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Sample records for beta-mediated human telomerase

  1. Human telomerase: biogenesis, trafficking, recruitment, and activation.

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    Schmidt, Jens C; Cech, Thomas R

    2015-06-01

    Telomerase is the ribonucleoprotein enzyme that catalyzes the extension of telomeric DNA in eukaryotes. Recent work has begun to reveal key aspects of the assembly of the human telomerase complex, its intracellular trafficking involving Cajal bodies, and its recruitment to telomeres. Once telomerase has been recruited to the telomere, it appears to undergo a separate activation step, which may include an increase in its repeat addition processivity. This review covers human telomerase biogenesis, trafficking, and activation, comparing key aspects with the analogous events in other species.

  2. Telomerase activity in human cancer

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    Griffith, J.

    2000-10-01

    The overall goal of this collaborative project was to investigate the role in malignant cells of both chromosome telomeres, and telomerase, the enzyme that replicates telomeres. Telomeres are highly conserved nucleoprotein complexes located at the ends of eucaryotic chromosomes. Telomere length in somatic cells is reduced by 40--50 nucleotide pairs with every cell division due to incomplete replication of terminal DNA sequences and the absence of telomerase, the ribonucleoprotein that adds telomere DNA to chromosome ends. Although telomerase is active in cells with extended proliferative capacities, including more than 85% of tumors, work performed under this contract demonstrated that the telomeres of human cancer cells are shorter than those of paired normal cells, and that the length of the telomeres is characteristic of particular types of cancers. The extent of telomere shortening ostensibly is related to the number of cell divisions the tumor has undergone. It is believed that ongoing cell proliferation leads to the accumulation and fixation of new mutations in tumor cell lineages.Therefore, it is not unreasonable to assume that the degree of phenotypic variability is related to the proliferative history of the tumor, and therefore to telomere length, implying a correlation with prognosis. In some human tumors, short telomeres are also correlated with genomic instabilities, including interstitial chromosome translocation, loss of heterozygosity, and aneuoploidy. Moreover, unprotected chromosome ends are highly recombinogenic and telomere shortening in cultured human cells correlates with the formation of dicentric chromosomes, suggesting that critically short telomeres not only identify, but also predispose, cells to genomic instability, again implying a correlation with prognosis. Therefore, telomere length or content could be an important predictor of metastatic potential or responsiveness to various therapeutic modalities.

  3. An Alternate Splicing Variant of the Human Telomerase Catalytic Subunit Inhibits Telomerase Activity

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    Xiaoming Yi

    2000-09-01

    Full Text Available Telomerase, a cellular reverse transcriptase, adds telomeric repeats to chromosome ends. In normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to proliferative senescence. Introduction of the telomerase (hTERT cDNA is sufficient to produce telomerase activity and immortalize normal human cells, suggesting that the repression of telomerase activity is transcriptional. The telomerase transcript has been shown to have at least six alternate splicing sites (four insertion sites and two deletion sites, and variants containing both or either of the deletion sites are present during development and in a panel of cancer cell lines we surveyed. One deletion (β site and all four insertions cause premature translation terminations, whereas the other deletion (α site is 36 by and lies within reverse transcriptase (RT motif A, suggesting that this deletion variant may be a candidate as a dominant-negative inhibitor of telomerase. We have cloned three alternately spliced hTERT variants that contain the α,β or both α and,β deletion sites. These alternate splicing variants along with empty vector and wild-type hTERT were introduced into normal human fibroblasts and several telomerase-positive immortal and tumor cell lines. Expression of the α site deletion variant (hTERT α− construct was confirmed by Western blotting. We found that none of the three alternate splicing variants reconstitutes telomerase activity in fibroblasts. However, hTERT α− inhibits telomerase activities in telomerase-positive cells, causes telomere shortening and eventually cell death. This alternately spliced dominant-negative variant may be important in understanding telomerase regulation during development, differentiation and in cancer progression.

  4. Telomerase activity and human telomerase reverse transcriptase expression in colorectal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jian-Lun Liu; Lian-Ying Ge; Gui-Nian Zhang

    2006-01-01

    AIM: To study the activity of telomerase and the expression of human telomerase reverse transcriptase(hTERT) in colorectal carcinoma and its adjacent tissues,normal mucosa and adenomatoid polyp, and to evaluate their relation with carcinogenesis and progression of colorectal carcinoma.METHODS: Telomerase activity and hTERT expression were determined in 30 samples of colorectal carcinoma and its adjacent tissues, normal mucosa and 20samples of adenomatoid polyp by modified telomeric repeat amplification protocol (TRAP), enzyme-linked immunosorbent assay (ELISA) and immunohistochemical method.RESULTS: Telomerase activity and hTERT expression were 83.33% (25/30) and 76.67% (23/30) respectively in colorectal carcinoma, which were obviously higher than those in paracancerous tissues (13.33%, 16.67%),normal mucosa (3.33%, 3.33%) and adenomatoid polyp(10%, 10%). There was a significant difference between colorectal carcinoma and other tissues (P=0.027). The telomerase activity and hTERT expression were higher in colorectal carcinoma with lymphatic metastasis than in that without lymphatic metastasis (P=0.034). When the histological classification and clinical stage were greater,the telomerase activity and hTERT expression increased,but there was no significant difference between them.In colorectal carcinoma, the telomerase activity was correlated with hTERT expression (positive vs negative expression of telomerase activity and hTERT, P=0.021).CONCLUSION: Telomerase activity is closely correlated with the occurrence, development and metastasis of colorectal carcinoma. Overexpression of hTERT may play a critical role in the regulation of telomerase activity.

  5. Functional Assessment of Pharmacological Telomerase Activators in Human T Cells

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    Rita B. Effros

    2013-01-01

    Full Text Available Telomeres are structures at the ends of chromosomes that shorten during cell division and eventually signal an irreversible state of growth arrest known as cellular senescence. To delay this cellular aging, human T cells, which are critical in the immune control over infections and cancer, activate the enzyme telomerase, which binds and extends the telomeres. Several different extracts from the Astragalus membranaceus root have been documented to activate telomerase activity in human T cells. The objective of this research was to compare two extracts from Astragalus membranaceus, TA-65 and HTA, for their effects on both telomerase and proliferative activity of human CD4 and CD8 T cells. Our results demonstrate that, TA-65 increased telomerase activity significantly (1.3 to 3.3-fold relative to controls in T cell cultures from six donors tested, whereas HTA only increased telomerase levels in two out of six donors. We also demonstrate that TA-65 activates telomerase by a MAPK- specific pathway. Finally, we determine that during a three-day culture period, only the T cells treated with the TA-65 extract showed a statistically significant increase in proliferative activity. Our results underscore the importance of comparing multiple telomerase activators within the same experiment, and of including functional assays in addition to measuring telomerase activity.

  6. Actions of human telomerase beyond telomeres

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    Yusheng Cong; Jerry W Shay

    2008-01-01

    Telomerase has fundamental roles in bypassing cellular aging and in cancer progression by maintaining telomere homeostasis and integrity. However, recent studies have led some investigators to suggest novel biochemical properties of telomerase in several essential cell signaling pathways without apparent involvement of its well established function in telomere maintenance. These observations may further enhance our understanding of the molecular actions of telomerase in aging and cancer. This review will provide an update on the extracurricular activities of telomerase in apoptosis, DNA repair, stem cell function, and in the regulation of gene expression.

  7. A human telomerase holoenzyme protein required for Cajal body localization and telomere synthesis.

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    Venteicher, Andrew S; Abreu, Eladio B; Meng, Zhaojing; McCann, Kelly E; Terns, Rebecca M; Veenstra, Timothy D; Terns, Michael P; Artandi, Steven E

    2009-01-30

    Telomerase is a ribonucleoprotein (RNP) complex that synthesizes telomere repeats in tissue progenitor cells and cancer cells. Active human telomerase consists of at least three principal subunits, including the telomerase reverse transcriptase, the telomerase RNA (TERC), and dyskerin. Here, we identify a holoenzyme subunit, TCAB1 (telomerase Cajal body protein 1), that is notably enriched in Cajal bodies, nuclear sites of RNP processing that are important for telomerase function. TCAB1 associates with active telomerase enzyme, established telomerase components, and small Cajal body RNAs that are involved in modifying splicing RNAs. Depletion of TCAB1 by using RNA interference prevents TERC from associating with Cajal bodies, disrupts telomerase-telomere association, and abrogates telomere synthesis by telomerase. Thus, TCAB1 controls telomerase trafficking and is required for telomere synthesis in human cancer cells.

  8. Telomere and telomerase stability in human diseases and cancer.

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    Chiodi, Ilaria; Mondello, Chiara

    2016-01-01

    Telomeres are the nucleoprotein structures at the end of linear eukaryotic chromosomes required for genome stability. Telomerase is the specialized enzyme deputed to their elongation. Maintenance of a proper telomere structure, an accurate regulation of telomerase biogenesis and activity, as well as a correct telomere-telomerase interaction and a faithful telomeric DNA replication are all processes that a cell has to precisely control to safeguard its functionality. Here, we review key factors that play a role in the development of these processes and their relationship with human health.

  9. Expression and effects of human telomerase RNA in testicular tumor

    Institute of Scientific and Technical Information of China (English)

    叶哲伟; 陈晓春; 杨述华; 杨秀萍; 曾汉青; 谷龙杰; 鲁功成

    2004-01-01

    @@ Human telomerase RNA (hTR) plays an important role in determining repeated telomere sequence and the expression of an antisense telomerase RNA that leads to telomere shortening and cell death.1 Using highly sensitive in situ nucleic acid hybridisation, we investigated the expression of hTR in human testicular tumours and located its cellular expression. Our study may help in elucidating the role of hTR in human testicular tumours, finding a highly sensitive diagnostic method and a target for gene therapy of testicular tumours.

  10. Telomerase RNA accumulates in Cajal bodies in human cancer cells.

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    Zhu, Yusheng; Tomlinson, Rebecca L; Lukowiak, Andrew A; Terns, Rebecca M; Terns, Michael P

    2004-01-01

    Telomerase synthesizes telomeric DNA repeats at the ends of eukaryotic chromosomes. The RNA component of the enzyme (hTR) provides the template for telomere synthesis, which is catalyzed by telomerase reverse transcriptase (hTERT). Little is known regarding the subcellular localization of hTR and hTERT and the pathway by which telomerase is assembled. Here we report the first glimpse of the detailed subcellular localization of endogenous hTR in human cells, which we obtained by fluorescence in situ hybridization (FISH). Our studies have revealed a distinctive hTR localization pattern in cancer cells. We have found that hTR accumulates within intranuclear foci called Cajal bodies in all typical tumor-derived cell lines examined (in which telomerase is active), but not in primary or ALT cells (where little or no hTERT is present). Accumulation of hTR in the Cajal bodies of primary cells is induced when hTERT is ectopically expressed. Moreover, we report that hTERT is also found in Cajal bodies. Our data suggest that Cajal bodies are involved in the assembly and/or function of human telomerase.

  11. Telomerase efficiently elongates highly transcribing telomeres in human cancer cells.

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    Benjamin O Farnung

    Full Text Available RNA polymerase II transcribes the physical ends of linear eukaryotic chromosomes into a variety of long non-coding RNA molecules including telomeric repeat-containing RNA (TERRA. Since TERRA discovery, advances have been made in the characterization of TERRA biogenesis and regulation; on the contrary its associated functions remain elusive. Most of the biological roles so far proposed for TERRA are indeed based on in vitro experiments carried out using short TERRA-like RNA oligonucleotides. In particular, it has been suggested that TERRA inhibits telomerase activity. We have exploited two alternative cellular systems to test whether TERRA and/or telomere transcription influence telomerase-mediated telomere elongation in human cancer cells. In cells lacking the two DNA methyltransferases DNMT1 and DNMT3b, TERRA transcription and steady-state levels are greatly increased while telomerase is able to elongate telomeres normally. Similarly, telomerase can efficiently elongate transgenic inducible telomeres whose transcription has been experimentally augmented. Our data challenge the current hypothesis that TERRA functions as a general inhibitor of telomerase and suggest that telomere length homeostasis is maintained independently of TERRA and telomere transcription.

  12. Telomerase reverse transcriptase is required for the localization of telomerase RNA to cajal bodies and telomeres in human cancer cells.

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    Tomlinson, Rebecca L; Abreu, Eladio B; Ziegler, Tania; Ly, Hinh; Counter, Christopher M; Terns, Rebecca M; Terns, Michael P

    2008-09-01

    Telomere maintenance by telomerase is critical for the unlimited division potential of most human cancer cells. The two essential components of human telomerase, telomerase RNA (hTR) and telomerase reverse transcriptase (hTERT), are recruited from distinct subnuclear sites to telomeres during S phase. Throughout the remainder of the cell cycle hTR is found primarily in Cajal bodies. The localization of hTR to Cajal bodies and telomeres is specific to cancer cells where telomerase is active and is not observed in primary cells. Here we show that the trafficking of hTR to both telomeres and Cajal bodies depends on hTERT. RNA interference-mediated depletion of hTERT in cancer cells leads to loss of hTR from both Cajal bodies and telomeres without affecting hTR levels. In addition, expression of hTERT in telomerase-negative cells (including primary and ALT cancer cell lines) induces hTR to localize to both sites. Factors that did not stimulate hTR localization in our experiments include increased hTR RNA levels and Cajal body numbers, and expression of SV40 large T antigen and oncogenic Ras. Our findings suggest that the trafficking of telomerase to Cajal bodies and telomeres in cancer cells correlates with and depends on the assembly of the enzyme.

  13. Expression and alternative splicing pattern of human telomerase reverse transcriptase in human lung cancer cells.

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    Fujiwara, Masachika; Kamma, Hiroshi; Wu, Wenwen; Hamasaki, Makoto; Kaneko, Setsuko; Horiguchi, Hisashi; Matsui-Horiguchi, Miwa; Satoh, Hiroaki

    2004-04-01

    Telomerase activity is generally considered to be necessary for cancer cells to avoid senescence. The expression of human telomerase reverse transcriptase (hTERT) is believed to be a rate-limiting step in telomerase activation. Recently, it has been proposed that the alternative splicing of hTERT is also involved in regulation of telomerase activity. However, the regulatory mechanism of telomerase in cancer cells has not been thoroughly investigated. To clarify it in lung cancer cells, we measured the expression of the hTERT transcript, analyzed its alternative splicing by RT-PCR, and compared it with telomerase activity and telomere length. The expression of the hTERT transcript was positively correlated with telomerase activity in lung cancer cells. Cancer cells with high telomerase activity contained 4 splicing variants of hTERT, and the full-length variant was 31.3-54.2% of the total transcripts. Cells of the TKB-20 cell line, which has extremely low telomerase activity, showed a different splicing pattern of hTERT in addition to low expression. The functional full-length variant was scarcely detected in TKB-20 cells, suggesting that the telomerase activity was repressed by alternative splicing of hTERT. Telomere length was not necessarily correlated with telomerase activity or hTERT expression in lung cancer cells. Cells of the TKB-4 cell line that also showed relatively low telomerase activity (as TKB-20 cells) had long telomeres. In conclusion, hTERT expression is regulated at both the transcriptional and post-transcriptional levels in lung cancer cells, and the alternative splicing of hTERT is involved in the control of telomerase activity.

  14. TELOMERASE ACTIVITY IN HUMAN GASTRIC AND COLORECTAL CANCER AND SURROUNDING TISSUES

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    CHEN Wen; ZHANG Qiao; WAN De-sen; CUN Ling-yun; WU Cheng-qiu; PAN Zhi-zhong

    1999-01-01

    Objective: To study the telomerase activities in human gastric and colorectal tumors. Methods: The telomerase activity was assayed by the telomeric repeat amplification protocol (TRAP) technique. Forty human tumor samples including 9 colonic, 20 rectal and 11gastric carcinomas and their surrounding tissues were used for the detection. Results: Thirty-six out of 40human tumor samples exhibited telomerase activity regardless of the stages or the differentiation of the tumors. However, only 1 out of 39 tumor surrounding tissues showed telomerase activity. Conclusion: Telomerase may be a good diagnosis biomarker for tumor detection.

  15. The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts.

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    Vidale, Pamela; Magnani, Elisa; Nergadze, Solomon G; Santagostino, Marco; Cristofari, Gael; Smirnova, Alexandra; Mondello, Chiara; Giulotto, Elena

    2012-10-01

    Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli's zebra and Grevy's zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.

  16. Modulation of interleukin-1beta mediated inflammatory response in human astrocytes by flavonoids: implications in neuroprotection.

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    Sharma, Vivek; Mishra, Mamata; Ghosh, Soumya; Tewari, Richa; Basu, Anirban; Seth, Pankaj; Sen, Ellora

    2007-06-15

    The proinflammatory cytokine interleukin-1beta (IL-1beta) contributes to inflammation and neuronal death in CNS injuries and neurodegenerative pathologies, and astrocytes have been implicated as the primary mediators of IL-1beta induced neuronal death. As astrocytes play an important role in supporting the survival and functions of neurons, we investigated the effect of plant flavonoids quercetin and luteolin, with known anti-inflammatory properties in modulating the response of human astrocytes to IL-1beta for therapeutic intervention. Flavonoids significantly decreased the release of reactive oxygen species (ROS) from astrocytes stimulated with IL-1beta. This decrease was accompanied by an increase in expression of superoxide dismutase (SOD-1) and thioredoxin (TRX1)-mediators associated with protection against oxidative stress. Flavonoids not only modulated the expression of astrocytes specific molecules such as glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), and ceruloplasmin (CP) both in the presence and absence of IL-1beta but also decreased the elevated levels of proinflammatory cytokine interleukin-6 (IL-6) and chemokines interleukin-8 (IL-8), interferon-inducible protein (IP-10), monocyte-chemoattractant protein-1 (MCP-1), and RANTES from IL-1beta activated astrocytes. Significant decrease in neuronal apoptosis was observed in neurons cultured in conditioned medium obtained from astrocytes treated with a combination of IL-1beta and flavonoids as compared to that treated with IL-1beta alone. Our result suggests that by (i) enhancing the potential of activated astrocytes to detoxify free radical, (ii) reducing the expression of proinflammatory cytokines and chemokines, and (iii) modulating expression of mediators associated with enhanced physiological activity of astrocyte in response to injury, flavonoids confer (iv) protection against IL-1beta induced astrocyte mediated neuronal damage.

  17. Telomere Lengths and Telomerase Activity in Dog Tissues: A Potential Model System to Study Human Telomere and Telomerase Biology

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    Lubna Nasir

    2001-01-01

    Full Text Available Studies on telomere and telomerase biology are fundamental to the understanding of aging and age-related diseases such as cancer. However, human studies have been hindered by differences in telomere biology between humans and the classical murine animal model system. In this paper, we describe basic studies of telomere length and telomerase activity in canine normal and neoplastic tissues and propose the dog as an alternative model system. Briefly, telomere lengths were measured in normal canine peripheral blood mononuclear cells (PBMCs, a range of normal canine tissues, and in a panel of naturally occurring soft tissue tumours by terminal restriction fragment (TRF analysis. Further, telomerase activity was measured in canine cell lines and multiple canine tissues using a combined polymerase chain reaction/enzyme-linked immunosorbent assay method. TRF analysis in canine PBMCs and tissues demonstrated mean TRF lengths to range between 12 and 23 kbp with heterogeneity in telomere lengths being observed in a range of normal somatic tissues. In soft tissue sarcomas, two subgroups were identified with mean TRFs of 22.2 and 18.2 kbp. Telomerase activity in canine tissue was present in tumour tissue and testis with little or no activity in normal somatic tissues. These results suggest that the dog telomere biology is similar to that in humans and may represent an alternative model system for studying telomere biology and telomerase-targeted anticancer therapies.

  18. Dynamics of Human Telomerase Holoenzyme Assembly and Subunit Exchange across the Cell Cycle.

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    Vogan, Jacob M; Collins, Kathleen

    2015-08-28

    Human telomerase acts on telomeres during the genome synthesis phase of the cell cycle, accompanied by its concentration in Cajal bodies and transient colocalization with telomeres. Whether the regulation of human telomerase holoenzyme assembly contributes to the cell cycle restriction of telomerase function is unknown. We investigated the steady-state levels, assembly, and exchange dynamics of human telomerase subunits with quantitative in vivo cross-linking and other methods. We determined the physical association of telomerase subunits in cells blocked or progressing through the cell cycle as synchronized by multiple protocols. The total level of human telomerase RNA (hTR) was invariant across the cell cycle. In vivo snapshots of telomerase holoenzyme composition established that hTR remains bound to human telomerase reverse transcriptase (hTERT) throughout all phases of the cell cycle, and subunit competition assays suggested that hTERT-hTR interaction is not readily exchangeable. In contrast, the telomerase holoenzyme Cajal body-associated protein, TCAB1, was released from hTR in mitotic cells coincident with TCAB1 delocalization from Cajal bodies. This telomerase holoenzyme disassembly was reversible with cell cycle progression without any change in total TCAB1 protein level. Consistent with differential cell cycle regulation of hTERT-hTR and TCAB1-hTR protein-RNA interactions, overexpression of hTERT or TCAB1 had limited if any influence on hTR assembly of the other subunit. Overall, these findings revealed a cell cycle regulation that disables human telomerase association with telomeres while preserving the co-folded hTERT-hTR ribonucleoprotein catalytic core. Studies here, integrated with previous work, led to a unifying model for telomerase subunit assembly and trafficking in human cells.

  19. Telomerase contributes to fludarabine resistance in primary human leukemic lymphocytes.

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    Shawi, May; Chu, Tsz Wai; Martinez-Marignac, Veronica; Yu, Y; Gryaznov, Sergei M; Johnston, James B; Lees-Miller, Susan P; Assouline, Sarit E; Autexier, Chantal; Aloyz, Raquel

    2013-01-01

    We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR), can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1) if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2) the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo) on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku) to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells.

  20. Telomerase contributes to fludarabine resistance in primary human leukemic lymphocytes.

    Directory of Open Access Journals (Sweden)

    May Shawi

    Full Text Available We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR, can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1 if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2 the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells.

  1. Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

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    Ren, He, E-mail: herenrh@yahoo.com.cn [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China); Zhao, Tiansuo; Wang, Xiuchao; Gao, Chuntao; Wang, Jian; Yu, Ming [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China); Hao, Jihui, E-mail: jihuihao@yahoo.com [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China)

    2010-03-26

    The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breast cancer.

  2. Human cells lacking coilin and Cajal bodies are proficient in telomerase assembly, trafficking and telomere maintenance

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    Chen, Yanlian; Deng, Zhiqiang; Jiang, Shuai; Hu, Qian; Liu, Haiying; Songyang, Zhou; Ma, Wenbin; Chen, Shi; Zhao, Yong

    2014-01-01

    The RNA component of human telomerase (hTR) localizes to Cajal bodies, and it has been proposed that Cajal bodies play a role in the assembly of telomerase holoenzyme and telomerase trafficking. Here, the role of Cajal bodies was examined in Human cells deficient of coilin (i.e. coilin-knockout (KO) cells), in which no Cajal bodies are detected. In coilin-KO cells, a normal level of telomerase activity is detected and interactions between core factors of holoenzyme are preserved, indicating t...

  3. The HSP90 inhibitor alvespimycin enhances the potency of telomerase inhibition by imetelstat in human osteosarcoma

    OpenAIRE

    Hu, Yafang; Bobb, Daniel; He, Jianping; Hill, D. Ashley; Dome, Jeffrey S.

    2015-01-01

    The unsatisfactory outcomes for osteosarcoma necessitate novel therapeutic strategies. This study evaluated the effect of the telomerase inhibitor imetelstat in pre-clinical models of human osteosarcoma. Because the chaperone molecule HSP90 facilitates the assembly of telomerase protein, the ability of the HSP90 inhibitor alvespimycin to potentiate the effect of the telomerase inhibitor was assessed. The effect of single or combined treatment with imetelstat and alvespimycin on long-term grow...

  4. Human cells lacking coilin and Cajal bodies are proficient in telomerase assembly, trafficking and telomere maintenance.

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    Chen, Yanlian; Deng, Zhiqiang; Jiang, Shuai; Hu, Qian; Liu, Haiying; Songyang, Zhou; Ma, Wenbin; Chen, Shi; Zhao, Yong

    2015-01-01

    The RNA component of human telomerase (hTR) localizes to Cajal bodies, and it has been proposed that Cajal bodies play a role in the assembly of telomerase holoenzyme and telomerase trafficking. Here, the role of Cajal bodies was examined in Human cells deficient of coilin (i.e. coilin-knockout (KO) cells), in which no Cajal bodies are detected. In coilin-KO cells, a normal level of telomerase activity is detected and interactions between core factors of holoenzyme are preserved, indicating that telomerase assembly occurs in the absence of Cajal bodies. Moreover, dispersed hTR aggregates and forms foci specifically during S and G2 phase in coilin-KO cells. Colocalization of these hTR foci with telomeres implies proper telomerase trafficking, independent of Cajal bodies. Therefore, telomerase adds similar numbers of TTAGGG repeats to telomeres in coilin-KO and controls cells. Overexpression of TPP1-OB-fold blocks cell cycle-dependent formation of hTR foci and inhibits telomere extension. These findings suggest that telomerase assembly, trafficking and extension occur with normal efficiency in Cajal bodies deficient human cells. Thus, Cajal bodies, as such, are not essential in these processes, although it remains possible that non-coilin components of Cajal bodies and/or telomere binding proteins (e.g. TPP1) do play roles in telomerase biogenesis and telomere homeostasis.

  5. Detection of telomerase activity in malignant neoplasms and nonmalignantepithelial tissues of human esophagus

    Institute of Scientific and Technical Information of China (English)

    Shah Min Yang; Tian Jiao Wang; Bao Yu Li; Yuan Huan Wu

    2000-01-01

    AIM To study the expression of telomerase activity in malignant esophageal neoplasms and normal humanesophageal epithelia.METHODS Telomerase activity was assayed by the telomere repeat amplification protocol (TRAP)method. All the neoplasms and epithelia of esophagus were confirmed by routine pathological diagnosis.RESULTS Telomerase activity was assayed in 18 normal esophageal epithelial tissues and in 35 malignantneoplasms of esophagus, including 27 cases of esophageal carcinoma and 8 cases of cardiac carcinoma.Telomerase activity was detected in most of malignant neoplasms of esophagus (91.4%, 32/35) and in allthe normal esophageal epithelial tissues except one (18/19).CONCLUSION The results suggest that in addition to contributing to proliferation of immortal blast cellsand neoplastic cells, telomerase activity may also play a similar role in regeneration of normal epithelia ofhuman esophagus. The potential use of telomerase activity as a diagnostic marker in human esophagealneoplasm might not be suitable.

  6. Telomerase activity, estrogen receptors (α, β), Bcl-2 expression in human breast cancer and treatment response

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    Murillo-Ortiz, Blanca; Astudillo-De la Vega, Horacio; Castillo-Medina, Sebastian; Malacara, JM; Benitez-Bribiesca, Luis

    2006-01-01

    Background The mechanism for maintaining telomere integrity is controlled by telomerase, a ribonucleoprotein enzyme that specifically restores telomere sequences, lost during replication by means of an intrinsic RNA component as a template for polymerization. Among the telomerase subunits, hTERT (human telomerase reverse transcriptase) is expressed concomitantly with the activation of telomerase. The role of estrogens and their receptors in the transcriptional regulation of hTERT has been demonstrated. The current study determines the possible association between telomerase activity, the expression of both molecular forms of estrogen receptor (ERα and ERβ) and the protein bcl-2, and their relative associations with clinical parameters. Methods Tissue samples from 44 patients with breast cancer were used to assess telomerase activity using the TRAP method and the expression of ERα, ERβ and bcl-2 by means of immunocytochemical techniques. Results Telomerase activity was detected in 59% of the 44 breast tumors examined. Telomerase activity ranged from 0 to 49.93 units of total product generated (TPG). A correlation was found between telomerase activity and differentiation grade (p = 0.03). The only significant independent marker of response to treatment was clinical stage. We found differences between the frequency of expression of ERα (88%) and ERβ (36%) (p = 0.007); bcl-2 was expressed in 79.5% of invasive breast carcinomas. We also found a significant correlation between low levels of telomerase activity and a lack of ERβ expression (p = 0.03). Conclusion Lower telomerase activity was found among tumors that did not express estrogen receptor beta. This is the first published study demonstrating that the absence of expression of ERβ is associated with low levels of telomerase activity. PMID:16911782

  7. Telomerase activity, estrogen receptors (α, β, Bcl-2 expression in human breast cancer and treatment response

    Directory of Open Access Journals (Sweden)

    Malacara JM

    2006-08-01

    Full Text Available Abstract Background The mechanism for maintaining telomere integrity is controlled by telomerase, a ribonucleoprotein enzyme that specifically restores telomere sequences, lost during replication by means of an intrinsic RNA component as a template for polymerization. Among the telomerase subunits, hTERT (human telomerase reverse transcriptase is expressed concomitantly with the activation of telomerase. The role of estrogens and their receptors in the transcriptional regulation of hTERT has been demonstrated. The current study determines the possible association between telomerase activity, the expression of both molecular forms of estrogen receptor (ERα and ERβ and the protein bcl-2, and their relative associations with clinical parameters. Methods Tissue samples from 44 patients with breast cancer were used to assess telomerase activity using the TRAP method and the expression of ERα, ERβ and bcl-2 by means of immunocytochemical techniques. Results Telomerase activity was detected in 59% of the 44 breast tumors examined. Telomerase activity ranged from 0 to 49.93 units of total product generated (TPG. A correlation was found between telomerase activity and differentiation grade (p = 0.03. The only significant independent marker of response to treatment was clinical stage. We found differences between the frequency of expression of ERα (88% and ERβ (36% (p = 0.007; bcl-2 was expressed in 79.5% of invasive breast carcinomas. We also found a significant correlation between low levels of telomerase activity and a lack of ERβ expression (p = 0.03. Conclusion Lower telomerase activity was found among tumors that did not express estrogen receptor beta. This is the first published study demonstrating that the absence of expression of ERβ is associated with low levels of telomerase activity.

  8. An antiapoptotic role for telomerase RNA in human immune cells independent of telomere integrity or telomerase enzymatic activity.

    Science.gov (United States)

    Gazzaniga, Francesca S; Blackburn, Elizabeth H

    2014-12-11

    Telomerase is a ribonucleoprotein complex that adds telomeric DNA to the ends of linear chromosomes. It contains two core canonical components: the essential RNA component, hTR, which provides the template for DNA synthesis, and the reverse transcriptase protein component, hTERT. Low telomerase activity in circulating peripheral blood mononuclear cells has been associated with a variety of diseases. It is unknown, however, whether telomerase, in addition to its long-term requirement for telomere maintenance, is also necessary for short-term immune cell proliferation and survival. We report that overexpression of enzymatically inactive hTR mutants protected against dexamethasone-induced apoptosis in stimulated CD4 T cells. Furthermore, hTR knockdown reproducibly induced apoptosis in the absence of any detectable telomere shortening or DNA damage response. In contrast, hTERT knockdown did not induce apoptosis. Strikingly, overexpression of hTERT protein caused apoptosis that was rescued by overexpression of enzymatically inactive hTR mutants. Hence, we propose that hTR can function as a noncoding RNA that protects from apoptosis independent of its function in telomerase enzymatic activity and long-term telomere maintenance in normal human immune cells. These results imply that genetic or environmental factors that alter hTR levels can directly affect immune cell function to influence health and disease.

  9. Human telomerase reverse transcriptase (hTERT Q169 is essential for telomerase function in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Haley D M Wyatt

    Full Text Available BACKGROUND: Telomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT, and an RNA subunit, the telomerase RNA (TR. Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: To understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT Q169 to create neutral (Q169A, conservative (Q169N, and non-conservative (Q169D mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function. CONCLUSIONS/SIGNIFICANCE: We provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme

  10. Is telomerase reactivation associated with the down-regulation of TGF β receptor-II expression in human breast cancer?

    Directory of Open Access Journals (Sweden)

    Thomas Valene

    2003-07-01

    Full Text Available Abstract Background Telomerase is a ribonucleoprotein that synthesizes telomeres and plays an important role in chromosomal stability and cellular immortalisation. Telomerase activity is detectable in most human cancers but not in normal somatic cells. TGF beta (transforming growth factor beta is a member of a family of cytokines that are essential for cell survival and seems to be down-regulated in human cancer. Recent in vitro work using human breast cancer cell lines has suggested that TGF beta down-regulates the expression of hTERT (human telomerase reverse transcriptase : the catalytic subunit of telomerase. We have therefore hypothesised that telomerase reactivation is associated with reduced immunohisto-chemical expression of TGF beta type II receptor (RII in human breast cancer. Methods TGF beta RII immunohistochemical expression was determined in 24 infiltrating breast carcinomas with known telomerase activity (17 telomerase-positive and 7 telomerase-negative. Immunohistochemical expression of TGF beta RII was determined by a breast pathologist who was blinded to telomerase data. Results TGF beta RII was detected in all lesions. The percentage of stained cells ranged from 1–100%. The difference in TGF beta RII expression between telomerase positive and negative tumours was not statistically significant (p = 1.0. Conclusion The results of this pilot study suggest that there is no significant association between telomerase reactivation and TGF-beta RII down-regulation in human breast cancer.

  11. Telomerase repeat amplification protocol (TRAP) activity upon recombinant expression and purification of human telomerase in a bacterial system.

    Science.gov (United States)

    Hansen, Debra T; Thiyagarajan, Thirumagal; Larson, Amy C; Hansen, Jeffrey L

    2016-07-01

    Telomerase biogenesis is a highly regulated process that solves the DNA end-replication problem. Recombinant expression has so far been accomplished only within a eukaryotic background. Towards structural and functional analyses, we developed bacterial expression of human telomerase. Positive activity by the telomerase repeat amplification protocol (TRAP) was identified in cell extracts of Escherichia coli expressing a sequence-optimized hTERT gene, the full-length hTR RNA with a self-splicing hepatitis delta virus ribozyme, and the human heat shock complex of Hsp90, Hsp70, p60/Hop, Hsp40, and p23. The Hsp90 inhibitor geldanamycin did not affect post-assembly TRAP activity. By various purification methods, TRAP activity was also obtained upon expression of only hTERT and hTR. hTERT was confirmed by tandem mass spectrometry in a ∼120 kDa SDS-PAGE fragment from a TRAP-positive purification fraction. TRAP activity was also supported by hTR constructs lacking the box H/ACA small nucleolar RNA domain. End-point TRAP indicated expression levels within 3-fold of that from HeLa carcinoma cells, which is several orders of magnitude below detection by the direct assay. These results represent the first report of TRAP activity from a bacterium and provide a facile system for the investigation of assembly factors and anti-cancer therapeutics independently of a eukaryotic setting.

  12. Attenuation of Telomerase Activity by siRNA Targeted Telomerase RNA Leads to Apoptosis and Inhibition of Proliferation in Human Renal Carcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    Rumin Wen; Junjie Liu; Wang Li; Wenfa Yang; Lijun Mao; Junnian Zheng

    2006-01-01

    OBJECTIVE Telomerase is an attractive molecular target for cancer therapy because the activation of telomerase is one of the key steps in cell immortalization and carcinogenesis. RNA interference using small-interfering RNA (siRNA) has been demonstrated to be an effective method for inhibiting the expression of a given gene in human cells. The aim of the present study was to investigate whether inhibition of telomerase activity by siRNA targeted against human telomerase RNA (hTR) can inhibit proliferation and induce apoptotic cell death in human renal carcinoma cells(HRCCs).METHODS The siRNA duplexes for hTR were synthesized and 786-O HRCCs were transfected with different concentrations of hTR-siRNA. The influence on the hTR mRNA level, telomerase activity, as well as the effect on cell proliferation and apoptosis was examined.RESULTS Anti-hTR siRNA treatment of HRCCs resulted in specific reduction of hTR mRNA and inhibition of telomerase activity. Additionally,significant inhibition of proliferation and induction of apoptosis were observed.CONCLUSION siRNA against the hTR gene can inhibit proliferation and induce apoptosis by blocking telomerase activity of HRCCs. Specific hTR inhibition by siRNA represents a promising new option for renal cancer treatment.

  13. Telomerase: The Devil Inside

    Directory of Open Access Journals (Sweden)

    Mukesh Kumar

    2016-07-01

    Full Text Available High telomerase activity is detected in nearly all human cancers but most human cells are devoid of telomerase activity. There is well-documented evidence that reactivation of telomerase occurs during cellular transformation. In humans, tumors can rely in reactivation of telomerase or originate in a telomerase positive stem/progenitor cell, or rely in alternative lengthening of telomeres, a telomerase-independent telomere-length maintenance mechanism. In this review, we will focus on the telomerase positive tumors. In this context, the recent findings that telomerase reverse transcriptase (TERT promoter mutations represent the most common non-coding mutations in human cancer have flared up the long-standing discussion whether cancer originates from telomerase positive stem cells or telomerase reactivation is a final step in cellular transformation. Here, we will discuss the pros and cons of both concepts in the context of telomere length-dependent and telomere length-independent functions of telomerase. Together, these observations may provoke a re-evaluation of telomere and telomerase based therapies, both in telomerase inhibition for cancer therapy and telomerase activation for tissue regeneration and anti-ageing strategies.

  14. Quantification of human telomerase RNA (Htr) and human telomerase reverse transcriptase (Htert)Mrna in testicular tissue of infertile patients

    Institute of Scientific and Technical Information of China (English)

    Mark Schrader; Markus Miller; Ridiger Heicappell; Bernd Straub; Kurt Miller

    2001-01-01

    Aim: To evaluate the quantitative detection of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA as diagnostic parameters in the workup of testicular tissue specimens from patients presenting with non-obstructive azoospemia. Methods: hTR and hTERT mRNA expression were quantified in 38 cryopreserved 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 additional semithin sectioning preparation in cases with maturation arrest ( n = 12) and Sertoli-cell-only syndrome ( n = 12). Results: The average normalized hTERT expression (NhTERT) Was 131.9 ± 48.0 copies (mean ± SD) in tissue specimens with full spermatogenesis, NhTERT = 51.2 ± 17.2 copies in those with maturation arrest and NhTERT = 2.7 ± 2.4 copies in those with Sertoli-cell-only syndrome (SCOS). The discriminant analysis showed that detection of NhTERT (NhTR) 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 enables a molecular-diagnostic classification of gametogenesis. Quantitative detection of hTERT in testicular biopsies is thus well suited for supplementing the histopathological evaluation.

  15. Human telomerase and Cajal body ribonucleoproteins share a unique specificity of Sm protein association

    OpenAIRE

    Fu, Dragony; Collins, Kathleen

    2006-01-01

    Cajal bodies are nuclear structures that host RNA modification and assembly reactions. Some RNAs transit Cajal bodies, while others must concentrate in Cajal bodies to function. Here we report that at least a subfraction of human telomerase RNA and individual resident Cajal body RNAs is associated with Sm proteins. Surprisingly, of seven Sm proteins assembled into a heteroheptameric ring, only a subset copurifies telomerase and Cajal body ribonucleoproteins. We show that a Cajal body RNA loca...

  16. Protein composition of catalytically active human telomerase from immortal cells

    DEFF Research Database (Denmark)

    Cohen, Scott B; Graham, Mark E; Lovrecz, George O;

    2007-01-01

    on the enzyme's ability to catalyze nucleotide addition onto a DNA oligonucleotide of telomeric sequence, thereby providing specificity for catalytically active telomerase. Mass spectrometric sequencing of the protein components and molecular size determination indicated an enzyme composition of two molecules...

  17. Inhibition of telomerase with human telomerase reverse transcriptase antisense enhances tumor necrosis factor-a-induced apoptosis in bladder cancer cells

    Institute of Scientific and Technical Information of China (English)

    GAO Xiao-dong; CHEN Yi-rong

    2007-01-01

    Background Telomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells.Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that plays an important role in telomerase activity. This study investigated the effect of the telomerase inhibition with an hTERT antisense oligodeoxynucleotide (ODN) in bladder cancer cells (T24) on tumor necrosis factor-o (TNF-α)-induced apoptosis.Methods Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR) assay and a gel-image system.hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by a morphological method and determined by flow cytometry.Results AS PS-ODN significantly inhibited telomerase activity and decreased the levels of hTERT mRNA which preceded the decline in the telomerase activity. AS PS-ODN significantly reduced the percentage of positive cells expressing hTERT protein following the decline of hTERT mRNA levels. There was no difference seen in the telomerase activity, hTERT mRNA expression or the protein levels between the sense phosphorothioate oligodeoxynucleotide (SPS-ODN) and the control group. AS PS-ODN treatment significantly decreased the cell viability and enhanced the apoptotic rate of T24 cells in response to TNF-α while there was no difference in cell viability and apoptotic rate between the S PS-ODN and the control group.Conclusions AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression. Treatment with AS PS-ODN may be a potential and most promising strategy for bladder cancer with telomerase

  18. Studying the Anti-aging Effect of Human Growth Hormone on Human Fibroblast Cells via Telomerase Activity

    Directory of Open Access Journals (Sweden)

    Nader Chaparzadeh

    2010-01-01

    Full Text Available Objective: In recent years, studies have focused on the telomerase for cancer treatmentby repressing telomerase in cancerous cells or prevent cell aging by activating it in theaged cells. Thus, in these studies natural and synthetic agents have been used to repressor activate telomerase. In this research, we investigated the effects of human growth hormone(hGH on aging via evaluation of telomerase activity.Materials and Methods: Primary human foreskin fibroblast cells were isolated, culturedand treated with different concentrations of hGH. BrdU and MTT cell proliferation assaysand cells number counting. Cell aging was assayed by the senescence sensitivegalactosidase staining method. Telomerase activity was measured with a telomerasePCR ELISA kit.Data were analyzed with SPSS software (one-way ANOVA and univariateANOVA.Results: Our results indicated that cells treated with a lower concentration (0.1, 1 ng/mlof hGH had more green color cells (aged cells. Furthermore, cell proliferation increasedwith increasing hGH concentrations (10 to 100 ng/ml which was significant in comparisonwith untreated control cells. TRAP assay results indicated that telomerase activityincreased with increasing hGH concentration, but there was no significant difference. Additionally,more rapid cell growth and telomerase activity was noted in the absence of H2O2when compared with the presence of H2O2, which was significantly different.Conclusion: Although increasing cell proliferation along with increasing hGH concentrationwas confirmed by all cell proliferation assays, only the cell counting test was statisticallysignificant. Thus, it is inconclusive that hGH (up to 100 ng/ml has an anti-agingeffect. Also, because there was no significant difference in the telomerase activity results(in spite of increasing progress along with increasing hGH concentration we can not certainlyconclude that hGH (up to 100 ng/ml impacts telomerase activity.

