma (HBL) development. In HBL, CTNNB1 coding ß-catenin protein with mutated or deleted at hot-spot regions involving exon 3 stimulates this pathway. Previously we reported that the levels of telomerase reverse transcriptase (TERT) mRNA and telomerase activities were correlated with outcomes of the patients with HBL. In this study, we examined the correlation between Wnt/β-catenin signaling and telomerase activation in HBL. And, we investigated the location of β-catenin and TERT in HBL and the expression levels of the Wnt signal genes downstream of ß -catenin. Methods: Tumors derived from 212 HBL cases treated with the JPLT-2 protocol were analyzed for oncogenic mutations (missense mutations and interstitial deletions in the third exon) of the CTNNB1 gene encoding β-catenin and mutations of Wnt signal genes including APC, AXIN1, and 2. Moreover, these tumors were analyzed for the expression levels of TERT (telomerase reverse transcriptase) and the Wnt signal genes downstream of ß -catenin (MYC, cyclin D1 and MMP7) using Real time RT-PCR and immunohistochemistry. Results: Oncogenic mutations of CTNNB1 were detected in 163 cases (76.4%) and more than 80% showed abnormalities of Wnt signal genes. The expression levels of TERT were significantly higher in 49 cases without mutation (P &amp;lt; 0.05). Interestingly, Wnt/β-catenin target genes including MYC, cyclin D1 and MMP7 were significantly activated in the tumors with telomerase activation (P = 0.01). ß -catenin staining distinguished the CTNNB1 mutated tumors from others but cyclin D1 and MMP-7 did not. In advanced HBLs, double stain analysis of TERT and β-catenin showed that both were located in the same site in nucleus, suggesting that both proteins were co-localized in nucleus in hepatoblastoma.Conclusions: Wnt/β-catenin signaling in the HBLs without CNNB1 mutations was activated by telomerase activation. The clinical courses in HBLs with mutated Wnt signal gens and high TERT expression seemed to be unfavorable due to highly activated Wnt/β-catenin signaling. This correlated pathway might become molecular targets for advanced hepatoblastoma. Citation Format: Eiso Hiyama, Yuka Ueda, Arata Kamimatsuse, Yoshiuki Onitake, Kaoru Ogura, Keiko Hiyama. Wnt signaling and telomerase activation in hepatoblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3820. doi:10.1158/1538-7445.AM2013-3820

Wnt signaling and telomerase activation of hepatoblastoma: correlation with chemosensitivity and surgical resectability

Purpose Recently, it became apparent that telomerase directly modulated Wnt signaling as a cofactor in a β-catenin transcriptional complex. In this study, we investigated Wnt/ β-catenin signaling and telomerase activation in hepatoblastoma (HBL). Methods Tumors derived from 56 HBL cases treated with the Japanese Study Group for Pediatric Liver Tumors (JPLT) Protocol-2 were analyzed for oncogenic mutations (missense mutations and interstitial deletions in the third exon) of the CTNNB1 gene-encoding β-catenin and for the expression levels of telomerase reverse transcriptase ( TERT). Results Oncogenic mutations of CTNNB1 were detected in 42 cases (75%). The expression levels of TERT were significantly higher in 14 cases without mutation ( P < .05) and in 8 cases with metastasis ( P < .01). Interestingly, Wnt/ β-catenin target genes were significantly activated in the tumors without mutations ( P = .013). In cases with mutations, preoperative chemotherapy was more effective ( P = .008), and complete resection rate was higher ( P = .034). Consequently, 2 patients with mutations and 4 patients without mutations died of disease ( P = .013). High expression of TERT was detected in all tumors of these dead patients. Conclusions Wnt/ β-catenin signaling in the HBLs without CTNNB1 mutations was activated by high expression of TERT. The clinical courses in HBLs without CTNNB1 mutations seemed to be unfavorable because of chemoresistance and low rates of resectability.