  19. The HSP90 inhibitor alvespimycin enhances the potency of telomerase inhibition by imetelstat in human osteosarcoma.

    Science.gov (United States)

    Hu, Yafang; Bobb, Daniel; He, Jianping; Hill, D Ashley; Dome, Jeffrey S

    2015-01-01

    The unsatisfactory outcomes for osteosarcoma necessitate novel therapeutic strategies. This study evaluated the effect of the telomerase inhibitor imetelstat in pre-clinical models of human osteosarcoma. Because the chaperone molecule HSP90 facilitates the assembly of telomerase protein, the ability of the HSP90 inhibitor alvespimycin to potentiate the effect of the telomerase inhibitor was assessed. The effect of single or combined treatment with imetelstat and alvespimycin on long-term growth was assessed in osteosarcoma cell lines (143B, HOS and MG-63) and xenografts derived from 143B cells. Results indicated that imetelstat as a single agent inhibited telomerase activity, induced telomere shortening, and inhibited growth in all 3 osteosarcoma cell lines, though the bulk cell cultures did not undergo growth arrest. Combined treatment with imetelstat and alvespimycin resulted in diminished telomerase activity and shorter telomeres compared to either agent alone as well as higher levels of γH2AX and cleaved caspase-3, indicative of increased DNA damage and apoptosis. With dual telomerase and HSP90 inhibition, complete growth arrest of bulk cell cultures was achieved. In xenograft models, all 3 treatment groups significantly inhibited tumor growth compared with the placebo-treated control group, with the greatest effect seen in the combined treatment group (imetelstat, p = 0.045, alvespimycin, p = 0.034; combined treatment, p = 0.004). In conclusion, HSP90 inhibition enhanced the effect of telomerase inhibition in pre-clinical models of osteosarcoma. Dual targeting of telomerase and HSP90 warrants further investigation as a therapeutic strategy.

  20. Relationship between the Expression of Telomerase and Human Papillomavirus Infection in Invasive Uterine Cervical Carcinoma

    Institute of Scientific and Technical Information of China (English)

    SIMA Ni; CAI Liping; ZHU Yuanfang; WANG Wei; WANG Shixuan; MA Ding

    2007-01-01

    Telomerase activity was examined in invasive cervical carcinoma to assess whether it is activated during cervical malignant transformation and to look for its possible association with human papillomavirus (HPV) infection. Histologically confirmed invasive cervical carcinomas and benign cervices were assayed for telomerase activity by using a modified telomere repeat amplification protocol (TRAP). The same cases were subjected to polymerase chain reaction (PCR) detection of HPV by using consensus primers and type-specific (HPV types 16 and 18) primers. Telomerase activity was detected in 40 of 45 (88.9%) invasive cervical carcinomas and 2 (all chronic cervicitis) of 50 (4%) benign cervical lesions. HPV was detected in 36 (24 HPV-16 and 4 HPV-18 cases) of 45 (80%) invasive cervical carcinomas and 20 (11 HPV-16 and 1 HPV-18 cases) of 50 (40%) benign cervical changes. There was a significant correlation between the expression of telomerase with histological grade (φ=0.44, P<0.005), but no correlation was found between telomerase expression and HPV-18 (P>0.05). Although larger sample studies are needed, there seems to be a clear association between telomerase upregulation and HPV status, mainly HPV-16 infection.

  1. Transcription Regulation of the Human Telomerase Reverse Transcriptase (hTERT Gene

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    Muhammad Khairul Ramlee

    2016-08-01

    Full Text Available Embryonic stem cells and induced pluripotent stem cells have the ability to maintain their telomere length via expression of an enzymatic complex called telomerase. Similarly, more than 85%–90% of cancer cells are found to upregulate the expression of telomerase, conferring them with the potential to proliferate indefinitely. Telomerase Reverse Transcriptase (TERT, the catalytic subunit of telomerase holoenzyme, is the rate-limiting factor in reconstituting telomerase activity in vivo. To date, the expression and function of the human Telomerase Reverse Transcriptase (hTERT gene are known to be regulated at various molecular levels (including genetic, mRNA, protein and subcellular localization by a number of diverse factors. Among these means of regulation, transcription modulation is the most important, as evident in its tight regulation in cancer cell survival as well as pluripotent stem cell maintenance and differentiation. Here, we discuss how hTERT gene transcription is regulated, mainly focusing on the contribution of trans-acting factors such as transcription factors and epigenetic modifiers, as well as genetic alterations in hTERT proximal promoter.

  2. A human breast cancer model for the study of telomerase inhibitors based on a new biotinylated-primer extension assay

    OpenAIRE

    1999-01-01

    Telomerase is an RNA-dependent polymerase that synthesizes telomeric DNA (TTAGGG)n repeats. The overall goal of our work was to establish human cancer models that can be used to design clinical trials with telomerase inhibitors. The objectives of this study were (1) to set up a human breast cancer system that allows evaluation of the effects of telomerase inhibitors in cultured cells using a non-amplified telomerase assay and (2) to test this system using two drugs (cisplatin and TMPyP4) that...

  3. Telomerase inhibition and telomere loss in BEL-7404 human hepatoma cells treated with doxorubicin

    Institute of Scientific and Technical Information of China (English)

    Ru-Gang Zhang; Li-Xia Guo; Xing-Wang Wang; Hong Xie

    2002-01-01

    AIM: To study the effects of doxorubicin on telomeraseactivity and telomere length in hepatocellular carcinoma.METHODS: Telomerase activity was assayed with a non-radioisotopic quantitative telomerase repeat amplificationprotocal-based method. The effect of doxorubicin (DOX) onthe growth of BEL-7404 human hepatoma cells wasdetermined by microculture tetrazolium assay. Meantelomere length (terminal restriction fragment) was detectedby Southern blot method. The expression of telomerasesubunits genes was investigated by RT-PCR. Cell apoptosisand cell cycle distribution were evaluated by flow cytometry.RESULTS: Telomerase activity was inhibited in a dose andtime-dependent manner in BEL-7404 human hepatoma cellstreated with DOX for 24, 48 or 72 h in concentrations from0.156 to 2.5 μM which was crrelated with the inhibition ofcell growth. No changes were found in the mRNA expressionof three telomerase subunits (hTERT, hTR and TP1) afterdrug exposure for 72 h with indicated concentrations. Thecells treated with DOX showed shortened mean telomerelength and accumulated at the G2/M phase. However, therewas almost no effects on cell apoptosis by DOX.CONCLUSION: The telomerase inhibition and the telomereshortening by DOX may contribute to its efficiency in thetreatment in hepatocellular carcinoma.

  4. Genome-wide identification of expression quantitative trait loci for human telomerase

    Science.gov (United States)

    Kim, Hanseol; Ryu, Jihye; Lee, Chaeyoung

    2016-01-01

    Abstract A genome-wide association study was conducted to identify expression quantitative trait loci (eQTL) for human telomerase. We tested the genetic associations of nucleotide variants with expression of the genes encoding human telomerase reverse transcriptase (hTERT) and telomerase RNA components (TERC) in lymphoblastoid cell lines derived from 373 Europeans. Our results revealed 6 eQTLs associated with hTERT (P planar cell polarity protein 2 (PRICKLE2) gene important for the Wnt signaling pathway. This concurs with previous studies in which significant expressional relationships between hTERT and some genes (β-catenin and Wnt-3a) in the Wnt signaling pathway have been observed. This study suggested 6 novel eQTLs for hTERT and the association of hTERT with the Wnt signaling pathway. Further studies are needed to understand their underlying mechanisms to improve our understanding of the role of hTERT in cancer. PMID:27759658

  5. Minimized human telomerase maintains telomeres and resolves endogenous roles of H/ACA proteins, TCAB1, and Cajal bodies.

    Science.gov (United States)

    Vogan, Jacob M; Zhang, Xiaozhu; Youmans, Daniel T; Regalado, Samuel G; Johnson, Joshua Z; Hockemeyer, Dirk; Collins, Kathleen

    2016-08-15

    We dissected the importance of human telomerase biogenesis and trafficking pathways for telomere maintenance. Biological stability of human telomerase RNA (hTR) relies on H/ACA proteins, but other eukaryotes use other RNP assembly pathways. To investigate additional rationale for human telomerase assembly as H/ACA RNP, we developed a minimized cellular hTR. Remarkably, with only binding sites for telomerase reverse transcriptase (TERT), minimized hTR assembled biologically active enzyme. TERT overexpression was required for cellular interaction with minimized hTR, indicating that H/ACA RNP assembly enhances endogenous hTR-TERT interaction. Telomere maintenance by minimized telomerase was unaffected by the elimination of the telomerase holoenzyme Cajal body chaperone TCAB1 or the Cajal body scaffold protein Coilin. Surprisingly, wild-type hTR also maintained and elongated telomeres in TCAB1 or Coilin knockout cells, with distinct changes in telomerase action. Overall, we elucidate trafficking requirements for telomerase biogenesis and function and expand mechanisms by which altered telomere maintenance engenders human disease.

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

    Institute of Scientific and Technical Information of China (English)

    RUI-JUAN GAO; YUE-XIN LIANG; DIAN-DONG LI; HONG-YIN ZHANG; YONG-SU ZHEN

    2007-01-01

    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-β-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-β-gal activity and the changes of senescence-related protein expression.Telomerase activity markedly decreased (P<0.01)in LDM-treated hepatoma BEL-7402 cells. Conelusion 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.

  7. Effects of cisplatin on telomerase activity and telomere length in BEL-7404 human hepatoma cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Telomerase activity was inhibited in a dose and time-dependent manner with the treatment of cisplatin for 24, 48, or 72 h in a concentration ranged from 0.8 to 50 μM in BEL-7404 human hepatoma cells. There were no changes in expression pattern of three telomerase subunits, its catalytic reverse transcriptase subunit (hTERT), its RNA component (hTR) or the associated protein subunit (TP1), after cisplatin treated for 72 h with indicated concentrations. Mean telomere lengths were decreased by the cisplatin treatment. Cell growth inhibition and cell cycle accumulation in G2/M phase were found to be correlated with telomerase inhibition in the present study, but percentages of cell apoptosis did not change markedly during the process.

  8. Human telomerase and Cajal body ribonucleoproteins share a unique specificity of Sm protein association.

    Science.gov (United States)

    Fu, Dragony; Collins, Kathleen

    2006-03-01

    Cajal bodies are nuclear structures that host RNA modification and assembly reactions. Some RNAs transit Cajal bodies, while others must concentrate in Cajal bodies to function. Here we report that at least a subfraction of human telomerase RNA and individual resident Cajal body RNAs is associated with Sm proteins. Surprisingly, of seven Sm proteins assembled into a heteroheptameric ring, only a subset copurifies telomerase and Cajal body ribonucleoproteins. We show that a Cajal body RNA localization motif determines this specificity. These discoveries expand the cellular repertoire of Sm protein assemblies and their involvement in ribonucleoprotein localization and function.

  9. Telomerase promoter reprogramming and interaction with general transcription factors in the human mesenchymal stem cell

    DEFF Research Database (Denmark)

    Serakinci, Nedime; Hoare, Stacey F.; Kassem, Moustapha

    2006-01-01

    The human adult mesenchymal stem cell (hMSC) does not express telomerase and has been shown to be the target for neoplastic transformation after transduction with hTERT. These findings lend support to the stem cell hypothesis of cancer development but by supplying hTERT, the molecular events...... and that modifications of the chromatin environment lead to reactivation of telomerase gene expression. It is shown that repression of hTERT expression in hMSCs is due to promoter-specific histone hypoacetylation coupled with low Pol II and TFIIB trafficking. This repression is overcome by treatment with Trichostatin...

  10. Inhibiting effect of antisense hTRT on telomerase activity of human liver cancer cell line SMMC-7721

    Institute of Scientific and Technical Information of China (English)

    牟娇; 李晓冬; 杨庆; 贾凤岐; 卫立辛; 郭亚军; 吴孟超

    2003-01-01

    Objective: To induce changes in biological character of human liver cancer cell line SMMC-7721 by blocking the expression of telomerase genes hTRT and to explore its value in cancer gene therapy. Methods: The vehicle for eukaryotic expression of antisense hTRT was constructed and then transfected into SMMC-7721 cells. The effects of antisense hTRT gene on telomerase activity, cancer cell growth and malignant phenotypes were analyzed. Results: The obtained transfectants that could express antisense hTRT gene stably showed marked decrease in telomerase activity; the shortening of telomere was obvious; cells presented contact growth inhibition; in nude mice transplantation, the rate of tumor induction dramatically decreased. Conclusion: Antisense hTRT gene expression can significantly inhibit telomerase activity of cancer cells and decrease malignant phenotypes in vitro and in vivo. Therefore, as a telomerase inhibitor, antisense hTRT gene may be a new pathway for cancer therapy.

  11. Efficient inhibition of human telomerase activity by antisense oligonucleotides sensitizes cancer cells to radiotherapy

    Institute of Scientific and Technical Information of China (English)

    Xue-mei JI; Cong-hua XIE; Ming-hao FANG; Fu-xiang ZHOU; Wen-jie ZHANG; Ming-sheng ZHANG; Yun-feng ZHOU

    2006-01-01

    Aim: To investigate the effect of the antisense oligonucleotides (ASODN) specific for human telomerase RNA (hTR) on radio sensitization and proliferation inhibition in human neurogliocytoma cells (U251). Methods: U251 cells were transfected with hTR ASODN or nonspecific oligonucleotides (NSODN). Before and after irradiation of 60Co-γray, telomerase activity was assayed by telomeric repeat amplification protocol (TRAP-PCR-ELISA), and DNA damage and repair were examined by the comet assay. The classical colony assay was used to plot the cell-survival curve, to detect the D0 value. Results: hTR antisense oligonucleotides could downregulate the telomerase activity, increase radiation induced DNA damage and reduce the subsequent repair. Furthermore, it could inhibit the proliferation and decrease the D0 value which demonstrates rising radiosensitivity. However, telomere length was unchanged over a short period of time. Conclusion: These findings suggest that an ASODN-based strategy may be used to develop telomerase inhibitors, which can efficiently sensitize radiotherapy.

  12. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Simonsen, Janne Lytoft; Rosada, Cecilia; Serakinci, Nedime;

    2002-01-01

    Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells......, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions....

  13. Culture of Human Tendon Cell Transfected by Human Telomerase Reverse Transcriptase Plasmid and their Biological Characteristics In Vitro

    Institute of Scientific and Technical Information of China (English)

    Hui-Qi XIE; Zhi-Ming YANG; Fan LIN; Yi QU

    2005-01-01

    @@ 1 Introduction The proliferative and functional characteristics of tendon cell are the key issue in the research of tissue engineered tendon. The standard tendon cell line, which has normal functional characteristics, and can be subcultured continuously and permanently, will not only meet the demands of seeding cell in tissue engineered tendon,but also control the variable of tendon cell. In our previous study, it showed that the proliferative ability of human tendon cell cultured in vitro is depressed gradually after subcultured to the 13th passage, which can not meet the demands of tendon tissue engineering. It has been proved that replicative senescence of cells was closely related to the activity of telomerase. We constructed pGRN145 plasmid which contain total human telomerase reverse transcriptase (hTERT) cDNA. We transfected it into human tendon cells to investigate the feasibility of life span extension by reconstitution of the telomerase activity.

  14. Telomerase activity and telomere length in human tumor cells with acquired resistance to anticancer agents.

    Science.gov (United States)

    Smith, V; Dai, F; Spitz, M; Peters, G J; Fiebig, H H; Hussain, A; Burger, A M

    2009-11-01

    Telomeres and telomerase are targets for anticancer drug development and specific inhibitors are currently under clinical investigation. However, it has been reported that standard cytotoxic agents can affect telomere length and telomerase activity suggesting that they also have of a role in drug resistance. in this study, telomere lengths and telomerase activity as well as drug efflux pump expression, glutathione (GSH) levels and polyadenosine-ribose polymerase (PARP) cleavage were assessed in a panel of human tumor cell lines made resistant to vindesine, gemcitabine and cisplatin. these included two lung cancer cell lines resistant to vindesine (LXFL 529L/Vind, LXFA 526L/Vind), a renal cancer cell line (RXF944L/Gem) and an ovarian cancer cell line (AG6000) resistant to gemcitabine, and one resistant to cisplatin (ADDP). The resistant clones were compared to their parental lines and evaluated for cross resistance to other cytotoxic agents. Several drug specific resistance patterns were found, and various complex patterns of cross resistance emerged from some cell lines, but these mechanisms of resistance could not be related to drug efflux pump expression, GSH levels or pARp cleavage. However, all displayed changes in telomerase activity and/or telomere length. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics.

  15. Transcript Regulation of Human Telomerase Reverse Transcriptase by c-myc and mad1

    Institute of Scientific and Technical Information of China (English)

    Lin ZOU; Peng-Hui ZHANG; Chun-Li LUO; Zhi-Guang TU

    2005-01-01

    Telomerase activity is highly positive correlated to most malignant neoplasms. Human telomerase reverse transcriptase (hTERT) is the rate-limiting factor of telomerase activity. Recent studies have shown that the expression of hTERT is mainly determined by its transcript regulation. Among the transcript regulation factors of hTERT, c-myc and mad1 are well known. Here, we constructed c-myc and mad1 eukaryotic expression vectors, then co-transfected them with the wild-type (Tw) or mutant hTERT promoter (Td)luciferase reporter plasmid, which were double-mutated in the E-box sequences from CACGTG to CACCTG of Tw. The change of luciferase activity in different cells was detected. The results showed that Tw was obviously activated in T24 and EJ bladder cancer cells, but not in normal fibrocytes. c-myc and mad1 had positive and negative effects respectively on the Tw transcript in a dose-dependent manner, while the roles of c-myc and mad1 in regulating the Td transcript were reversed. c-myc combined with mad1 can downregulate Tw but not Td. These observations indicate that c-myc and mad1 can regulate the hTERT transcript in a different manner in hTERT positive cells, but not in normal cells. This may provide an insight into some telomerase-related carcinogenesis mechanisms.

  16. Delivering Antisense Morpholino Oligonucleotides to Target Telomerase Splice Variants in Human Embryonic Stem Cells.

    Science.gov (United States)

    Radan, Lida; Hughes, Chris S; Teichroeb, Jonathan H; Postovit, Lynne-Marie; Betts, Dean H

    2016-01-01

    Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) Δα and Δβ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells.

  17. The effect of β-ionone on telomerase activity in the human leukemia cell line K562

    Directory of Open Access Journals (Sweden)

    Zohreh Faezizadeh

    2015-06-01

    Full Text Available Background: Telomerase is highly activated in most human cancer cells, therefore, its inhibition has been proposed as a novel and promising strategy for cancer therapy. Many plant-derived anticancer agents act through inhibition of telomerase activity and induction of apoptosis. β-ionone, a carotenoid compound isolated from Roseaceae, has been reported to possess anticancer properties. The present study was undertaken to examine the mechanism of β-ionone-induced apoptosis in human leukemia cell line K562 with special emphasis on its role in telomerase inhibition. Method: In this study the anti-proliferation effect of β-ionone on K562 cells was evaluated by MTT assay. Apoptosis rate was detected by Hoechst staining and flow cytometry analysis. Telomerase activity was measured by (TRAP ELISA assay. Results: Exposure of K562 cells to β-ionone caused a dose-dependent decrease in proliferation. Flow cytometry analysis and Hoechst staining showed that percentage of apoptotic cells markedly increased with an increase in β-ionone concentration. Compared to control cells, treatment of K562 cells with β-ionone resulted in a significant decrease of telomerase activity. Moreover, a positive correlation was detected between telomerase inhibition and apoptosis induction in the treated K562 cells. Conclusion: Based on these results, β-ionone is an appropriate candidate for inhibiting telomerase activity in K562 cells. Therefore, it may be utilized as a novel drug against some leukemia cell lines.

  18. EFFECT OF SeO2 ON TELOMERASE ACTIVITY IN HUMAN LUNG CARCINOMA CELL LINE GLC-82

    Institute of Scientific and Technical Information of China (English)

    陈维香; 曹晓哲; 朱任之

    2003-01-01

    Objective: To observe the effect of inhibition of telomerase activity by selenium dioxide (SeO2) on lung carcinoma cell line GLC-82. Methods: TRAP-PCR-ELISA was used to study the changes of telomerase activity in human pulmonary adenocarcinoma cell line GLC-82 treated by SeO2 at the different concentrations (3, 10, 30 μmol/L) and for different times (24, 48, and 72 h). Results: SeO2 inhibited the telomerase activity of GLC-82 at the different concentrations after treatment of 24, 48 and 72 h. Conclusion: SeO2 inhibits from telomerase activity of human lung carcinoma line GLC-82. The effect of inhibition is dose-dependant and time-dependant.

  19. Promotion of cell proliferation by HBXIP via upregulation of human telomerase reverse transcriptase in human mesenchymal stem cells1

    Institute of Scientific and Technical Information of China (English)

    Feng-ze WANG; Li SHA; Li-hong YE; Xiao-dong ZHANG

    2008-01-01

    Aim: We previously found that the hepatitis B X-interacting protein (HBXIP) was able to promote the proliferation of cells. Telomerase activity is known to be critical in cellular senescence and its level is modulated by the regulation of the telomerase catalytic subunit, telomerase reverse transcriptase (TERT), at both the transcriptional and post-transcriptional levels. To investigate the mechanism of promoting proliferation by HBXIP, the effect of HBXIP on human TERT (hTERT) was investigated in human mesenchymal stem cells (hMSC). Methods: BMMS-03 cells and hMSC from the bone marrow of a 4-month-old elicited fetus, were tran-siently transfected with the pcDNA3-hbxip plasmid encoding the HBXIP gene and pSilencer-hbxip plasmid encoding RNA interference (RNAi) targeting HBXIP mRNA, followed by the examination of the hTERT promoter reporter gene by luciferase assay, and the detection of telomerase activity by telomeric repeat amplication protocol, respectively, as well as the expression levels of hTERT, c-Myc, and Bcl-2 by Western blot analysis. Results: The overexpression of HBXIP led to a significant upregulation of hTERT promoter activity, telomerase activity, and the expression levels of hTERT, c-Myc, and Bcl-2 in BMMS-03 cells. RNAi target-ing HBXIP mRNA produced the opposite results completely. Conclusion: Our data demonstrated that HBXIP significantly stimulated the transcription and ex-pression of hTERT and increased the activity of telomerase in BMMS-03 cells, which provides a new insight into the mechanism of promoting cell proliferation by HBXIP.

  20. Telomerase-specific oncolytic virotherapy for human hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    AIM: To evaluate the therapeutic efficiency of replicative adenovirus CNHK300 targeted in telomerase-positive hepatocellular carcinoma. METHODS: CNHK300, ONYX-015 (55 kDa protein deleted adenovirus) and wtAd5 (wild type adenovirus 5) were compared, and virus proliferation assay, cell viability assay, Western blot and fluorescence microscopy were used to evaluate the proliferation and cytolysis selectivity of CNHK300.RESULTS:The replicative multiples in Hep3B and HepG after 48 h of CNHK300 proliferation were 40625and 65326 fold, respectively, similar to that of wtAd5..However, CNHK300 exhibited attenuated replicative ability in normal fibroblast cell line BJ.CNHK300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection (MOI),but could not affect growth of normal cells even at a high MOI.CONCLUSION:CNHK300 is a cancer-selective replication-competent adenovirus which can cause oncolysis of liver cancer cells as well as wtAd5 (wild type adenovirus 5),but had severely attenuated replicative and cytolytic ability in normal cells. This novel strategy of cancer treatment offers a promising treatment platform.

  1. Tissue distribution and engraftment of human mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene

    DEFF Research Database (Denmark)

    Bentzon, J F; Stenderup, K; Hansen, F D

    2005-01-01

    Engraftment of mesenchymal stem cells (MSC) in peripheral tissues for replenishing of local stem cell function has been proposed as a therapeutic approach to degenerative diseases. We have previously reported the development of an immortalized human telomerase reverse transcriptase transduced MSC...... line (hMSC-TERT). In the present study, we co-transduced hMSC-TERT with enhanced green fluorescent protein gene, and studied tissue distribution, engraftment, and cell survival after intracardiac and intravenous injections in immunodeficient mice. The pattern of organ distribution suggested...... that infused cells were efficiently arrested in microvasculature during first-pass, but only for a fraction of the infused cells was arrest followed by vascular emigration and tissue engraftment. Few engrafted cells in lungs, heart, and kidney glomeruli remained after 4 weeks. These observations are consistent...

  2. Therapeutic Targeting of Telomerase

    Directory of Open Access Journals (Sweden)

    Kathrin Jäger

    2016-07-01

    Full Text Available Telomere length and cell function can be preserved by the human reverse transcriptase telomerase (hTERT, which synthesizes the new telomeric DNA from a RNA template, but is normally restricted to cells needing a high proliferative capacity, such as stem cells. Consequently, telomerase-based therapies to elongate short telomeres are developed, some of which have successfully reached the stage I in clinical trials. Telomerase is also permissive for tumorigenesis and 90% of all malignant tumors use telomerase to obtain immortality. Thus, reversal of telomerase upregulation in tumor cells is a potential strategy to treat cancer. Natural and small-molecule telomerase inhibitors, immunotherapeutic approaches, oligonucleotide inhibitors, and telomerase-directed gene therapy are useful treatment strategies. Telomerase is more widely expressed than any other tumor marker. The low expression in normal tissues, together with the longer telomeres in normal stem cells versus cancer cells, provides some degree of specificity with low risk of toxicity. However, long term telomerase inhibition may elicit negative effects in highly-proliferative cells which need telomerase for survival, and it may interfere with telomere-independent physiological functions. Moreover, only a few hTERT molecules are required to overcome senescence in cancer cells, and telomerase inhibition requires proliferating cells over a sufficient number of population doublings to induce tumor suppressive senescence. These limitations may explain the moderate success rates in many clinical studies. Despite extensive studies, only one vaccine and one telomerase antagonist are routinely used in clinical work. For complete eradication of all subpopulations of cancer cells a simultaneous targeting of several mechanisms will likely be needed. Possible technical improvements have been proposed including the development of more specific inhibitors, methods to increase the efficacy of vaccination

  3. Current Insights to Regulation and Role of Telomerase in Human Diseases

    Science.gov (United States)

    Ozturk, Mert Burak; Li, Yinghui; Tergaonkar, Vinay

    2017-01-01

    The telomerase ribonucleoprotein complex has a pivotal role in regulating the proliferation and senescence of normal somatic cells as well as cancer cells. This complex is comprised mainly of telomerase reverse transcriptase (TERT), telomerase RNA component (TERC) and other associated proteins that function to elongate telomeres localized at the end of the chromosomes. While reactivation of telomerase is a major hallmark of most cancers, together with the synergistic activation of other oncogenic signals, deficiency in telomerase and telomeric proteins might lead to aging and senescence-associated disorders. Therefore, it is critically important to understand the canonical as well as non-canonical functions of telomerase through TERT to develop a therapeutic strategy against telomerase-related diseases. In this review, we shed light on the regulation and function of telomerase, and current therapeutic strategies against telomerase in cancer and age-related diseases. PMID:28264499

  4. Inhibitory effect of human telomerase antisense oligodeoxyribonucleotides on the growth of gastric cancer cell lines in variant tumor pathological subtype

    Institute of Scientific and Technical Information of China (English)

    Jing Ye; Yun-Lin Wu; Shu Zhang; Zi Chen; Li-Xia Guo; Ruo-Yu Zhou; Hong Xie

    2005-01-01

    AIM: To investigate the inhibitory effect of specialized human telomerase antisense oligodeoxyribonucleotides on the growth of well (MKN-28), moderately (SGC-7901)and poorly (MKN-45) differentiated gastric cancer cell lines under specific conditions and its inhibition mechanism,and to observe the correlation between the growth inhibition ratio and the tumor pathologic subtype of gastric cancer cells.METHODS: Telomerase activity in three gastric cancer cell lines of variant tumor pathologic subtype was determined by modified TRAP assay before and after the specialized human telomerase antisense oligodeoxyribonucleotides were dealt with under specific conditions. Effect of antisense oligomer under specific conditions of the growth and viability of gastric cancer cell lines was explored by using trypan blue dye exclusion assay, and cell apoptosis was detected by cell morphology observation, flow cytometry and TUNEL assay.RESULTS: Telomerase activity was detected in well,moderately and poorly differentiated gastric cancer cell lines (the quantification expression of telomerase activity was 43.7TPG, 56.5TPG, 76.7TPG, respectively).Telomerase activity was controlled to 30.2TPG, 36.3TPG and 35.2TPG for MKN-28, SGC-7901 and MKN-45 cell lines respectively after treatment with human telomerase antisense oligomers at the concentration of 5 μmol/L, and was entirely inhibited at 10 μmol/L, against the template region of telomerase RNA component, whereas no inhibition effect was detected in missense oligomers (P<0.05). After treatment with antisense oligomers at different concentrations under specific conditions for 96 h, significant growth inhibition effects were found in MKN-45 and SGC-7901gastric cancer cell lines (the inhibition ratio was 40.89%and 71.28%), but not in MKN-28 cell lines (15.86%). The ratio of inactive SGC-7901 cells increased according to the prolongation of treatment from 48 to 96 h. Missense oligomers could not lead to the same effect (P<0

  5. Effect of antisense human telomerase RNA on malignant behaviors of gastric carcinoma cell line SGC-7901

    Institute of Scientific and Technical Information of China (English)

    YANG Jin-liang; FANG Dian-chun; YANG Shi-ming; LUO Yuan-hui; LUO Kun-lun; LU Rong; LIU Wei-wen

    2001-01-01

    Objective: To study the effects of antisense human telomerase RNA (ahTR) transfection on the malignant behaviors of gastric carcinoma cell line SGC-7901 and its potential role in gene therapy for tumor. Methods: An antisense hTR eukaryotic expression vector containing the sequence of template region of telomere repeats was transfected into gastric carcinoma cell line SGC-7901 with liposome DOTAP. The expressions of hTR RNA and antisense hTR RNA were observed with RT-PCR, telomerase activity with PCR-ELISA. Telomere length was measured with Southern blot. Cell morphology and cellular proliferation capacity were studied with MTT assay. Cell cycle distribution and apoptotic state were observed with flow cytometry. Efficiency of clone formation in soft agar and tumorigencity in nude mice were examined and evaluated in ahTR-transfected 7901 cells, and plasmid pCL-neo transfected 7901 cells and parental 7901 cells served as control. Results: An antisense hTR eukaryotic expression vector was transfected into 7901 cells successfully. The telomerase activity in ahTR-transfected 7901 cells was decreased from 100% to about 25%, and telomere length in the cells shortened from 4.08 kb to 3.35 kb at 60 population doublings (PDs). Compared with parental 7901 and pCL-neo transfected 7901 cells, ahTR-transfected 7901 cells displayed some morphological changes, including decreased cell atypia and nucleus/cytoplasm ratio under light microscope. Furthermore, ahTR-transfected 7901 cells displayed growth inhibition, decreased invasive capacity in Borden's chamber invasive model, increased G0/G1 phase rate and apoptotic rate, and restored contact inhibition and density inhibition. Surprisingly, ahTR-transfected 7901 cells lost their capacity of clone formation in soft agar and carcinogensis in nude mice. Conclusion: Antisense hTR transfection can induce 7901 cell differentiation and reverse its malignant phenotype. This study provides an exciting approach for cancer therapy through the

  6. Antiproliferative effect of rapamycin on human T-cell leukemia cell line Jurkat by cell cycle arrest and telomerase inhibition

    Institute of Scientific and Technical Information of China (English)

    Yan-min ZHAO; Qian ZHOU; Yun XU; Xiao-yu LAI; He HUANG

    2008-01-01

    Aim:To examine the ability of rapamycin to suppress growth and regulate telomerase activity in the human T-cell leukemia cell line Jurkat. Methods:Cell proliferation was assessed after exposure to rapamycin by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle progression and apoptosis were determined by flow cytometry. The proteins important for cell cycle progres-sion and Akt/mammalian target of rapamycin signaling cascade were assessed by Western blotting. Telomerase activity was quantified by telomeric repeat amplication protocol assay. The human telomerase reverse transcriptase (hTERT) mRNA levels were determined by semi-quantitative RT-PCR. Results:Rapamycin inhibited the proliferation of Jurkat, induced G1 phase arrest, unregulated the pro-tein level of p21 as well as p27, and downregulated cyclinD3, phospho-p70s6k, and phospho-s6, but had no effect on apoptosis. Treatment with rapamycin reduced telomerase activity, and reduced hTERT mRNA and protein expression. Conclusion:Rapamycin displayed a potent antileukemic effect in the human T-cell leukemia cell line by inhibition of cell proliferation through G1 cell cycle arrest and also through the suppression of telomerase activity, suggesting that rapamycin may have potential clinical implications in the treatment of some leukemias.

  7. The Effect of Nano-apatite on the Expression of Telomerase Gene of Human Hepatocellular Carcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the effect of nano- apatite on the expression of the telomerase gene of human hepatocellular carcinoma cell lines and further explore the mechanism of the nano-apatite inhibiting cancer cells. Using the hybridization in situ method to detect the expression of the telomerase gene of human hepatocellular carcinoma cells treated with the nano- apatite for 4 h at 37 ℃. The hybridization in situ showed that the cytoplasm of the positive cells was stained in nigger-brown. The positive cell rate of the control group was 88.49% , the cisplatin group was 25.6% , the nano-apatite group was 63.6% . The activity oftelomerase gene was both obviously declined comparing with the control group and the difference had significance (p < 0.05, p < 0.01 ). The nanoapatite obviously inhabit the expression of the telomerase gene of human hepatocellular carcinoma cells.

  8. Cryptolepine Derivatives:Quadruplex-Interactive Agents as Inhibitors of Human Telomerase

    Institute of Scientific and Technical Information of China (English)

    HUANG,Zhi-Shu; ZHOU,Jin-Lin; LU,Yu-Jing; GU,Lian-Quan

    2004-01-01

    @@ Alkaloids are very important natural products. Most of them have biologic activity. Many novel drugs have been developed based on alkaloids, such as camptothecin, taxol, vinblastine. A series of novel cryptolepine derivatives were synthesized (Figure 1). The interaction of cryptolepine derivatives with G-quadruplex (Figure 2) was studied by CD and UV spectra.[1] Most of these compounds can induce the formation of G-quadruplex and stabilize the formed G-quadruplex, resulting in the inhibitory effect on telomerase. Most of these cryptolepine derivatives have potent cytotoxicity in vitro against human tumor cell line.

  9. Relationship between Dyskerin Expression and Telomerase Activity in Human Breast Cancer

    Directory of Open Access Journals (Sweden)

    Lorenzo Montanaro

    2008-01-01

    Full Text Available The nucleolar protein dyskerin is involved in the modification of specific uridine residues to pseudouridine on ribosomal and small nuclear RNAs and in the stabilization of the telomerase RNA component (TERC. In this study we investigated for the first time the relationship between dyskerin expression and telomerase activity in a series of 61 primary breast carcinomas. We found that when dyskerin mRNA values were very low the telomerase activity was markedly reduced, independently of the expression of other important components of the telomerase complex such as telomerase reverse transcriptase (TERT. In vitro experiments showed that reduction of dyskerin expression affect telomerase activity through the reduction of TERC. Only when TERC levels were strongly reduced telomerase activity was hindered. Retroviral mediated over-expression of TERC abolished the telomerase impairment due to dyskerin knock down. In conclusion, our results indicated that, beside its effect on ribosome biogenesis, the levels of dyskerin in cancer cells modulate telomerase activity through the regulation of TERC levels, independently of TERT expression. This should be taken into consideration when utilizing TERT expression as a surrogate indicator of telomerase activity in tumour pathology.

  10. Ectopic expression of telomerase enhances osteopontin and osteocalcin expression during osteogenic differentiation of human mesenchymal stem cells from elder donors

    Directory of Open Access Journals (Sweden)

    Machado CB

    2009-01-01

    Full Text Available Age related bone loss is one of the most prevalent diseases in the elder population. The osteoblasts are the effectors cells of bone formation and regeneration. With the aging the osteoblasts become senescent reducing their ability to produce bone. Cellular replicative senescence is triggered by telomers shortening. Telomerase elongate the telomers length and maintain the cell proliferative capacity. Here, we demonstrated that the expression of human telomerase reverse transcriptase mediated by an adenovirus vector increases the levels of osteopontin and osteocalcin mRNA during the in vitro osteogenic differentiation of elderly human mesenchymal stem cells. Bone marrow human mesenchymal stem cells were obtained from old donors (>65 years and induced to differentiate into osteoblasts for 14 days. The levels of mRNA of human telomerase reverse transcriptase, osteopontin and osteocalcin during the differentiation were assessed by semi-quantitative PCR before and during the differentiation on days 7 and 14. Infected cells showed 1.5 fold increase in telomerase expression. Also telomerized cells exhibit 1.5 fold increase in osteopontin and 0.5 fold increase in osteocalcin expression compared to primary osteoblasts isolated from the same donors. The transformed cells were not able to form tumours in NUDE mice.

  11. PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK)

    Energy Technology Data Exchange (ETDEWEB)

    Senthilkumar, P.K. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Robertson, L.W. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA (United States); Ludewig, G., E-mail: Gabriele-ludewig@uiowa.edu [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA (United States)

    2012-02-15

    Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24 days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism. -- Highlights: ► Human immortal (HaCaT) and primary (NFK) keratinocytes were exposed to PCB153. ► PCB153 significantly reduced telomerase activity and telomere length in HaCaT. ► No effect on telomere length and

  12. Telomerase Activation in Hematological Malignancies

    Directory of Open Access Journals (Sweden)

    Joana Ropio

    2016-09-01

    Full Text Available Telomerase expression and telomere maintenance are critical for cell proliferation and survival, and they play important roles in development and cancer, including hematological malignancies. Transcriptional regulation of the rate-limiting subunit of human telomerase reverse transcriptase gen (hTERT is a complex process, and unveiling the mechanisms behind its reactivation is an important step for the development of diagnostic and therapeutic applications. Here, we review the main mechanisms of telomerase activation and the associated hematologic malignancies.

  13. The MAPK pathway signals telomerase modulation in response to isothiocyanate-induced DNA damage of human liver cancer cells.

    Directory of Open Access Journals (Sweden)

    Evelyn Lamy

    Full Text Available 4-methylthiobutyl isothiocyanate (MTBITC, an aliphatic, sulphuric compound from Brassica vegetables, possesses in vitro and in vivo antitumor activity. Recently we demonstrated the potent growth inhibitory potential of the DNA damaging agent MTBITC in human liver cancer cells. Here we now show that MTBITC down regulates telomerase which sensitizes cells to apoptosis induction. This is mediated by MAPK activation but independent from production of reactive oxygen species (ROS. Within one hour, MTBITC induced DNA damage in cancer cells correlating to a transient increase in hTERT mRNA expression which then turned into telomerase suppression, evident at mRNA as well as enzyme activity level. To clarify the role of MAPK for telomerase regulation, liver cancer cells were pre-treated with MAPK-specific inhibitors prior to MTBITC exposure. This clearly showed that transient elevation of hTERT mRNA expression was predominantly mediated by the MAPK family member JNK. In contrast, activated ERK1/2 and P38, but not JNK, signalled to telomerase abrogation and consequent apoptosis induction. DNA damage by MTBITC was also strongly abolished by MAPK inhibition. Oxidative stress, as analysed by DCF fluorescence assay, electron spin resonance spectroscopy and formation of 4-hydroxynonenal was found as not relevant for this process. Furthermore, N-acetylcysteine pre-treatment did not impact MTBITC-induced telomerase suppression or depolarization of the mitochondrial membrane potential as marker for apoptosis. Our data therefore imply that upon DNA damage by MTBITC, MAPK are essential for telomerase regulation and consequent growth impairment in liver tumor cells and this detail probably plays an important role in understanding the potential chemotherapeutic efficacy of ITC.

  14. TELOMERE AND TELOMERASE MODULATION BY BERGAMOT POLYPHENOLIC FRACTION IN EXPERIMENTAL PHOTOAGEING IN HUMAN KERATINOCYTES.

    Science.gov (United States)

    Nisticò, S; Ehrlich, J; Gliozzi, M; Maiuolo, J; Del Duca, E; Muscoli, C; Mollace, V

    2015-01-01

    Photoageing represents the addition of extrinsic chronic ultraviolet radiation-induced damage on intrinsic ageing and accounts for most age-associated changes in skin appearance. In this study, we evaluated the effect of 38% BPF, a highly concentrated extract of the bergamot fruit (Citrus bergamia) on UVB-induced photoageing by examining inflammatory cytokine expression, telomere length/telomerase alterations and cellular viability in human immortalized HaCaT keratinocytes. Our results suggest that 38% BPF protects HaCaT cells against UVB-induced oxidative stress and markers of photoageing in a dose-dependent manner and could be a useful supplement in skin care products. Together with antioxidant properties, BPF, a highly concentrated extract of the bergamot fruit, appears to modulate basic cellular signal transduction pathways leading to anti-proliferative, anti-aging and immune modulating responses.

  15. Glucose metabolite glyoxal induces senescence in telomerase-immortalized human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Kassem, Moustapha; Rattan, Suresh

    2012-01-01

    Background Various by-products of the cellular metabolism, such as reactive carbonyl species (RCS) are potentially harmful to cells and tissues, and play a role in many physiological and pathological processes. Among various RCS is the highly reactive dicarbonyl glyoxal (GO), which is a natural......, multinucleation, induction of senescence associated beta-galactosidase, cell cycle arrest, and increased levels of a cell cycle inhibitor p16. These changes were accompanied by increased extent of DNA breaks as measured by the comet assay, and increased levels of the AGE product, carboxymethyl-lysine (CML...... physiological metabolite produced by the auto-oxidation of glucose, and can form covalent adducts known as advanced glycation endproducts (AGE). We have previously reported that GO accelerates ageing and causes premature senescence in normal human skin fibroblasts. Results Using a bone marrow-derived telomerase...

  16. In vitro and ex vivo inhibition of human telomerase by anti-HIV nucleoside reverse transcriptase inhibitors (NRTIs but not by non-NRTIs.

    Directory of Open Access Journals (Sweden)

    Kyle R Hukezalie

    Full Text Available Telomerase is a specialized reverse transcriptase responsible for the de novo synthesis of telomeric DNA repeats. In addition to its established reverse transcriptase and terminal transferase activities, recent reports have revealed unexpected cellular activities of telomerase, including RNA-dependent RNA polymerization. This telomerase characteristic, distinct from other reverse transcriptases, indicates that clinically relevant reverse transcriptase inhibitors might have unexpected telomerase inhibition profiles. This is particularly important for the newer generation of RT inhibitors designed for anti-HIV therapy, which have reported higher safety margins than older agents. Using an in vitro primer extension assay, we tested the effects of clinically relevant HIV reverse transcriptase inhibitors on cellular telomerase activity. We observed that all commonly used nucleoside reverse transcriptase inhibitors (NRTIs, including zidovudine, stavudine, tenofovir, didanosine and abacavir, inhibit telomerase effectively in vitro. Truncated telomere synthesis was consistent with the expected mode of inhibition by all tested NRTIs. Through dose-response experiments, we established relative inhibitory potencies of NRTIs on in vitro telomerase activity as compared to the inhibitory potencies of the corresponding dideoxynucleotide triphosphates. In contrast to NRTIs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs nevirapine and efavirenz did not inhibit the primer extension activity of telomerase, even at millimolar concentrations. Long-term, continuous treatment of human HT29 cells with select NRTIs resulted in an accelerated loss of telomere repeats. All tested NRTIs exhibited the same rank order of inhibitory potencies on telomerase and HIV RT, which, according to published data, were orders-of-magnitude more sensitive than other DNA polymerases, including the susceptible mitochondria-specific DNA polymerase gamma. We concluded that

  17. Human telomerase reverse transcriptase binds to a pre-organized hTR in vivo exposing its template.

    Science.gov (United States)

    Zemora, Georgeta; Handl, Stefan; Waldsich, Christina

    2016-01-08

    Telomerase is a specialized reverse transcriptase that is responsible for telomere length maintenance. As in other organisms, the minimal components required for an active human telomerase are the template-providing telomerase RNA (hTR) and the enzymatic entity telomerase reverse transcriptase (hTERT). Here, we explored the structure of hTR and the hTERT-induced conformational changes within hTR in living cells. By employing an in vivo DMS chemical probing technique, we showed that the pseudoknot and associated triple helical scaffold form stably in vivo independently of hTERT. In fact, the dimethyl-sulfate (DMS) modification pattern suggests that hTR alone is capable of adopting a conformation that is suited to interact with hTERT. However, in the absence of hTERT the template region of hTR is only weakly accessible to DMS-modifications. The predominant change after binding of hTERT to hTR is the exposure of the template region.

  18. Relationship between the telomerase activity and the growth kinetics of the human umbilical cord derived mesenchymal stem cells

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    Leila Hosseinzadeh Anvar

    2016-08-01

    Full Text Available Background: Telomerase as an enzyme with reverse transcriptase activity has an essential role in telomere maintenance by adding a telomere repeat sequence to the 3' end of chromosome and is important for regulating of many processes in embryonic development including cell proliferation and differentiation. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs with a self-renewal capacity are cells that can differentiate into various germ layer derivatives including neural cells and cardiomyocytes, and undergo biological changes during long-term cultivation. Hence, the passage number in which the cells expanded seems to be very important for proliferating and differentiating. This study was aimed at investigating the relationship between the telomerase activity and the growth rate of (hUC-MSCs at different passages. Methods: This experimental study was performed in Ardabil University of Medical Sciences, Iran, from March 2014 to December 2014. The umbilical cord samples were obtained from full-term neonate hospitalized in Alavi’s Hospital in Ardabil under sterile conditions. The umbilical vessels were clear off and the small pieces of the umbilical cord were cultured in Dulbecco's modified eagle's medium (DMEM supplemented with 20% fetal bovine serum (FBS. Then, the hUC-MSCs were harvested from passage one to three to calculate the population doubling time (PDT and extract proteins by using CHAPS lysis buffer. Finally, the telomerase activity of the cells at different passages was measured by telomeric repeat amplification protocol (TRAP and qRT-TRAP assays. Results: The hUC-MSCs population doubling time at passage from 1 to 3 were calculated as the average of 54.68±1.92, 55.03±1.71 and 69.41±2.54 hours, respectively, suggesting the higher cell passage number, the more extended PDT. The threshold cycles (CTs for the telomerase activity also showed 30.58±0.51, 27.24±0.74 and 32.13±0.75 for the cell passage from one to three

  19. Dynamics of human telomerase RNA structure revealed by antisense oligonucleotide technique.

    Science.gov (United States)

    Vasilkova, Daria V; Azhibek, Dulat M; Zatsepin, Timofei S; Naraikina, Yulia V; Prassolov, Vladimir S; Prokofjeva, Maria M; Zvereva, Maria I; Rubtsova, Maria P

    2013-12-01

    Telomeres are the nucleoprotein complexes that cap the linear chromosome ends. Telomerase is a ribonucleoprotein that maintains telomere length in stem, embryonic and cancer cells. Somatic cells don't contain active telomerase and telomere function as mitotic clock and telomere length determines the number of cell divisions. Telomerase RNA (TER) contains the template for telomere synthesis and serves as a structural scaffold for holoenzyme assembly. We compared different oligonucleotide based methods for telomerase RNA inhibition, such as antisense oligonucleotides, knockdown by transient siRNA transfection and silencing by miRNA derived from short expressed RNA hairpin in HEK293 cells. All of these methods were applied to different TER regions. Our results revealed that CR2/CR3 domain of TER is accessible in vitro and in vivo and could serve as an optimal site for oligonucleotide-based telomerase silencing.

  20. Using a non-radioisotopic, quantitative TRAP-based me thod detecting telomerase activities in human hepatoma cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A non-radioisotopic, quantitative TRAP-based telom erase activity assay was established mainly by using SYBR Green-I staining instead of radioisotope. Comparing with conventional radioisotope based method, it was better in reproducibility and accuracy. Using this method, we found telomerase activities were absent in normal human liver cells, while detected in all of four human hepatoma cell lines (BEL-7404, SMMC-7721, QGY-7903 and HCCM) without significant differences.

  1. Human telomerase gene amplification and high-risk human papillomavirus infection in women with cervical intra-epithelial neoplasia.

    Science.gov (United States)

    Takac, Iztok; Arko, D; Kodric, T; Poljak, M; Zagorac, A; Erjavec-Skerget, A; Kokalj-Vokac, N

    2009-01-01

    This study was designed to investigate whether a correlation exists between amplification of the human telomerase gene (human telomerase RNA component [TERC]) and high-risk human papillomavirus (HR-HPV) infection in 101 women with cervical intra-epithelial neoplasia (CIN). Eight patients (7.9%) had CIN 1, 24 (23.8%) had CIN 2 and 69 (68.3%) had CIN 3. TERC was amplified in 31.7% of all CIN patients. The difference in frequency of TERC amplification between patients with low-grade CIN (CIN 1) and those with high-grade CIN (CIN 2 and CIN 3) was not significant. HR-HPV infection was detected in 88.1% of all CIN cases and was significantly more frequent in patients with CIN 2 and CIN 3 than in patients with CIN 1. There was no significant difference in the frequency of HR-HPV infection between groups of patients with and without TERC amplification. In conclusion, this study found no correlation between TERC amplification and HR-HPV infection in patients with CIN.

  2. Telomere and telomerase in oncology

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Shortening of the telomeric DNA at the chromosome ends is presumed to limit the lifespan of human cells and elicit a signal for the onset of cellular senescence. To continually proliferate across the senescent checkpoint, cells must restore and preserve telomere length. This can be achieved by telomerase, which has the reverse transcriptase activity. Telomerase activity is negative in human normal somatic cells but can be detected in most tumor cells. The enzyme is proposed to be an essential factor in cell immortalization and cancer progression. In this review we discuss the structure and function of telomere and telomerase and thefr roles in cell immortalization and oncogenesis. Simultaneously the experimental studies of telomerase assays for cancer detection and diagnosis are reviewed. Finally, we discuss the potential use of inhibitors of telomerase in anti-cancer therapy.

  3. MiRNA profile associated with replicative senescence, extended cell culture, and ectopic telomerase expression in human foreskin fibroblasts.

    Directory of Open Access Journals (Sweden)

    Laura N Bonifacio

    Full Text Available Senescence is a highly regulated process that limits cellular replication by enforcing a G1 arrest in response to various stimuli. Replicative senescence occurs in response to telomeric DNA erosion, and telomerase expression can offset replicative senescence leading to immortalization of many human cells. Limited data exists regarding changes of microRNA (miRNA expression during senescence in human cells and no reports correlate telomerase expression with regulation of senescence-related miRNAs. We used miRNA microarrays to provide a detailed account of miRNA profiles for early passage and senescent human foreskin (BJ fibroblasts as well as early and late passage immortalized fibroblasts (BJ-hTERT that stably express the human telomerase reverse transcriptase subunit hTERT. Selected miRNAs that were differentially expressed in senescence were assayed for expression in quiescent cells to identify miRNAs that are specifically associated with senescence-associated growth arrest. From this group of senescence-associated miRNAs, we confirmed the ability of miR-143 to induce growth arrest after ectopic expression in young fibroblasts. Remarkably, miR-143 failed to induce growth arrest in BJ-hTERT cells. Importantly, the comparison of late passage immortalized fibroblasts to senescent wild type fibroblasts reveals that miR-146a, a miRNA with a validated role in regulating the senescence associated secretory pathway, is also regulated during extended cell culture independently of senescence. The discovery that miRNA expression is impacted by expression of ectopic hTERT as well as extended passaging in immortalized fibroblasts contributes to a comprehensive understanding of the connections between telomerase expression, senescence and processes of cellular aging.

  4. Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen

    Institute of Scientific and Technical Information of China (English)

    BIAN Chang; ZHAO Kui; TONG Guo-xin; ZHU Yong-liang; CHEN Peng

    2005-01-01

    Objective: To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen. Methods:Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination.Results: The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor,hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-α (tumor necrosis factor-α), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found. Conclusion: Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.

  5. Telomerase inhibitor Imetelstat (GRN163L limits the lifespan of human pancreatic cancer cells.

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    Katrina M Burchett

    Full Text Available Telomerase is required for the unlimited lifespan of cancer cells. The vast majority of pancreatic adenocarcinomas overexpress telomerase activity and blocking telomerase could limit their lifespan. GRN163L (Imetelstat is a lipid-conjugated N3'→P5' thio-phosphoramidate oligonucleotide that blocks the template region of telomerase. The aim of this study was to define the effects of long-term GRN163L exposure on the maintenance of telomeres and lifespan of pancreatic cancer cells. Telomere size, telomerase activity, and telomerase inhibition response to GRN163L were measured in a panel of 10 pancreatic cancer cell lines. The cell lines exhibited large differences in levels of telomerase activity (46-fold variation, but most lines had very short telomeres (2-3 kb in size. GRN163L inhibited telomerase in all 10 pancreatic cancer cell lines, with IC50 ranging from 50 nM to 200 nM. Continuous GRN163L exposure of CAPAN1 (IC50 = 75 nM and CD18 cells (IC50 = 204 nM resulted in an initial rapid shortening of the telomeres followed by the maintenance of extremely short but stable telomeres. Continuous exposure to the drug eventually led to crisis and to a complete loss of viability after 47 (CAPAN1 and 69 (CD18 doublings. Crisis In these cells was accompanied by activation of a DNA damage response (γ-H2AX and evidence of both senescence (SA-β-galactosidase activity and apoptosis (sub-G1 DNA content, PARP cleavage. Removal of the drug after long-term GRN163L exposure led to a reactivation of telomerase and re-elongation of telomeres in the third week of cultivation without GRN163L. These findings show that the lifespan of pancreatic cancer cells can be limited by continuous telomerase inhibition. These results should facilitate the design of future clinical trials of GRN163L in patients with pancreatic cancer.

  6. Telomerase inhibitor Imetelstat (GRN163L) limits the lifespan of human pancreatic cancer cells.

    Science.gov (United States)

    Burchett, Katrina M; Yan, Ying; Ouellette, Michel M

    2014-01-01

    Telomerase is required for the unlimited lifespan of cancer cells. The vast majority of pancreatic adenocarcinomas overexpress telomerase activity and blocking telomerase could limit their lifespan. GRN163L (Imetelstat) is a lipid-conjugated N3'→P5' thio-phosphoramidate oligonucleotide that blocks the template region of telomerase. The aim of this study was to define the effects of long-term GRN163L exposure on the maintenance of telomeres and lifespan of pancreatic cancer cells. Telomere size, telomerase activity, and telomerase inhibition response to GRN163L were measured in a panel of 10 pancreatic cancer cell lines. The cell lines exhibited large differences in levels of telomerase activity (46-fold variation), but most lines had very short telomeres (2-3 kb in size). GRN163L inhibited telomerase in all 10 pancreatic cancer cell lines, with IC50 ranging from 50 nM to 200 nM. Continuous GRN163L exposure of CAPAN1 (IC50 = 75 nM) and CD18 cells (IC50 = 204 nM) resulted in an initial rapid shortening of the telomeres followed by the maintenance of extremely short but stable telomeres. Continuous exposure to the drug eventually led to crisis and to a complete loss of viability after 47 (CAPAN1) and 69 (CD18) doublings. Crisis In these cells was accompanied by activation of a DNA damage response (γ-H2AX) and evidence of both senescence (SA-β-galactosidase activity) and apoptosis (sub-G1 DNA content, PARP cleavage). Removal of the drug after long-term GRN163L exposure led to a reactivation of telomerase and re-elongation of telomeres in the third week of cultivation without GRN163L. These findings show that the lifespan of pancreatic cancer cells can be limited by continuous telomerase inhibition. These results should facilitate the design of future clinical trials of GRN163L in patients with pancreatic cancer.

  7. Inhibition of human telomerase enhances the effect of chemotherapeutic agents in lung cancer cells.

    Science.gov (United States)

    Misawa, Masafumi; Tauchi, Tetsuzo; Sashida, Goro; Nakajima, Akihiro; Abe, Kenji; Ohyashiki, Junko H; Ohyashiki, Kazuma

    2002-11-01

    Telomerase is a ribonucleoprotein enzyme that maintains protective structures at the ends of eukaryotic chromosomes. Earlier studies have reported that the presence of telomerase activity in tumors of patients with non-small cell lung cancer patients correlates with a high proliferation rate and advanced pathological stage. Thus, the modification of telomerase activity may be a potential therapeutic modality for the treatment of lung and other cancers. We introduced vectors encoding dominant negative (DN)-hTERT, or wild-type (WT)-hTERT, or a control vector expressing only a drug-resistance marker, into the A549 lung cancer cell line, and assessed the biological effect of telomerase inhibition on cellular immortality. Ectopic expression of DN-hTERT resulted in complete inhibition of telomerase activity and reduction of telomere length. The entire population of telomerase-inhibited A549 cells exhibited cytoplasmic blebbling and chromatin condensation, which are features of apoptosis. In contrast, A549 cells expressing wild-type hTERT, which differs from the mutants by only two amino acids, exhibited normal morphology. Evidence for apoptosis in the telomerase-inhibited cells was provided by flow cytometric analysis with APO2.7 monoclonal antibody. We also observed enhanced induction of apoptosis by chemotherapeutic reagents, including cisplatin, docetaxel and etoposide, in DN-hTERT-expressing A549 cells, as compared with WT-hTERT-expressing cells. These results demonstrate that disruption of telomere maintenance limits the cellular lifespan of lung cancer cells, and show that the combined use of chemotherapeutic agents and telomere maintenance inhibition may be effective in the treatment of patients with non-small cell lung cancer.

  8. Structural and functional characterization of human telomerase RNA processing and cajal body localization signals.

    Science.gov (United States)

    Theimer, Carla A; Jády, Beáta E; Chim, Nicholas; Richard, Patricia; Breece, Katherine E; Kiss, Tamás; Feigon, Juli

    2007-09-21

    The RNA component of human telomerase (hTR) includes H/ACA and CR7 domains required for 3' end processing, localization, and accumulation. The terminal loop of the CR7 domain contains the CAB box (ugAG) required for targeting of scaRNAs to Cajal bodies (CB) and an uncharacterized sequence required for accumulation and processing. To dissect out the contributions of the CR7 stem loop to hTR processing and localization, we solved the solution structures of the 3' terminal stem loops of hTR CR7 and U64 H/ACA snoRNA, and the 5' terminal stem loop of U85 C/D-H/ACA scaRNA. These structures, together with analysis of localization, processing, and accumulation of hTRs containing nucleotide substitutions in the CR7 domain, identified the sequence and structural requirements of the hTR processing and CB localization signals and showed that these signals are functionally independent. Further, 3' end processing was found to be a prerequisite for translocation of hTR to CBs.

  9. Implication of human telomerase reverse transcriptase in cervical carcinogenesis and cancer recurrence.

    Science.gov (United States)

    Wang, P-H; Ko, J-L

    2006-01-01

    The objective of this study was to evaluate the implication of human telomerase reverse transcriptase (hTERT) in cervical carcinogenesis and cancer recurrence. One hundred three cases of uterine cervix, including 20 normal, 13 low-grade squamous intraepithelial lesion (LSIL), 30 high-grade squamous intraepithelial lesion (HSIL), and 40 squamous cell carcinoma (SCC) tissues, were evaluated for hTERT immunoreactivity. The expressions of hTERT in normal, LSIL, HSIL, and SCC tissues were compared by Fisher exact or Chi-square test. The relationships between hTERT and clinicopathologic variables of SCC were also assessed. Furthermore, SCC patients were subdivided into negative and positive hTERT expression subgroups, and Kaplan-Meier curves were used to plot the cumulative recurrence hazard for 5 years. There was a significant difference for hTERT expression between LSIL and HSIL subgroups (P recurrence hazard for 5 years was about 29% in positive hTERT expression subgroup compared to 0% in negative hTERT subgroup (P = 0.2866). In conclusion, a point stage of HSIL exists in the progression of cervical carcinogenesis when the hTERT expression increases significantly. Moreover, SCC patients with positive hTERT expression may have higher cumulative recurrence hazard.

  10. In vitro anti-telomerase activity of novel lycopene-loaded nanospheres in the human leukemia cell line K562

    Science.gov (United States)

    Gharib, Amir; Faezizadeh, Zohreh

    2014-01-01

    Background: Lycopene, a plant carotenoid, has potent effects against the various types of cancer cells. To date, the effect of lycopene in the free and encapsulated forms on the telomerase activity in human leukemia cell line K562 have not been investigated. The aim of the present study was to prepare a novel lycopene-loaded nanosphere and compare its anti-telomearse activity in K562 cell line with those of free lycopene. Materials and Methods: The lycopene-loaded nanospheres were prepared by nanoprecipitation method. The lycopene entrapment efficacy was measured by high-performance liquid chromatography (HPLC) method. The anti-proliferation effect of the lycopene in the free and encapsulated forms in the different times (0-72 h) and the different doses (0-100 μg/ml) on K562 cell line was studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The changes of telomerase activity, following treatment with the lycopene in the free and encapsulated forms, were detected using the telomeric repeat amplification protocol-enzyme-linked immunosorbent assay. Results: The entrapment efficacy of lycopene was 78.5% ± 2. Treatment of the K562 cell line with lycopene, in particular in encapsulated form, resulted in a significant inhibition of the cell growth and increasing of percentage of apoptotic cells. It has also been observed that the telomerase activity in the lycopene-loaded nanospheres-treated cells was significantly inhibited in a dose and time-dependent manner. Conclusion: Our data suggest a novel mechanism in the anti-cancer activity of the lycopene, in particular in encapsulated form, and could be provided a basis for the future development of anti-telomerase therapies. PMID:24914298

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

    Institute of Scientific and Technical Information of China (English)

    Lingping Kong; Lingzhi Wu; Jie Zhang; Yaping Liao; Huaqiao Wang

    2009-01-01

    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 (Aβ25-35).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-Cdk5 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 μmol/L Aβ25-35 for 24 hours.Normal human embryonic cortical neurons treated with 10 μmol/L Aβ25-35 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

  12. Genomic amplification of the human telomerase gene (hTERC associated with human papillomavirus is related to the progression of uterine cervical dysplasia to invasive cancer

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    Liu Hongqian

    2012-10-01

    Full Text Available Abstract Background Human papillomavirus (HPV infection plays an etiological role in the development of cervical dysplasia and cancer. Amplification of human telomerase gene (hTERC and over expression of telomerase were found to be associated with cervical tumorigenesis. This study was performed to analyze genomic amplification of hTERC gene, telomerase activity in association with HPV infection in different stages of cervical intraepithelial neoplasia (CIN and cervical cancer. We were studying the role of hTERC in the progression of uterine cervical dysplasia to invasive cancer, and proposed an adjunct method for cervical cancer screening. Methods Exfoliated cervical cells were collected from 114 patients with non neoplastic lesion (NNL, n=27, cervical intraepithelial neoplasia (CIN1, n=26, CIN2, n=16, CIN3, n=24 and cervical carcinoma (CA, n=21, and analyzed for amplification of hTERC with two-color fluorescence in situ hybridization (FISH probe and HPV-DNA with Hybrid Capture 2. From these patients, 53 were taken biopsy to analyze telomerase activity by telomeric repeat amplification protocol (TRAP and expression of human telomerase reverse transcriptase (hTERT, with immunohistochemistry (IHC. All biopsies were clinically confirmed by phathologists. Results Amplification of hTERC was significantly associated with the histologic diagnoses (p Conclusions hTERC ampliffication can be detected with FISH technique on exfoliated cervical cells. Amplification of hTERC and HPV infection are associated with more progressive CIN3 and CA. The testing of hTERC amplification might be a supplementary to cytology screening and HPV test, especially high-risk patients. Virtual slides The virtual slide(s for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1857134686755648.

  13. PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST HUMAN TELOMERASE REVERSE TRANSCRIPTASE

    Institute of Scientific and Technical Information of China (English)

    王俊梅; 张波; 杨邵敏; 韩继生; 李冰思; 侯琳

    2003-01-01

    Objective. To develop monoclonal antibodies against the catalytic subunit of human telomerase reverse transcriptase (hTERT) for its expression detection of human tumors. Methods. A dominant epitope in hTERT (peptide hTERT7)was automatically synthesized based on Fmoc method, and was used to immunize Balb/c mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and the characterization was performed by Western blotting and immunohistochemical staining. The heavy chain variable region of antibody was cloned by RT-PCR and sequenced. Results. Antigenic peptide hTERT7 was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis. One hybridoma cell line secreting anti-hTERT7 antibodies designated as M2 was established after primary screening and consequent 3 rounds of limited dilution. M2 was IgG1 in isotyping. The competi tive assay showed that the M2 antibody was hTERT7 -specific, and the affinity constant was about 1×106 mol-1. The antibody reacted with cell extracts from HeLa cancer cells but not with those from normal 2BS cells in ELISA assay. For in situ staining of immunohistochemistry, the positive staining presented in the nuclear compartment of HeLa, while 2BS was negative. The heavy chain variable region from M2 re vealed that the monoclonal antibody was mouse origin. Conclusions. The developed mouse monoclonal antibody is hTERT-specific and able to recognize native cellular hTERT in ELISA and immunohistochemistry, which makes the immuno-detection of telom erase hTERT expression in cancer cells or tissues possible.

  14. Human telomerase reverse transcriptase regulates vascular endothelial growth factor expression via human papillomavirus oncogene E7 in HPV-18-positive cervical cancer cells.

    Science.gov (United States)

    Li, Fang; Cui, Jinquan

    2015-07-01

    Human papillomavirus (HPV) infection induces chronic and precancerous lesions and results in invasive cervical cancer. Human telomerase as well as inflammatory and angiogenic factors such as telomerase reverse transcriptase (hTERT) or vascular endothelial growth factor (VEGF) could play a role in regulating HPV-induced cervical cancer. This study investigated underlying molecular events in HPV-induced HPV-positive cervical cancer through hTERT and VEGF in vitro. Expressions of hTERT, a rate-limiting subunit of telomerase, and VEGF mRNA and proteins were, respectively, assessed by qRT-PCR, ELISA, and TRAP-ELISA in HPV-positive tissue samples and cervical cancer cell lines. To assess hTERT and VEGF secretion, hTERT overexpression and knockdown were conducted in HPV-18-positive Hela cells by hTERT cDNA and shRNA transfection, respectively. Then, the effect of HPV E6 and E7 on VEGF expressions was assessed in HPV-negative cervical cancer cells. Data have shown that VEGF expression levels are associated with hTERT expressions and telomerase activity in HPV-positive cervical cancer tissues and cells. Knockdown of hTERT expression down-regulated VEGF expressions, whereas overexpression of hTERT up-regulated VEGF expressions in HPV-18-positive Hela cells. Furthermore, HPV E7 oncoprotein was necessary for hTERT to up-regulate VEGF expressions in HPV-negative cervical cancer cells. Data from this current study indicate that HPV oncoproteins up-regulated hTERT and telomerase activity and in turn promoted VEGF expressions, which could be a key mechanism for HPV-induced cervical cancer development and progression.

  15. Leptin as a critical regulator of hepatocellular carcinoma development through modulation of human telomerase reverse transcriptase

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    Stefanou Nikolaos

    2010-08-01

    Full Text Available Abstract Background Numerous epidemiological studies have documented that obesity is associated with hepatocellular carcinoma (HCC. The aim of this study was to investigate the biological actions regulated by leptin, the obesity biomarker molecule, and its receptors in HCC and the correlation between leptin and human telomerase reverse transcriptase (hTERT, a known mediator of cellular immortalization. Methods We investigated the relationship between leptin, leptin receptors and hTERT mRNA expression in HCC and healthy liver tissue samples. In HepG2 cells, chromatin immunoprecipitation assay was used to study signal transducer and activator of transcription-3 (STAT3 and myc/mad/max transcription factors downstream of leptin which could be responsible for hTERT regulation. Flow cytometry was used for evaluation of cell cycle modifications and MMP1, 9 and 13 expression after treatment of HepG2 cells with leptin. Blocking of leptin's expression was achieved using siRNA against leptin and transfection with liposomes. Results We showed, for the first time, that leptin's expression is highly correlated with hTERT expression levels in HCC liver tissues. We also demonstrated in HepG2 cells that leptin-induced up-regulation of hTERT and TA was mediated through binding of STAT3 and Myc/Max/Mad network proteins on hTERT promoter. We also found that leptin could affect hepatocellular carcinoma progression and invasion through its interaction with cytokines and matrix mettaloproteinases (MMPs in the tumorigenic microenvironment. Furthermore, we showed that histone modification contributes to leptin's gene regulation in HCC. Conclusions We propose that leptin is a key regulator of the malignant properties of hepatocellular carcinoma cells through modulation of hTERT, a critical player of oncogenesis.

  16. Real-time determination of human telomerase reverse transcriptase mRNA in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Li-Hua Hu; Feng-Hua Chen; Yi-Rong Li; Lin Wang

    2004-01-01

    AIM: To set up a real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay,to detect human telomerase reverse transcriptase (hTERT)messenger RNA in gastric carcinomas, and to evaluate quantitative determination of hTERT mRNA in the diagnostic value of gastric carcinomas, and to analyze the correlation between the expression level of hTERT mRNA and dinicopathological parameters in patients with gastric cancer.METHODS: A real-time quantitative RT-PCR (RQ-PCR)based on TaqMan fluorescence methodoloogy and the LightCyder system was used to quantify the full range of hTERT mRNA copy numbers in 35 samples of gastric carcinomas and corresponding adjacent non-cancerous tissues. The normalized hTERT (NhTERT) was standardized by quantifying the number of GAPDH transcripts as internal control and expressed as 100× (hTERT/GAPDH) ratio. Variables were analyzed by the Student's t-test, χ2 test and Fisher's exact test.RESULTS: NhTERT from gastric carcinomas and corresponding adjacent non-cancerous tissues was 6.27±0.89 and 0.93±0.18,respectively (t= 12.76, P<0.001). There was no significant association between gastric cancer hTERT mRNA expression level and patient's age, gender, tumor size, location and stage (pTNM), but a significant correlation was found between hTERT mRNA expression level in gastric carcinomas and the degree of differentiation.CONCLUSION: Quantitative determination of hTERT mRNA by RQ-PCR is a rapid and sensitive method. hTERT might be a potential biomarker for the early detection of gastric cancer.

  17. Antimetastatic Effects of a Novel Telomerase Inhibitor, GRN163L, on Human Prostate Cancer

    Science.gov (United States)

    2010-05-01

    Menlo Park, CA) for providing the imetelstat telomerase inhibitor, Ms. Erin Kitten for technical support and Ms. Angela Diehl for the graphic design...appear to vary signifi- cantly in their morphology, invasiveness, and tumor- igenicity. ACKNOWLEDGMENTS Wewould like to thank Gail Marian and Erin Kitten

  18. Correlation of Bmi-1 expression and telomerase activity in human ovarian cancer

    NARCIS (Netherlands)

    Zhang, F. B.; Sui, L. H.; Xin, T.

    2008-01-01

    This study investigates the correlation between the oncoprotein Bmi-1 and telomerase activity in ovarian cancer. A real-time polymerase chain reaction (PCR) method is used to detect the messenger RNA (mRNA) expression of Bmi-1 protein in 47 ovarian epithelial cancer cases, and immunohistochemistry i

  19. [The expression of human telomerase reverse transcriptase mRNA and its significance in acute leukemia].

    Science.gov (United States)

    Meng, Xiao-Li; Lin, Mao-Fang; Jin, Jie

    2003-02-01

    To investigate the expression of hTERT mRNA in bone marrow mononuclear cells (MNCs) from acute leukemia patients, the method of semi-quntitative RT-PCR was used to examine the expression of hTERT mRNA in marrow MNCs, and the telomerase activity of marrow MNCs was determined with the method of TRAP-PCR-ELISA by using a commercial kit. The results indicated that the expression of hTERT mRNA of marrow MNCs in 30 untreated AL patients was markedly higher than that in 12 CR cases (0.71 +/- 0.34 vs 0.43 +/- 0.25, P < 0.05) and 6 normal volunteers (0.71 +/- 0.34 vs 0.22 +/- 0.21, P < 0.01), respectively. Telomerase activity of marrow MNCs in 30 untreated AL patients was significantly higher than that in 12 CR cases (0.235 +/- 0.395 vs 0.012 +/- 0.015, P = 0.007). Moreover, there was a positive correlation between the hTERT mRNA synthesis and telomerase activity in AL cells (r = 0.421, P < 0.01). The pencentage of blast cells in marrow smear of the untreated AL patients was positively correlated with both the expression of hTERT mRNA and the telomerase activity of bone marrow MNCs (r = 0.457, P < 0.05 and r = 0.411, P < 0.05), respectively. It is concluded that the expression of hTERT mRNA in bone marrow MNCs from untreated AL patients was correlated with their telomerase activity. It is suggested that the expression of hTERT mRNA leukemic cells indicates their higher proliferation ability.

  20. Telomere and Telomerase Therapeutics in Cancer

    Directory of Open Access Journals (Sweden)

    Yucheng Xu

    2016-05-01

    Full Text Available Telomerase is a reverse transcriptase capable of utilizing an integrated RNA component as a template to add protective tandem telomeric single strand DNA repeats, TTAGGG, to the ends of chromosomes. Telomere dysfunction and telomerase reactivation are observed in approximately 90% of human cancers; hence, telomerase activation plays a unique role as a nearly universal step on the path to malignancy. In the past two decades, multiple telomerase targeting therapeutic strategies have been pursued, including direct telomerase inhibition, telomerase interference, hTERT or hTERC promoter driven therapy, telomere-based approaches, and telomerase vaccines. Many of these strategies have entered clinical development, and some have now advanced to phase III clinical trials. In the coming years, one or more of these new telomerase-targeting drugs may be expected to enter the pharmacopeia of standard care. Here, we briefly review the molecular functions of telomerase in cancer and provide an update about the preclinical and clinical development of telomerase targeting therapeutics.

  1. Study on the immune responses against pancreatic cancer induced by mucin 4 and human telomerase reverse transcriptase mRNA co-transfected dendritic cells in vitro

    Institute of Scientific and Technical Information of China (English)

    陈江

    2014-01-01

    Objective To investigate the anti-tumor immune response induced by human pancreatic cancer mucin 4mRNA and human telomerase reverse transcriptase(hTERT)mRNA cotransfected dendritic cells(DC),and to provide the experimental evidences for the treatment of pancreatic cancer with multi-epitope loaded DC vaccine.Methods DC were isolated from peripheral DC.

  2. Zoning of mucosal phenotype, dysplasia, and telomerase activity measured by telomerase repeat assay protocol in Barrett's esophagus

    NARCIS (Netherlands)

    Going, JJ; Fletcher-Monaghan, AJ; Neilson, L; Wisman, BA; van der Zee, A; Stuart, RC; Keith, WN

    2004-01-01

    Glandular dysplasia in Barrett's esophagus may regress spontaneously but can also progress to cancer. The human telomerase RNA template and the human telomerase reverse transcriptase enzyme which do not, of themselves, correlate strongly with telomerase activity, are too often overexpressed in Barre

  3. ES micro-environment enhances stemness and inhibits apoptosis in human limbal stem cells via the maintenance of telomerase activity.

    Directory of Open Access Journals (Sweden)

    Zhiping Liu

    Full Text Available Our previous work had found that telomerase rejuvenated in the cytoplasm of corneal epithelial cells cultured in embryonic stem cell-conditioned medium, the functional properties of stem-like corneal epithelial cells can be enhanced by co-culturing with embryonic stem cells (ESCs via activation of the integrinβ1-FAK-PI3K/Akt signaling pathway. The goal of this study was to explore the potential molecular mechanisms of the ES micro-environment that enhance the stem cell-like phenotype and inhibit apoptosis in human limbal stem cells (LSC. The LSC were cultured in different media, either CnT-20 medium or CnT-20 +20% ES culture supernatant (ESC-CM. We observed that LSC cultured in ESC-CM had an increased proliferative capacity, greater serial passage capacity, higher colony-forming efficiency (CFE and higher levels of stem cell-associated marker than those cultured in CnT-20. Compared with CnT-20, ESC-CM enhanced the undifferentiated status and inhibited apoptosis in the LSC by promoting the maintenance of telomerase activity, which could reduce the generation of reactive oxygen species (ROS, maintain the membrane potential (Δψm at higher levels and reduce the expression of the p21 protein. Our findings indicated that ESC-CM system induced LSC to maintain a stem cell phenotype and inhibit the process of apoptosis. These effects might partially be achieved via the telomerase-p21-mitochondrial axis and the activation of the FAK/Wnt signaling pathways. This study may have high impact and clinic implication on the expansion of LSC in regenerative medicine, especially for ocular surface reconstruction.

  4. Homologous recombination in human telomerase-positive and ALT cells occurs with the same frequency

    OpenAIRE

    Bechter, Oliver E.; Zou, Ying; Shay, Jerry W.; Woodring E. Wright

    2003-01-01

    Homologous recombination is thought to be the molecular mechanism for maintaining telomere length in alternative lengthening of telomeres (ALT) cells. We used a recombination reporter system to show that the frequency of homologous recombination is the same for ALT- and telomerase-positive cells, suggesting that if ALT cells have a recombination defect it specifically involves telomeric sequences. We compared internal and telomere-adjacent positions of our ...

  5. Effect of Estrogen on Telomerase Activity in Human Breast Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    高金波; 陈道达; 田元; 张锦辉; 蔡开琳

    2003-01-01

    To investigate the effects of estrogen(E2 ) on telomerase activity and its mechanism inhuman breast cancer cells, estrogen receptor positive MCF-7 cells were treated with different con-centrations of E2. Telomerase activity was measured by using TRAP-ELISA method, the cell cyclephases analyzed by using flow cytometry, and the expression of Cyclin D1 detected by using immu-nohistochemistry method. The results showed that telomerase activity levels were increased inMCF-7 cells treated with 10-8 mol/L E2 during the observed period (P<0.05), and E2 increasedtelomerase activity levels in a dose-dependent manner(10-10- 10-8 mol/L); Simultaneously, thecell cycle phases of MCF-7 cells treated with 10-8 mol/L E2 were changed significantly: G0/G1phase decreased from 60.52% to 50. 93%, S phase increased from 29.03% to 30.83%; However,the expression of Cyclin D1 was decreased. It was concluded that estrogen can upregulate telomeraseactivity of MCF-7 cells, and the effect can be blocked by antiestrogen tamoxifen. Its mechanismmay be closely associated with modulation of cell cycle phases.

  6. Telomerase prevents accelerated senescence in glucose-6-phosphate dehydrogenase (G6PD-deficient human fibroblasts

    Directory of Open Access Journals (Sweden)

    Wu Yi-Hsuan

    2009-02-01

    Full Text Available Abstract Fibroblasts derived from glucose-6-phosphate dehydrogenase (G6PD-deficient patients display retarded growth and accelerated cellular senescence that is attributable to increased accumulation of oxidative DNA damage and increased sensitivity to oxidant-induced senescence, but not to accelerated telomere attrition. Here, we show that ectopic expression of hTERT stimulates telomerase activity and prevents accelerated senescence in G6PD-deficient cells. Stable clones derived from hTERT-expressing normal and G6PD-deficient fibroblasts have normal karyotypes, and display no sign of senescence beyond 145 and 105 passages, respectively. Activation of telomerase, however, does not prevent telomere attrition in earlier-passage cells, but does stabilize telomere lengths at later passages. In addition, we provide evidence that ectopic expression of hTERT attenuates the increased sensitivity of G6PD-deficient fibroblasts to oxidant-induced senescence. These results suggest that ectopic expression of hTERT, in addition to acting in telomere length maintenance by activating telomerase, also functions in regulating senescence induction.

  7. Specific expression of short-interfering RNA driven by human telomerase reverse transcriptase promoter in tumor cells

    Institute of Scientific and Technical Information of China (English)

    Xuejing Luan; Limin Guo; Zuozhen Yang; Min Liu; Xin Li; Hua Tang

    2008-01-01

    RNA interference (RNAi) has been shown to be an effective method for inhibiting the expression of a given gene in human cells by targeting with short duplex RNA (short-interfering RNA or siRNA). However, more and more studies suggest that non-specific effects can be induced by siRNAs, such as off-target inhibition, activation of interferon response, and saturation of cellular silencing machinery. It has been known that more than 90% of human tumors exhibit teiomerase activity. Consequently, teiomerase is believed to be a broadspectrum molecular marker of malignancies. In the present study we attempt to develop a tumor-specific RNAi system using the human telomerase reverse transcriptase promoter.This system may provide a basis for RNAi therapy.

  8. Telomerase and the endocrine system.

    Science.gov (United States)

    Pacini, Furio; Cantara, Silvia; Capezzone, Marco; Marchisotta, Stefania

    2011-03-29

    Telomeres are nucleoprotein complexes located at the ends of chromosomes that have a critical role in the maintenance of chromosomal integrity. This involvement is based on complex secondary and tertiary structures that rely on DNA-DNA, DNA-protein and protein-protein interactions. De novo synthesis and maintenance of telomere repeats is controlled by telomerase, a specialized complex that consists of a telomerase RNA component and a protein component--telomerase reverse transcriptase. When telomerase is silent (its default state in differentiated somatic cells), chromosomes shorten with every cell division, thus limiting the lifespan of the cells (the process of senescence) and preventing unlimited cell proliferation, which might eventually lead to the development of cancer. During this process, occasionally, a cell can activate telomerase, which stabilizes short telomeres and enables immortalization-a process essential for malignant transformation. Thus, although telomere erosion is a barrier to malignant progression, paradoxically, in certain circumstances it might also trigger tumorigenesis. A number of studies have demonstrated unequivocally that reactivation of telomerase in the presence of short telomeres is one of the most common features of human cancers, including those of the endocrine system.

  9. Telomerase - et potensielt angrepspunkt i kampen mot kreft

    OpenAIRE

    Karlstad, Ida Marie; Tell, Nicolai

    2012-01-01

    Background: Telomerase is a necessity for infinite cell division in cancer cells, and high telomerase activity is present in as much as 90 % of human cancers. Hence telomerase has become an attractive target in cancer therapy because it suggests a universal cure for cancer. We summarized research done on the vaccine GV1001 and the telomerase inhibitor Imetelstat in prostate, pancreas and lung cancer. Methods: We reviewed finished and ongoing clinical trials on GV1001 and Imetelstat; and precl...

  10. Dynamics of telomerase activity in response to acute psychological stress

    Science.gov (United States)

    Epel, Elissa S.; Lin, Jue; Dhabhar, Firdaus S.; Wolkowitz, Owen M.; Puterman, E; Karan, Lori; Blackburn, Elizabeth H.