Characterization of telomeres and telomerase expression in Xiphophorus

Research investigating telomere lengths and telomerase expression in vertebrates has progressively become important due to the association of these two biological endpoints with cellular aging and cancer in humans. Studies that rely upon the traditional use of laboratory mice have been faced with limitations largely due to inbred mice possessing large telomeres and ubiquitous expression of telomerase. Recently, a number of small fish species have been shown to provide potentially informative models for examining the role of telomeres and telomerase within intact vertebrate animals. Xiphophorus fishes represent a new world live-bearing genus that has not previously been assessed for telomere length or telomerase expression. To add to the knowledge base of telomere and telomerase biology in vertebrates we assessed telomere length and telomerase expression among several species of Xiphophorus. The telomere lengths in several organs (gill, brain, eyes, testis, ovary and liver) in three species ( Xiphophorus hellerii, Xiphophorus maculatus, Xiphophorus couchianus) and also in F 1 interspecies hybrids were approximately 2–6 kb. This size was consistent within the same organs of the same species, as well as between species and F 1 hybrids. Despite possessing relatively short telomere lengths compared to humans, the consistency of size among Xiphophorus species and organs may allow experimental detection of telomere shortening. The relative expression of telomerase reverse transcriptase (TERT) was determined by quantitative real-time PCR. Expression levels of TERT was measured in seven organs (ovary, testis, liver, gill, brain, heart, skin) from X. maculatus, X. hellerii and in control and ultraviolet light (UVB) exposed skin samples from X. maculatus, X. hellerii, and F 1 interspecies hybrids. TERT gene expression was significantly higher in ovary and testis, while all other organs showed low relative TERT expression. Detectable increases in TERT expression were found in skin samples upon UVB exposure. Our findings suggest that Xiphophorus may serve as a suitable model for future studies investigating the association of telomere length and telomerase expression in regard to aging and disease.

Telomerase reverse transcriptase catalytic subunit expression and proliferation index in Wilms tumor

Telomerase activity provides telomere maintenance in chromosomes. It prevents cells from entering senescence. Telomerase activity is one of the crucial steps in various cancers. Wilms tumor (nephroblastoma) is one of the most common solid tumors of childhood. Hitherto, telomerase reverse transcriptase (TERT) catalytic subunit expression in Wilms tumor has not been investigated widely. The aim of this study was to explore the expression level of human TERT in Wilms tumor and to correlate with some clinical prognosis factors such as tumor weight, stage, histology, and Ki67 expression. This study included 41 nephroblastoma cases of childhood. The telomerase catalytic subunit expression and proliferation index was determined using an immunohistochemical method on archival paraffin-embedded tissue sections. Statistical analysis was done on SPSS 9.05 by Mann-Whitney U test and Spearman's correlation analysis. TERT expression was negative in 11 cases (26.8%), weakly positive in 14 cases (34.1%), and strongly positive in 16 cases (39%). The proliferation index was found to be 20 to 90 (mean 58.9 ± 26.8). Using Spearman correlation analysis, both the TERT expression (p=0.032) and Ki67 index (p=0.048) were found to be correlated with survival rate. Similarly, both the telomerase expression (p=0.011) and the Ki67 index (0.040) were correlated with the weight and dimension of the tumor. But there was no relationship between telomerase expression and Ki67 index (p=0.429). The mean survival time for telomerase negative cases was 56.6 ± 27.3 months, while it was 34.67 ± 28.36 months for positive cases. The Mann-Whitney U test revealed that levels of telomerase (p=0.040) significantly affected the survival rate. In the present study, we showed that the presence of TERT expression correlated with both tumor size and survival time. These findings suggest that senescence may play an important role in WT evolution, and determination of telomere maintenance will be useful to predict survival and follow-up of patients with Wilms tumor.

Abstract 3079: Loss of β2-spectrin may lead to the activation of telomerase reverse transcriptase (TERT) and hepatocellular cancer formation