    2010-01-01

    Telomerase activity plays an essential role in cel0l survival, by lengthening telomeres and promoting cell growth and longevity. It is now possible to quantify the low levels of telomerase activity in human leukocytes. Low basal telomerase activity has been related to chronic stress in people and to chronic glucocorticoid exposure in vitro. Here we test whether leukocyte telomerase activity changes under acute psychological stress. We exposed 44 elderly women, including 22 high stress dementia caregivers and 22 matched low stress controls, to a brief laboratory psychological stressor, while examining changes in telomerase activity of peripheral blood mononuclear cells (PBMC). At baseline, caregivers had lower telomerase activity levels than controls, but during stress telomerase activity increased similarly in both groups. Across the entire sample, subsequent telomerase activity increased by 18% one hour after the end of the stressor (p<0.01). The increase in telomerase activity was independent of changes in numbers or percentages of monocytes, lymphocytes, and specific T cell types, although we cannot fully rule out some potential contribution from immune cell redistribution in the change in telomerase activity. Telomerase activity increases were associated with greater cortisol increases in response to the stressor. Lastly, psychological response to the tasks (greater threat perception) was also related to greater telomerase activity increases in controls. These findings uncover novel relationships of dynamic telomerase activity with exposure to an acute stressor, and with two classic aspects of the stress response -- perceived psychological stress and neuroendocrine (cortisol) responses to the stressor. PMID:20018236

  11. Telomerase in (pre)neoplastic cervical disease

    NARCIS (Netherlands)

    Wisman, GBA; De Jong, S; Meersma, GJ; Helder, MN; Hollema, H; de Vries, EGE; Keith, WN; van der Zee, AGJ

    2000-01-01

    This study was performed to determine upregulation of the human telomerase RNA component (hTR) and mRNA of the catalytic subunit of telomerase (hTERT) in (pre)malignant cervical lesions, to analyze possible intralesional heterogeneity of hTR expression, and to relate hTR and hTERT mRNA levels to tel

  12. Telomeres and telomerase: Biological and clinical importance in dogs.

    Science.gov (United States)

    Nasir, Lubna

    2008-02-01

    In recent years in human oncology the enzyme telomerase has emerged as an ideal target for cancer therapy. This has led to the assessment of telomerase in cancers in companion animals, mainly dogs and these studies confirm that in dogs, like humans, telomere maintenance by telomerase is the primary mechanism by which cancer cells overcome their mortality and extend their lifespan. This review aims to provide an introduction to the biology of telomeres and telomerase and to discuss some of the telomere/telomerase directed therapeutic methodologies currently under development which may be of benefit to the canine cancer patient.

  13. Quantification of human telomerase reverse transcriptase mRNA in testicular germ cell tumors by quantitative fluorescence real-time RT-PCR.

    Science.gov (United States)

    Schrader, Mark; Burger, Angelika M; Müller, Markus; Krause, Hans; Straub, Bernd; Smith, Gilian L; Newlands, Eward S; Miller, Kurt

    2002-01-01

    Telomerase is a ribonucleoprotein enzyme which is endogenously expressed in germ, stem and tumor cells, but absent in benign somatic cells. The two major telomerase components are human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). It has been shown that hTERT is rate-limiting for telomerase activity and that it plays a central role in human carcinogenesis. Here, we investigated the potential of hTERT and hTR gene expression as diagnostic markers in testicular germ cell tumors (TGCT). hTERT mRNA and hTR expression were quantified in 55 testicular germ cell tumors comprising 36 primary and 19 germ cell tumors from retroperitonal sides by fluorescence real-time RT-PCR using the LightCycler technology. Porphobilinogen deaminase (PBGD) was used as housekeeping gene and to enable relative quantification. For comparison to TGCTs, 38 benign testicular biopsies from patients with fertility disorders were assayed. hTERT expression was detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content (N(hTERT) 38-127). In contrast, mature teratomas from primary and post-chemotherapy masses, which are characterized by well-differentiated tissue components showed a nearly complete downregulation of hTERT expression (N(hTERT) 2-4, pmRNA is expressed in all undifferentiated TGCTs but repressed in mature teratomas. This suggests an inverse correlation between the differentiation status of germ cell tumors and hTERT expression. Thus, detection of hTERT expression in tumors histopathologically classified as mature teratomas enables a molecular-diagnostic confirmation and might aid decision making for treatment of patients presenting with this tumor subtype.

  14. Ectopically hTERT expressing adult human mesenchymal stem cells are less radiosensitive than their telomerase negative counterpart

    DEFF Research Database (Denmark)

    Serakinci, Nedime; Graakjaer, Jesper; Cairney, Claire J

    2007-01-01

    radiosensitivity to other mammalian cells so far tested. In this study, we investigated the genetic effects of ionizing radiation (2.5-15 Gy) on normal human mesenchymal stem cells and their telomerised counterpart hMSC-telo1. We evaluated overall genomic integrity, DNA damage/repair by applying a fluorescence...... in the two cell lines. The telomeres were extensively destroyed after irradiation in both cell types suggesting that telomere caps are especially sensitive to radiation. The TERT-immortalized hMSCs showed higher stability at telomeric regions than primary hMSCs indicating that cells with long telomeres...... and high telomerase activity have the advantage of re-establishing the telomeric caps. Udgivelsesdato: 2007-Mar-10...

  15. Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Burns, Jorge S; Abdallah, Basem M; Guldberg, Per

    2005-01-01

    Long-term cultures of telomerase-transduced adult human mesenchymal stem cells (hMSC) may evolve spontaneous genetic changes leading to tumorigenicity in immunodeficient mice (e.g., hMSC-TERT20). We wished to clarify whether this unusual phenotype reflected a rare but dominant subpopulation...... or if the stem cell origin allowed most cells to behave as cancer stem cells. Cultures of the hMSC-TERT20 strain at population doubling 440 were highly clonogenic (94%). From 110 single-cell clones expanded by 20 population doublings, 6 underwent detailed comparison. Like the parental population, each clone had...... tumorigenicity correlated with good viability plus capillary morphogenesis on serum starvation and high cyclin D1 expression. Thus, hMSC-TERT20 clones represent cancer stem cells with hierarchical tumorigenicity, providing new models to explore the stem cell hypothesis for cancer....

  16. Oxoisoaporphine as Potent Telomerase Inhibitor

    Directory of Open Access Journals (Sweden)

    Zu-Zhuang Wei

    2016-11-01

    Full Text Available Two compounds previously isolated from traditional Chinese medicine, Menispermum dauricum (DC, 6-hydroxyl-oxoisoaporphine (H-La, and 4,6-di(2-pyridinylbenzo[h]isoindolo[4,5,6-de]quinolin-8(5H-one (H-Lb, were known to have in vitro antitumor activity and to selectively bind human telomeric, c-myc, and bcl-2 G-quadruplexes (G4s. In this study, the binding properties of these two compounds to telomerase were investigated through molecular docking and telomeric repeat amplication protocol and silver staining assay (TRAP-silver staining assay. The binding energies bound to human telomerase RNA were calculated by molecular docking to be −6.43 and −9.76 kcal/mol for H-La and H-Lb, respectively. Compared with H-La, the ligand H-Lb more strongly inhibited telomerase activity in the SK-OV-3 cells model.

  17. Relationship between the expression of human telomerase reverse transcriptase gene and cell cycle regulators in gastric cancer and its significance

    Institute of Scientific and Technical Information of China (English)

    Jin-Chen Shao; Ji-Feng Wu; Dao-Bin Wang; Rong Qin; Hong Zhang

    2003-01-01

    AIM: To investigate the expression of human telomerase reverse transcriptase gene (hTRT) in gastric cancer (GC)and its relevance with cell cycle regulators including P16INK4,cyclin and P53.METHODS: In situ hybridization (ISH) for hTRT mRNA was performed in 53 cases of gastric cancer and adjacent cancerous tissues. Immunohistochemical staining (S-Pmethod) for hTRT protein, P16INK4, cyclinD1 and P53 was performed in 53 cases of GC and adjacent cancerous tissues.RESULTS: Of 53 cases of GC, the expression of hTRT mRNA and hTRT protein was significantly higher than the expression of hTRT mRNA and hTRT protein in adjacent canerous tissues (P<0.01), the positive rates of hTRTmRNA and hTRT protein were 79.2 % and 88.6 %. There was a stastical difference of the expression of hTRT protein among well differentiated adenocarcinoma, poorly differentiated adenocarcinoma and mucoid carcinoma. And there was a highly significant positive correlation between the expression of hTRT mRNA and hTRT protein (r=0.625, P<0.01). However, the expression of hTRT mRNA and its protein in GC were not related with other clinicopathological parameters including gender, age, location and size of neoplasm, invasion depth, lymph node metastasis and clinical stage. There was a significant positive correlation between the expression of hTRT mRNA and cyclinD1 protein (r=0.350, P<0.01). There was a significant positive correlation between the expression of cyclinD1 protein and hTRT protein (r=0.549, P<0.01), so was between P53 and hTRT protein (r=0.319, P<0.05).CONCLUSION: The expression of hTRT gene is correlated significantly to the specific defects of cell cycle on G1/S check point; telomerase activity may depend on cell cycle in gastric cancer and it is available to clarify the molecular mechanism of telomerase activity regulation. The expression of hTRT mRNA and hTRT protein in GC is significantly different from the expression of hTRT mRNA and hTRT protein in adjacent cancerous tissue

  18. Inositol hexaphosphate represses telomerase activity and translocates TERT from the nucleus in mouse and human prostate cancer cells via the deactivation of Akt and PKCalpha.

    Science.gov (United States)

    Jagadeesh, Shankar; Banerjee, Partha P

    2006-11-03

    Inositol hexaphosphate (IP6) has anti-proliferative effects on a variety of cancer cells, including prostate cancer. However, the molecular mechanism of anti-proliferative effects of IP6 is not entirely understood. Since the activation of telomerase is crucial for cells to gain immortality and proliferation ability, we examined the role of IP6 in the regulation of telomerase activity in prostate cancer cells. Here, we show that IP6 represses telomerase activity in mouse and human prostate cancer cells dose-dependently. In addition, IP6 prevents the translocation of TERT to the nucleus. Since phosphorylation of TERT by Akt and/or PKCalpha is necessary for nuclear translocation, we examined phosphorylation of Akt and PKCalpha after IP6 treatments. Our results show that IP6 inhibits phosphorylation of Akt and PKCalpha. These results show for the first time that IP6 represses telomerase activity in prostate cancer cells by posttranslational modification of TERT via the deactivation of Akt and PKCalpha.

  19. Fucoidan Induces ROS-Dependent Apoptosis in 5637 Human Bladder Cancer Cells by Downregulating Telomerase Activity via Inactivation of the PI3K/Akt Signaling Pathway.

    Science.gov (United States)

    Han, Min Ho; Lee, Dae-Sung; Jeong, Jin-Woo; Hong, Su-Hyun; Choi, Il-Whan; Cha, Hee-Jae; Kim, Suhkmann; Kim, Heui-Soo; Park, Cheol; Kim, Gi-Young; Moon, Sung-Kwon; Kim, Wun-Jae; Hyun Choi, Yung

    2017-02-01

    Preclinical Research Fucoidan, a sulfated polysaccharide, is a compound found in various species of seaweed that has anti-viral, anti-bacterial, anti-oxidant, anti-inflammatory, and immunomodulatory activities; however, the underlying relationship between apoptosis and anti-telomerase activity has not been investigated. Here, we report that fucoidan-induced apoptosis in 5637 human bladder cancer cells was associated with an increase in the Bax/Bcl-2 ratio, the dissipation of the mitochondrial membrane potential (MMP, Δψm), and cytosolic release of cytochrome c from the mitochondria. Under the same experimental conditions, fucoidan-treatment decreased hTERT (human telomerase reverse transcriptase) expression and the transcription factors, c-myc and Sp1. This was accompanied by decreased telomerase activity. Fucoidan-treatment also suppressed activation of the PI3K/Akt signaling pathway. Inhibition of PI3K/Akt signaling enhanced fucoidan-induced apoptosis and anti-telomerase activity. Meanwhile, fucoidan treatment increased the generation of intracellular ROS, whereas the over-elimination of ROS by N-acetylcysteine, an anti-oxidant, attenuated fucoidan-induced apoptosis, inhibition of hTERT, c-myc, and Sp1 expression, and reversed fucoidan-induced inactivation of the PI3K/Akt signaling pathway. Collectively, these data indicate that the induction of apoptosis and the inhibition of telomerase activity by fucoidan are mediated via ROS-dependent inactivation of the PI3K/Akt pathway. Drug Dev Res 78 : 37-48, 2017.   © 2016 Wiley Periodicals, Inc.

  20. Designed modulation of sex steroid signaling inhibits telomerase activity and proliferation of human prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Verma, Vikas; Sharma, Vikas; Singh, Vishal [Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow 226 031 (India); Sharma, Siddharth; Bishnoi, Ajay Kumar [Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226 031 (India); Chandra, Vishal; Maikhuri, J.P.; Dwivedi, Anila [Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow 226 031 (India); Kumar, Atul [Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226 031 (India); Gupta, Gopal, E-mail: g_gupta@cdri.res.in [Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow 226 031 (India)

    2014-10-15

    The predominant estrogen-receptor (ER)-β signaling in normal prostate is countered by increased ER-α signaling in prostate cancer (CaP), which in association with androgen-receptor (AR) signaling results in pathogenesis of the disease. However CaP treatments mostly target AR signaling which is initially effective but eventually leads to androgen resistance, hence simultaneous targeting of ERs has been proposed. A novel series of molecules were designed with multiple sex-steroid receptor modulating capabilities by coalescing the pharmacophores of known anti-CaP molecules that act via modulation of ER(α/β) and/or AR, viz. 3,3′diindolylmethane (DIM), mifepristone, toremifene, tamoxifen and raloxifene. N,N-diethyl-4-((2-(4-methoxyphenyl)-1H-indol-3-yl)methyl) aniline (DIMA) was identified as the most promising structure of this new series. DIMA increased annexin-V labelling, cell-cycle arrest and caspase-3 activity, and decreased expression of AR and prostate specific antigen in LNCaP cells, in vitro. Concurrently, DIMA increased ER-β, p21 and p27 protein levels in LNCaP cells and exhibited ∼ 5 times more selective binding for ER-β than ER-α, in comparison to raloxifene. DIMA exhibited a dose-dependent ER-β agonism and ER-α antagonism in classical gene reporter assay and decreased hTERT (catalytic subunit of telomerase) transcript levels in LNCaP at 3.0 μM (P < 0.05). DIMA also dose-dependently decreased telomerase enzyme activity in prostate cancer cells. It is thus concluded that DIMA acts as a multi-steroid receptor modulator and effectively inhibits proliferation of prostate cancer cells through ER-β mediated telomerase inhibition, by countering actions of ER-α and AR. Its unique molecular design can serve as a lead structure for generation of potent agents against endocrine malignancies like the CaP.

  1. Telomerase Is Essential for Zebrafish Heart Regeneration

    Directory of Open Access Journals (Sweden)

    Dorota Bednarek

    2015-09-01

    Full Text Available After myocardial infarction in humans, lost cardiomyocytes are replaced by an irreversible fibrotic scar. In contrast, zebrafish hearts efficiently regenerate after injury. Complete regeneration of the zebrafish heart is driven by the strong proliferation response of its cardiomyocytes to injury. Here we show that, after cardiac injury in zebrafish, telomerase becomes hyperactivated, and telomeres elongate transiently, preceding a peak of cardiomyocyte proliferation and full organ recovery. Using a telomerase-mutant zebrafish model, we found that telomerase loss drastically decreases cardiomyocyte proliferation and fibrotic tissue regression after cryoinjury and that cardiac function does not recover. The impaired cardiomyocyte proliferation response is accompanied by the absence of cardiomyocytes with long telomeres and an increased proportion of cardiomyocytes showing DNA damage and senescence characteristics. These findings demonstrate the importance of telomerase function in heart regeneration and highlight the potential of telomerase therapy as a means of stimulating cell proliferation upon myocardial infarction.

  2. Human telomerase RNA component (hTERC gene amplification detected by FISH in precancerous lesions and carcinoma of the larynx

    Directory of Open Access Journals (Sweden)

    Liu Yu

    2012-03-01

    Full Text Available Abstract Background Gain of 3q26 is frequently observed in squamous cell carcinomas of mucosal origin, including those originating in the head and neck region. The human telomerase RNA component (hTERC gene, which is located on chromosome 3q26, encodes for an RNA subunit of telomerase that maintains the length of telomeres through cellular divisions, and is activated in malignant diseases. The present study was designed to detect hTERC amplification in laryngeal lesions and evaluate whether this might serve as a supportive biomarker in histopathological analysis for in the diagnosis of laryngeal lesions. Methods Fluorescent in situ hybridization (FISH was applied on formalin-fixed paraffin-embedded blocks of 93 laryngeal specimens, including 14 normal epithelium (NE, 15 mild dysplasia (Md, 18 moderate dysplasia (MD, 16 severe dysplasia (SD, 9 carcinoma in situ (CIS, and 21 invasive carcinoma (IC. Results By histopathologic examination, hTERC amplification rates in NE, Md, MD, SD, CIS and IC cases were 0% (0/14, 13.33% (2/15, 72.22% (13/18, 81.25% (13/16, 100% (9/9 and 100% (21/21, respectively. Amplification of hTERC was significantly associated with histopathologic diagnosis (P Conclusions The hTERC amplification is important in the development of laryngeal squamous cell carcinoma (LSCC. FISH detection of hTERC amplification may provide an effective approach in conjunction with histopathologic evaluation for differential diagnosis of laryngeal lesions. Virtual Slides The virtual slide(s for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2226606266791985

  3. Telomerase:a novel target of antitumor agents

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Telomerase activity was found to be high in various human cancers, but absent in most normal tissues. Its expression pattern made it a novel target for antitumor agents. Several strategies against telomerase were presented in this review. Targeting the telomerase RNA component by oligonucleotide/ribozyme was considered to be one of the most hopeful approaches. Some progresses were made in this area, such as the use of PANs and 2- 5A antisense compounds. The relationships among telomerase activity and cell differentiation, signal transduction, oncogene, tumor suppressor gene as well as cell cycle modulation also provided a series of valuable ideas in designing anti-telomerase drugs for cancer therapy. In conclusion, although there is still a long way in understanding the mechanism and regulation of telomerase, the advance of studies on telomerase has allowed the development of numerous strategies for the treatment of cancer.

  4. The conserved Est1 protein stimulates telomerase DNA extension activity

    Science.gov (United States)

    DeZwaan, Diane C.; Freeman, Brian C.

    2009-01-01

    The first telomerase cofactor identified was the budding yeast protein Est1, which is conserved through humans. While it is evident that Est1 is required for telomere DNA maintenance, understanding its mechanistic contributions to telomerase regulation has been limited. In vitro, the primary effect of Est1 is to activate telomerase-mediated DNA extension. Although Est1 displayed specific DNA and RNA binding, neither activity contributed significantly to telomerase stimulation. Rather Est1 mediated telomerase upregulation through direct contacts with the reverse transcriptase subunit. In addition to intrinsic Est1 functions, we found that Est1 cooperatively activated telomerase in conjunction with Cdc13 and that the combinatorial effect was dependent upon a known salt-bridge interaction between Est1 (K444) and Cdc13 (E252). Our studies provide insights into the molecular events used to control the enzymatic activity of the telomerase holoenzyme. PMID:19805136

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

    Institute of Scientific and Technical Information of China (English)

    Steffen Weikert; Frank Christoph; Wolfgang Schulze; Hans Krause; Carsten Kempkensteffen; Martin Schostak; Kurt Miller; Mark Schrader

    2006-01-01

    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.

  6. Human Telomerase Reverse Transcriptase (hTERT) Transcription Requires Sp1/Sp3 Binding to the Promoter and a Permissive Chromatin Environment.

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    Cheng, De; Zhao, Yuanjun; Wang, Shuwen; Jia, Wenwen; Kang, Jiuhong; Zhu, Jiyue

    2015-12-11

    The transcription of human telomerase gene hTERT is regulated by transcription factors (TFs), including Sp1 family proteins, and its chromatin environment. To understand its regulation in a relevant chromatin context, we employed bacterial artificial chromosome reporters containing 160 kb of human genomic sequence containing the hTERT gene. Upon chromosomal integration, the bacterial artificial chromosomes recapitulated endogenous hTERT expression, contrary to transient reporters. Sp1/Sp3 expression did not correlate with hTERT promoter activity, and these TFs bound to the hTERT promoters in both telomerase-positive and telomerase-negative cells. Mutation of the proximal GC-box resulted in a dramatic decrease of hTERT promoter activity, and mutations of all five GC-boxes eliminated its transcriptional activity. Neither mutations of GC-boxes nor knockdown of endogenous Sp1 impacted promoter binding by other TFs, including E-box-binding proteins, and histone acetylation and trimethylation of histone H3K9 at the hTERT promoter in telomerase-positive and -negative cells. The result indicated that promoter binding by Sp1/Sp3 was essential, but not a limiting step, for hTERT transcription. hTERT transcription required a permissive chromatin environment. Importantly, our data also revealed different functions of GC-boxes and E-boxes in hTERT regulation; although GC-boxes were essential for promoter activity, factors bound to the E-boxes functioned to de-repress hTERT promoter.

  7. Premature aging in telomerase-deficient zebrafish

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    Monique Anchelin

    2013-09-01

    The study of telomere biology is crucial to the understanding of aging and cancer. In the pursuit of greater knowledge in the field of human telomere biology, the mouse has been used extensively as a model. However, there are fundamental differences between mouse and human cells. Therefore, additional models are required. In light of this, we have characterized telomerase-deficient zebrafish (Danio rerio as the second vertebrate model for human telomerase-driven diseases. We found that telomerase-deficient zebrafish show p53-dependent premature aging and reduced lifespan in the first generation, as occurs in humans but not in mice, probably reflecting the similar telomere length in fish and humans. Among these aging symptoms, spinal curvature, liver and retina degeneration, and infertility were the most remarkable. Although the second-generation embryos died in early developmental stages, restoration of telomerase activity rescued telomere length and survival, indicating that telomerase dosage is crucial. Importantly, this model also reproduces the disease anticipation observed in humans with dyskeratosis congenita (DC. Thus, telomerase haploinsufficiency leads to anticipation phenomenon in longevity, which is related to telomere shortening and, specifically, with the proportion of short telomeres. Furthermore, p53 was induced by telomere attrition, leading to growth arrest and apoptosis. Importantly, genetic inhibition of p53 rescued the adverse effects of telomere loss, indicating that the molecular mechanisms induced by telomere shortening are conserved from fish to mammals. The partial rescue of telomere length and longevity by restoration of telomerase activity, together with the feasibility of the zebrafish for high-throughput chemical screening, both point to the usefulness of this model for the discovery of new drugs able to reactivate telomerase in individuals with DC.

  8. Imetelstat (GRN163L)--telomerase-based cancer therapy.

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    Röth, Alexander; Harley, Calvin B; Baerlocher, Gabriela M

    2010-01-01

    Telomeres and telomerase play essential roles in the regulation of the lifespan of human cells. While normal human somatic cells do not or only transiently express telomerase and therefore shorten their telomeres with each cell division, most human cancer cells typically express high levels of telomerase and show unlimited cell proliferation. High telomerase expression allows cells to proliferate and expand long-term and therefore supports tumor growth. Owing to the high expression and its role, telomerase has become an attractive diagnostic and therapeutic cancer target. Imetelstat (GRN163L) is a potent and specific telomerase inhibitor and so far the only drug of its class in clinical trials. Here, we report on the structure and the mechanism of action of imetelstat as well as about the preclinical and clinical data and future prospects using imetelstat in cancer therapy.

  9. Evaluation of telomerase expression in chronic periodontitis

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    Balaji T

    2010-01-01

    Full Text Available Background : Human telomerase is a multi subunit ribonucleoprotein enzyme concerned with telomeric lengthening and homeostasis in man. This enzyme has been found to be elevated in inflammatory conditions like rheumatoid arthritis and silica injury lung. Since chronic periodontitis is also an inflammatory condition where immune cells and cytokines mediate tissue destruction, we set out to evaluate telomerase in gingival tissue samples from healthy subjects and chronic periodontitis patients by reverse transcriptase polymerase chain reaction. Materials and Methods : Gingival biopsies were obtained from eight healthy subjects and eight chronic periodontitis patients. Reverse transcriptase polymerase chain reaction (RTPCR was carried out to evaluate telomerase gene expression in the samples. Results : None of the healthy gingival tissue samples expressed the telomerase gene while all the chronic periodontitis samples expressed it. The severe chronic periodontitis samples expressed the gene more intensely than the moderate chronic periodontitis samples. Conclusion : Various mechanisms have been explained to account for telomerase elevation in chronic periodontitis .This study helps us understand the role of telomerase in the pathogenesis of periodontal disease. It could be concluded that telomerase could be used as a marker to assess the severity of inflammation in chronic periodontitis.

  10. Molecular Detection of Human Telomerase mRNA (hTERT-mRNA in Egyptian Patients with Hepatocellular Carcinoma

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    Gahan Kamal El-Saeed

    2009-06-01

    Full Text Available Background and Aims: Diagnostic modalities for hepatocellular carcinoma (HCC as markers, sonography, and CT have contributed to the early detection of HCC but are still not sensitive enough. Human telomerase RNA subunit (hTERT-mRNA has been identified in many cancers and claimed to be reactivated in HCC. To investigate hTERT-mRNA in the peripheral blood of HCC and chronic liver disease (CLD patients and correlate its level with alpha feto protein (AFP, the serological marker for HCC.Methods: The study was conducted on 44 patients selected from the National Liver Institute. Patients included Group I (22 patients diagnosed to have HCC, Group II (22 patients with CLD, and 12 apparently healthy volunteers as controls (Group III. All selected individuals were subjected to history taking, a clinical examination, abdominal sonography and laboratory investigations as liver function tests (LFTs, cell blood count (CBC, hepatitis viral markers, AFP, and real-time polymerase chain reaction (PCR Quantitative detection of -mRNA expression, encoding for telomerase catalytic subunit.Results: There was a significant elevation of AFP levels in the HCC group compared to both the CLD and control groups (P < 0.00, P < 0.001. The mean hTERT-mRNA expression in HCC patients was significantly higher than both CLD patients and controls (P < 0.001, P < 0.001. hTERT-mRNA was correlated with AFP and tumor size (P < 0.05, P < 0.001. The AFP cutoff level (185 ng/ml resulted in a 63.6% sensitivity, a 85.3% specificity; a 89.3% positive predictive value (PPV level, a 76.2 % negative predictive value (NPV level and a 83.4% accuracy for HCC prediction. The hTERT-mRNA cutoff level (112.5 copies/ml showed a 77.3% sensitivity, a 97.1% specificity, a 98% PPV level, a 79.2 % NPV level, and an accuracy of 84% for HCC prediction. Combining hTERT-mRNA and AFP increased diagnostic accuracy to 90.5%. Both markers had a 84.1% sensitivity, a 86.4% specificity, a 86.4% PPV level, and a 88

  11. Inhibition of telomerase in tumor cells by ribozyme targeting telomerase RNA component

    Institute of Scientific and Technical Information of China (English)

    LIU; Bailin(刘柏林); QU; Yi(屈艺); LIU; Shuqiu(刘菽秋); OUYANG; Xuesong(欧阳雪松)

    2002-01-01

    Telomerase plays an important role in cell proliferation and carcinogenesis and is believed to be a good target for anti-cancer drugs. Elimination of template function of telomerase RNA may repress the telomerase activity. A hammer-headed ribozyme(telomerase ribozyme, teloRZ) directed against the RNA component of human telomerase(hTR) was designed and synthesized. TeloRZ showed a specific cleavage activity against the hTR. The cleavage efficacy reached 60%. A eukaryotic expression plasmid containing teloRZ gene was inducted into HeLa cells by lipofectamine, the telomerase activity in HeLa cells expressing teloRZ decreased to one eighth of that in the control cells. The doubling time increased significantly and the apoptosis ratio was elevated with increasing population doublings(PDS). After 19-20 PDS 95% cells were apoptotic. To further investigate the effect of teloRZ on tumor growth, the eukaryotic expression plasmid containing teloRZ was injected into transplanted tumor of nude mouse. The teloRZ effectively inhibited the telomerase activity in transplanted tumor, promoted apoptosis of the transplanted tumor cells, and decreased the tumor size significantly. These results indicate that teloRZ can effectively inhibit telomerase activity and growth of tumor cells, and suggest the potential use of this ribozyme in anti-cancer therapy.

  12. Telomerase Regulation from Beginning to the End

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    Deanna Elise MacNeil

    2016-09-01

    Full Text Available The vast body of literature regarding human telomere maintenance is a true testament to the importance of understanding telomere regulation in both normal and diseased states. In this review, our goal was simple: tell the telomerase story from the biogenesis of its parts to its maturity as a complex and function at its site of action, emphasizing new developments and how they contribute to the foundational knowledge of telomerase and telomere biology.

  13. Telomerase-immortalized human mammary stem/progenitor cells with ability to self-renew and differentiate.

    Science.gov (United States)

    Zhao, Xiangshan; Malhotra, Gautam K; Lele, Subodh M; Lele, Manjiri S; West, William W; Eudy, James D; Band, Hamid; Band, Vimla

    2010-08-10

    There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults, or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5, K14, and vimentin), luminal (E-cadherin, K8, K18, or K19), and stem/progenitor (CD49f, CD29, CD44, and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types--a K5(+)/K19(-) type and a K5(+)/K19(+) type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways, such as Notch, Wnt, Hedgehog, and LIF, in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers, these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer.

  14. Telomerase recruitment requires both TCAB1 and Cajal bodies independently.

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    Stern, J Lewis; Zyner, Katherine G; Pickett, Hilda A; Cohen, Scott B; Bryan, Tracy M

    2012-07-01

    The ability of most cancer cells to grow indefinitely relies on the enzyme telomerase and its recruitment to telomeres. In human cells, recruitment depends on the Cajal body RNA chaperone TCAB1 binding to the RNA subunit of telomerase (hTR) and is also thought to rely on an N-terminal domain of the catalytic subunit, hTERT. We demonstrate that coilin, an essential structural component of Cajal bodies, is required for endogenous telomerase recruitment to telomeres but that overexpression of telomerase can compensate for Cajal body absence. In contrast, recruitment of telomerase was sensitive to levels of TCAB1, and this was not rescued by overexpression of telomerase. Thus, although Cajal bodies are important for recruitment, TCAB1 has an additional role in this process that is independent of these structures. TCAB1 itself localizes to telomeres in a telomerase-dependent but Cajal body-independent manner. We identify a point mutation in hTERT that largely abolishes recruitment yet does not affect association of telomerase with TCAB1, suggesting that this region mediates recruitment by an independent mechanism. Our results demonstrate that telomerase has multiple independent requirements for recruitment to telomeres and that the function of TCAB1 is to directly transport telomerase to telomeres.

  15. Telomeres and telomerase: a modern fountain of youth?

    Science.gov (United States)

    de Magalhães, João Pedro; Toussaint, Olivier

    2004-01-01

    Since ageing is a universal human feature, it is not surprising that, from the Babylonian epic of Gilgamesh to Ponce de Leon seeking the "Fountain of Youth," countless people have dreamed of finding a way to avoid ageing, to no avail. Yet the search continues. In this review, we present one of the latest candidates: the enzyme telomerase, capable of elongating the tips of chromosomes, the telomeres. Research into the causes of cellular ageing established the telomeres as the molecular clock that counts the number of times cells divide and triggers cellular senescence. Herein, we review arguments both in favor and against the use of telomerase as an anti-ageing therapy. The importance of the telomeres in cellular ageing, the low or non-existent levels of telomerase activity in human tissues, and the ability of telomerase to immortalize human cells suggest that telomerase can be used as an anti-ageing therapy. On the other hand, recent experiments in mice have raised doubts whether telomerase affects organismal ageing. Results from human cells expressing telomerase have also suggested telomerase may promote tumorigenesis. We conclude that, though telomerase may be used in regenerative medicine and to treat specific diseases, it is unlikely to become a source of anti-ageing therapies.

  16. Nutrition and lifestyle in healthy aging: the telomerase challenge.

    Science.gov (United States)

    Boccardi, Virginia; Paolisso, Giuseppe; Mecocci, Patrizia

    2016-01-01

    Nutrition and lifestyle, known to modulate aging process and age-related diseases, might also affect telomerase activity. Short and dysfunctional telomeres rather than average telomere length are associated with longevity in animal models, and their rescue by telomerase maybe sufficient to restore cell and organismal viability. Improving telomerase activation in stem cells and potentially in other cells by diet and lifestyle interventions may represent an intriguing way to promote health-span in humans.

  17. Cancer-Specific Telomerase Reverse Transcriptase (TERT Promoter Mutations: Biological and Clinical Implications

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    Tiantian Liu

    2016-07-01

    Full Text Available The accumulated evidence has pointed to a key role of telomerase in carcinogenesis. As a RNA-dependent DNA polymerase, telomerase synthesizes telomeric DNA at the end of linear chromosomes, and attenuates or prevents telomere erosion associated with cell divisions. By lengthening telomeres, telomerase extends cellular life-span or even induces immortalization. Consistent with its functional activity, telomerase is silent in most human normal somatic cells while active only in germ-line, stem and other highly proliferative cells. In contrast, telomerase activation widely occurs in human cancer and the enzymatic activity is detectable in up to 90% of malignancies. Recently, hotspot point mutations in the regulatory region of the telomerase reverse transcriptase (TERT gene, encoding the core catalytic component of telomerase, was identified as a novel mechanism to activate telomerase in cancer. This review discusses the cancer-specific TERT promoter mutations and potential biological and clinical significances.

  18. Study on the Detection of Telomerase Activity by Combining DNA Sequence Analysis with TRAP

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Telomeric repeat amplification protocol (TRAP) is now aconventional assay for detecting telomerase activity. However, this method presents problems owing to tedious quantitation, radioisotopic handling. In order to alleviate these inconveniences, a novel telomerase DNA sequencing assay together with TRAP to detect human telomerase activity was developed. It was used to detect telomerase activity in Hela, HLF, MCF, K562, SMMC-7721 cells, Leukocytes and RNase-pretreated or heat-treated cells as control. Telomerase activity assayed by this method was positive when the number of K562 cells examined was 102,103, and 104. The telomerase activity depended on the number of K562 cells used in the assay. Telomerase activity of Rnase-pretreated cells or heat-treated cells, and human normal peripheral blood leukocyte(Leu) were negative. The result of this method was available within a few hours and was handled without radioisotope. Further studies should be taken to detect telomerase activity in quantitation.

  19. Comparison of Inhibitory Effect of Curcumin Nanoparticles and Free Curcumin in Human Telomerase Reverse Transcriptase Gene Expression in Breast Cancer

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    Nosratollah Zarghami

    2013-02-01

    Full Text Available Purpose: Telomerase is expressed in most cancers, including breast cancer. Curcumin, a polyphenolic compound that obtained from the herb of Curcuma longa, has many anticancer effects. But, its effect is low due to poor water solubility. In order to improve its solubility and drug delivery, we have utilized a β-cyclodextrin-curcumin inclusion complex. Methods: To evaluate cytotoxic effects of cyclodextrin-curcumin and free curcumin, MTT assay was done. Cells were treated with equal concentration of cyclodextrin-curcumin and free curcumin. Telomerase gene expression level in two groups was compared by Real-time PCR. Results: MTT assay demonstrated that β-cyclodextrin-curcumin enhanced curcumin delivery in T47D breast cancer cells. The level of telomerase gene expression in cells treated with cyclodextrin-curcumin was lower than that of cells treated with free curcumin (P=0.001. Conclusion: Results are suggesting that cyclodextrin-curcumin complex can be more effective than free curcumin in inhibition of telomerase expression.

  20. Interferon-inducible IFI16, a negative regulator of cell growth, down-regulates expression of human telomerase reverse transcriptase (hTERT gene.

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    Lynda Li Song

    Full Text Available BACKGROUND: Increased levels of interferon (IFN-inducible IFI16 protein (encoded by the IFI16 gene located at 1q22 in human normal prostate epithelial cells and diploid fibroblasts (HDFs are associated with the onset of cellular senescence. However, the molecular mechanisms by which the IFI16 protein contributes to cellular senescence-associated cell growth arrest remain to be elucidated. Here, we report that increased levels of IFI16 protein in normal HDFs and in HeLa cells negatively regulate the expression of human telomerase reverse transcriptase (hTERT gene. METHODOLOGY/PRINCIPAL FINDINGS: We optimized conditions for real-time PCR, immunoblotting, and telomere repeat amplification protocol (TRAP assays to detect relatively low levels of hTERT mRNA, protein, and telomerase activity that are found in HDFs. Using the optimized conditions, we report that treatment of HDFs with inhibitors of cell cycle progression, such as aphidicolin or CGK1026, which resulted in reduced steady-state levels of IFI16 mRNA and protein, was associated with increases in hTERT mRNA and protein levels and telomerase activity. In contrast, knockdown of IFI16 expression in cells increased the expression of c-Myc, a positive regulator of hTERT expression. Additionally, over-expression of IFI16 protein in cells inhibited the c-Myc-mediated stimulation of the activity of hTERT-luc-reporter and reduced the steady-state levels of c-Myc and hTERT. CONCLUSIONS/SIGNIFICANCE: These data demonstrated that increased levels of IFI16 protein in HDFs down-regulate the expression of hTERT gene. Our observations will serve basis to understand how increased cellular levels of the IFI16 protein may contribute to certain aging-dependent diseases.

  1. Interferon-Inducible IFI16, a Negative Regulator of Cell Growth, Down-Regulates Expression of Human Telomerase Reverse Transcriptase (hTERT) Gene

    Science.gov (United States)

    Shen, Hui; Duan, Xin; Alimirah, Fatouma; Choubey, Divaker

    2010-01-01

    Background Increased levels of interferon (IFN)-inducible IFI16 protein (encoded by the IFI16 gene located at 1q22) in human normal prostate epithelial cells and diploid fibroblasts (HDFs) are associated with the onset of cellular senescence. However, the molecular mechanisms by which the IFI16 protein contributes to cellular senescence-associated cell growth arrest remain to be elucidated. Here, we report that increased levels of IFI16 protein in normal HDFs and in HeLa cells negatively regulate the expression of human telomerase reverse transcriptase (hTERT) gene. Methodology/Principal Findings We optimized conditions for real-time PCR, immunoblotting, and telomere repeat amplification protocol (TRAP) assays to detect relatively low levels of hTERT mRNA, protein, and telomerase activity that are found in HDFs. Using the optimized conditions, we report that treatment of HDFs with inhibitors of cell cycle progression, such as aphidicolin or CGK1026, which resulted in reduced steady-state levels of IFI16 mRNA and protein, was associated with increases in hTERT mRNA and protein levels and telomerase activity. In contrast, knockdown of IFI16 expression in cells increased the expression of c-Myc, a positive regulator of hTERT expression. Additionally, over-expression of IFI16 protein in cells inhibited the c-Myc-mediated stimulation of the activity of hTERT-luc-reporter and reduced the steady-state levels of c-Myc and hTERT. Conclusions/Significance These data demonstrated that increased levels of IFI16 protein in HDFs down-regulate the expression of hTERT gene. Our observations will serve basis to understand how increased cellular levels of the IFI16 protein may contribute to certain aging-dependent diseases. PMID:20052289

  2. Oxoisoaporphine as Potent Telomerase Inhibitor.

    Science.gov (United States)

    Wei, Zu-Zhuang; Qin, Qi-Pin; Chen, Jia-Nian; Chen, Zhen-Feng

    2016-11-14

    Two compounds previously isolated from traditional Chinese medicine, Menispermum dauricum (DC), 6-hydroxyl-oxoisoaporphine (H-L(a)), and 4,6-di(2-pyridinyl)benzo[h]isoindolo[4,5,6-de]quinolin-8(5H)-one (H-L(b)), were known to have in vitro antitumor activity and to selectively bind human telomeric, c-myc, and bcl-2 G-quadruplexes (G4s). In this study, the binding properties of these two compounds to telomerase were investigated through molecular docking and telomeric repeat amplication protocol and silver staining assay (TRAP-silver staining assay). The binding energies bound to human telomerase RNA were calculated by molecular docking to be -6.43 and -9.76 kcal/mol for H-L(a) and H-L(b), respectively. Compared with H-L(a), the ligand H-L(b) more strongly inhibited telomerase activity in the SK-OV-3 cells model.

  3. Telomeres and telomerase: Pharmacological targets for new anticancer strategies?

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    Pendino, F; Tarkanyi, I; Dudognon, C; Hillion, J; Lanotte, M; Aradi, J; Ségal-Bendirdjian, E

    2006-03-01

    Telomeres are located at the ends of eukaryotic chromosomes. Human telomerase, a cellular reverse transcriptase, is a ribonucleoprotein enzyme that catalyzes the synthesis and extension of telomeric DNA. It is composed of at least, a template RNA component (hTR; human Telomerase RNA) and a catalytic subunit, the telomerase reverse transcriptase (hTERT). The absence of telomerase is associated with telomere shortening and aging of somatic cells, while high telomerase activity is observed in over 85% of human cancer cells, strongly indicating its key role during tumorigenesis. Several details regarding telomere structure and telomerase regulation have already been elucidated, providing new targets for therapeutic exploitation. Further support for anti-telomerase approaches comes from recent studies indicating that telomerase is endowed of additional functions in the control of growth and survival of tumor cells that do not depend only on the ability of this enzyme to maintain telomere length. This observation suggests that inhibiting telomerase or its synthesis may have additional anti-proliferative and apoptosis inducing effect, independently of the reduction of telomere length during cell divisions. This article reviews the basic information about the biology of telomeres and telomerase and attempts to present various approaches that are currently under investigation to inhibit its expression and its activity. We summarize herein distinct anti-telomerase approaches like antisense strategies, reverse transcriptase inhibitors, and G-quadruplex interacting agents, and also review molecules targeting hTERT expression, such as retinoids and evaluate them for their therapeutic potential. "They conceive a certain theory, and everything has to fit into that theory. If one little fact will not fit it, they throw it aside. But it is always the facts that will not fit in that are significant". "Death on the Nile". Agatha Christie.

  4. Telomerase activity coevolves with body mass, not lifespan

    Science.gov (United States)

    Seluanov, Andrei; Chen, Zhuoxun; Hine, Christopher; Sasahara, Tais H. C.; Ribeiro, Antonio A. C. M.; Catania, Kenneth C.; Presgraves, Daven C.; Gorbunova, Vera

    2009-01-01

    Summary In multicellular organisms, telomerase is required to maintain telomere length in the germline but is dispensable in the soma. Mice, for example, express telomerase in somatic and germline tissues, while humans express telomerase almost exclusively in the germline. As a result, when telomeres of human somatic cells reach a critical length the cells enter irreversible growth arrest called replicative senescence. Replicative senescence is believed to be an anticancer mechanism that limits cell proliferation. The difference between mice and humans led to the hypothesis that repression of telomerase in somatic cells has evolved as a tumor-suppressor adaptation in large, long-lived organisms. We tested whether regulation of telomerase activity coevolves with lifespan and body mass using comparative analysis of 15 rodent species with highly diverse lifespans and body masses. Here we show that telomerase activity does not coevolve with lifespan but instead coevolves with body mass: larger rodents repress telomerase activity in somatic cells. These results suggest that large body mass presents a greater risk of cancer than long lifespan, and large animals evolve repression of telomerase activity to mitigate that risk. PMID:17173545

  5. Gain of human telomerase RNA gene is associated with progression of cervical intraepithelial neoplasia grade Ⅰ or Ⅱ

    Institute of Scientific and Technical Information of China (English)

    LAN Yong-lian; YU Lan; JIA Chan-wei; WU Yu-mei; WANG Shu-yu

    2012-01-01

    Background The 3q26 chromosome region,where the human telomerase RNA gene (hTERC) is located,is a biomarker for cervical cancer and precancerous lesions.The aim of this study was to confirm the value of measuring hTERC gene gain in predicting the progression of cervical intraepithelial neoplasia grade Ⅰ or Ⅱ (CIN-Ⅰ and -Ⅱ,respectively) to CIN-Ⅲ and cervical cancer.Methods Liquid-based cytological samples from 54 patients with CIN-Ⅰ or CIN-Ⅱ lesions were enrolled in this study.Follow-up was performed with colposcopy and biopsy within 24 months after the diagnosis of CIN-Ⅰ or CIN-Ⅱ.Copy numbers of the hTERC gene were measured by fluorescence in situ hybridization with a dual-color probe mix containing the hTERC gene probe (labeled red) and the control,the chromosome 3 centromere-specific probe (labeled green).Results All patients whose lesions progressed from CIN-Ⅰ or CIN-Ⅱ to CIN-Ⅲ displayed a gain of the hTERC gene,whereas patients where the hTERC gene was not amplified did not subsequently progress to CIN-Ⅲ or cervical cancer.The signal ratio pattem per cell was recorded as N∶N (green∶ red).The numbers of cells with the signal ratio pattern of 4∶4or N∶≥5 in patients whose lesions progressed to CIN-Ⅲ were significantly higher than those whose lesions did not progress.Significantly,none of the patients with a 4∶4 signal ratio pattern regressed spontaneously.Conclusions In conclusion,measurement of hTERC gene gain in CIN-Ⅰ or CIN-Ⅱ patients using liquid-based cytological samples could be a useful biomarker to predict the progression of such cervical lesions.In addition,a 4∶4 or N∶≥5 signal ratio pattern may indicate the unlikeness of spontaneous regression of CIN-Ⅰ or CIN-Ⅱ lesions.

  6. Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b

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    Brosens Jan J

    2009-07-01

    Full Text Available Abstract Background Coordinated differentiation of the endometrial compartments in the second half of the menstrual cycle is a prerequisite for the establishment of pregnancy. Endometrial stromal cells (ESC decidualize under the influence of ovarian progesterone to accommodate implantation of the blastocyst and support establishment of the placenta. Studies into the mechanisms of decidualization are often hampered by the lack of primary ESC. Here we describe a novel immortalized human ESC line. Methods Primary ESC were immortalized by the transduction of telomerase. The resultant cell line, termed St-T1b, was characterized for its morphological and biochemical properties by immunocytochemistry, RT-PCR and immunoblotting. Its progestational response was tested using progesterone and medroxyprogesterone acetate with and without 8-Br-cAMP, an established inducer of decidualization in vitro. Results St-T1b were positive for the fibroblast markers vimentin and CD90 and negative for the epithelial marker cytokeratin-7. They acquired a decidual phenotype indistinguishable from primary ESC in response to cAMP stimulation. The decidual response was characterized by transcriptional activation of marker genes, such as PRL, IGFBP1, and FOXO1, and enhanced protein levels of the tumor suppressor p53 and the metastasis suppressor KAI1 (CD82. Progestins alone had no effect on St-T1b cells, but medroxyprogesterone acetate greatly enhanced the cAMP-stimulated expression of IGFBP-1 after 3 and 7 days. Progesterone, albeit more weakly, also augmented the cAMP-induced IGFBP-1 production but only after 7 days of treatment. The cell line remained stable in continuous culture for more than 150 passages. Conclusion St-T1b express the appropriate phenotypic ESC markers and their decidual response closely mimics that of primary cultures. Decidualization is efficiently induced by cAMP analog and enhanced by medroxyprogesterone acetate, and, to a lesser extent, by natural

  7. Telomerase Structure Paves the way for New Cancer Therapies

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    Skordalakes, S.

    2009-01-01

    Inappropriate activation of a single enzyme, telomerase, is associated with the uncontrollable proliferation of cells observed in as many as 90% of all of human cancers. Since the mid-1990s, when telomerase activity was detected in human tumors, scientists have eyed the enzyme as an ideal target for developing broadly effective anticancer drugs. One of the missing links in the effort to identify such therapies has been the high-resolution structure of the enzyme, a powerful tool used for the identification and development of clinical drugs. A recent structure of the catalytic subunit of teleomerase from the Skordalakes laboratory, a major advancement in the field of telomeres, has opened the door to the development of new, broadly effective cancer drugs, as well as anti-aging therapies. Here we present a brief description of telomerase biology, current efforts to identify telomerase function modulators and the potential importance of the telomerase structure in future drug development.

  8. Antiproliferative effect of gold(I compound auranofin through inhibition of STAT3 and telomerase activity in MDA-MB 231 human breast cancer cells

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    Nam-Hoon Kim

    2013-01-01

    Full Text Available Signal transducer and activator of transcription 3 (STAT3 andtelomerase are considered attractive targets for anticancertherapy. The in vitro anticancer activity of the gold(I compoundauranofin was investigated using MDA-MB 231 human breastcancer cells, in which STAT3 is constitutively active. In cellculture, auranofin inhibited growth in a dose-dependent manner,and N-acetyl-L-cysteine (NAC, a scavenger of reactive oxygenspecies (ROS, markedly blocked the effect of auranofin.Incorporation of 5-bromo-2’-deoxyuridine into DNA andanchorage-independent cell growth on soft agar were decreasedby auranofin treatment. STAT3 phosphorylation and telomeraseactivity were also attenuated in cells exposed to auranofin, butNAC pretreatment restored STAT3 phosphorylation andtelomerase activity in these cells. These findings indicate thatauranofin exerts in vitro antitumor effects in MDA-MB 231 cellsand its activity involves inhibition of STAT3 and telomerase.Thus, auranofin shows potential as a novel anticancer drug thattargets STAT3 and telomerase. [BMB Reports 2013; 46(1: 59-64

  9. Evaluation of human first trimester decidual and telomerase-transformed endometrial stromal cells as model systems of in vitro decidualization

    Directory of Open Access Journals (Sweden)

    Saleh Leila

    2011-12-01

    Full Text Available Abstract Background Decidualization, the differentiation process of maternal uterine stromal cells into secretory decidual cells, is a prerequisite for successful implantation and progression of pregnancy. For in vitro differentiation mostly primary human endometrial stromal cells (HESC isolated from uterine samples after hysterectomy for benign gynaecological diseases are utilised. However, a continuous supply of endometrial tissue is often lacking. Hence, we analysed whether cultivated human decidual stromal cells (HDSC prepared from first trimester pregnancy terminations may represent an alternative model system for in vitro decidualization. Moreover, based on the expression of critical marker genes these cells were compared to a previously established endometrial stromal cell line during in vitro differentiation. Methods HDSC isolated from decidual tissue attached to first trimester placentae, and telomerase-transformed human endometrial stromal cells (THESC were characterised by immunofluorescence and differentiated in vitro using either cyclic adenosine monophosphate (cAMP and/or estrogen (E2/progesterone (P4. Proliferation was measured by analyzing cumulative cell numbers. Expression of mRNAs encoding progesterone receptor (PR, prolactin (PRL, insulin-like growth factor binding protein-1 (IGFBP1, and Dickkopf-1 (DKK1 was evaluated using quantitative PCR after 3, 6, 9 and 12 days of in vitro differentiation. PRL and IGFBP-1 protein expression was investigated by enzyme-linked immunosorbent assay (ELISA and Western blotting, respectively. Furthermore, forkhead box O1A (FOXO1A, a critical transcription factor in decidualization, was analysed by immunofluorescence and Western blotting at two different time points of differentiation. Results Treatment with cAMP provoked morphological changes and growth arrest of THESC and HDSC, the latter showing loss of cells after 6 days of treatment. E2P4 stimulation did neither affect cell morphology nor

  10. Effective siRNA targets screening for human telomerase reverse transcriptase

    Institute of Scientific and Technical Information of China (English)

    Yun Xia; Ru-Xian Lin; Su-Jun Zheng; Ying Yang; Xiao-Chen Bo; Dao-Yin Zhu; Sheng-Qi Wang

    2005-01-01

    AIM: To study the inhibitory effects of siRNAs targeting different hTERT sequences and to screen the effective siRNA sequence.METHODS: Five double-stranded siRNAs targeting coding and non-coding regions of hTERT gene were designed and synthesized by T7 transcription system in vitro. siRNA4sequence was screened by full length gene targeting technique and the rest of the siRNA sequences were selected randomly. After being purified by ethanol precipitation, the siRNAs were transfected to the human hepatocellular carcinoma cell (HepG2) by Lipofectamine 2000TM. At 48-72 h after siRNAs transfection, MTT assay,RT-PCR and Western-blot were applied to evaluate the effects of siRNAs on cell growth, mRNA and protein expression level of hTERT gene, respectively.RESULTS: Compared to the control cells, the cells treated with the five double-stranded siRNAs exhibited different degrees of inhibition of cell proliferation in a dose-dependent manner. siRNA2 and siRNA4, exhibited obvious effects of inhibiting hTERT mRNA and protein expression in HepG2cells.CONCLUSION: siRNAs targeting different hTERT sequences have significantly various inhibitory effects on hTERT gene expression. The siRNA sequence screened by full length gene targeting technique has comparable inhibitory effect with the rest siRNA sequences screened by random selection, suggesting that siRNAs and antisense oligonucleic acids may have the same effective target sites. Compared with chemical synthesis method,synthesizing double-stranded siRNA by T7 transcription system in vitro is a rapid, simple, and inexpensive method suitable for screening high-effect siRNA targeting site for specific gene.

  11. TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe.

    Science.gov (United States)

    Moravec, Martin; Wischnewski, Harry; Bah, Amadou; Hu, Yan; Liu, Na; Lafranchi, Lorenzo; King, Megan C; Azzalin, Claus M

    2016-07-01

    Telomerase-mediated telomere elongation provides cell populations with the ability to proliferate indefinitely. Telomerase is capable of recognizing and extending the shortest telomeres in cells; nevertheless, how this mechanism is executed remains unclear. Here, we show that, in the fission yeast Schizosaccharomyces pombe, shortened telomeres are highly transcribed into the evolutionarily conserved long noncoding RNA TERRA A fraction of TERRA produced upon telomere shortening is polyadenylated and largely devoid of telomeric repeats, and furthermore, telomerase physically interacts with this polyadenylated TERRA in vivo We also show that experimentally enhanced transcription of a manipulated telomere promotes its association with telomerase and concomitant elongation. Our data represent the first direct evidence that TERRA stimulates telomerase recruitment and activity at chromosome ends in an organism with human-like telomeres.

  12. Expression of Telomerase Activity in Gastric Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective To study the relationship between telomerase activity and biological behavior in human gastric cells and appraise the clinical significance of detecting telomerase activity. Methods The telomerase activity in 47 gastric cancer tissue samples,their matched nomal tissues,7 gastric ulcer and 2 gastric cancer cell lines was detected using a PCR-based non-radioisotopic telomeric repeat amplification protocol(TRAP) assay. Results None of the 47 samples from normal gastric tissues expressed telomerase activity.The 41 of 47 cases of gastric cancer presented telomerase activity with an 87.2% positive rate (P<0.001). 2/2 gastric cancer cell lines and 0/7 gastric ulcer line were also positive for telmerase activity.The activity of telomerase was associated with the pathological differentiation of gastric cancer. Conclusion Telomerase activity may be related to the biological behavior of gastric cancer and can help in assessing the malignant poten-tial of gastric cancer.Telomerase activity will be a good diagnostic marker for the detection of gastric cancer.

  13. Expression of human telomerase (hTERT) does not prevent stress-induced senescence in normal human fibroblasts but protects the cells from stress-induced apoptosis and necrosis.

    Science.gov (United States)

    Gorbunova, Vera; Seluanov, Andrei; Pereira-Smith, Olivia M

    2002-10-11

    Cells subjected to sub-lethal doses of stress such as irradiation or oxidative damage enter a state that closely resembles replicative senescence. What triggers stress-induced premature senescence (SIPS) and how similar this mechanism is to replicative senescence are not well understood. It has been suggested that stress-induced senescence is caused by rapid telomere shortening resulting from DNA damage. In order to test this hypothesis directly, we examined whether overexpression of the catalytic subunit of human telomerase (hTERT) can protect cells from SIPS. We therefore analyzed the response of four different lines of normal human fibroblasts with and without hTERT to stress induced by UV, gamma-irradiation, and H(2)O(2). SIPS was induced with the same efficiency in normal and hTERT-immortalized cells. This suggests that SIPS is not triggered by telomere shortening and that nonspecific DNA damage serves as a signal for induction of SIPS. Although telomerase did not protect cells from SIPS, fibroblasts expressing hTERT were more resistant to stress-induced apoptosis and necrosis. We hypothesize that healing of DNA breaks by telomerase inhibits the induction of cell death, but because healing does not provide legitimate DNA repair, it does not protect cells from SIPS.

  14. Telomerase and telomere length in pulmonary fibrosis.

    Science.gov (United States)

    Liu, Tianju; Ullenbruch, Matthew; Young Choi, Yoon; Yu, Hongfeng; Ding, Lin; Xaubet, Antoni; Pereda, Javier; Feghali-Bostwick, Carol A; Bitterman, Peter B; Henke, Craig A; Pardo, Annie; Selman, Moises; Phan, Sem H

    2013-08-01

    In addition to its expression in stem cells and many cancers, telomerase activity is transiently induced in murine bleomycin (BLM)-induced pulmonary fibrosis with increased levels of telomerase transcriptase (TERT) expression, which is essential for fibrosis. To extend these observations to human chronic fibrotic lung disease, we investigated the expression of telomerase activity in lung fibroblasts from patients with interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF). The results showed that telomerase activity was induced in more than 66% of IPF lung fibroblast samples, in comparison with less than 29% from control samples, some of which were obtained from lung cancer resections. Less than 4% of the human IPF lung fibroblast samples exhibited shortened telomeres, whereas less than 6% of peripheral blood leukocyte samples from patients with IPF or hypersensitivity pneumonitis demonstrated shortened telomeres. Moreover, shortened telomeres in late-generation telomerase RNA component knockout mice did not exert a significant effect on BLM-induced pulmonary fibrosis. In contrast, TERT knockout mice exhibited deficient fibrosis that was independent of telomere length. Finally, TERT expression was up-regulated by a histone deacetylase inhibitor, while the induction of TERT in lung fibroblasts was associated with the binding of acetylated histone H3K9 to the TERT promoter region. These findings indicate that significant telomerase induction was evident in fibroblasts from fibrotic murine lungs and a majority of IPF lung samples, whereas telomere shortening was not a common finding in the human blood and lung fibroblast samples. Notably, the animal studies indicated that the pathogenesis of pulmonary fibrosis was independent of telomere length.

  15. 端粒、端粒酶与人类衰老和肿瘤%Telomeres, telomerase and human senility, cancer

    Institute of Scientific and Technical Information of China (English)

    沈兰; 左伋

    2000-01-01

    @@ 近年来,不少学者注意到染色体端粒(telomere)的长度和端粒酶(telomerase)活性与细胞的衰老、细胞的永生化以及细胞的恶性变有着密切的关系,显示它们在细胞的增殖调控中起着重要的作用,本文仅对此作一综述.

  16. Catalytically active telomerase holoenzyme is assembled in the dense fibrillar component of the nucleolus during S phase.

    Science.gov (United States)

    Lee, Ji Hoon; Lee, Yang Sin; Jeong, Sun Ah; Khadka, Prabhat; Roth, Jürgen; Chung, In Kwon

    2014-02-01

    The maintenance of human telomeres requires the ribonucleoprotein enzyme telomerase, which is composed of telomerase reverse transcriptase (TERT), telomerase RNA component, and several additional proteins for assembly and activity. Telomere elongation by telomerase in human cancer cells involves multiple steps including telomerase RNA biogenesis, holoenzyme assembly, intranuclear trafficking, and telomerase recruitment to telomeres. Although telomerase has been shown to accumulate in Cajal bodies for association with telomeric chromatin, it is unclear where and how the assembly and trafficking of catalytically active telomerase is regulated in the context of nuclear architecture. Here, we show that the catalytically active holoenzyme is initially assembled in the dense fibrillar component of the nucleolus during S phase. The telomerase RNP is retained in nucleoli through the interaction of hTERT with nucleolin, a major nucleolar phosphoprotein. Upon association with TCAB1 in S phase, the telomerase RNP is transported from nucleoli to Cajal bodies, suggesting that TCAB1 acts as an S-phase-specific holoenzyme component. Furthermore, depletion of TCAB1 caused an increase in the amount of telomerase RNP associated with nucleolin. These results suggest that the TCAB1-dependent trafficking of telomerase to Cajal bodies occurs in a step separate from the holoenzyme assembly in nucleoli. Thus, we propose that the dense fibrillar component is the provider of active telomerase RNP for supporting the continued proliferation of cancer and stem cells.

  17. Oxoisoaporphine as Potent Telomerase Inhibitor

    OpenAIRE

    Zu-Zhuang Wei; Qi-Pin Qin; Jia-Nian Chen; Zhen-Feng Chen

    2016-01-01

    Two compounds previously isolated from traditional Chinese medicine, Menispermum dauricum (DC), 6-hydroxyl-oxoisoaporphine (H-La), and 4,6-di(2-pyridinyl)benzo[h]isoindolo[4,5,6-de]quinolin-8(5H)-one (H-Lb), were known to have in vitro antitumor activity and to selectively bind human telomeric, c-myc, and bcl-2 G-quadruplexes (G4s). In this study, the binding properties of these two compounds to telomerase were investigated through molecular docking and telomeric repeat amplication protocol a...

  18. Human adipose tissue possesses a unique population of pluripotent stem cells with nontumorigenic and low telomerase activities: potential implications in regenerative medicine.

    Science.gov (United States)

    Ogura, Fumitaka; Wakao, Shohei; Kuroda, Yasumasa; Tsuchiyama, Kenichiro; Bagheri, Mozhdeh; Heneidi, Saleh; Chazenbalk, Gregorio; Aiba, Setsuya; Dezawa, Mari

    2014-04-01

    In this study, we demonstrate that a small population of pluripotent stem cells, termed adipose multilineage-differentiating stress-enduring (adipose-Muse) cells, exist in adult human adipose tissue and adipose-derived mesenchymal stem cells (adipose-MSCs). They can be identified as cells positive for both MSC markers (CD105 and CD90) and human pluripotent stem cell marker SSEA-3. They intrinsically retain lineage plasticity and the ability to self-renew. They spontaneously generate cells representative of all three germ layers from a single cell and successfully differentiate into targeted cells by cytokine induction. Cells other than adipose-Muse cells exist in adipose-MSCs, however, do not exhibit these properties and are unable to cross the boundaries from mesodermal to ectodermal or endodermal lineages even under cytokine inductions. Importantly, adipose-Muse cells demonstrate low telomerase activity and transplants do not promote teratogenesis in vivo. When compared with bone marrow (BM)- and dermal-Muse cells, adipose-Muse cells have the tendency to exhibit higher expression in mesodermal lineage markers, while BM- and dermal-Muse cells were generally higher in those of ectodermal and endodermal lineages. Adipose-Muse cells distinguish themselves as both easily obtainable and versatile in their capacity for differentiation, while low telomerase activity and lack of teratoma formation make these cells a practical cell source for potential stem cell therapies. Further, they will promote the effectiveness of currently performed adipose-MSC transplantation, particularly for ectodermal and endodermal tissues where transplanted cells need to differentiate across the lineage from mesodermal to ectodermal or endodermal in order to replenish lost cells for tissue repair.

  19. Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Zhen-Liang Qu; Sheng-Quan Zou; Nai-Qiang Cui; Xian-Zhong Wu; Ming-Fang Qin; Di Kong; Zhen-Li Zhou

    2005-01-01

    AIM: To study the changes of human telomerase reverse transcriptase (hTERT) mRNA expression in human hepatocarcinoma cell lines (HepG2) and cholangiocarcinoma cell lines (QBC939) after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis.METHODS: HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency.Cells were harvested and total RNA was extracted using TRIzol() reagent. The expression of hTERT mRNA in HepG2and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting.RESULTS: Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector.Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939cells only when transfected with HBx gene.CONCLUSION: HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection.

  20. Telomerer og telomerase

    DEFF Research Database (Denmark)

    Bendix, Laila; Kølvraa, Steen

    2010-01-01

    In 2009 the Nobel Prize in Medicine was awarded to EH Blackburn, CW Greider and JW Szostak for their work on "How chromosomes are protected by telomeres and the enzyme telomerase". Telomeres are specialized DNA structures localized at the end of linear chromosomes. Telomeres are known...... as the biological clock of the cell, since they shorten with each cell division. Telomerase can elongate telomeres. Telomeres protect chromosome ends against being recognized as double stranded DNA breaks, and are thought to be a guard against cancer. It has furthermore been suggested that telomeres may play a role...

  1. Notch1 signaling regulates the proliferation and self-renewal of human dental follicle cells by modulating the G1/S phase transition and telomerase activity.

    Directory of Open Access Journals (Sweden)

    Xuepeng Chen

    Full Text Available Multipotent human dental follicle cells (HDFCs have been intensively studied in periodontal regeneration research, yet the role of Notch1 in HDFCs has not been fully understood. The aim of the current study is to explore the role of Notch1 signaling in HDFCs self-renewal and proliferation. HDFCs were obtained from the extracted wisdom teeth from adolescent patients. Regulation of Notch1 signaling in the HDFCs was achieved by overexpressing the exogenous intracellular domain of Notch1 (ICN1 or silencing Notch1 by shRNA. The regulatory effects of Notch1 on HDFC proliferation, cell cycle distribution and the expression of cell cycle regulators were investigated through various molecular technologies, including plasmid construction, retrovirus preparation and infection, qRT-PCR, western blot, RBP-Jk luciferase reporter and cell proliferation assay. Our data clearly show that constitutively activation of Notch1 stimulates the HDFCs proliferation while inhibition of the Notch1 suppresses their proliferation in vitro. In addition, the HDFCs proliferation is associated with the increased expression of cell cycle regulators, e.g. cyclin D1, cyclin D2, cyclin D3, cyclin E1, CDK2, CDK4, CDK6, and SKP2 and the decreased expression of p27 (kip1. Moreover, our data show that the G1/S phase transition (indicating proliferation and telomerase activity (indicating self-renewal can be enhanced by overexpression of ICN1 but halted by inhibition of Notch1. Together, the current study provides evidence for the first time that Notch1 signaling regulates the proliferation and self-renewal capacity of HDFCs through modulation of the G1/S phase transition and the telomerase activity.

  2. A telomerase immortalized human proximal tubule cell line with a truncation mutation (Q4004X in polycystin-1.

    Directory of Open Access Journals (Sweden)

    Brittney-Shea Herbert

    Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is associated with a variety of cellular phenotypes in renal epithelial cells. Cystic epithelia are secretory as opposed to absorptive, have higher proliferation rates in cell culture and have some characteristics of epithelial to mesenchymal transitions. In this communication we describe a telomerase immortalized cell line that expresses proximal tubule markers and is derived from renal cysts of an ADPKD kidney. These cells have a single detectable truncating mutation (Q4004X in polycystin-1. These cells make normal appearing but shorter cilia and fail to assemble polycystin-1 in the cilia, and less uncleaved polycystin-1 in membrane fractions. This cell line has been maintained in continuous passage for over 35 passages without going into senescence. Nephron segment specific markers suggest a proximal tubule origin for these cells and the cell line will be useful to study mechanistic details of cyst formation in proximal tubule cells.

  3. [Telomerase reverse transcriptase (TERT) promoter mutations in the tumors of human endocrine organs: Biological and prognostic value].

    Science.gov (United States)

    Selivanova, L S; Volganova, K S; Abrosimov, A Y U

    2016-01-01

    The analysis of the data available in the literature has shown that telomerase reverse transcriptase TERT promoter may serve as promising markers of malignancy, aggressive disease course, and poor prognosis for malignant tumors of endocrine organs. Considering the established association of mutations with tumors having a poor prognosis (high-grade and anaplastic carcinoma of the thyroid), it is reasonable to perform prognostic-value investigations in a group of low-grade thyroid carcinomas that may occasionally recur and may be resistant to radioactive iodine therapy, i.e. can demonstrate a poor course and prognosis. TERT promoter mutations may be a specific marker of the clinically aggressive forms of adrenocortical carcinoma, but the determination of its diagnostic value calls for additional investigations that will have the larger number cases and establish the association with clinical features and survival rates.

  4. Coevolution of telomerase activity and body mass in mammals: from mice to beavers.

    Science.gov (United States)

    Gorbunova, Vera; Seluanov, Andrei

    2009-01-01

    Telomerase is repressed in the majority of human somatic tissues. As a result human somatic cells undergo replicative senescence, which plays an important role in suppressing tumorigenesis, and at the same time contributes to the process of aging. Repression of somatic telomerase activity is not a universal phenomenon among mammals. Mice, for example, express telomerase in somatic tissues, and mouse cells are immortal when cultured at physiological oxygen concentration. What is the status of telomerase in other animals, beyond human and laboratory mouse, and why do some species evolve repression of telomerase activity while others do not? Here we discuss the data on telomere biology in various mammalian species, and a recent study of telomerase activity in a large collection of wild rodent species, which showed that telomerase activity coevolves with body mass, but not lifespan. Large rodents repress telomerase activity, while small rodents maintain high levels of telomerase activity in somatic cells. We discuss a model that large body mass presents an increased cancer risk, which drives the evolution of telomerase suppression and replicative senescence.

  5. Dysfunctional telomeres promote genomic instability and metastasis in the absence of telomerase activity in oncogene induced mammary cancer.

    Science.gov (United States)

    Bojovic, Bojana; Crowe, David L

    2013-02-01

    Telomerase is a ribonucleoprotein that maintains the ends of chromosomes (telomeres). In normal cells lacking telomerase activity, telomeres shorten with each cell division because of the inability to completely synthesize the lagging strand. Critically shortened telomeres elicit DNA damage responses and limit cellular division and lifespan, providing an important tumor suppressor function. Most human cancer cells express telomerase which contributes significantly to the tumor phenotype. In human breast cancer, telomerase expression is predictive of clinical outcomes such as lymph node metastasis and survival. In mouse models of mammary cancer, telomerase expression is also upregulated. Telomerase overexpression resulted in spontaneous mammary tumor development in aged female mice. Increased mammary cancer also was observed when telomerase deficient mice were crossed with p53 null mutant animals. However, the effects of telomerase and telomere length on oncogene driven mammary cancer have not been completely characterized. To address these issues we characterized neu proto-oncogene driven mammary tumor formation in G1 Terc-/- (telomerase deficient with long telomeres), G3 Terc-/- (telomerase deficient with short telomeres), and Terc+/+ mice. Telomerase deficiency reduced the number of mammary tumors and increased tumor latency regardless of telomere length. Decreased tumor formation correlated with increased apoptosis in Terc deficient tumors. Short telomeres dramatically increased lung metastasis which correlated with increased genomic instability, and specific alterations in DNA copy number and gene expression. We concluded that short telomeres promote metastasis in the absence of telomerase activity in neu oncogene driven mammary tumors.

  6. Telomerase regulation and the intimate relationship with aging

    Directory of Open Access Journals (Sweden)

    Morgan G

    2013-06-01

    Full Text Available Garrett Morgan1–31Department of Physiology, University of Utah School of Medicine, University of Utah, 2George E. Wahlen Department of Veterans Affairs Medical Center, 3Geriatric Research, Education and Clinical Center, VA Salt Lake City Health Care System, Salt Lake City, UT, USAAbstract: Advancing age in humans is associated with a decline in function of physiological systems, as well as the onset and progression of many chronic diseases. An ongoing focus of basic aging research is to identify biomarkers that could be used to assess the severity of age-related diseases or that could also be modulated to treat them. Telomerase is an enzyme with mechanistic links to telomere length homeostasis and cell viability. Human telomerase is a ribonucleoprotein complex that consists of its catalytic subunit (hTERT and ribonucleic acid template (hTERC. Telomerase adds de novo TTAGGG repeats to the ends of telomeres, which may prevent telomere shortening and subsequent dysfunction in certain cell compartments. Telomerase function within the cell represents a growing area of interest among biologists who study human aging, due to its potential use as a biomarker and therapeutic target for chronic disease. Here I review the basic biology of telomerase and the consequences of telomerase overexpression and deficiency in human cells and animal models. I discuss the putative role of telomerase in telomere-length homeostasis with advancing age in humans. Finally, I describe novel nontelomeric activities of telomerase related to cell viability and mitochondrial function.Keywords: chronic disease, inflammation, oxidative stress, hTERT, hTERC, mitochondria

  7. Culture of Human Tendon Cell Transfected by Human Telomerase Reverse Transcriptase Plasmid and their Biological CharacteristicsIn Vitro

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    1 IntroductionThe proliferative and functional characteristics of tendon cell are the key issue in the research of tissue engineered tendon. The standard tendon cell line, which has normal functional characteristics, and can be subcultured continuously and permanently, will not only meet the demands of seeding cell in tissue engineered tendon, but also control the variable of tendon cell. In our previous study, it showed that the proliferative ability of human tendon cell cultured in vitro is depressed grad...

  8. 人TR基因真核表达载体的构建%Construction of eukaryotic expression vector of human telomerase RNA component and its function

    Institute of Scientific and Technical Information of China (English)

    营孙阳; 熊加秀; 麦洪旭; 林佳佳; 姜丽娜; 程龙; 叶棋浓

    2016-01-01

    目的 构建人端粒酶RNA组分( hTR)的真核表达载体,并对其生物学功能进行初步检测. 方法 以人胚肾293 T细胞RNA逆转录得到的cDNA为模板,采用PCR技术扩增出hTR编码序列,将其插入到pCDNA 3 .0载体中;将重组质粒与空载体分别转染293T细胞,实时荧光定量PCR(real-time quantitative PCR, qRT-PCR)检测其表达情况,在HepG2细胞中构建此基因的稳定细胞系并用qRT-PCR检测其效果,通过RNA结合免疫共沉淀( RIP )实验验证其和人端粒酶逆转录酶( hTERT )及角化不良蛋白( dyskerin )的相互作用,通过端粒酶重复序列扩增技术( telomerase repeat amplification protocol ,TRAP)检测过表达hTR后HepG2细胞的端粒酶活性. 结果 双酶切和测序结果表明,pCDNA3.0-hTR的真核表达载体构建成功;转染293T细胞用qRT-PCR证明成功表达;qRT-PCR证实HepG2 pCDNA3.0-hTR稳定细胞系筛选成功;RIP实验证实了hTR与hTERT和角化不良蛋白之间存在相互作用;TRAP实验发现过表达hTR并不影响HepG2细胞的端粒酶活性. 结论 成功构建了pCDNA 3.0-hTR真核表达载体,为进一步研究以针对hTR为靶标的癌症治疗奠定了基础.%Objective To construct the eukaryotic expression vector of human telomerase RNA component ( hTR) and study its biological function tentatively .Methods hTR Gene was obtained by PCR from cDNA template , which was reverse transcribed from 293T mRNA and cloned into pCDNA3.0 vector.The recombinant plasmid and empty vector were trans-fected into 293T cells, and hTR expression was identified by qRT-PCR.HepG2 cells that stably transfected with pCDNA3.0-hTR were constructed and identified by qRT-PCR.These cells were used to assess the interaction of hTR with human telomerase revese transcriptase ( hTERT ) and dyskerin .Telomerase activity was also detected in HepG 2 cells transfected with pCDNA3.0-hTR.Results pCDNA3.0-hTR eukaryotic expression vector was successfully constructed by double digestion

  9. Kaempferol increases apoptosis in human cervical cancer HeLa cells via PI3K/AKT and telomerase pathways.

    Science.gov (United States)

    Kashafi, Elham; Moradzadeh, Maliheh; Mohamadkhani, Ashraf; Erfanian, Saiedeh

    2017-02-28

    Cervical cancer is one of the most frequent cancers in women worldwide. Defects in the apoptotic pathways are responsible for both the disease pathogenesis and its therapy resistance. It is thus a good candidate for treatment by pro-apoptotic agents. Kaempferol as a flavonoid has antioxidant and anti-tumor properties. Kaempferol has been shown to induce apoptosis and cell death in cancer cells. However, due to the problems in the treatment of cervical cancer, this study is designed to investigate the molecular mechanism by which kaempferol suppresses the growth of cervical cancer HeLa cell as compared with HFF cells (normal cells). Cells treated with kaempferol (12-100μM) and 5-FU (1-10μM), as the positive control, up to 72h. Cell viability was determined by MTT assay and real time PCR was used to investigate apoptosis and telomerase genes expression. The results showed that kaempferol decreased cell viability as concentration- and time-dependently. IC50 values were 10.48μM for HeLa and 707.00μM for HFF cells, as compared with 1.40μM and 16.38μM for 5-FU after 72h treatment, respectively. Also, kaempferol induced cellular apoptosis and aging through down-regulating the PI3K/AKT and hTERT pathways. This study suggests that kaempferol may be a useful adjuvant therapeutic agent in the treatment of cervical cancer.

  10. Inhibition of Leukemic Cell Telomerase Activity by Antisense Phosphorothioate Oligodeoxynucleotides

    Institute of Scientific and Technical Information of China (English)

    HEDongmei; ZHANGYuan

    2002-01-01

    Objective To evaluate the effect of human telomerase reverse transcriptase(hTERT) gene antisense oligodeoxynucleotide (ASON) on telomerase activity in K562 cells.Methods Telomerase activity was detemined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA) in K562 cells treated with ASODN and hTERTmRNA expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The hTERTmRNA level was decreased,and telomerase activity was significantly inhibited when the K562 cells were treated with ASODN for 48 h. Conclusion It is suggested that hTETR ASODN might specifically inhibit telomrase activity of K562 cells at translation level,and it is further proved that hTERT gene has significant correlation with telopmerase activity.

  11. Expression of telomerase hTERT in human non-small cell lung cancer and its correlation with c-myc gene

    Institute of Scientific and Technical Information of China (English)

    耿志华; 张敦华; 刘银坤

    2003-01-01

    Objective To investigate the expression of human telomerase catalytic subunit, hTERT, in human non-small cell lung cancer (NSCLC) and its correlations to c-myc gene.Methods hTERT and c-myc mRNA expressions were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Statistical correlation analysis was made to estimate whether there was interrelation between them.Results Positive rate of hTERT expression in 51 surgically resected lung cancer specimens was 86.3%, significantly higher than that in adjacent non-neoplastic lung tissues and benign lesions, which were 14.3% and 27.3% respectively. No statistical significance was observed between the frequency of hTERT expression and histologic types, degree of differentiation, TNM stages, tumor size or lymph nodes metastases. Correlation analysis revealed that the expression of c-myc gene was significantly related to that of hTERT (correlation coefficient, r=0.633, P<0.001).Conclusions hTERT may be a useful tumor marker in diagnosing lung cancer. Significant correlation between the expression of hTERT and c-myc mRNA indicates that the activation and up-regulation of hTERT might be conferred by over-expression of c-myc gene.

  12. Telomerase expression in sebaceous carcinoma of the eyelid

    Institute of Scientific and Technical Information of China (English)

    李彬; 李宁东; 顼晓琳; 郑邦和; 孙宪丽; 李辽青; 陈长喜

    2004-01-01

    Background In humans telomerase is expressed in most cancers and immortal cell lines, and astivation of telomerase may play important roles in tumorigenesis and immortalization. This study was to investigate the roles of telomerase activity (TA) and human telomerase RNA (hTR) in sebaceous carcinoma of the eyelid.Methods The telomerase repeated amplification protocal (TRAP) was used to demonstrate telomerase activity in 12 cases of sebaceous carcinoma of the eyelid. In situ hybridization (ISH) was used to demonstrate the expression of hTR in 55 cases of paraffin-embedded sebaceous carcinoma of the eyelid, and the results were compared with the proliferative index determined by Mib-1 immuno-labeling, histological patterns and recurrence of the tumor.Results Different telomerase activity was shown in the 12 cases of sebaceous carcinoma of the eyelid. The positive expression of hTR was 85.5% (47/55) in tumor cells, but not in the adjacent tissues. The positive expression of hTR was correlated with the proliferative activity (as assessed by Mib-1 immunolabelling, r=0.942, P<0.001) and the differentiation of sebaceous carcinoma of the eyelid (χ2=17.621, P<0.001), but not significantly related to tumor recurrence. The level of hTR expression increased with the decrease of differentiation of sebaceous carcinoma of the eyelid.Conclusions The results suggest that the up-regulation of telomerase expression plays some roles in tarsal gland carcinogenesis, and the expression of hTR is a useful marker for malignant degree of sebaceous carcinoma of the eyelid.

  13. Brief Report: Differential Effects of Tenofovir, Abacavir, Emtricitabine, and Darunavir on Telomerase Activity In Vitro.

    Science.gov (United States)

    Stella-Ascariz, Natalia; Montejano, Rocío; Pintado-Berninches, Laura; Monge, Susana; Bernardino, José I; Pérez-Valero, Ignacio; Montes, María L; Mingorance, Jesús; Perona, Rosario; Arribas, José R

    2017-01-01

    In vitro, tenofovir and abacavir induced a significant dose-dependent inhibition of telomerase activity at therapeutic concentrations in peripheral blood mononuclear cells of healthy subjects. Median inhibition of telomerase activity by tenofovir at 0.5 and 1 μM was 29% [Interquartile range (IQR) 29%-34%, P = 0.042] and 28% (IQR 28%-41%, P = 0.042), respectively. Abacavir inhibition was 12% (IQR 9%-13%, P = 0.043) at 3 μM and 14% (IQR 10%-29%, P = 0.043) at 10 μM. Tenofovir and abacavir did not change human telomerase reverse transcriptase (hTERT) levels or mRNA levels of other telomerase complex genes. Exposure to emtricitabine or darunavir did not affect telomerase activity, hTERT protein levels, or mRNA levels of telomerase/shelterin genes.

  14. [Telomerase activity in esophageal carcinoma and lesions unstained with Lugol's solution].

    Science.gov (United States)

    Yoneyama, K; Aoyama, N; Koizumi, H; Tamai, S

    1998-05-01

    Telomerase is a specific enzyme required for the replication of telomeres. Its activity is detected in almost human cancers. We examined in esophageal carcinoma and lesions unstained with Lugol's solution telomerase activity by using telomeric repeat amplification protocol (TRAP) assay. Telomerase activity was detected in all 22 esophageal carcinomas, regardless of histopathological findings. In unstained lesions, telomerase activity was detected in 15 of 22; 10 squamous cell carcinomas, four dysplasia, one regenerative epithelium, no telomerase activity was found in seven; four normal esophageal epithelia, two Barrett's esophagi, one regenerative epithelium. These results suggest that telomerase activity may be a useful molecular marker for the diagnosis of esophageal carcinoma and of the early esophageal carcinoma in area unstained with Lugol's solution.

  15. Stem cell function and maintenance - ends that matter: Role of telomeres and telomerase

    Indian Academy of Sciences (India)

    Hamid Saeed; Mehwish Iqtedar

    2013-09-01

    Stem cell research holds a promise to treat and prevent age-related degenerative changes in humans. Literature is replete with studies showing that stem cell function declines with aging, especially in highly proliferative tissues/organs. Among others, telomerase and telomere damage is one of the intrinsic physical instigators that drive agerelated degenerative changes. In this review we provide brief overview of telomerase-deficient aging affects in diverse stem cells populations. Furthermore, potential disease phenotypes associated with telomerase dysregulation in a specific stem cell population is also discussed in this review. Additionally, the role of telomerase in stem cell driven cancer is also briefly touched upon.

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

    Science.gov (United States)

    Gandhi, Puneet; Khare, Richa; Niraj, Kavita; Garg, Nitin; Sorte, Sandeep K; Gulwani, Hanni

    2016-09-16

    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 (hTERT) 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 neutrophil-lymphocyte ratio, are the other parameters assessed. Findings suggest that hTERT 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.

  17. Structural Mechanism Underpinning Cross-reactivity of a CD8+ T-cell Clone That Recognizes a Peptide Derived from Human Telomerase Reverse Transcriptase*

    Science.gov (United States)

    Cole, David K.; van den Berg, Hugo A.; Lloyd, Angharad; Crowther, Michael D.; Beck, Konrad; Ekeruche-Makinde, Julia; Miles, John J.; Bulek, Anna M.; Dolton, Garry; Schauenburg, Andrea J.; Wall, Aaron; Fuller, Anna; Clement, Mathew; Laugel, Bruno; Rizkallah, Pierre J.; Wooldridge, Linda; Sewell, Andrew K.

    2017-01-01

    T-cell cross-reactivity is essential for effective immune surveillance but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focused antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8+ T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase. The ILA1 TCR contacted its pMHC with a broad peptide binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding, the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity with important implications for pathogen surveillance, autoimmunity, and transplant rejection. PMID:27903649

  18. A model for triple helix formation on human telomerase reverse transcriptase (hTERT) promoter and stabilization by specific interactions with the water soluble perylene derivative, DAPER.