Abstract Background and Aims: Telomerase is a specialized reverse transcriptase (TERT) that synthesizes repeat DNA sequences at telomeres- the specialized ends of chromosomes is suppressed in normal cells by multiple proteins that include TGF-β and Smad3 resulting in the progressive shortening of telomeres with each cell division, ultimately leading to chromosomal instability and cellular replicative senescence or growth arrest. Emerging evidence indicates that β2-spectrin (β2SP), a Smad3/Smad4 adaptor protein required for TGF-β signaling, is a powerful tumor suppressor. β2SP+/−, β2SP+/−/Smad3+/− and β2SP+/−/Smad4+/− mice dramatically develop Hepatic cancers. Because TERT is markedly activated in β2SP+/−/Smad3+/− hepatocellular cancer tissues compared to Smad3−/− tissues, we hypothesized that β2SP interaction could be involved in the interaction with Smad3 to suppress TERT expression in normal tissues, and the loss of β2SP may lead to telomerase gene activation resulting in HCC. Results: We tested human TERT (hTERT) expression levels in several HCC cell lines that have different levels of β2SP. hTERT expression levels negatively correlate with β2SP by Western Blot analysis. Loss of β2SP in β2SP+/− and β2SP−/− mouse embryonic fibroblasts (MEFs) significantly activated mTERT expression. Co-transfection of a β2SP expression plasmid and hTERT promoter-luciferase construct significantly inhibited the hTERT promoter in β2SP-deficient SNU-398 cells. Depletion of Smad3 by in vitro RNAi and the expression of hTERT-luciferase in Smad3 depleted cells upon co-transfection with Smad3 and/or β2SP expression plasmids suggest that both Smad3 and β2SP are required for the suppression of hTERT. Furthermore, chromatin immunoprecipitation (ChIP) assay suggests binding of Smad3/β2SP protein to hTERT promoter. Conclusions: Taken together our data suggest that divergent pathways converge on β2SP and Smad3 for the regulation of TERT. Activation of the TGF-β signaling pathway with suppression of TERT provides a strong strategy for generating targeted therapeutics to these lethal human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3079.

Nuclear translocation of telomerase reverse transcriptase: a critical process in chemical induced hepatocellular carcinogenesis

Telomerase, a ribonuclearprotein complex, functions not only in cancer development but also in apoptosis, and senescence. As a catalytic subunit of telomerase, telomerase reverse transcriptase (TERT) has been confirmed to regulate telomerase activity in a rate-limiting manner. Although a lot of work has been done, the dynamic state of TERT protein and the relationship with telomerase have not been delineated systematically in cancer development. The purpose of this study was to do such an exploration. To investigate the role of TERT in the in vivo carcinogenesis, we performed immunofluorescence and Western blot analysis, respectively, to detect the alteration of TERT status as well as telomeric repeat amplification protocol (TRAP) assay to detect telomerase activity in diethyl nitrosoamine (DENA) induced rat hepatocellular carcinoma (HCC). The course of cancer development was divided into three main stages, which were inflammation ( <12 weeks), hepatocirrhosis (15 weeks), and hepatocarcinoma (18-21 weeks). In normal liver and its early inflammatory stage, concomitant with a weak positive TERT signal, which was detected exclusively in the cytoplasm, telomerase activity was very low at this stage. However, in late hepatocirrhosis and particularly cancer stage, high levels of TERT expression (P = 0.044 and P = 0.001, respectively) and telomerase activity (P = 0.02 and P = 0.01, respectively) were observed following TERT nuclear translocation. Our data suggest that TERT protein might regulate telomerase activity. TERT translocation from cytoplasm to the nucleus might be a turning point in cancer development. Therefore, TERT translocation might be more useful than TERT expression level and telomerase activity in predicting the progression of HCC.

Telomerase activity is a biomarker for high grade malignant peripheral nerve sheath tumors in neurofibromatosis type 1 individuals

Telomerase activity (TA) and the expression of its enzymatic subunits, which have been demonstrated in many tumors, remain poorly investigated in tumors associated with neurofibromatosis type 1 (NF1). In this study, we analysed the association of TA and the expression of telomerase RNA (TR) and telomerase reverse transcriptase (TERT) in 23 malignant peripheral nerve sheath tumors (MPNST) (17 high grade and 6 low grade tumors), 11 plexiform neurofibromas (PNF) and 6 dermal neurofibromas (DNF). TA was studied using telomerase repeat amplification protocol (TRAP) assay and expression of TR and TERT was investigated using reverse transcription PCR (RT-PCR) and real-time PCR. TA was detected in 14 out of 17 (82%) high grade MPNST, whereas all 6 low grade MPNST and 17 benign tumors were telomerase negative. The TERT transcripts were detected in all high grade MPNST, 50% of the low grade MPNST, and 4 benign tumors. However, the expression level of the TERT strikingly correlated with TA and high grade MPNST. Thus, while TERT expression was similar in both low grade MPNST and PNF (P = 0.115), it was significantly higher in high grade MPNST when compared to either low grade MPNST (P = 0.042), PNF (P = 0.001) or DNF tumors (P = 0.010). These findings indicate that TA and expression level of TERT are potential markers for high grade malignancy in NF1 patients.