    Science.gov (United States)

    Rossetti, Luigi; D'Isa, Giuliana; Mauriello, Clementina; Varra, Michela; De Santis, Pasquale; Mayol, Luciano; Savino, Maria

    2007-08-01

    The promoter of human telomerase reverse transcriptase (hTERT) gene, in the region from -1000 to +1, contains two homopurine-homopyrimidine sequences (-835/-814 and -108/-90), that can be considered as potential targets to triple helix forming oligonucleotides (TFOs) for applying antigene strategy. We have chosen the sequence (-108/-90) on the basis of its unfavorable chromatin organization, evaluated by theoretical nucleosome positioning and nuclease hypersensitive sites mapping. On this sequence, anti-parallel triplex with satisfactory thermodynamic stability is formed by two TFOs, having different lengths. Triplex stability is significantly increased by specific interactions with the perylene derivative N,N'-bis[3,3'-(dimethylamino) propylamine]-3,4,9,10-perylenetetracarboxylic diimide (DAPER). Since DAPER is a symmetric molecule, the induced Circular Dichroism (CD) spectra in the range 400-600 nm allows us to obtain information on drug binding to triplex and duplex DNA. The drug-induced ellipticity is significantly higher in the case of triplex with respect to duplex and, surprisingly, it increases at decreasing of DNA. A model is proposed where self-stacked DAPER binds to triplex or to duplex narrow grooves.

  19. TCAB1: driving telomerase to Cajal bodies.

    Science.gov (United States)

    Venteicher, Andrew S; Artandi, Steven E

    2009-05-01

    Telomerase supports the proliferation of progenitor cells and tumor cells by adding telomere repeats to chromosome ends. The low abundance and restricted expression pattern of telomerase have limited our knowledge of this important enzyme. A new telomerase protein, TCAB1, sheds light on the pathway that governs telomerase holoenzyme assembly and function in vivo. TCAB1 is a component of active telomerase and is required for the telomerase holoenzyme to accumulate in Cajal bodies and to elongate telomeres. These findings provide important new insights into how telomerase functions in cancer and in stem cell biology.

  20. Relationship between cervical cancer and human telomerase gene%宫颈癌与人端粒酶基因关系的研究

    Institute of Scientific and Technical Information of China (English)

    李亚波; 王卓; 杨红英

    2013-01-01

    Objective: To explore the correlation between cervical cancer and the expression of human telomerase RNA component (TERC) gene and evaluate the significance of detecting TERC gene expression by fluorescence in situ hybridization (FISH) technique for predicting the progress from cervical intraepithelial neoplasia (CIN) to cervical cancer. Methods; TERC gene expression was detected by the method of FISH in uterine epithelial exfoliated cells from 81 patients including 20 patients in normal group, 28 patients in CIN1 group, 12 patients in CIN2 group, 9 patients in CIN3 group, and 12 patients in cervical cancer group. The correlation between cervical cancer and TERC gene was analyzed. Results: The positive rate of TERC gene test in normal group was significant lower than CIN1 group, CIN2 group (P0. 05) . There was also significant difference between CIN3 group and cervical cancer group (P<0. 05) . The higher malignant degree was, the more significant difference would be (P<0. 01) . Conclusions: The abnormal expression of TERC gene is closely related with the process of cervical cancer. The FISH technique for detecting TERC gene may be an effective method for helping to diagnose early cervical cancer.%目的 通过研究宫颈癌人端粒酶基因(Human telomerase RNA component gene,TERC gene)的表达情况,探讨宫颈癌与TERC基因异常表达的相关性,评估荧光原位杂交(Fluorescence in situ hybridization,FISH)技术检测TERC基因的表达对子宫颈上皮内瘤样病变(Cervical intraepithelial neoplasia,CIN)发展到宫颈癌的预测价值. 方法 采用FISH技术进行子宫颈上皮脱落细胞TERC基因的检测,实验对象81例,病理活检正常组20例,CIN1组28例,CIN2组12例,CIN3组9例,宫颈癌组1 2例.并进行宫颈癌与TERC基因相关性分析. 结果 在CIN1组、CIN2组、CIN3组和宫颈癌组中,TERC基因的表达均显著高于正常组(P<0.05);CIN1组和CIN2组之间无显著差异(P>0.05);CIN3组

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    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.

  2. Metastases suppressor NME2 associates with telomere ends and telomerase and reduces telomerase activity within cells.

    Science.gov (United States)

    Kar, Anirban; Saha, Dhurjhoti; Purohit, Gunjan; Singh, Ankita; Kumar, Parveen; Yadav, Vinod Kumar; Kumar, Pankaj; Thakur, Ram Krishna; Chowdhury, Shantanu

    2012-03-01

    Analysis of chromatin-immunoprecipitation followed by sequencing (ChIP-seq) usually disregards sequence reads that do not map within binding positions (peaks). Using an unbiased approach, we analysed all reads, both that mapped and ones that were not included as part of peaks. ChIP-seq experiments were performed in human lung adenocarcinoma and fibrosarcoma cells for the metastasis suppressor non-metastatic 2 (NME2). Surprisingly, we identified sequence reads that uniquely represented human telomere ends in both cases. In vivo presence of NME2 at telomere ends was validated using independent methods and as further evidence we found intranuclear association of NME2 and the telomere repeat binding factor 2. Most remarkably, results demonstrate that NME2 associates with telomerase and reduces telomerase activity in vitro and in vivo, and sustained NME2 expression resulted in reduced telomere length in aggressive human cancer cells. Anti-metastatic function of NME2 has been demonstrated in human cancers, however, mechanisms are poorly understood. Together, findings reported here suggest a novel role for NME2 as a telomere binding protein that can alter telomerase function and telomere length. This presents an opportunity to investigate telomere-related interactions in metastasis suppression.

  3. Cancer Immunotherapy Targeting the Telomerase Reverse Transcriptase

    Institute of Scientific and Technical Information of China (English)

    Longfei Huo; Janice WS Tang; Junjian Huang; Peitang Huang; Cuifen Huang; Hsiang-fu Kung; Marie C. Lin

    2006-01-01

    The human telomerase reverse transcriptase (hTERT) is expressed in more than 85% of tumor cells but is usually not found in normal cells, which makes hTERT as an ideal tumor-associate antigen (TAA) to develop potential vaccine specifically destroying cancers without impairing normal tissues in human cancer immunotherapy. Here are reviewed the fundamental advances of studies on immunogenicity of hTERT or its peptides and the early clinical trials using the hTERT vaccine approach in the last decades.

  4. Effect of newly identified hTERT-interacting proteins on telomerase activity

    Institute of Scientific and Technical Information of China (English)

    Lina Zhou; Bing Chen; Xing Hua; Ping Zhou; Lian Guo; Yong Peng; Kunhua Qiu

    2013-01-01

    There is a close relationship between telomeres-telomerase and age-related disease.Human telomerase reverse transcriptase (hTERT) is both the catalytic component of human telomerase and the rate-limiting determinant of telomerase activity.Its transcriptional regulation is the primary mode of control of telomerase activity.It is critical to find the proteins interacting with hTERT for exploring the regulatory mechanisms of the hTERT expression and the telomerase activity.In this study,the yeast two-hybrid system was used to screen the potential interactive proteins of hTERT.Six proteins were obtained,among which TSTAR,LOXL3,HKR3,and Par-4 were further confirmed as the interacting proteins of hTERT by co-immunopreci-pitation.Then the sense and antisense gene eukaryotic expression vectors containing these four genes were constructed and transfected into tumor cell lines.The correlations among the expression levels of these four proteins,the expression level of hTERT,and the telomerase activity were analyzed.Results showed that the up-regulation of TSTAR expression and down-regulation of HKR3 expression led to the increase of hTERT expression and telomerase activity,while the up-and down-regulation of LOXL3 and Par-4 expressions had no obvious effect.Our results suggested that T-STAR has a positive correlation with the telomerase activity while HKR3 may be a negative regulator.This conclusion is important to further explore the influencing factors or regulation pathways of human telomerase activity,which may be of great importance for the potential clinical application.

  5. ARSENIC TRIOXIDE DOWNREGULATES TELOMERASE ACTIVITY IN HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    何冬梅; 张洹

    2002-01-01

    Objective: To evaluate whether arsenic trioxide (AS2O3) could downregulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity during induction of apoptosis of HL-60 cells. Methods: Apoptosis was detected by morphological observation and flow cytomertric cell cycle analysis. The expression of hTERT at mRNA and protein levels was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence using fluoresce isothiocyanate (FITC) label, respectively. Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Results: Treatment of 2 μmol/L at As2O3 could induce apoptosis of HL-60 cells. hTERT was decreased at both mRNA and protein levels during apoptosis of HL-60 cells. Telomerase activity of HL-60 cells was significantly inhibited. Conclusion:It is suggested that telomerase activity of HL-60 cells might be specifically inhibited by AS2O3 through the downregulation of hTERT gene expression.

  6. Progressive Increase in Telomerase Activity From Benign Melanocytic Conditions to Malignant Melanoma

    Directory of Open Access Journals (Sweden)

    Ruben D. Ramirez

    1999-04-01

    Full Text Available The expression of telomerase activity and the in situ localization of the human telomerase RNA component (hTR in melanocytic skin lesions was evaluated in specimens from sixty-three patients. Specimens of melanocytic nevi, primary melanomas and subcutaneous metastases of melanoma were obtained from fifty-eight patients, whereas metastasized lymph nodes were obtained from five patients. Telomerase activity was determined in these specimens by using a Polymerase Chain Reaction—based assay (TRAP. High relative mean telomerase activity levels were detected in metastatic melanoma (subcutaneous metastasess = 54.5, lymph node metastasess = 56.5. Much lower levels were detected in primary melanomas, which increased with advancing levels of tumor cell penetration (Clark II = 0.02, Clark III = 1.1, and Clark IV = 1.9. Twenty-six formalin-fixed, paraffin-embedded melanocytic lesions were sectioned and analyzed for telomerase RNA with a radioactive in situ hybridization assay. In situ hybridization studies with a probe to the template RNA component of telomerase confirmed that expression was almost exclusively confined to tumor cells and not infiltrating lymphocytes. These results indicate that levels of telomerase activity and telomerase RNA in melanocytic lesions correlate well with clinical stage and could potentially assist in the diagnosis of borderline lesions.

  7. Relationship between human telomerase gene amplification and infection of human papillomavirus in cervical disease between Uygur and Han patients in Xinjiang%新疆维吾尔族、汉族妇女宫颈病变人类染色体端粒酶基因扩增与人乳头瘤病毒感染相关性研究

    Institute of Scientific and Technical Information of China (English)

    韩英; 艾星子·艾里; 段玲; 陈志芳; 苏晶; 丁岩

    2011-01-01

    Objective To investigate the difference of human telomerase gene amplification in cervical exfoliated cells between Uygur and Hah patients with cervical diseases, and their relationship with high-risk subtypes of human papillomavirus infection.Methods Fifty Uygur and 52 Han patients with cervical diseases were detected with fluorescence in situ hybridization to compare the amplification of human telomerase gene, and human papillomavirus infection was detected.Results Human telomerase gene amplification rates were 62.00% and 51.92% in Uygur and Han patients with cervical disease.With the progression of disease, the human telomerase gene amplification rate increased significantly.There were significant differences in average human telomerase gene multiples amplification between multiple infection and single infection(P<0.05), and no significant differences among the infections of each human papillomavirus subtype in Uygur and Han patients.However,there was significant difference in the total of human telomerase gene multiple amplification between Uygur and Han patients ( P< 0.05 ).Conclusion Human telomerase gene amplification is correlated with high-risk human papillomavirus infection, and it might be a predictor of progression of cervical cancer.Multiple high-risk subtype of human papillomavirus infection may be one of causes of the high incidence of cervical cancer in Uygur patients.%目的:探讨新疆地区维吾尔族(维族)、汉族妇女宫颈病变脱落细胞中人类染色体端粒酶基因(human telomerase gene,hTERC)的表达差异及其与人乳头瘤病毒(human papillomavirus,HPV)高危亚型感染的相关性.方法:采用荧光原位杂交技术,比较50例维族和52例汉族宫颈病变患者hTERC基因扩增情况,同时检测HPV感染情况.结果:维族宫颈病变患者hTERC基因扩增率为62.00%,汉族宫颈病变hTERC基因扩增率为51.92%.随病变进展.维、汉族官颈病变患者hTERC基因扩增均明显增加.维、汉

  8. Determination of estrogen receptor {beta}-mediated estrogenic potencies of hydroxylated PCBS by a yeast two-hybrid assay

    Energy Technology Data Exchange (ETDEWEB)

    Kuroki, H.; Kumate, M.; Nakaoka, H.; Yonekura, S. [Daiichi Coll. of Pharmaceutical Sciences, Fukuoka (Japan); Nishikawa, J.; Nishihara, T. [Osaka Univ., Osaka (Japan)

    2004-09-15

    Several environmental phenolic chemicals such as Nonylphenol and Bisphenol A (BPA) have been previously shown to possess estrogenic properties. In the previous paper, we have investigated the estrogenic activity of a series of hydroxylated PCBs (OH-PCBs) by a yeast two-hybrid assay (estrogen receptor{alpha} (ER{alpha}) -TIF2), in which the expression of estrogenic activity is based on the interaction of chemicals with ER{alpha}, and demonstrated that 4'-OH-CB30 and 4'-OH-CB61 are more estrogenic than BPA, one of the environmental estrogens. We have showed that one chlorine substitution adjacent to 4-OH at 3- or 5-position significantly reduces the ER{alpha}-mediated estrogenic activity of 4-OH-PCBs. Thus, 4'-OH-CB25 and 4-OH-CB56 showed a very weak estrogenicity. We have also showed that 4-OH-PCBs with two chlorine substitutions adjacent to 4-OH at 3- and 5-position such as 4'-OH-CB79 (hydroxylated metabolite of CB77) and persistent 4-OH-PCBs retained in human blood (4-OH-CB107, 4-OH-CB146 and 4-OH-CB187) have no ER{alpha}-mediated estrogenic activity. ER is known to have two subtypes, namely ER{alpha} and ER{beta} and it is reported that ligand, some agonist and antagonist have a different binding affinity for ER{alpha} and ER{beta}. However, there is limited information on ER{beta}-mediated endocrine disrupting potency. In this study, we examined the ER{beta}-mediated estrogenic activity of a series of OH-PCBs, including environmentally relevant 4-OH-PCBs by a yeast two-hybrid assay (ER{beta}-TIF2).

  9. TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site.

    Science.gov (United States)

    Azhibek, Dulat; Skvortsov, Dmitry; Andreeva, Anna; Zatsepin, Timofei; Arutyunyan, Alexandr; Zvereva, Maria; Dontsova, Olga

    2016-06-01

    Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G-rich sequence, and with noncoding RNA, Telomeric repeat-containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2'-OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G-quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate - the 3'-end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3'-end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

  10. Interleukin-1beta mediates ischemic injury in the rat retina.

    Science.gov (United States)

    Yoneda, S; Tanihara, H; Kido, N; Honda, Y; Goto, W; Hara, H; Miyawaki, N

    2001-11-01

    Two types of experiment were performed to examine the role of interleukin-1beta in ischemia-induced damage in the rat retina. In the in vivo study, enzyme-linked immunosorbent assay was used to investigate the expression of immunoreactive interleukin-1beta in the rat retina following a hypertension-induced ischemia/reperfusion, while the effect of a recombinant human interleukin-1 receptor antagonist or an anti-interleukin-1beta neutralizing antibody on the ischemia-induced damage was examined histologically. A transient increase in the expression of immunoreactive interleukin-1beta was observed in the retina 3-12 hr after reperfusion, and morphometric evaluation at 7 days after the ischemia showed a decrease in cell numbers in the ganglion cell layer and a decreased thickness of the inner plexiform layer with no change in the other retinal layers. Intravitreal injection of interleukin-1 receptor antagonist (1 or 10 ng per eye) or anti-interleukin-1beta antibody (50 or 500 ng per eye) 5 min before the onset of the ischemia reduced the damage. In the in vitro study, interleukin-1 receptor antagonist (500 ng ml(-1)) significantly reduced glutamate-induced neurotoxicity in rat cultured retinal neurons. These results suggest that interleukin-1 plays an important role in mediating ischemic and excitotoxic damage in the retina, and that interleukin-1 inhibitors may be therapeutically useful against neuronal injury caused by optic nerve or retinal diseases such as glaucoma and central retinal artery or vein occlusion.

  11. Diagnosis of pancreatic cancer by cytology and telomerase activity in exfoliated cells obtained by pancreatic duct brushing during endoscopy

    Institute of Scientific and Technical Information of China (English)

    Guo-Xiong Zhou; Jie-Fei Huang; Hong Zhang; Jian-Ping Chen

    2007-01-01

    BACKGROUND:Telomerase activity is reported to be speciifc and frequent in human pancreatic cancer. We conducted this study to assess the usefulness of monitoring telomerase activity in exfoliated cells obtained by pancreatic duct brushing during endoscopic retrograde cholangiopancreatography (ERCP) for the diagnosis of pancreatic cancer. METHODS:Exfoliated cells obtained by pancreatic duct brushing during ERCP from 21 patients (18 with pancreatic cancer, 3 with chronic pancreatitis) were examined. Telomerase activity was detected by polymerase chain reaction and telomeric repeat ampliifcation protocol assay (PCR-TRAP-ELISA). RESULTS:D450 values of telomerase activity were 0.446± 0.2700 in pancreatic cancer and 0.041±0.0111 in chronic pancreatitis. 77.8% (14/18) of patients with pancreatic cancer had cells with telomerase activity. None of the samples from patients with chronic pancreatitis showed telomerase activity, when the cutoff value of telomerase activity was set at 2.0. Cytological examination showed cancer cells in 66.7%(12/18) of the patients. CONCLUSIONS:Telomerase activity may be an early malignant event in pancreatic cancer development. Cytology and telomerase activity in cells obtained by pancreatic duct brushing may complement each other for the diagnosis of pancreatic cancer.

  12. A three-state model for the regulation of telomerase by TERRA and hnRNPA1.

    Science.gov (United States)

    Redon, Sophie; Zemp, Ivo; Lingner, Joachim

    2013-10-01

    Telomeres, the physical ends of eukaryotic chromosomes, are transcribed into telomeric repeat-containing RNA (TERRA), a large non-coding RNA, which forms an integral part of telomeric heterochromatin. In vitro, naked TERRA molecules are efficient inhibitors of human telomerase, base-pairing via their 5'-UUAGGG-3' repeats with the template sequence of telomerase RNA, in addition to contacting the telomerase reverse transcriptase protein subunit. In vivo, however, TERRA-mediated inhibition of telomerase can be prevented by unknown mechanisms. Also, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) has been implicated in telomere length control. In vivo, TERRA is partially associated with hnRNPA1, and hnRNPA1 is also detected at telomeres. We demonstrate that on binding of TERRA, hnRNPA1 can alleviate the TERRA-mediated inhibition of telomerase. However, when in excess over TERRA, hnRNPA1 becomes itself an inhibitor of telomere extension, on binding of the telomeric DNA substrate. Yet, hnRNPA1 has no notable direct effects on the telomerase catalysis. Our in vitro results suggest that TERRA-mediated telomerase inhibition may be prevented by hnRNPA1 in vivo. Telomere extension by telomerase may require balanced levels of TERRA and hnRNPA1 at telomeres. Thus, TERRA and hnRNPA1 can function as a bimolecular regulator to turn telomerase and the telomere on and off.

  13. DETECTION OF TELOMERASE ACTIVITY IN BREAST CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    Yang Wentao; Xu Liangzhong; Zhang Taiming; Zhu weiping; Li Xiaomei; Jin Aiping

    1998-01-01

    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 16benign 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 nodenegative 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.

  14. Identification of Telomerase Components and Telomerase Regulating Factors in Yeast

    Science.gov (United States)

    1998-07-01

    required for telomere function. To further explore the mechanism of telomere replication, I identified CDCl3 and EST2 interacting proteins via yeast two...hybrid screens. Characterization of the EST2 interacting proteins may reveal additional telomerase components or regulators. STNl, a high-copy

  15. Telomerase functions beyond telomere maintenance in primary cutaneous T-cell lymphoma.

    Science.gov (United States)

    Chevret, Edith; Andrique, Laetitia; Prochazkova-Carlotti, Martina; Ferrer, Jacky; Cappellen, David; Laharanne, Elodie; Idrissi, Yamina; Boettiger, Anna; Sahraoui, Wafa; Ruiz, Florian; Pham-Ledard, Anne; Vergier, Beatrice; Belloc, Francis; Dubus, Pierre; Beylot-Barry, Marie; Merlio, Jean-Philippe

    2014-03-20

    Telomere erosion may be counteracted by telomerase. Here we explored telomere length (TL) and telomerase activity (TA) in primary cutaneous T-cell lymphoma (CTCL) by using quantitative polymerase chain reaction and interphase quantitative fluorescence in situ hybridization assays. Samples from patients with Sézary syndrome (SS), transformed mycosis fungoides (T-MF), and cutaneous anaplastic large cell lymphoma were studied in parallel with corresponding cell lines to evaluate the relevance of TL and TA as target candidates for diagnostic and therapeutic purposes. Compared with controls, short telomeres were observed in aggressive CTCL subtypes such as SS and T-MF and were restricted to neoplastic cells in SS. While no genomic alteration of the hTERT (human telomerase catalytic subunit) locus was observed in patients' tumor cells, TA was detected. To understand the role of telomerase in CTCL, we manipulated its expression in CTCL cell lines. Telomerase inhibition rapidly impeded in vitro cell proliferation and led to cell death, while telomerase overexpression stimulated in vitro proliferation and clonogenicity properties and favored tumor development in immunodeficient mice. Our data indicate that, besides maintenance of TL, telomerase exerts additional functions in CTCL. Therefore, targeting these functions might represent an attractive therapeutic strategy, especially in aggressive CTCL.

  16. Activation of p53, inhibition of telomerase activity and induction of estrogen receptor beta are associated with the anti-growth effects of combination of ovarian hormones and retinoids in immortalized human mammary epithelial cells

    Directory of Open Access Journals (Sweden)

    Smith-Schneider Sallie

    2005-03-01

    Full Text Available Abstract Background A full-term pregnancy has been associated with reduced risk for developing breast cancer. In rodent models, the protective effect of pregnancy can be mimicked with a defined regimen of estrogen and progesterone combination (E/P. However, the effects of pregnancy levels of E/P in humans and their underlying mechanisms are not fully understood. In this report, we investigated the growth inhibitory effects of pregnancy levels of E/P and both natural and synthetic retinoids in an immortalized human mammary epithelial cell line, 76N TERT cell line. Results We observed that cell growth was modestly inhibited by E/P, 9-cis-retinoic acid (9-cis RA or all-trans-retinoic acid (ATRA, and strongly inhibited by N-(4-hydroxyphenyl retinamide (HPR. The growth inhibitory effects of retinoids were further increased in the presence of E/P, suggesting their effects are additive. In addition, our results showed that both E/P and retinoid treatments resulted in increased RARE and p53 gene activity. We further demonstrated that p53 and p21 protein expression were induced following the E/P and retinoid treatments. Furthermore, we demonstrated that while the telomerase activity was moderately inhibited by E/P, 9-cis RA and ATRA, it was almost completely abolished by HPR treatment. These inhibitions on telomerase activity by retinoids were potentiated by co-treatment with E/P, and correlated well with their observed growth inhibitory effects. Finally, this study provides the first evidence that estrogen receptor beta is up-regulated in response to E/P and retinoid treatments. Conclusion Taken together, our studies show that part of the anti-growth effects of E/P and retinoids is p53 dependent, and involve activation of p53 and subsequent induction of p21 expression. Inhibition of telomerase activity and up-regulation of estrogen receptor beta are also associated with the E/P- and retinoid-mediated growth inhibition. Our studies also demonstrate that

  17. FISH技术检测宫颈组织TERC基因扩增%Expression of the human telomerase gene in cervical tissue detected with FISH

    Institute of Scientific and Technical Information of China (English)

    刘爽; 李亚里; 姜淑芳; 胡凌云; 邹杰; 张咏梅; 张艾芃

    2012-01-01

    目的 应用荧光原位杂交技术(FISH)检测子宫颈病变组织中人端粒酶RNA (TERC)基因异常扩增的临床意义.方法 应用双色荧光原位杂交技术(FISH)检测195例宫颈组织TERC基因的异常扩增.结果 ①195例各类宫颈病变中,慢性宫颈炎33例,CIN1 34例,CIN2/3(包括原位癌)37例,宫颈鳞状细胞癌30例,宫颈腺癌61例,用FISH检测TERC基因的阳性表达率分别是3.03% (1/33)、29.41% (10/34)、72.97% (27/37)、100% (30/30)、91.80% (56/61),子宫颈鳞癌与腺癌TERC基因的表达较宫颈上皮内瘤变各组差异有统计学意义(P<0.05).TERC基因的扩增阳性率随着宫颈病变程度增加呈逐渐上升趋势,且TERC扩增在子宫颈鳞癌与腺癌患者中无明显差异性;②TERC基因异常扩增与高危型HPV感染呈正相关.结论 应用F ISH技术检测TERC基因的异常扩增可以作为组织学诊断困难的病变确诊、病变预测及治疗后风险评估的手段.%Objective To detect the human telomerase gene (TERC) amplification by dualcol or interphase DNA probe fluorescence in situ hybridization (FISH) in cervical carcinoma and CIN. Methods 195 cases of cervical lesion were detected the TERC expression by FISH. Results Among 195 cases, the positive rate of TERC gene in chronic cervicitis, CIN1, CIN2/3, cervical squamous cell carcinoma, cervical adenocarcinoma was 3. 03% (1/33), 29.41% (10/34), 72.97% (27/37), 100% (30/30) and 91. 80% (56/61), respectively; the difference between cervical carcinoma and chronic cervicitis and CIN groups was significant (P<0. 05). The positive rate of TERC gene amplification increasing with the grade of CIN increased; but it has no difference between cervical squamous cell carcinoma and cervical adenoeareinoma. Conclusion The detection of amplification of TERC by FISH can assist to identifying cervical lesions with high sensitivity and specificity.

  18. Design and synthesis of new benzimidazole-carbazole conjugates for the stabilization of human telomeric DNA, telomerase inhibition, and their selective action on cancer cells.

    Science.gov (United States)

    Maji, Basudeb; Kumar, Krishan; Kaulage, Mangesh; Muniyappa, K; Bhattacharya, Santanu

    2014-08-28

    Cell-permeable small molecules that enhance the stability of the G-quadruplex (G4) DNA structures are currently among the most intensively pursued ligands for inhibition of the telomerase activity. Herein we report the design and syntheses of four novel benzimidazole-carbazole conjugates and demonstrate their high binding affinity to G4 DNA. S1 nuclease assay confirmed the ligand mediated G-quadruplex DNA protection. Additional evidence from Telomeric Repeat Amplification Protocol (TRAP-LIG) assay demonstrated efficient telomerase inhibition activity by the ligands. Two of the ligands showed IC50 values in the sub-micromolar range in the TRAP-LIG assay, which are the best among the benzimidazole derivatives reported so far. The ligands also exhibited cancer cell selective nuclear internalization, nuclear condensation, fragmentation, and eventually antiproliferative activity in long-term cell viability assays. Annexin V-FITC/PI staining assays confirm that the cell death induced by the ligands follows an apoptotic pathway. An insight into the mode of ligand binding was obtained from the molecular dynamics simulations.

  19. New dimeric carbazole-benzimidazole mixed ligands for the stabilization of human telomeric G-quadruplex DNA and as telomerase inhibitors. A remarkable influence of the spacer.

    Science.gov (United States)

    Maji, Basudeb; Kumar, Krishan; Muniyappa, K; Bhattacharya, Santanu

    2015-08-14

    The development of G-quadruplex (G4) DNA binding small molecules has become an important strategy for selectively targeting cancer cells. Herein, we report the design and evolution of a new kind of carbazole-based benzimidazole dimers for their efficient telomerase inhibition activity. Spectroscopic titrations reveal the ligands high affinity toward the G4 DNA with significantly higher selectivity over duplex-DNA. The electrophoretic mobility shift assay shows that the ligands efficiently promote the formation of G4 DNA even at a lower concentration of the stabilizing K(+) ions. The TRAP-LIG assay demonstrates the ligand's potential telomerase inhibition activity and also establishes that the activity proceeds via G4 DNA stabilization. An efficient nuclear internalization of the ligands in several common cancer cells (HeLa, HT1080, and A549) also enabled differentiation between normal HFF cells in co-cultures of cancer and normal ones. The ligands induce significant apoptotic response and antiproliferative activity toward cancer cells selectively when compared to the normal cells.

  20. Increased in vitro glial fibrillary acidic protein expression, telomerase activity, and telomere length after productive human immunodeficiency virus-1 infection in murine astrocytes.

    Science.gov (United States)

    Ojeda, Diego; López-Costa, Juan José; Sede, Mariano; López, Ester María; Berria, María Isabel; Quarleri, Jorge

    2014-02-01

    Although HIV-associated neurocognitive disorders (HAND) result from injury and loss of neurons, productive infection routinely takes place in cells of macrophage lineage. In such a complex context, astrocytosis induced by local chemokines/cytokines is one of the hallmarks of HIV neuropathology. Whether this sustained astrocyte activation is able to alter telomere-aging process is unknown. We hypothesized that interaction of HIV with astrocytes may impact astrocyte telomerase activity (TA) and telomere length in a scenario of astrocytic activation measured by expression of glial fibrillary acidic protein (GFAP). To test this hypothesis, cultured murine astrocytes were challenged with pseudotyped HIV/vesicular stomatitis virus (HIV/VSV) to circumvent the absence of viral receptors; and GFAP, telomerase activity, and telomere length were quantified. As an early and transient event after HIV infection, both TA activity and telomere length were significantly augmented (P telomere length, that may attenuate cell proliferation and enhance the astrocyte dysregulation, contributing to HIV neuropathogenesis. Understanding the mechanisms involved in HIV-mediated persistence by altering the telomere-related aging processes could aid in the development of therapeutic modalities for neurological complications of HIV infection.

  1. 16型人乳头状瘤病毒DNA及端粒酶在 口腔粘膜病变中的表达%Research on expression of human papillomavirus type 16 and telomerase in oral lesions

    Institute of Scientific and Technical Information of China (English)

    帕提曼; 张志愿; 曹俊; 张铃

    2001-01-01

    目的探讨人乳头状瘤病毒16型(human papillomavirus type 16, HPV16)感染与端粒酶活化在口腔粘膜癌变过程中的关系。方法对口腔粘膜不同阶段病变的82例标本进行HPV16 DNA的聚合酶链反应检测及端粒酶活性催化亚单位( human telomerase reverse transcriptase, hTRT)mRNA的原位杂交检测。结果正常口腔粘膜、上皮单纯性增生、上皮异常增生与鳞状细胞癌的HPV16感染率分别为14.3%(1/7)、42.0%(3/7)、66.6%(20/30)及92.1%(35/38);hTRTmRNA阳性表达率分别为0.0%(0/7)、0.0%(0/7)、30.0%(9/30)及81.6%(31/38)。hTRTmRNA与HPV16协同表达率为67.0%(55/82)。结论 HPV16感染和端粒酶的激活与口腔粘膜恶变关系密切,HPV16可能通过激活端粒酶使口腔上皮细胞癌变。%Objective To define a correlation between human papilllomavirus (HPV) type 16 and telomerase activity during the carcinogenesis of oral mucosa. Methods HPV16 DNA and human telomerase reverse transcriptase (hTRT) mRNA were detected in 82 cases of paraffin embedded tissues including 7 cases of normal oral mucosa, 7 cases of hyperplasia lesions, 30 cases of oral dysplasia lesions and 38 cases of oral squamous cell carcinomas (OSCCs) by PCR and in situ hybridization (ISH) respectively. Results HPV16DNA was positive in 14.3% (1/7) of normal oral mucosa, 42.9% (3/7) of hyperplasia lesions, 66.6% (20/30) of dysplasia lesions and 92.1% (35/38) of OSCCs. hTRTmRNA was detectable in 30.0% (9/30) of oral dysplasia lesions and 81.6% (31/35) of OSCCs while normal oral mucosa and hyperplasia lesions were negative. Expression of HPV16DNA and hTRTmRNA were co-ordinate in 67.0% (55/82) cases. Conclusions HPV16 infection may play an important role in carcinogenesis of oral mucosa by activation of telomerase.

  2. TELOMERASE ACTIVITY AND hTERT mRNA EXPRESSION IN ACUTE LEUKEMIA

    Institute of Scientific and Technical Information of China (English)

    何冬梅; 张洹

    2004-01-01

    Objective: To investigate the clinical implications of telomerase activity and human telomerase reverse transcriptase (hTERT) expression as useful diagnostic marker in acute leukemia. Methods: Expression of hTERT was detected by reverse transcription- polymerase chain reaction (RT-PCR) in 24 cases with acute leukemia and in 12 normal persons. Quantitative levels of telomerase activity were examined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Results: In the bone marrow and peripheral blood of 24 acute leukemia, telomerase activity was detected in 75% of the samples, with absorbances (A) of 0.538(0.062 and 0.463(0.054, respectively. Whereas in 12 normal peripheral blood, telomerase activity had only a positive rate of 8.3%, with A value of 0.16(0.012. telomerase activities in the bone marrow and peripheral blood of acute leukemia were significantly higher than in normal control (P<0.05). RT-PCR analysis revealed that hTERT mRNA was expressed in 79.17%(19/24) of acute leukemia, but in only 1 of 12 normal peripheral blood. In 24 acute leukemias, 17 cases had both positive telomerse activity and hTERT mRNA expression. The expression of hTERT mRNA is correlated with telomerase activity (P<0.01). Conclusion: Telomerase and hTERT mRNA could be useful in diagnosis of acute leukemia. hTERT gene expression was strongly associated with telomerase activity in acute leukemia.

  3. A Cajal body-independent pathway for telomerase trafficking in mice.

    Science.gov (United States)

    Tomlinson, Rebecca L; Li, Jian; Culp, Bradley R; Terns, Rebecca M; Terns, Michael P

    2010-10-15

    The intranuclear trafficking of human telomerase involves a dynamic interplay between multiple nuclear sites, most notably Cajal bodies and telomeres. Cajal bodies are proposed to serve as sites of telomerase maturation, storage, and assembly, as well as to function in the cell cycle-regulated delivery of telomerase to telomeres in human cells. Here, we find that telomerase RNA does not localize to Cajal bodies in mouse cells, and instead resides in separate nuclear foci throughout much of the cell cycle. However, as in humans, mouse telomerase RNA (mTR) localizes to subsets of telomeres specifically during S phase. The localization of mTR to telomeres in mouse cells does not require coilin-containing Cajal bodies, as mTR is found at telomeres at similar frequencies in cells from wild-type and coilin knockout mice. At the same time, we find that human TR localizes to Cajal bodies (as well as telomeres) in mouse cells, indicating that the distinct trafficking of mTR is attributable to an intrinsic property of the RNA (rather than a difference in the mouse cell environment such as the properties of mouse Cajal bodies). We also find that during S phase, mTR foci coalesce into short chains, with at least one of the conjoined mTR foci co-localizing with a telomere. These findings point to a novel, Cajal body-independent pathway for telomerase biogenesis and trafficking in mice.

  4. A Cajal body-independent pathway for telomerase trafficking in mice

    Energy Technology Data Exchange (ETDEWEB)

    Tomlinson, Rebecca L.; Li, Jian; Culp, Bradley R.; Terns, Rebecca M., E-mail: rterns@bmb.uga.edu; Terns, Michael P., E-mail: mterns@bmb.uga.edu

    2010-10-15

    The intranuclear trafficking of human telomerase involves a dynamic interplay between multiple nuclear sites, most notably Cajal bodies and telomeres. Cajal bodies are proposed to serve as sites of telomerase maturation, storage, and assembly, as well as to function in the cell cycle-regulated delivery of telomerase to telomeres in human cells. Here, we find that telomerase RNA does not localize to Cajal bodies in mouse cells, and instead resides in separate nuclear foci throughout much of the cell cycle. However, as in humans, mouse telomerase RNA (mTR) localizes to subsets of telomeres specifically during S phase. The localization of mTR to telomeres in mouse cells does not require coilin-containing Cajal bodies, as mTR is found at telomeres at similar frequencies in cells from wild-type and coilin knockout mice. At the same time, we find that human TR localizes to Cajal bodies (as well as telomeres) in mouse cells, indicating that the distinct trafficking of mTR is attributable to an intrinsic property of the RNA (rather than a difference in the mouse cell environment such as the properties of mouse Cajal bodies). We also find that during S phase, mTR foci coalesce into short chains, with at least one of the conjoined mTR foci co-localizing with a telomere. These findings point to a novel, Cajal body-independent pathway for telomerase biogenesis and trafficking in mice.

  5. Structure of the Tribolium castaneum Telomerase Catalytic Subunit TERT

    Energy Technology Data Exchange (ETDEWEB)

    Gillis,A.; Schuller, A.; Skordalakes, E.

    2008-01-01

    A common hallmark of human cancers is the overexpression of telomerase, a ribonucleoprotein complex that is responsible for maintaining the length and integrity of chromosome ends. Telomere length deregulation and telomerase activation is an early, and perhaps necessary, step in cancer cell evolution. Here we present the high-resolution structure of the Tribolium castaneum catalytic subunit of telomerase, TERT. The protein consists of three highly conserved domains, organized into a ring-like structure that shares common features with retroviral reverse transcriptases, viral RNA polymerases and B-family DNA polymerases. Domain organization places motifs implicated in substrate binding and catalysis in the interior of the ring, which can accommodate seven to eight bases of double-stranded nucleic acid. Modelling of an RNA-DNA heteroduplex in the interior of this ring demonstrates a perfect fit between the protein and the nucleic acid substrate, and positions the 3'-end of the DNA primer at the active site of the enzyme, providing evidence for the formation of an active telomerase elongation complex.

  6. RADIATION-INDUCED PROGRESSIVE DECREASINGIN THE EXPRESSION OF REVERSE TRANSCRIPTASE GENE OF hEST2 AND TELOMERASE ACTIVITY

    Institute of Scientific and Technical Information of China (English)

    朱涵能; 熊思东; 程文英

    2001-01-01

    Objectites. 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 tranacriptase(RT) seg-ment in the expression of telomerase activity. Methods. Tumor FIeLa cells, KB cells and A431 cells were employed to measure the change in telomeraseactivity after 60Co-ray irradiation at RNA level and protein level. Quantitative PCR and Northern blotting wereused to determine the expression of bEST2 RT segment that encodes seven motifs of the human telomeres, a PCR-besed telomeric repeat amplification protocol (TRAP)was used to assay telomerase activity after exposure toradiation. Results. Both of telomerase activity and the expression hEST2 RT segment were decreased with increasingdosage of radiation. In addition, testing the expression of motifs domain is similar to the measurement of telomerase activity. Conclusion. The detection of the hEST2 BT segment by Northern blotting and quantitative PCR are new methods for testing Uflomerase 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 ceils after irradiation.

  7. Telomerase Cajal body protein 1 depletion inhibits telomerase trafficking to telomeres and induces G1 cell cycle arrest in A549 cells.

    Science.gov (United States)

    Yuan, Ping; Wang, Zhitian; Lv, Wang; Pan, Hui; Yang, Yunhai; Yuan, Xiaoshuai; Hu, Jian

    2014-09-01

    Telomerase Cajal body protein 1 (TCAB1) is a telomerase holoenzyme, which is markedly enriched in Cajal bodies (CBs) and facilitates the recruitment of telomerase to CBs in the S phase of the cell cycle. This recruitment is dependent on TCAB1 binding to a telomerase RNA component. The majority of cancer cells are able to grow indefinitely due to telomerase and its mechanism of trafficking to telomeres. In the present study, a certain level of TCAB1 expression in A549 human lung cells was identified and TCAB1 knockdown exhibited a potent antiproliferative effect on these cells, which was coupled with a decrease in the cell density and activity of the cellular enzymes. In addition, TCAB1-depletion was demonstrated to inhibit telomerase trafficking to telomeres in the A549 cells, leading to subsequent G1 cell cycle arrest without inducing apoptotic cell death. Overall, these observations indicated that TCAB1 may be essential for A549 cell proliferation and cell cycle regulation, and may be a potential candidate for the development of a therapeutic target for lung adenocarcinomas.

  8. Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding.

    Science.gov (United States)

    Webb, Christopher J; Zakian, Virginia A

    2015-09-08

    The stem terminus element (STE), which was discovered 13 y ago in human telomerase RNA, is required for telomerase activity, yet its mode of action is unknown. We report that the Schizosaccharomyces pombe telomerase RNA, TER1 (telomerase RNA 1), also contains a STE, which is essential for telomere maintenance. Cells expressing a partial loss-of-function TER1 STE allele maintained short stable telomeres by a recombination-independent mechanism. Remarkably, the mutant telomere sequence was different from that of wild-type cells. Generation of the altered sequence is explained by reverse transcription into the template boundary element, demonstrating that the STE helps maintain template boundary element function. The altered telomeres bound less Pot1 (protection of telomeres 1) and Taz1 (telomere-associated in Schizosaccharomyces pombe 1) in vivo. Thus, the S. pombe STE, although distant from the template, ensures proper telomere sequence, which in turn promotes proper assembly of the shelterin complex.

  9. Thermodynamic characterization of the Saccharomyces cerevisiae telomerase RNA pseudoknot domain in vitro.

    Science.gov (United States)

    Liu, Fei; Kim, Yoora; Cruickshank, Charmion; Theimer, Carla A

    2012-05-01

    Recent structural and functional characterization of the pseudoknot in the Saccharomyces cerevisiae telomerase RNA (TLC1) has demonstrated that tertiary structure is present, similar to that previously described for the human and Kluyveromyces lactis telomerase RNAs. In order to biophysically characterize the identified pseudoknot secondary and tertiary structures, UV-monitored thermal denaturation experiments, nuclear magnetic resonance spectroscopy, and native gel electrophoresis were used to investigate various potential conformations in the pseudoknot domain in vitro, in the absence of the telomerase protein. Here, we demonstrate that alternative secondary structures are not mutually exclusive in the S. cerevisiae telomerase RNA, tertiary structure contributes 1.5 kcal mol(-1) to the stability of the pseudoknot (≈ half the stability observed for the human telomerase pseudoknot), and identify additional base pairs in the 3' pseudoknot stem near the helical junction. In addition, sequence conservation in an adjacent overlapping hairpin appears to prevent dimerization and alternative conformations in the context of the entire pseudoknot-containing region. Thus, this work provides a detailed in vitro characterization of the thermodynamic features of the S. cerevisiae TLC1 pseudoknot region for comparison with other telomerase RNA pseudoknots.

  10. Telomere and telomerase in the initial stage of immortalization of esophageal epithelial cell

    Institute of Scientific and Technical Information of China (English)

    Zhong-Ying Shen; Li-Yan Xu; En-Min Li; Wei-Jia Cai; Min-Hua Chen; Jian Shen; Yi Zeng

    2002-01-01

    AIM: To search for the biomarker of cellular immortalization,the telomere length, telomerase activity and its subunits incultured epithelial cells of human fetal esophagus in theprocess of immortalization.METHODS: The transgenic cell line of human fetalesophageal epithelium (SHEE) was established with E6 E7genes of bt man papillomavirus (HPV) type 18 in ourlaboratory. Morphological phenotype of cultured SHEE cellsfrom the 6th to 30th passages, was examined by phasecontrast microscopy, the telomere length was assayed bySouthern blot method, and the activity of telomerase wasanalyzed by telomeric repeat amplification protocol (TRAP).Expressions of subunits of telomerase, hTR and hTERT,were assessed by RT-PCR. DNA content in cell cycle wasdetected by flow cytometry. The cell apoptosis wasexamined by electron microscopy (EM) and TUNEL label.RESULTS: SHEE cells from the 6 th to 10 th passagesshowed cellular proliferation with a good differentiation.From the 12 th to the 16 th passages, many senescent andapoptotic cells appeared, and the telomere length sharplyshortened from 23 kb to 17 kb without expression of hTERTand telornerase activity. At the 20 th passage, SHEE cellsovercame the senescence and apoptosis and restored theirproliferative activity with expression of telomerase andhTERT at low levels, but the telomere length shortenedcontinuously to the lowest of 3 kb. After the 30 th passagecells proliferation was restored by increment of cells at S andG2M phase in the cell cycle end telomerase activity expressedat high levels and with maintenance of telomere length.CONCLUSION: At the early stage of SHEE cells, telomeresare shortened without expression of telomerase and hTERTcausing cellular senescence and cell death. From the 20 thto the 30 th passages, the activation of telomerase andmaintenance of telomere length show a progressive processfor immortalization of esophageal epithelial cells. Theexpression of telomerase may constitute a biomarker fordetection of

  11. Ionizing Radiation Promotes Migration and Invasion of Cancer Cells Through Transforming Growth Factor-Beta-Mediated Epithelial-Mesenchymal Transition

    Energy Technology Data Exchange (ETDEWEB)

    Zhou Yongchun [Department of Radiation Oncology, Xijing Hospital Fourth Military Medical University, Xi' an (China); Department of Radiation Medicine, College of Preventive Medicine, Xijing Hospital Fourth Military Medical University, Xi' an (China); Liu Junye; Li Jing; Zhang Jie [Department of Radiation Medicine, College of Preventive Medicine, Xijing Hospital Fourth Military Medical University, Xi' an (China); Xu Yuqiao [Department of Pathology, Xijing Hospital Fourth Military Medical University, Xi' an (China); Zhang Huawei; Qiu Lianbo; Ding Guirong [Department of Radiation Medicine, College of Preventive Medicine, Xijing Hospital Fourth Military Medical University, Xi' an (China); Su Xiaoming [Department of Radiation Oncology, 306th Hospital of PLA, Beijing (China); Mei Shi [Department of Radiation Oncology, Xijing Hospital Fourth Military Medical University, Xi' an (China); Guo Guozhen, E-mail: guozhenguo@hotmail.com [Department of Radiation Medicine, College of Preventive Medicine, Xijing Hospital Fourth Military Medical University, Xi' an (China)

    2011-12-01

    Purpose: To examine whether ionizing radiation enhances the migratory and invasive abilities of cancer cells through transforming growth factor (TGF-{beta})-mediated epithelial-mesenchymal transition (EMT). Methods and Materials: Six cancer cell lines originating from different human organs were irradiated by {sup 60}Co {gamma}-ray at a total dose of 2 Gy, and the changes associated with EMT, including morphology, EMT markers, migration and invasion, were observed by microscope, Western blot, immunofluorescence, scratch assay, and transwell chamber assay, respectively. Then the protein levels of TGF-{beta} in these cancer cells were detected by enzyme-linked immunosorbent assay, and the role of TGF-{beta} signaling pathway in the effect of ionizing radiation on EMT was investigate by using the specific inhibitor SB431542. Results: After irradiation with {gamma}-ray at a total dose of 2 Gy, cancer cells presented the mesenchymal phenotype, and compared with the sham-irradiation group the expression of epithelial markers was decreased and of mesenchymal markers was increased, the migratory and invasive capabilities were strengthened, and the protein levels of TGF-{beta} were enhanced. Furthermore, events associated with EMT induced by IR in A549 could be reversed through inhibition of TGF-{beta} signaling. Conclusions: These results suggest that EMT mediated by TGF-{beta} plays a critical role in IR-induced enhancing of migratory and invasive capabilities in cancer cells.

  12. Role of Telomerase in the Cardiovascular System

    Directory of Open Access Journals (Sweden)

    Mark Zurek

    2016-06-01

    Full Text Available Aging is one major risk factor for the incidence of cardiovascular diseases and the development of atherosclerosis. One important enzyme known to be involved in aging processes is Telomerase Reverse Transcriptase (TERT. After the discovery of the enzyme in humans, TERT had initially only been attributed to germ line cells, stem cells and cancer cells. However, over the last few years it has become clear that TERT is also active in cells of the cardiovascular system including cardiac myocytes, endothelial cells, smooth muscle cells and fibroblasts. Interference with the activity of this enzyme greatly contributes to cardiovascular diseases. This review will summarize the findings on the role of TERT in cardiovascular cells. Moreover, recent findings concerning TERT in different mouse models with respect to cardiovascular diseases will be described. Finally, the extranuclear functions of TERT will be covered within this review.

  13. Behaviour of telomere and telomerase during aging and regeneration in zebrafish.

    Directory of Open Access Journals (Sweden)

    Monique Anchelin

    Full Text Available Telomere length and telomerase activity are important factors in the pathobiology of human diseases. Age-related diseases and premature aging syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase. The zebrafish (Danio rerio offers multiple experimental manipulation advantages over other vertebrate models and, therefore, it has been recently considered as a potential model for aging, cancer, and regeneration studies. However, it has only partially been exploited to shed light on these fundamental biological processes. The aim of this study was, therefore, to investigate telomere length and telomerase expression and activity in different strains of zebrafish obtained from different stock centres to determine whether they undergo any changes during aging and regeneration. We found that although both telomerase expression and telomere length increased from embryo to adulthood stages, they drastically declined in aged fish despite telomerase activity was detected in different tissues of old fish. In addition, we observed a weaker upregulation of telomerase expression in regenerating fins of old fish, which well correlates with their impaired regeneration capacity. Strikingly, telomeres were elongated or maintained during the fin regeneration process at all ages and after repeated amputations, likely to support high cell proliferation rates. We conclude that the expression of telomerase and telomere length are closely related during the entire life cycle of the fish and that these two parameters can be used as biomarkers of aging in zebrafish. Our results also reveal a direct relationship between the expression of telomerase, telomere length and the efficiency of tissue regeneration.

  14. Molecular regulation of telomerase activity in aging

    Institute of Scientific and Technical Information of China (English)

    Craig Nicholls; He Li; Jian-Qiu Wang; Jun-Ping Liu

    2011-01-01

    The process of aging is mitigated by the maintenance and repair of chromosome ends (telomeres),resulting in extended lifespan.This review examines the molecular mechanisms underlying the actions and regulation of the enzyme telomerase reverse transcriptase (TERT),which functions as the primary mechanism of telomere maintenance and regulates cellular life expectancy.Underpinning increased cell proliferation,telomerase is also a key factor in facilitating cancer cell immortalization.The review focuses on aspects of hormonal regulations of telomerase,and the intraceilular pathways that converge to regulate telomerase activity with an emphasis on molecular interactions at protein and gene levels.In addition,the basic structure and function of two key telomerase enzyme components-the catalytic subunit TERT and the template RNA (TERC) are discussed briefly.

  15. Fundamental mechanisms of telomerase action in yeasts and mammals: understanding telomeres and telomerase in cancer cells

    Science.gov (United States)

    Armstrong, Christine A.

    2017-01-01

    Aberrant activation of telomerase occurs in 85–90% of all cancers and underpins the ability of cancer cells to bypass their proliferative limit, rendering them immortal. The activity of telomerase is tightly controlled at multiple levels, from transcriptional regulation of the telomerase components to holoenzyme biogenesis and recruitment to the telomere, and finally activation and processivity. However, studies using cancer cell lines and other model systems have begun to reveal features of telomeres and telomerase that are unique to cancer. This review summarizes our current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts. Finally, we discuss the differences in telomere homeostasis between normal cells and cancer cells, which may provide a foundation for telomere/telomerase targeted cancer treatments. PMID:28330934

  16. Influence of anti-keratin autoantibodies on telomerase activity of squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    FU Meng; ZHANG Yan-guo; LIU Yu-feng; CHEN Yan; WANG Qiu-feng; LI Wei

    2002-01-01

    Objective: To investigate the influence of anti-keratin autoantibodies (AK auto Abs) on telomerase activity of squamous cell carcinoma cultured in vitro and the mechanisms of the inhibitory effects of AK auto Abs on squamous cell carcinoma. Methods: Influence of AK auto Abs on the proliferation of Tca cells was observed by MTT colorimetry. Telomerase activity of cultured Tca cells and human keratinocytes was determined by telomeric repeat amplication protocol-ELISA (TRAP-ELISA) and polyacrylamide gel electrophoresis (PAGE). After being treated with AK auto Abs for 36 h at a concentration of 4, 8, 16 mg/L respectively, the changes of telomerase activity of Tca cells were also detected by TRAP-ELISA and PAGE.Results: MTT colorimetric determination showed that the capacity of proliferation of Tca cells correlated negatively with the concentration of AK auto Abs (r=-0. 74, P<0. 01). TRAP-ELISA and PAGE showed that telomerase activity of Tca cells increased significantly compared to that of cultured human keratinocytes(t=3. 5396, P<0. 01). AK auto Abs at a concentrations of 4, 8, 16 mg/L had significant dose-dependent inhibitory effects on telomerase activity of Tca cells (r=- 0. 8358, P<0. 01). Conclusion: AK auto Abs have a significant dose-dependent inhibitory effect on the proliferation of cultured Tca cells. AK auto Abs inhibit telomerase activity of cultured Tca cells with dose-dependent pattern. It suggests that decrease of telomerase activity may play an important role in the inhibitory effects of AK auto Aba on squamous cell carcinoma.

  17. Telomerase activity in cervical intraepithelial neoplasia

    Institute of Scientific and Technical Information of China (English)

    王淑珍; 孙建衡; 张伟; 金顺钱; 王洪平; 金玉生; 曲萍; 刘毅; 李茉

    2004-01-01

    Background It was reported that telomerase expression is closely associated with cellular immortality and cancer. This study was designed to investigate the relationship between telomerase expression and the carcinogenesis of cervical cancer, the possible use of telomerase as a marker of cervical intraepithelial neoplasia (CIN) progression or regression, and the natural history of CIN. Methods Telomeric repeat amplification protocol (TRAP) assay was used to measure telomerase activity in cervical scrapings and biopsy samples obtained from 105 cases affected with various cervical conditions, including chronic cervicitis (n=20), CIN (n=64, 16 cases of CIN Ⅰ , 20 cases of CIN Ⅱ, and 28 cases of CIN Ⅲ ), and invasive squamous cell carcinoma (n =21 ).Results In exfoliated cell samples, telomerase activity was detected in 5 of 20 (25. 0% ) cases of cervicitis, 10 of 16 (62.5%) cases of CIN Ⅰ , 11 of 20 (55.0%) cases of CIN Ⅱ, 23 of 28 (82.1%) cases of CIN Ⅲ, and 13 of 21 (61.9%) cases of carcinoma. In cervical biopsy samples, telomerase activity was detected in 6 of 20 (30. 0%) cases of cervicitis, 8 of 16 (50. 0%) cases of CIN Ⅰ , 9 of 20 (45.0%) cases of (CIN Ⅱ, 27 of 28 (96. 4%) cases of CIN Ⅲ, and 20 of 21 (95. 2%) cases of carcinoma. Telomerase activation was significantly higher in CIN samples than in cervicitis samples. Telomerase activity was detected at similar frequency in samples from cervical scrapings and cervical biopsies.Conclusion These results seem to suggest that telomerase expression may be associated with carcinogenesis of the cervix. TRAP assay of cervical scraping samples could be used to monitor and predict the development of CIN in clinical practice.

  18. Telomerase activity and telomere length in Daphnia.

    Science.gov (United States)

    Schumpert, Charles; Nelson, Jacob; Kim, Eunsuk; Dudycha, Jeffry L; Patel, Rekha C

    2015-01-01

    Telomeres, comprised of short repetitive sequences, are essential for genome stability and have been studied in relation to cellular senescence and aging. Telomerase, the enzyme that adds telomeric repeats to chromosome ends, is essential for maintaining the overall telomere length. A lack of telomerase activity in mammalian somatic cells results in progressive shortening of telomeres with each cellular replication event. Mammals exhibit high rates of cell proliferation during embryonic and juvenile stages but very little somatic cell proliferation occurs during adult and senescent stages. The telomere hypothesis of cellular aging states that telomeres serve as an internal mitotic clock and telomere length erosion leads to cellular senescence and eventual cell death. In this report, we have examined telomerase activity, processivity, and telomere length in Daphnia, an organism that grows continuously throughout its life. Similar to insects, Daphnia telomeric repeat sequence was determined to be TTAGG and telomerase products with five-nucleotide periodicity were generated in the telomerase activity assay. We investigated telomerase function and telomere lengths in two closely related ecotypes of Daphnia with divergent lifespans, short-lived D. pulex and long-lived D. pulicaria. Our results indicate that there is no age-dependent decline in telomere length, telomerase activity, or processivity in short-lived D. pulex. On the contrary, a significant age dependent decline in telomere length, telomerase activity and processivity is observed during life span in long-lived D. pulicaria. While providing the first report on characterization of Daphnia telomeres and telomerase activity, our results also indicate that mechanisms other than telomere shortening may be responsible for the strikingly short life span of D. pulex.

  19. Telomerase activity in solid transitional cell carcinoma, bladder washings, and voided urine.

    Science.gov (United States)

    Lance, R S; Aldous, W K; Blaser, J; Thrasher, J B

    1998-03-04

    Telomerase activity has been detected in a wide variety of human malignancies. It appears to be one of the fundamental ingredients necessary for cellular immortality. We sought to determine the incidence of telomerase activity in solid transitional cell carcinoma (TCC) specimens, benign urothelium, bladder washings, and voided urine from patients with TCC identified cystoscopically compared with controls. Telomerase activity was measured in 26 solid bladder cancers and 13 benign urothelial specimens using the telomere repeat amplification protocol (TRAP), a polymerase chain reaction (PCR) based assay. Telomerase activity was further measured in the centrifuged cellular material obtained from the bladder washings of 26 patients with TCC and 40 with benign urologic disease found to have a normal cystoscopy. All patients with hematuria were additionally evaluated with an upper tract radiographic examination and found to be free of malignancy. Voided urine was likewise evaluated in 11 patients with TCC, 12 with benign urologic diseases, and 56 asymptomatic control subjects. Telomerase activity was detected in 25 of 26 (96%) solid specimens, 21 of 26 (81%) bladder washings, and 6 of 11 (54%) voided urine specimens from patients with histologically confirmed TCC. In the control group, 2 of 13 (15%) benign urothelial specimens and 2 of 56 (4%) voided urine specimens from the asymptomatic volunteer group demonstrated telomerase activity. Of those with benign urologic disease, 16 of 40 (40%) bladder barbotage specimens and 6 of 12 (50%) voided urine specimens demonstrated telomerase activity. Sensitivity and specificity of telomerase as a marker for TCC were 81% and 60%, respectively, in the bladder washings group and 54% and 50%, respectively, in voided urine. These data indicate that activation of telomerase is frequent in solid TCC and appears to be a sensitive marker in bladder washings of patients with TCC. We noted an unexpectedly high false positive detection rate in

  20. trt-1 is the Caenorhabditis elegans catalytic subunit of telomerase.

    Directory of Open Access Journals (Sweden)

    Bettina Meier

    2006-02-01

    Full Text Available Mutants of trt-1, the Caenorhabditis elegans telomerase reverse transcriptase, reproduce normally for several generations but eventually become sterile as a consequence of telomere erosion and end-to-end chromosome fusions. Telomere erosion and uncapping do not cause an increase in apoptosis in the germlines of trt-1 mutants. Instead, late-generation trt-1 mutants display chromosome segregation defects that are likely to be the direct cause of sterility. trt-1 functions in the same telomere replication pathway as mrt-2, a component of the Rad9/Rad1/Hus1 (9-1-1 proliferating cell nuclear antigen-like sliding clamp. Thus, the 9-1-1 complex may be required for telomerase to act at chromosome ends in C. elegans. Although telomere erosion limits replicative life span in human somatic cells, neither trt-1 nor telomere shortening affects postmitotic aging in C. elegans. These findings illustrate effects of telomere dysfunction in C. elegans mutants lacking the catalytic subunit of telomerase, trt-1.

  1. DETECTION OF TELOMERASE ACTIVITY IN PATIENTS WITH MYCOSIS FUNGOIDES

    Institute of Scientific and Technical Information of China (English)

    应作霖; 孙建方; 刘珊

    2003-01-01

    Objectives. To detect telomerase activity in patients with mycosis fungoides (MF) and to study therole of telomerase in the tumorigenesis of MF.Methods. The technique of PCR-ELISA was employed to detect telomerase activity in 35 patientswith various stages of MF.Results. 92.3% tumor stage of MF, 78.6% plaque stage of MF and 75.0% patch stage of MF hadpositive telomerase activity. The control samples had no telomerase activity. Telomerase activity in tumorstage of MF was significantly higher than that in plaque stage, while the latter was higher than that inpatch stage. Telomerase activity was correlated with the stage of MF.Conclusion. High level of telomerase activity frequently occurred in patients with MF, suggestingthat telomerase might play an important role in the tumorigenesis of MF and is a useful marker for thediagnosis of MF possibly.

  2. siRNA Downregulates Human Telomerase Reverse Transcriptase Gene and Telomere Length and Telomerase Activity in Human Colon Cancer Colo320 Cells%c-myc靶向siRNA抑制人结直肠癌Colo320细胞的增殖及下调hTERT基因表达的研究

    Institute of Scientific and Technical Information of China (English)

    黄浩; 李秀; 肖宏; 傅雷; 余兰才; 林世和; 易艳东

    2009-01-01

    Objective To study transfected shRNA of c-myc as therapeutic agent in target cell Colo320 which expressed more totomerase activity and to investigate the effect of inhibition on telomerase activity and telomere lengths and tumor cell growth. Methods A plasmid-based polymerase Ⅲ promoter system was used to deliver and express short interfering RNA (siRNA) targeting c-myc in 12o1o320 cells. The c-myc and hTERT mRNA levels were monitored by fluorescence real time reverse transcription-polymerase chain reaction, the protein levels of c-myc and hTERT were examined by Western blot analysis. Mean-while, telomere lengths and telomerase activity were measured by Southern analysis of telomere restric-tion fragment (TRF) length and PCR-ELISA. We also assessed the effects of c-myc silencing on tumor growth by DNA synthesis (3H-thymidine). Results Our data showed that expressions of c-myc and hTERT were decreased in shRNA-transfected cells, and down-regulations of c-myc and hTERT inhibited cell growth, reduced cell telomere lengths, telomerase activity. Conclusion shRNA of c-myc has the a-bility to inhibit telomerase activity, telomere lengths and cell growth with a dose dependent pattern.%目的 探讨发夹状shRNA封阻c-mye基因,抑制人结直肠癌Colo320细胞增殖、生长的状况.方法 针对原癌基因e-myc构建发夹状shRNA的真核表达质粒,并转染人结直肠癌Colo320细胞.荧光定量RT-PCR检测c-myc及细胞端粒酶逆转录酶的mRNA表达,Westemn-blot检测c-myc、hTERT蛋白表达水平.Southern blot检测端粒的长度,PCR-ELISE法检测端粒酶活性.3H-thymidine实验分析DNA合成和细胞增殖.结果 转染细胞增殖、生长皆受到抑制.同时,c-myc和hTERT的mRNA和蛋白表达显著下降,端粒的长度明显缩短,端粒酶活性降低.结论 c-myc的shRNA对人结直肠癌Co1p320细胞的增殖、端粒长度、端粒酶活性有特异性抑制作用,并呈剂量依赖关系.

  3. Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes.

    Science.gov (United States)

    Härle-Bachor, C; Boukamp, P

    1996-06-25

    Cellular senescence is defined by the limited proliferative capacity of normal cultured cells. Immortal cells overcome this regulation and proliferate indefinitively. One step in the immortalization process may be reactivation of telomerase activity, a ribonucleoprotein complex, which, by de novo synthesized telomeric TTAGGG repeats, can prevent shortening of the telomeres. Here we show that immortal human skin keratinocytes, irrespective of whether they were immortalized by simian virus 40, human papillomavirus 16, or spontaneously, as well as cell lines established from human skin squamous cell carcinomas exhibit telomerase activity. Unexpectedly, four of nine samples of intact human skin also were telomerase positive. By dissecting the skin we could show that the dermis and cultured dermal fibroblasts were telomerase negative. The epidermis and cultured skin keratinocytes, however, reproducibly exhibited enzyme activity. By separating different cell layers of the epidermis this telomerase activity could be assigned to the proliferative basal cells. Thus, in addition to hematopoietic cells, the epidermis, another example of a permanently regenerating human tissue, provides a further exception of the hypothesis that all normal human somatic tissues are telomerase deficient. Instead, these data suggest that in addition to contributing to the permanent proliferation capacity of immortal and tumor-derived keratinocytes, telomerase activity may also play a similar role in the lifetime regenerative capacity of normal epidermis in vivo.

  4. 以端粒酶为靶标抗癌药物筛选模型建立及端粒酶抑制剂筛选%Determination of Telomerase from HeLa Cells as a Target for Screening Antitumor Agents

    Institute of Scientific and Technical Information of China (English)

    郑晓飞; 王升启; 孙志贤

    2002-01-01

    Telomerase, a ribonucleoprotein enzyme, has been found in immortalized but not in most sonatic adult human tissues, and thus emerged as a novel target for cancer chemotherapy. Recently it has been found that telomerase is a fruitful target for oncologic drug development. A new method for screening antitumor agents by using telomerase as a target has been established according to the phenomena that the enzyme activity ean be affected bv some types of antitumor agents or chemicals. The telomerase was extracted from HeLa cells. The telomeric repeat amplification protocol(TRAP) was used to measure enzyme activity. Telomerase activity can be inhibited by 4 kinds of chemical compounds.

  5. Novel Efficient Cell-Penetrating, Peptide-Mediated Strategy for Enhancing Telomerase Inhibitor Oligonucleotides.

    Science.gov (United States)

    Muñoz-Alarcón, Andrés; Eriksson, Jonas; Langel, Ülo

    2015-12-01

    At present, there are several therapeutic approaches for targeting telomerase in tumors. One in particular, currently undergoing clinical trials, is based on synthetic lipid-modified oligonucleotide antagonists aimed at inhibiting the ribonucleoprotein subunit of human telomerase. However, while enabling efficient uptake, the lipid modifications reduce the potency of the therapeutic oligonucleotides compared to nonmodified oligonucleotides. Moreover, lipid modification may increase oligonucleotide accumulation in the liver causing undesirable hepatotoxicity. Noncovalent complexation strategies for cell-penetrating peptide (CPP)-mediated delivery present an option to circumvent the need for potency-reducing modifications, while allowing for a highly efficient uptake, and could significantly improve the efficiency of telomerase-targeting cancer therapeutics. Delivery of a nonlipidated locked nucleic acid/2'-O-methyl mixmer significantly inhibits the telomerase activity in treated HeLa cells. The inhibitory effect was further improved through addition of a CPP. Furthermore, calculated IC50-values for the oligonucleotide delivered by CPPs into HeLa cells are more than 20 times lower than telomerase inhibitor Imetelstat, currently undergoing clinical trials. These results emphasize the potential of CPP-mediated delivery of future pharmaceuticals and provide means by which to enhance an already promising therapeutic strategy for cancer treatment.

  6. Imetelstat (a telomerase antagonist) exerts off‑target effects on the cytoskeleton.

    Science.gov (United States)

    Mender, Ilgen; Senturk, Serif; Ozgunes, Nuriman; Akcali, K Can; Kletsas, Dimitris; Gryaznov, Sergei; Can, Alp; Shay, Jerry W; Dikmen, Z Gunnur

    2013-05-01

    Telomerase is a cellular ribonucleoprotein reverse transcriptase that plays a crucial role in telomere maintenance. This enzyme is expressed in approximately 90% of human tumors, but not in the majority of normal somatic cells. imetelstat sodium (GRN163L), is a 13-mer oligonucleotide N3'→P5' thio-phosphoramidate lipid conjugate, which represents the latest generation of telomerase inhibitors targeting the template region of the human functional telomerase RNA (hTR) subunit. In preclinical trials, this compound has been found to inhibit telomerase activity in multiple cancer cell lines, as well as in vivo xenograft mouse models. Currently, GRN163L is being investigated in several clinical trials, including a phase II human non‑small cell lung cancer clinical trial, in a maintenance setting following standard doublet chemotherapy. In addition to the inhibition of telomerase activity in cancer cell lines, GRN163L causes morphological cell rounding changes, independent of hTR expression or telomere length. This leads to the loss of cell adhesion properties; however, the mechanism underlying this effect is not yet fully understood. In the present study, we observed that GRN163L treatment leads to the loss of adhesion in A549 lung cancer cells, due to decreased E-cadherin expression, leading to the disruption of the cytoskeleton through the alteration of actin, tubulin and intermediate filament organization. Consequently, the less adherent cancer cells initially cease to proliferate and are arrested in the G1 phase of the cell cycle, accompanied by decreased matrix metalloproteinase-2 (MMP-2) expression. These effects of GRN163L are independent of its telomerase catalytic activity and may increase the therapeutic efficacy of GRN163L by decreasing the adhesion, proliferation and metastatic potential of cancer cells in vivo.

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

    Institute of Scientific and Technical Information of China (English)

    程文; 位志峰; 高建平; 张征宇; 葛京平; 景抗震; 徐锋; 解鹏

    2010-01-01

    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 reg...

  8. Collapse of Telomere Homeostasis in Hematopoietic Cells Caused by Heterozygous Mutations in Telomerase Genes

    NARCIS (Netherlands)

    Aubert, Geraldine; Baerlocher, Gabriela M.; Vulto, Irma; Poon, Steven S.; Lansdorp, Peter M.

    2012-01-01

    Telomerase activity is readily detectable in extracts from human hematopoietic stem and progenitor cells, but appears unable to maintain telomere length with proliferation in vitro and with age in vivo. We performed a detailed study of the telomere length by flow FISH analysis in leukocytes from 835

  9. Effect of realgar on telomerase activity and hTERT-mRNA expression in NB4 cells

    Institute of Scientific and Technical Information of China (English)

    李静; 刘陕西; 张梅

    2003-01-01

    Objective: To evaluate whether realgar could down-regulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity in acute promyelocytic leukemia cell line-NB4 cells. Methods: The expression of hTERT-mRNA was analyzed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Flow cytometry using PI staining was applied to analyze the cell cycle and apoptosis. Results: Treatment of NB4 cells with 155, 300, 600 μg/L realgar reduced telomerase activity significantly accompanying with decrease of hTERT-mRNA and increasing cell apoptosis. G2/M phase arrest appeared when treated with realgar in 300, 600 μg/L. Conclusion: It is suggested that telomerase activity of NB4 cells can be specifically inhibited by realgar through the down-regulation of hTERT gene expression. G2/M phase arrest and apoptosis by realgar in NB4 cells might be related to the reduction of telomerase activity and hTERT-mRNA expression.

  10. EFFECTS OF ARSENITE IN TELOMERE AND TELOMERASE IN RELATION TO CELL PROLIFERATION AND APOPTOSIS IN HUMAN KERATINOCYTES AND LEUKEMIA CELLS IN VITRO

    Science.gov (United States)

    Telomeres are critical in maintaining chromosome and genomic stability. Arsenic, a human carcinogen as well as an anticancer agent, is known for its clastogenicity. To better understand molecular mechanisms of arsenic actions, we investigated arsenite effects on telomere and telo...

  11. Telomerase stimulates ribosomal DNA transcription under hyperproliferative conditions.

    Science.gov (United States)

    Gonzalez, Omar Garcia; Assfalg, Robin; Koch, Sylvia; Schelling, Adrian; Meena, Jitendra K; Kraus, Johann; Lechel, Andre; Katz, Sarah-Fee; Benes, Vladimir; Scharffetter-Kochanek, Karin; Kestler, Hans A; Günes, Cagatay; Iben, Sebastian

    2014-08-13

    In addition to performing its canonical function, Telomerase Reverse Transcriptase (TERT) has been shown to participate in cellular processes independent of telomerase activity. Furthermore, although TERT mainly localizes to Cajal bodies, it is also present within the nucleolus. Because the nucleolus is the site of rDNA transcription, we investigated the possible role of telomerase in regulating RNA polymerase I (Pol I). Here we show that TERT binds to rDNA and stimulates transcription by Pol I during liver regeneration and Ras-induced hyperproliferation. Moreover, the inhibition of telomerase activity by TERT- or TERC-specific RNA interference, the overexpression of dominant-negative-TERT, and the application of the telomerase inhibitor imetelstat reduce Pol I transcription and the growth of tumour cells. In vitro, telomerase can stimulate the formation of the transcription initiation complex. Our results demonstrate how non-canonical features of telomerase may direct Pol I transcription in oncogenic and regenerative hyperproliferation.

  12. Adenovirus-mediated and tumor-specific transgene expression of the sodium-iodide symporter from the human telomerase reverse transcriptase promoter enhances killing of lung cancer cell line in vitro

    Institute of Scientific and Technical Information of China (English)

    SHI Yi-zhen; ZHANG Jun; LIU Zeng-li; DU Shou-ying; SHEN Yong-mei

    2010-01-01

    Background The sodium-iodide symporter (NIS) protein can mediate the active radioiodine uptake.The human telomerase reverse transcriptase (hTERT) promoter is known to be selectively reactivated in majority of tumors and hence could be used for tumor targeting.We constructed a recombinant adenovirus containing the human sodium iodide symporter (hNIS) gene directed by the hTERT promoter, characterized the ability of infected cells in uptaking iodide, and explored the therapeutic efficacy of 131I in a lung cancer cell line in vitro.Methods The hTERT promoter was amplified by PCR from DNA isolated from log-phase HepG2 cells, subcloned into lineralized FL*-hNIS/pcDNA3, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack.The recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system.A positive control adenovirusAd-CMV-hNIS and a negative control adenovirus Ad-CMV were created similarly.A549 cells were transduced with recombinant adenoviruses.125I uptake studies and sodium perchlorate suppression studies were used to confirm hNIS expression and function.Toxic effects of 131I on tumor cells were studied by in vitro clonogenic assay.Results We first successfully constructed an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter.When infected with recombinant adenovirus constructs expressing hNIS directed by hTERTand CMV-promoters (Ad-hTERT-hNIS and Ad-CMV-hNIS, respectively), the lung cancer cell line A549 had increased ability to uptake radioiodide up to 23- and 30- fold compared to the control parental cells, respectively.The radioiodide uptake ability of both the Ad-CMV-hNIS and Ad-hTERT-hNIS transduced cell lines were repressed 11-fold by sodium perchlorate (NaCIO4).The subsequent in vitro clonogenic assay of the infected A549 cell line was further repressed to 23% (Ad-CMV-hNIS) and 30% (Ad-hTERT-hNIS) of the control group after receiving radioiodide for 7 hours (P <0.001).Conclusion

  13. Telomeres and Telomerase in the Radiation Response: implications for instability, reprogramming, and carcinogenesis

    Directory of Open Access Journals (Sweden)

    Brock James Sishc

    2015-11-01

    Full Text Available Telomeres are nucleoprotein complexes comprised of tandem arrays of repetitive DNA sequence that serve to protect chromosomal termini from inappropriate degradation, as well as to prevent these natural DNA ends from being recognized as broken DNA (double-strand breaks; DSBs and triggering of inappropriate DNA damage responses. Preservation of telomere length requires telomerase, the specialized reverse transcriptase capable of maintaining telomere length via template-mediated addition of telomeric repeats onto the ends of newly synthesized chromosomes. Loss of either end-capping function or telomere length maintenance has been associated with genomic instability or senescence in a variety of settings; therefore telomeres and telomerase have well-established connections to cancer and aging. It has long been recognized that oxidative stress promotes shortening of telomeres, and that telomerase activity is a radiation-inducible function. However, the effects of ionizing radiation (IR exposure on telomeres per se are much less well understood and appreciated. To gain a deeper understanding of the roles telomeres and telomerase play in the response of human cells to ionizing radiations of different qualities, we tracked changes in telomeric end-capping function, telomere length, and telomerase activity in panels of mammary epithelial and hematopoietic cell lines exposed to low linear energy transfer (LET gamma(γ-rays or high LET high charge, high energy (HZE particles, delivered either acutely or at low dose rates (LDR. In addition to demonstrating that dysfunctional telomeres contribute to IR-induced mutation frequencies and genome instability, we reveal non-canonical roles for telomerase, in that telomerase activity was required for IR-induced enrichment of mammary epithelial putative stem/progenitor cell populations, a finding also suggestive of cellular reprogramming. Taken together, the results reported here establish the critical importance of

  14. Rhodacyanine dye MKT-077 inhibits in vitro telomerase assay but has no detectable effects on telomerase activity in vivo.

    Science.gov (United States)

    Wadhwa, Renu; Colgin, Lorel; Yaguchi, Tomoko; Taira, Kazunari; Reddel, Roger R; Kaul, Sunil C

    2002-08-01

    MKT-077, a cationic rhodacyanine dye analogue, causes selective toxicity to cancer cells. Its cellular targets elucidated thus far include oncogenic Ras, F-actin, mortalin (hmot-2)/mthsp70, and telomerase. Here we report that MKT-077 causes growth arrest of cancer cells in culture independent of their Ras, p53, or telomerase status. Telomerase activity is inhibited in vitro by MKT-077 in the telomerase assay used. However, the in vivo toxicity seen in telomerase-positive cancer cells was not accompanied by inhibition of telomerase activity or telomere shortening. Furthermore, cells with an alternative mechanism for lengthening of telomeres were also sensitive to MKT-077 and showed enhanced formation of alternative mechanism for lengthening of telomeres-associated PML bodies in their nuclei. The data suggested that inhibition of telomerase activity is unlikely to be a prime cause of MKT-077-induced toxicity in cancer cells.

  15. ARSENIC EFFECTS ON TELOMERE AND TELOMERASE ACTIVITY

    Science.gov (United States)

    Arsenic effects on telomere and telomerase activity. T-C. Zhang, M. T. Schmitt, J. Mo, J. L. Mumford, National Research Council and U.S Environmental Protection Agency, NHEERL, Research Triangle Park, NC 27711Arsenic is a known carcinogen and also an anticancer agent for acut...

  16. Pyrimidine motif triple helix in the Kluyveromyces lactis telomerase RNA pseudoknot is essential for function in vivo.

    Science.gov (United States)

    Cash, Darian D; Cohen-Zontag, Osnat; Kim, Nak-Kyoon; Shefer, Kinneret; Brown, Yogev; Ulyanov, Nikolai B; Tzfati, Yehuda; Feigon, Juli

    2013-07-02

    Telomerase is a ribonucleoprotein complex that extends the 3' ends of linear chromosomes. The specialized telomerase reverse transcriptase requires a multidomain RNA (telomerase RNA, TER), which includes an integral RNA template and functionally important template-adjacent pseudoknot. The structure of the human TER pseudoknot revealed that the loops interact with the stems to form a triple helix shown to be important for activity in vitro. A similar triple helix has been predicted to form in diverse fungi TER pseudoknots. The solution NMR structure of the Kluyveromyces lactis pseudoknot, presented here, reveals that it contains a long pyrimidine motif triple helix with unexpected features that include three individual bulge nucleotides and a C(+)•G-C triple adjacent to a stem 2-loop 2 junction. Despite significant differences in sequence and base triples, the 3D shape of the human and K. lactis TER pseudoknots are remarkably similar. Analysis of the effects of nucleotide substitutions on cell growth and telomere lengths provides evidence that this conserved structure forms in endogenously assembled telomerase and is essential for telomerase function in vivo.

  17. Telomere Elongation and Naive Pluripotent Stem Cells Achieved from Telomerase Haplo-Insufficient Cells by Somatic Cell Nuclear Transfer

    Directory of Open Access Journals (Sweden)

    Li-Ying Sung

    2014-12-01

    Full Text Available Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs have been efficiently achieved by somatic cell nuclear transfer (SCNT. We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc+/− mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc+/− cells exhibit naive pluripotency as evidenced by generation of Terc+/− ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells.

  18. A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku

    2013-09-01

    Full Text Available An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR on magnetic beads (MBs and subsequent application of cycling probe technology (CPT is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGGn-3' of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity.

  19. Telomerase antagonist imetelstat inhibits esophageal cancer cell growth and increases radiation-induced DNA breaks.

    Science.gov (United States)

    Wu, Xuping; Smavadati, Shirin; Nordfjäll, Katarina; Karlsson, Krister; Qvarnström, Fredrik; Simonsson, Martin; Bergqvist, Michael; Gryaznov, Sergei; Ekman, Simon; Paulsson-Karlsson, Ylva

    2012-12-01

    Telomerase is mainly active in human tumor cells, which provides an opportunity for a therapeutic window on telomerase targeting. We sought to evaluate the potential of the thio-phosphoramidate oligonucleotide inhibitor of telomerase, imetelstat, as a drug candidate for treatment of esophageal cancer. Our results showed that imetelstat inhibited telomerase activity in a dose-dependent manner in esophageal cancer cells. After only 1 week of imetelstat treatment, a reduction of colony formation ability of esophageal cancer cells was observed. Furthermore, long-term treatment with imetelstat decreased cell growth of esophageal cancer cells with different kinetics regarding telomere lengths. Short-term imetelstat treatment also increased γ-H2AX and 53BP1 foci staining in the esophageal cancer cell lines indicating a possible induction of DNA double strand breaks (DSBs). We also found that pre-treatment with imetelstat led to increased number and size of 53BP1 foci after ionizing radiation. The increase of 53BP1 foci number was especially pronounced during the first 1h of repair whereas the increase of foci size was prominent later on. This study supports the potential of imetelstat as a therapeutic agent for the treatment of esophageal cancer.

  20. The level of telomere dysfunction determines the efficacy of telomerase-based therapeutics in a lung cancer cell line.

    Science.gov (United States)

    Pantic, Milena; Zimmermann, Stefan; Waller, Cornelius F; Martens, Uwe M

    2005-05-01

    Telomerase is the ribonucleoprotein enzyme that maintains telomeres of eukaryotic chromosomes. Activation of telomerase is a common feature of the majority of human cancers, and inhibition of this enzyme has been proposed as a novel target for cancer therapeutics. Here, we investigated the effects of telomerase inhibition in the non-small cell lung cancer cell line NCI-H460, using a genetic approach by ectopic expression of dominant-negative (DN)-hTERT. Five clones were selected in which telomerase activity was completely abolished. As a result, telomere erosion was observed leading to proliferation arrest after a lag period of 20-28 population doublings. Although overall telomere length was similar between the different clones as measured by quantitative fluorescence in situ hybridization (Q-FISH), striking differences were found in telomere length of individual chromosomes. In particular, lack of individual telomeres and formation of end-to-end fusions were variable. Interestingly, this level of individual telomere dysfunction was positively correlated with the remaining life span of the different clones in vitro. In addition, the amount of telomere dysfunction induced by DN-hTERT was twice as high compared to the small molecule telomerase inhibitor BIBR1532, which induced growth arrest after >100 population doublings. Thus, pharmacological strategies that aim at inhibition of telomerase in cancer cells should take into account that not only overall telomere shortening, but rapid induction of a high level telomere dysfunction appears to be the crucial surrogate parameter for the development of future telomerase-based therapeutics.

  1. The Clinical Study of Telomerase Activity in Gastric Tumor

    Institute of Scientific and Technical Information of China (English)

    XI Weihong; NI Xiaoqian; SHEN Yuqin; HUANG Qinmei

    2002-01-01

    Telomerase activity was detected with both telomeric repeat amplification protocol (TRAP) - silver stain and polymerase chain reaction (PCR) - enzyme linked immuno - sorbent assay (ELISA). We have studied the telomerase activity in the 68 gastric tumors and their neighboring tissues,25 gastric ulcer, and 3 tumor cell colonies. The positive rate of telomerase activity in gastric tumors was 86.8% (59/68) and which was obviously higher than 7.3% (5/68) in the normal tissues adjacent to the tumors and 4% (1/25) in gastric ulcer. The telomerase activity was 100% (3/3) in the tumor colonies. It allowed to be seen that higher telomerase activity was associated with the origin and development of the gastric tumor. We believe that telomerase activity may be a useful clinical diagnostic marker for the gastric tumor.

  2. A gene expression signature classifying telomerase and ALT immortalization reveals an hTERT regulatory network and suggests a mesenchymal stem cell origin for ALT

    DEFF Research Database (Denmark)

    Lafferty-Whyte, K; Cairney, C J; Will, M B;

    2009-01-01

    this signature revealed a regulatory signalling network consistent with a model of human telomerase reverse transcriptase (hTERT) repression in ALT cell lines and liposarcomas. This network expands on our existing knowledge of hTERT regulation and provides a platform to understand differential regulation of h......Telomere length is maintained by two known mechanisms, the activation of telomerase or alternative lengthening of telomeres (ALT). The molecular mechanisms regulating the ALT phenotype are poorly understood and it is unknown how the decision of which pathway to activate is made at the cellular...... level. We have shown earlier that active repression of telomerase gene expression by chromatin remodelling of the promoters is one mechanism of regulation; however, other genes and signalling networks are likely to be required to regulate telomerase and maintain the ALT phenotype. Using gene expression...

  3. Telomerase activity and the risk of lung cancer.

    Science.gov (United States)

    Jeon, Hyo-Sung; Choi, Jin Eun; Jung, Deuk Kju; Choi, Yi Young; Kang, Hyo Gyoung; Lee, Won-Kee; Yoo, Seung Soo; Lim, Jeong-Ok; Park, Jae Yong

    2012-02-01

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

  4. Telomerase inhibition targets clonogenic multiple myeloma cells through telomere length-dependent and independent mechanisms.

    Directory of Open Access Journals (Sweden)

    Sarah K Brennan

    Full Text Available BACKGROUND: Plasma cells constitute the majority of tumor cells in multiple myeloma (MM but lack the potential for sustained clonogenic growth. In contrast, clonotypic B cells can engraft and recapitulate disease in immunodeficient mice suggesting they serve as the MM cancer stem cell (CSC. These tumor initiating B cells also share functional features with normal stem cells such as drug resistance and self-renewal potential. Therefore, the cellular processes that regulate normal stem cells may serve as therapeutic targets in MM. Telomerase activity is required for the maintenance of normal adult stem cells, and we examined the activity of the telomerase inhibitor imetelstat against MM CSC. Moreover, we carried out both long and short-term inhibition studies to examine telomere length-dependent and independent activities. METHODOLOGY/PRINCIPAL FINDINGS: Human MM CSC were isolated from cell lines and primary clinical specimens and treated with imetelstat, a specific inhibitor of the reverse transcriptase activity of telomerase. Two weeks of exposure to imetelstat resulted in a significant reduction in telomere length and the inhibition of clonogenic MM growth both in vitro and in vivo. In addition to these relatively long-term effects, 72 hours of imetelstat treatment inhibited clonogenic growth that was associated with MM CSC differentiation based on expression of the plasma cell antigen CD138 and the stem cell marker aldehyde dehydrogenase. Short-term treatment of MM CSC also decreased the expression of genes typically expressed by stem cells (OCT3/4, SOX2, NANOG, and BMI1 as revealed by quantitative real-time PCR. CONCLUSIONS: Telomerase activity regulates the clonogenic growth of MM CSC. Moreover, reductions in MM growth following both long and short-term telomerase inhibition suggest that it impacts CSC through telomere length-dependent and independent mechanisms.

  5. Identification of telomerase RNAs from filamentous fungi reveals conservation with vertebrates and yeasts.

    Directory of Open Access Journals (Sweden)

    Paulius V Kuprys

    Full Text Available Telomeres are the nucleoprotein complexes at eukaryotic chromosomal ends. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which comprises a telomerase reverse transcriptase (TERT and a telomerase RNA (TER. TER contains a template for telomeric DNA synthesis. Filamentous fungi possess extremely short and tightly regulated telomeres. Although TERT is well conserved between most organisms, TER is highly divergent and thus difficult to identify. In order to identify the TER sequence, we used the unusually long telomeric repeat sequence of Aspergillus oryzae together with reverse-transcription-PCR and identified a transcribed sequence that contains the potential template within a region predicted to be single stranded. We report the discovery of TERs from twelve other related filamentous fungi using comparative genomic analysis. These TERs exhibited strong conservation with the vertebrate template sequence, and two of these potentially use the identical template as humans. We demonstrate the existence of important processing elements required for the maturation of yeast TERs such as an Sm site, a 5' splice site and a branch point, within the newly identified TER sequences. RNA folding programs applied to the TER sequences show the presence of secondary structures necessary for telomerase activity, such as a yeast-like template boundary, pseudoknot, and a vertebrate-like three-way junction. These telomerase RNAs identified from filamentous fungi display conserved structural elements from both yeast and vertebrate TERs. These findings not only provide insights into the structure and evolution of a complex RNA but also provide molecular tools to further study telomere dynamics in filamentous fungi.

  6. Critical telomerase activity for uncontrolled cell growth

    Science.gov (United States)

    Wesch, Neil L.; Burlock, Laura J.; Gooding, Robert J.

    2016-08-01

    The lengths of the telomere regions of chromosomes in a population of cells are modelled using a chemical master equation formalism, from which the evolution of the average number of cells of each telomere length is extracted. In particular, the role of the telomere-elongating enzyme telomerase on these dynamics is investigated. We show that for biologically relevant rates of cell birth and death, one finds a critical rate, R crit, of telomerase activity such that the total number of cells diverges. Further, R crit is similar in magnitude to the rates of mitosis and cell death. The possible relationship of this result to replicative immortality and its associated hallmark of cancer is discussed.

  7. Chronotherapy by Ribozyme Targeted to Telomerase in Nude Mice with a Transplanted Tumor: Model for Human Hepatic Cancer%端粒酶核酶时间治疗肝癌裸鼠移植瘤研究

    Institute of Scientific and Technical Information of China (English)

    屈艺; 王正荣; 万朝敏; 黄翔; 母得志; Germaine CORNELISSEN; Franz HALBERG

    2008-01-01

    Objective To investigate therapeutic autcomes of using telomerase inhibitors to treat cancer at the presumably most and least opportune circadian stages basing on our earlier study. Methods Twenty-four BALB/C nude mice were synchronized to a regimen of LD12:12 for 4 wk. Hepatic cancer cells (SMMC-7721) were implanted into both flanks of each mouse.Two weeks after transplantation,the hTERT-5'RZ was used to treat the hepatic cancer transplanted into the nude mice daily for two weeks,the injection times being either 9 or 21 HALO.Results The tumor inhibition ratio of mice treated at 21 HALO (65%) was statistically significantly higher than that of mice treated at 9 HALO (48%). Telomerase activity was also reduced to a greater extent in mice treated with hTERT-5'RZ at 21 than at 9 HALO, that was at the time of maximal circadian telomerase activity. Conclusion Injection of ribozyme targeted to telomerase during the tumor's DNA synthesis is associated with a better inhibition of tumor growth and a better therapeutic outcome in hepatic cancer.%目的 探讨在端粒酶峰值时相与谷值时相施以端粒酶抑制剂治疗肝癌裸鼠移植瘤的疗效差异.方法 24只BALB/C裸鼠进行为期4 wk的LD12: 12节律同步化.移植肝癌细胞(SMMC-7721)于裸鼠以建立肝癌裸鼠移植瘤.在移植后 2 wk,瘤体内注射端粒酶抑制剂hTERT-5'RZ以治疗肝癌裸鼠移植瘤,注射时间点为9 HALO或21 HALO,持续2 wk.结果 21 HALO治疗组的肿瘤抑制率(65%)和端粒酶抑制率(90%)高于9 HALO治疗组的肿瘤抑制率(48%)和端粒酶抑制率(70%),差异具有统计学意义. 结论 在肿瘤DNA合成期注射以端粒酶为作用靶点的核酶能更好地抑制肝癌生长,具有更好的治疗效果.

  8. 放射性皮肤溃疡中端粒酶逆转录晦表达与癌变及难愈合的机制%Differential expression of telomerase reverse transcriptase in chronic human skin ulcer induced by radiation and mechanism of cancer transformation and poor healing

    Institute of Scientific and Technical Information of China (English)

    赵坡; 刘武; 李志军; 吕亚莉; 钟梅; 谷庆阳; 王德文

    2003-01-01

    AIM: To study the expression of the catalytic subunit of telomerase, telomerase revere transcriptase(TRT) and explore the possible relationship between the TRT and cancer txanaformation or poor healing in radiation-induced chronic human skin ulcer. METHODS: Rabbit antibody to human TRT and SP immunohistochemical method were used to detect TRT expression in 24 cases of formalin-fixed, paraffin-embeded chronic human skin ulcer tissues induced by radiation, 5 cases of normal skin, 2 of burnt skin, and 8 of carcinoma. RESULTS: The TRT was detected positive in 14 of 24 (58.3%) chronic radiation ulcers, of which the stxongly positive was 10 of 24 (41.7%) and the weakly positive 4 of 24 (16. 7% ); in 0 of 5 normal and 0 of 2 burnt skins; and in 8 of 8 (100%) carcinomas. The expression of TRT was observed almost always strongly positive in the cytoplasm and nucleus of squamous epithelial cells of epidermis but negatively in the endoepithelial cells of capillaries and small blood vessels, or weakly in the cytoplasm of smooth myocytes of media and fibroblssts, of dermis. Chronic inflammtory cells, such an plasma cells and lymphocytes also showed weakly positive for TRT. CONCLUSION: The strong TRT expression in the epidermis could be involved in the cancer transformation from chronic radiation ulcer to scuamous carcinoma, whereas the negative or weak TRT expression in the capillaries, small blood vessels and fibroblasts of dermis might be responsible for the poor healing of chronic ulcers induced by radiation, caused by sclerosis of small blood vessels and lack of granulation tissue consisting of capillaries and fibroblasts.

  9. Defining the Regulation of Telomerase Through Identification of Mammary-Specific Telomerase Interacting Proteins

    Science.gov (United States)

    2007-06-01

    Studying Telomeres and Telomerase. Zebrafish , 1:349-355. Jones,K.R., L.W.Elmore, L.Povirk, S.E.Holt, and D.A.Gewirtz. 2005. Reciprocal regulation... Zebrafish Blastula Cell Line on Rainbow Trout Stromal Cells and Subsequent Development under Feeder-Free Conditions into a Cell Line, ZEB2J. Zebrafish 5: 49...63. Elmore,L.W., M.Norris, A.T.Bright, P.A.McChesney, R.Winn, and S.E.Holt. 2008. Upregulation of Telomerase Function during Tissue Regeneration in

  10. 人类染色体端粒酶基因在食管癌组织中的表达及其临床意义%Expression of human telomerase gene in esophageal cancer

    Institute of Scientific and Technical Information of China (English)

    陈强; 王新帅; 陈书昌; 高社干; 王玉峰; 冯笑山

    2011-01-01

    Objective To evaluate the dual-color interphase fluorescence in situ hybridization (FISH) of human telomerase gene (hTERC) in the esophageal cancer and preneoplastic lesions. Methods The fluorescence signals of hTERC in the esophageal cancer and preneoplastic lesions were detected by using interphase FISH. According to histology, biopsy and pathology certification, the samples include 45 cases of esophageal normal tissue, 27 cases of atypical hyperplasia Ⅰ , 22 cases of atypical hyperplasia Ⅱ / Ⅲ and 55 cases of squamous cell carcinomas (SCC) of the esophagus. Results None normal samples revealed copy number increases of 3q, while 25.9% of atypical hyperplasia Ⅰ , 54. 5% of atypical hyperplasia Ⅱ/Ⅲ, 90. 9% of the squamous esophageal cancer showed extra copies of 3q. The comparison between groups showed statistically significant difference ( P < 0. 05 ). The mean copy number was 2. 19 ±0. 11,2. 35 ± 0. 30, 2. 64 ± 0. 27 respectively. The positive amplification of the hTERC gene was not correlated with histological stages, lymph nodes metastasis and the grades of carcinoma differentiation in SCC (P > 0. 05 ). Conclusion The presence of hTERC gene may play an important role in the development of SCC.%目的 探讨人类染色体端粒酶(hTERC)基因在食管癌及各相关组织中的表达及其l临床意义.方法 选取食管正常组织45例、Ⅰ度不典型增生组织27例、Ⅱ/Ⅲ不典型增生组织22例、癌组织55例,应用荧光原位杂交(FISH)技术检测各组织中hTERC基因的表达.结果 hTERC基因在食管正常组织、Ⅰ度不典型增生组织、Ⅱ/Ⅲ度不典型增生组织及食管癌组织中表达率分别为0%、25.9%、54.5%及90.9%,各组间比较差异有统计学意义(P<0.05);癌前病变及癌基因平均拷贝数分别为2.19±0.11、2.35±0.30及2.64±0.27,显示出扩增趋势.食管癌组织中hTERC基因表达在组织分化程度、浸润深度、大体类型、淋巴结转移

  11. Anti-HIV-1 Activities of 4 Telomerase Restrictors

    Institute of Scientific and Technical Information of China (English)

    YU Xin; WANG Jinghui; de Giuli Morghen; Radaelli A; Zanotto C; Beggio P

    2007-01-01

    MTT Cell Proliferation Assay was used to optimize the concentration of Telomerase Restrictors(TRs) with minimum toxicity to the selected cells. FACSort flow cytometer and Innotest P24 HIV(Human immunodeficiency Virus) antigen mAb ELISA Kit were used to investigate the anti-HIV-1 activities of TRs. The results showed that TRs had low cytotoxicity to the PBMC (Peripheral Blood mononuclear cells) and CEM/GFP if the concentration of TRs was at 50 μmol/L or below, and the supernatant from PBMC pretreated with SHIV and TR1-001 /TR1-002 could not infect the PBMC, while can infect the C8166 with reduced infectivity, which suggested that the TRs may be one of the novel resources for screening anti-HIV-1 agents.

  12. Telomerase and telomere-associated proteins: structural insights into mechanism and evolution.

    Science.gov (United States)

    Lewis, Karen A; Wuttke, Deborah S

    2012-01-11

    Recent advances in our structural understanding of telomerase and telomere-associated proteins have contributed significantly to elucidating the molecular mechanisms of telomere maintenance. The structures of telomerase TERT domains have provided valuable insights into how experimentally identified conserved motifs contribute to the telomerase reverse transcriptase reaction. Additionally, structures of telomere-associated proteins in a variety of organisms have revealed that, across evolution, telomere-maintenance mechanisms employ common structural elements. For example, the single-stranded 3' overhang of telomeric DNA is specifically and tightly bound by an OB-fold in nearly all species, including ciliates (TEBP and Pot1a), fission yeast (SpPot1), budding yeast (Cdc13), and humans (hPOT1). Structures of the yeast Cdc13, Stn1, and Ten1 proteins demonstrated that telomere maintenance is regulated by a complex that bears significant similarity to the RPA heterotrimer. Similarly, proteins that specifically bind double-stranded telomeric DNA in divergent species use homeodomains to execute their functions (human TRF1 and TRF2 and budding yeast ScRap1). Likewise, the conserved protein Rap1, which is found in budding yeast, fission yeast, and humans, contains a structural motif that is known to be critical for protein-protein interaction. In addition to revealing the common underlying themes of telomere maintenance, structures have also elucidated the specific mechanisms by which many of these proteins function, including identifying a telomere-specific domain in Stn1 and how the human TRF proteins avoid heterodimerization. In this review, we summarize the high-resolution structures of telomerase and telomere-associated proteins and discuss the emergent common structural themes among these proteins. We also address how these high-resolution structures complement biochemical and cellular studies to enhance our understanding of telomere maintenance and function.

  13. System x(c)- activity and astrocytes are necessary for interleukin-1 beta-mediated hypoxic neuronal injury.

    Science.gov (United States)

    Fogal, Birgit; Li, Jun; Lobner, Doug; McCullough, Louise D; Hewett, Sandra J

    2007-09-19

    The purpose of this study was to elucidate the cellular/biochemical pathway(s) by which interleukin-1beta (IL-1beta) contributes to the pathogenesis of hypoxic-ischemic brain damage. In vivo, IL-1 receptor type I (IL-1RI)-deficient mice showed smaller infarcts and less neurological deficits than wild-type animals after a 90 min reversible middle cerebral artery occlusion. In vitro, IL-1beta mediated an enhancement of hypoxic neuronal injury in murine cortical cultures that was lacking in cultures derived from IL-1RI null mutant animals and was blocked by the IL-1 receptor antagonist or an IL-1RI blocking antibody. This IL-1beta-mediated potentiation of hypoxic neuronal injury was associated with an increase in both cellular cystine uptake ([cystine]i) and extracellular glutamate levels ([glutamate]e) and was prevented by either ionotropic glutamate receptor antagonism or removal of L-cystine, suggesting a role for the cystine/glutamate antiporter (System x(c)-). Indeed, dual System x(c)-/metabotropic glutamate receptor subunit 1 (mGluR1) antagonism but not selective mGluR1 antagonism prevented neuronal injury. Additionally, cultures derived from mGluR1-deficient mice exhibited the same potentiation in injury after treatment with IL-1beta as wild-type cultures, an effect prevented by System x(c)-/mGluR1 antagonism. Finally, assessment of System x(c)- function and kinetics in IL-1beta-treated cultures revealed an increase in velocity of cystine transport (Vmax), in the absence of a change in affinity (Km). Neither the enhancement in [cystine]i, [glutamate]e, or neuronal injury were observed in chimeric cultures consisting of IL-1RI(+/+) neurons plated on top of IL-1RI(-/-) astrocytes, highlighting the importance of astrocyte-mediated alterations in System x(c)- as a novel contributor to the development and progression of hypoxic neuronal injury.

  14. Eimeria tenella: 14-3-3 protein interacts with telomerase.

    Science.gov (United States)

    Zhao, Na; Gong, Pengtao; Cheng, Baiqi; Li, Jianhua; Yang, Zhengtao; Li, He; Yang, Ju; Zhang, Guocai; Zhang, Xichen

    2014-10-01

    Telomerase, consisting of telomerase RNA and telomerase reverse transcriptase (TERT), is responsible for the maintenance of the end of linear chromosomes. TERT, as the catalytic subunit of telomerase, plays a critical role in telomerase activity. Researches indicate TERT-associated proteins participate in the regulation of telomerase assembly, posttranslational modification, localization, and enzymatic function. Here, the telomerase RNA-binding domain of Eimeria tenella TERT (EtTRBD) was cloned into pGBKT7 and performed as the bait. α-Galactosidase assay showed that the bait plasmid did not activate Gal4 reporter gene. Further, we isolated an EtTRBD-associated protein, 14-3-3, by yeast two-hybrid screening using the constructed bait plasmid. To confirm the interaction, EtTRBD and 14-3-3 were expressed by prokaryotic and eukaryotic expression systems. Pull-down assays by purified proteins demonstrated a direct bind between EtTRBD and 14-3-3. Co-immunoprecipitation techniques successfully validated that 14-3-3 interacted with EtTRBD in 293T cells. The protein-protein interaction provides a starting point for more in-depth studies on telomerase and telomere regulation in E. tenella.

  15. Activity of telomerase and telomeric length in Apis mellifera.

    Science.gov (United States)

    Korandová, Michala; Frydrychová, Radmila Čapková

    2016-06-01

    Telomerase is an enzyme that adds repeats of DNA sequences to the ends of chromosomes, thereby preventing their shortening. Telomerase activity is associated with proliferative status of cells, organismal development, and aging. We report an analysis of telomerase activity and telomere length in the honeybee, Apis mellifera. Telomerase activity was found to be regulated in a development and caste-specific manner. During the development of somatic tissues of larval drones and workers, telomerase activity declined to 10 % of its level in embryos and remained low during pupal and adult stages but was upregulated in testes of late pupae, where it reached 70 % of the embryo level. Upregulation of telomerase activity was observed in the ovaries of late pupal queens, reaching 160 % of the level in embryos. Compared to workers and drones, queens displayed higher levels of telomerase activity. In the third larval instar of queens, telomerase activity reached the embryo level, and an enormous increase was observed in adult brains of queens, showing a 70-fold increase compared to a brain of an adult worker. Southern hybridization of terminal TTAGG fragments revealed a high variability of telomeric length between different individuals, although the same pattern of hybridization signals was observed in different tissues of each individual.

  16. DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

    Energy Technology Data Exchange (ETDEWEB)

    Cogan, Nicola [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Baird, Duncan M. [Department of Pathology School of Medicine, Cardiff University, Henry Wellcome Building for Biomedical Research in Wales, Heath Park, Cardiff, CF14 4XN (United Kingdom); Phillips, Ryan [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Crompton, Lucy A.; Caldwell, Maeve A. [Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, BS1 3NY (United Kingdom); Rubio, Miguel A. [Center of Regenerative Medicine in Barcelona, CMRB Dr. Aiguader, 88, 7th Floor, 08003 Barcelona (Spain); Newson, Roger [Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Dublin 2 (Ireland); Lyng, Fiona [National Heart and Lung Institute, Imperial College London, London, SW7 2AZ (United Kingdom); Case, C. Patrick, E-mail: c.p.case@bristol.ac.uk [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom)

    2010-01-05

    The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

  17. Transcriptional Regulation of Telomerase Reverse Transcriptase (TERT) by MYC

    Science.gov (United States)

    Khattar, Ekta; Tergaonkar, Vinay

    2017-01-01

    Telomerase elongates telomeres and is crucial for maintaining genomic stability. While stem cells and cancer cells display high telomerase activity, normal somatic cells lack telomerase activity primarily due to transcriptional repression of telomerase reverse transcriptase (TERT), the catalytic component of telomerase. Transcription factor binding, chromatin status as well as epigenetic modifications at the TERT promoter regulates TERT transcription. Myc is an important transcriptional regulator of TERT that directly controls its expression by promoter binding and associating with other transcription factors. In this review, we discuss the current understanding of the molecular mechanisms behind regulation of TERT transcription by Myc. We also discuss future perspectives in investigating the regulation of Myc at TERT promoter during cancer development.

  18. Telomerase and it's inhibition in caner: a review article

    Directory of Open Access Journals (Sweden)

    Noori-Daloii MR

    2009-12-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Telomere, by which is a terminal structure of eukaryotic chromosomes was discovered at first in 1938 and has a vital role in chromosome protection. Telomere in human and other vertebrates consists of thousands of 5′-TTAGGG-3′ tandem repeats at the end of the chromosome, has a main role in the chromosome stability. Telomere protects the end of the chromosome from degeneration, rearrangement and end to end fusion. There is a telomere loss at every cell division. Progressive loss in telomere length results in disassociation of telomere binding proteins and change in gene expression profiles. Adjacent genes are suppressed by the telomere effect so the telomere loss results in adjacent gene expressions. Apoptosis and replicative senescence are caused by progressive telomere loss. There are three mechanisms for increasing telomere length in eukaryotes and telomerase is the predominant mechanism. Telomerase can synthesize telomere, without the template. Telomerase is overexpressed In 90% of cancers. Therefore cancerous cells compensate the telomere loss in every cell division because of telomerase. In conclusion, telomerase is a proper target for cancer therapy and many methods including direct inhibition of telomerase

  19. Urine Telomerase: An Important Marker in the Diagnosis of Bladder Cancer

    Directory of Open Access Journals (Sweden)

    Maria Aurora Sanchini

    2004-05-01

    Full Text Available Telomerase activity is present in most human malignant tumors, whereas it is generally not detectable, with some exceptions, in normal cells. Therefore, it represents a potential tool for tumor detection. In the present study, telomerase activity was determined in urine from 79 healthy individuals and 121 previously untreated bladder cancer patients using a highly sensitive telomeric repeat amplification protocol (TRAP assay and the results were expressed as arbitrary enzymatic units (AEU. This approach enabled us to identify cutoff values characterized by high sensitivity (from 75% to 93% and specificity (from 72% to 92%. Moreover, analysis as a function of gender showed a higher accuracy of TRAP assay in males (93% sensitivity and 90% specificity at the cutoff of 50 AEU than in females. This sensitivity was confirmed in patients with nonassessable or negative cytology. In women, morphological and immunocytochemical determinations using a human telomerase reverse transcriptase monoclonal antibody (anti-hTERT recently developed in our laboratory showed a large fraction of immunoreactive inflammatory or nonbladder cells, which may justify the false-positive TRAP results. In conclusion, this assay represents an important noninvasive diagnostic tool to detect bladder cancer also in patients with negative or nonassessable urine cytology and with low-grade and early-stage lesions.

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

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-bin; ZHU Li; HUANG Jun; YIN Yi-gang; KONG Xiang-qing; RONG Qi-fei; SHI Ai-wu; CAO Ke-jiang

    2011-01-01

    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 μmol/L) or control for specified times.After in vitro cultivation,acidic β-galactosidase 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 Resveratrol 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.

  1. Porphyrin-aminoquinoline conjugates as telomerase inhibitors.

    Science.gov (United States)

    Maraval, Alexandrine; Franco, Sonia; Vialas, Corine; Pratviel, Geneviève; Blasco, Maria A; Meunier, Bernard

    2003-03-21

    A series of metalloporphyrins was prepared in order to target the G-quadruplex structure of telomeric DNA for the design of antitelomerase compounds. The initial cationic tetramethylpyridiniumyl porphyrin was modified by the replacement of one or two methylpyridiniumyl groups by one or two 4-aminoquinoline moieties, at the meso position, in order to increase the cell penetration and the quadruplex affinity. The porphyrins were either metallated by manganese or by nickel. The degradation of quadruplex DNA was assayed in vitro with the manganese redox-active derivatives. All porphyrins complexes were capable of inhibiting the telomerase enzyme with IC50 values in the micromolar range (TRAP assay).

  2. Telomeric overhang length determines structural dynamics and accessibility to telomerase and ALT associated proteins

    Science.gov (United States)

    Hwang, Helen; Kreig, Alex; Calvert, Jacob; Lormand, Justin; Kwon, Yongho; Daley, James M.; Sung, Patrick; Opresko, Patricia L.; Myong, Sua

    2014-01-01

    The G-rich single stranded DNA at the 3′ end of human telomeres can self-fold into G-quaduplex (GQ). However, telomere lengthening by telomerase or the recombination-based alternative lengthening of telomere (ALT) mechanism requires protein loading on the overhang. Using single molecule fluorescence spectroscopy we discovered that lengthening the telomeric overhang also increased the rate of dynamic exchanges between structural conformations. Overhangs with five to seven TTAGGG repeats, compared to four repeats, showed much greater dynamics and accessibility to telomerase binding and activity, and loading of the ALT-associated proteins RAD51, WRN and BLM. Although the eight repeats are highly dynamic, they can fold into two GQs, which limited protein accessibility. In contrast, the telomere-specific protein, POT1 is unique in that it binds independently of repeat number. Our results suggest that the telomeric overhang length and dynamics may contribute to the regulation of telomere extension via telomerase action and the ALT mechanism. PMID:24836024

  3. 雌二醇通过增加端粒酶活性影响人LECs生长动力学的实验研究%The regulation of estradiol on growth dynamics of human LECs affected by increasing telomerase activity

    Institute of Scientific and Technical Information of China (English)

    王杰; 康刚劲; 袁雪峰; 徐曼华; 蒋燕; 罗波

    2016-01-01

    Background Human LECs can express telomerase activity,which participates in the formation of cataract.It is reported that estrogen can increase the expression of telomerase activity in human endometrial cancer and breast cancer cells and play an important role in promoting proliferation and anti-apoptosis,but whether estrogen exerts its role on human LECs is still unclear.Objective This study aimed to investigate whether β-estradiol (β-E2) can increase the telomerase activity of human LECs and the influence of β-E2 on proliferation and apoptosis of human LECs.Method Human LECs line was cultured and passaged in vitro,and 1×10-6 mol/L β-E2 was added in the medium for 0,6,12,24 and 48 hours,and reverse transcription PCR was used to determine the optimal time of the expression of human telomerase reverse transcriptase (hTERT) mRNA in the cells.Cultured cells were divided into five groups.The cells in the blank contol group were cultured in the routin medium.Ethanol of 0.1% was added in the solvent control group,and 1 × 10-8,1 × 10-7 or 1 × 10-6 mol/L β-E2 was added in the medium in different contents of β-E2 groups,respectively.The relative expression level of hTERT mRNA in different groups was detected by reverse transcription PCR.Telomere repeat amplification protocol (TRAP)-ELISA was employed to determine the telomerase activity.The proliferative value of the cells was assayed by cell counting kit-8,and the apoptosis rate of the cells was examined by Hoechst33258 staining.Results The optimal time of β-E2 to rise the expression of hTERT mRNA (absorbance) was at 24 hours under the 1×10-6 mol/L.The relative expression levels of hTERT mRNA in the cells were 0.477±0.015,0.712±0.013 and 0.914±0.031 in the 1 ×10-8,1 ×10-7 and 1 ×10-6 mol/L β-E2 group,which were signifincatly higher than 0.428±0.010 in the blank control group and 0.426±0.010 in the solvent control group (all at P<0.05).The telomerase activity values (absorbance) were 0.711 ±0

  4. Phytoestrogens induce differential estrogen receptor alpha- or Beta-mediated responses in transfected breast cancer cells.

    Science.gov (United States)

    Harris, D M; Besselink, E; Henning, S M; Go, V L W; Heber, D

    2005-09-01

    Increased intake of phytoestrogens may be associated with a lower risk of cancer in the breast and several other sites, although there is controversy surrounding this activity. One of the mechanisms proposed to explain the activity of phytoestrogens is their ability to bind and activate human estrogen receptor alpha (ERalpha) and human estrogen receptor beta (ERbeta). Nine phytoestrogens were tested for their ability to transactivate ERalpha or ERbeta at a range of doses. Mammary adenocarcinoma (MCF-7) cells were co-transfected with either ERalpha or ERbeta, and an estrogen-response element was linked to a luciferase reporter gene. Dose-dependent responses were compared with the endogenous ligand 17beta-estradiol. Purified genistein, daidzein, apigenin, and coumestrol showed differential and robust transactivation of ERalpha- and ERbeta-induced transcription, with an up to 100-fold stronger activation of ERbeta. Equol, naringenin, and kaempferol were weaker agonists. When activity was evaluated against a background of 0.5 nM 17beta-estradiol, the addition of genistein, daidzein, and resveratrol superstimulated the system, while kaempferol and quercetin were antagonists at the highest doses. This transfection assay provides an excellent model to evaluate the activation of ERalpha and ERbeta by different phytoestrogens in a breast cancer context and can be used as a screening bioassay tool to evaluate the estrogenic activity of extracts of herbs and foods.

  5. Impact of Arsenic Trioxide on LS-174T Cell Growth in vitro and the Activity of Telomerase

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The therapeutic action of arsenic trioxide( As2O3 ) on solid tumors has aroused widespread interest among scholars. To study the impact of As2 O3 on human colorectal carcinoma cells( LS-174T cell) and the activity of telomerase,the methods of PCR-ELISA, flow cytometry (FCM) and MTT assay in vitro were utilized. The results show that ( 1 ) with an increase in the concentration of As2 O3, the ratio of living the cells to dead cells decreases significantly,and the IC50 value is 5. 23 μg/mL; (2) the cells of the experimental groups can endure a series of morphological changes similar to the features of apoptosis; (3) the apoptotic curves of FCM pictures appear after 24 h, and the cells show the apoptosis in a time-dependent manner; (4) As2O3 can inhibit the activity of telomerase of the cell extraction obviously in a concentration-dependent and time-dependent manner after 24 h. It can be concluded from the experiment results in vitro that As2O3 can induce the apoptosis of LS-174T cells and inhibit the telomerase activity. Therefore, it has been proposed, for the first time, that these two factors( the apoptosis of LS-174T cells and the inhibition to the telomerase activity) are important causes of the LS-174T cell death caused by As2O3.

  6. Telomerase reverse transcriptase promoter mutations in bladder cancer

    DEFF Research Database (Denmark)

    Allory, Yves; Beukers, Willemien; Sagrera, Ana

    2014-01-01

    BACKGROUND: Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. OBJECTIVES: To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential ...

  7. Telomere shortening and telomerase activity in ischaemic cardiomyopathy patients

    DEFF Research Database (Denmark)

    Sawhney, V; Campbell, N G; Brouilette, S W

    2016-01-01

    BACKGROUND: Implantable cardioverter defibrillators (ICDs) reduce mortality in patients with ischaemic cardiomyopathy at high risk of ventricular arrhythmias (VA). However, the current indication for ICD prescription needs improvement. Telomere and telomerase in leucocytes have been shown to asso...

  8. Telomerase promoter mutations in cancer: an emerging molecular biomarker?

    Science.gov (United States)

    Vinagre, João; Pinto, Vasco; Celestino, Ricardo; Reis, Marta; Pópulo, Helena; Boaventura, Paula; Melo, Miguel; Catarino, Telmo; Lima, Jorge; Lopes, José Manuel; Máximo, Valdemar; Sobrinho-Simões, Manuel; Soares, Paula

    2014-08-01

    Cell immortalization has been considered for a long time as a classic hallmark of cancer cells. Besides telomerase reactivation, such immortalization could be due to telomere maintenance through the "alternative mechanism of telomere lengthening" (ALT) but the mechanisms underlying both forms of reactivation remained elusive. Mutations in the coding region of telomerase gene are very rare in the cancer setting, despite being associated with some degenerative diseases. Recently, mutations in telomerase (TERT) gene promoter were found in sporadic and familial melanoma and subsequently in several cancer models, notably in gliomas, thyroid cancer and bladder cancer. The importance of these findings has been reinforced by the association of TERT mutations in some cancer types with tumour aggressiveness and patient survival. In the first part of this review, we summarize the data on the biology of telomeres and telomerase, available methodological approaches and non-neoplastic diseases associated with telomere dysfunction. In the second part, we review the information on telomerase expression and genetic alterations in the most relevant types of cancer (skin, thyroid, bladder and central nervous system) on record, and discuss the value of telomerase as a new biomarker with impact on the prognosis and survival of the patients and as a putative therapeutic target.

  9. Suppression of telomere-binding protein TPP1 resulted in telomere dysfunction and enhanced radiation sensitivity in telomerase-negative osteosarcoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Qiang, Weiguang [Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan (China); Department of Oncology, The Third Affiliated Hospital, Soochow University, Changzhou (China); Wu, Qinqin [Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan (China); Department of Radiation Oncology, Changzhou Tumor Hospital, Soochow University, Changzhou (China); Zhou, Fuxiang; Xie, Conghua [Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan (China); Wu, Changping, E-mail: wcpzlk@163.com [Department of Oncology, The Third Affiliated Hospital, Soochow University, Changzhou (China); Zhou, Yunfeng, E-mail: yfzhouwhu@163.com [Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan (China)

    2014-03-07

    Highlights: • Down-regulation of TPP1 shortened telomere length in telomerase-negative cells. • Down-regulation of TPP1 induced cell apoptosis in telomerase-negative cells. • Down-regulation of TPP1 increased radiosensitivity in telomerase-negative cells. - Abstract: Mammalian telomeres are protected by the shelterin complex that contains the six core proteins POT1, TPP1, TIN2, TRF1, TRF2 and RAP1. TPP1, formerly known as TINT1, PTOP, and PIP1, is a key factor that regulates telomerase recruitment and activity. In addition to this, TPP1 is required to mediate the shelterin assembly and stabilize telomere. Previous work has found that TPP1 expression was elevated in radioresistant cells and that overexpression of TPP1 led to radioresistance and telomere lengthening in telomerase-positive cells. However, the exact effects and mechanism of TPP1 on radiosensitivity are yet to be precisely defined in the ALT cells. Here we report on the phenotypes of the conditional deletion of TPP1 from the human osteosarcoma U2OS cells using ALT pathway to extend the telomeres.TPP1 deletion resulted in telomere shortening, increased apoptosis and radiation sensitivity enhancement. Together, our findings show that TPP1 plays a vital role in telomere maintenance and protection and establish an intimate relationship between TPP1, telomere and cellular response to ionizing radiation, but likely has the specific mechanism yet to be defined.

  10. Herpesvirus telomerase RNA (vTR with a mutated template sequence abrogates herpesvirus-induced lymphomagenesis.

    Directory of Open Access Journals (Sweden)

    Benedikt B Kaufer

    2011-10-01

    Full Text Available Telomerase reverse transcriptase (TERT and telomerase RNA (TR represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5 by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1 that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2 that this strategy could be used to generate novel vaccine candidates

  11. Cloning and molecular characterization of telomerase reverse transcriptase (TERT) and telomere length analysis of Peromyscus leucopus.

    Science.gov (United States)

    Zhao, Xin; Ueda, Yasutaka; Kajigaya, Sachiko; Alaks, Glen; Desierto, Marie J; Townsley, Danielle M; Dumitriu, Bogdan; Chen, Jichun; Lacy, Robert C; Young, Neal S

    2015-08-15

    Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase complex that regulates telomerase activity to maintain telomere length for all animals with linear chromosomes. As the Mus musculus (MM) laboratory mouse has very long telomeres compared to humans, a potential alternative animal model for telomere research is the Peromyscus leucopus (PL) mouse that has telomere lengths close to the human range and has the wild counterparts for comparison. We report the full TERT coding sequence (pTERT) from PL mice to use in the telomere research. Comparative analysis with eight other mammalian TERTs revealed a pTERT protein considerably homologous to other TERTs and preserved all TERT specific-sequence signatures, yet with some distinctive features. pTERT displayed the highest nucleotide and amino acid sequence homology with hamster TERT. Unlike human but similar to MM mice, pTERT expression was detected in various adult somatic tissues of PL mice, with the highest expression in testes. Four different captive stocks of PL mice and wild-captured PL mice each displayed group-specific average telomere lengths, with the longest and shortest telomeres in inbred and outbred stock mice, respectively. pTERT showed considerable numbers of synonymous and nonsynonymous mutations. A pTERT proximal promoter region cloned was homologous among PL and MM mice and rat, but with species-specific features. From PL mice, we further cloned and characterized ribosomal protein, large, P0 (pRPLP0) to use as an internal control for various assays. Peromyscus mice have been extensively used for various studies, including human diseases, for which pTERT and pRPLP0 would be useful tools.

  12. BLOOD TELOMERASE ACTIVITY AND ITS CORRELATIVITY WITH NON-SMALL CELL LUNG CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    胡坚; 李任远; 孙骊; 倪一鸣

    2004-01-01

    Objective: To study the correlativity between blood telomerase activity and Non-small cell lung carcinoma (NSCLC) through relative quantitative analysis of telomerase activity. Methods: Thirty-eight NSCLC and 25 inpatients with benign lung disease were selected. Telomerase repeat amplification protocol was adopted. PCR products were assayed with ELISA. Results: (a) Blood telomerase activity during operation was higher than that before or after operation (P0.05). (c) Blood telomerase activity of adenocarcinoma during and after operation was higher than that before operation (P0.05). Conclusion: The qualitative assay of blood telomerase activity can be adopted as an assistant index for diagnosis of NSCLC. Postoperative blood telomerase activity of adenocarcinoma is higher than that of squamous carcinoma. It may be an evidence for the likelihood of adenocarcinoma to metastase through blood. Blood telomerase activity increases significantly during operation, suggesting that operation may cause more cancer cells entering into circulation.

  13. Telomeric noncoding RNA TERRA is induced by telomere shortening to nucleate telomerase molecules at short telomeres.

    Science.gov (United States)

    Cusanelli, Emilio; Romero, Carmina Angelica Perez; Chartrand, Pascal

    2013-09-26

    Elongation of a short telomere depends on the action of multiple telomerase molecules, which are visible as telomerase RNA foci or clusters associated with telomeres in yeast and mammalian cells. How several telomerase molecules act on a single short telomere is unknown. Herein, we report that the telomeric noncoding RNA TERRA is involved in the nucleation of telomerase molecules into clusters prior to their recruitment at a short telomere. We find that telomere shortening induces TERRA expression, leading to the accumulation of TERRA molecules into a nuclear focus. Simultaneous time-lapse imaging of telomerase RNA and TERRA reveals spontaneous events of telomerase nucleation on TERRA foci in early S phase, generating TERRA-telomerase clusters. This cluster is subsequently recruited to the short telomere from which TERRA transcripts originate during S phase. We propose that telomere shortening induces noncoding RNA expression to coordinate the recruitment and activity of telomerase molecules at short telomeres.

  14. Construction of human telomerase reverse transcriptase-immortalized rat bone marrow mesenchemal stem cell strains%构建人端粒酶反转录酶修饰的永生化大鼠骨髓间充质干细胞株

    Institute of Scientific and Technical Information of China (English)

    刘慧萍; 钟晓龙; 赵晴; 黄文起; 安珂

    2015-01-01

    BACKGROUND:Because of convenient source, multi-lineage differentiation and low immunogenicity, bone marrow mesenchymal stem cels are the ideal cel type to serve as vectors of transgenic cels in pain management. However, the replicative senescence and smal amount of cels obtained from the bone marrow restrict the application of bone marrow mesenchymal stem cels in pain research. OBJECTIVE:To construct human telomerase reverse transcriptase (hTERT)-immortalized rat bone marrow mesenchymal stem cels as transgenic celular vectors for pain therapy. METHODS:Bone marrow mesenchymal stem cels were obtained from whole rat bone marrow, and then transfected with a lentivirus containing the hTERT (pLV-Puro-EF1α-hTERT) folowed by puromycin selection. hTERT expression and telomerase activity in these transfected cels were determined by RT-PCR and TRAP. Morphological changes, capacity of cel growth and multi-lineage differentiation, chromosome karyotype and tumorigenicity were observed in vitro. Moreover, the expression of cel surface molecule, Nestin, MHC-I and MHC-II in transfected cels were also detected by flow cytometry and immunocytochemistry. RESULTS AND CONCLUSION: The bone marrow mesenchymal stem cels geneticaly modified by hTERT could be cultured and passaged through 30 generations in vitro. Compared to the primary and negative transfected cels, the hTERT-modified bone marrow mesenchymal stem cels showed higher expression of hTERT mRNA, telomerase activity and cel proliferation. Most of transfected cels stayed at G2/M and S stages. The proliferation index of the transfected cels were increased dramaticaly. The positive rates of CD29, CD44 and CD90 were over 70%, but the positive rates of CD34 and CD45 were less than 5%. Transfected cels were positive for Nestin in the cytoplasm, but negative for MHC-1 and MHC-11. In addition, this cel line continued to exhibit the characteristics of fibroblastic bone marrow mesenchymal stem cels, including phenotype, differentiation

  15. Relationshap between human papillomavirus DNA testing and human telomerase RNA gene amplification detection in esophageal cancer tissue%食管癌组织人乳头瘤病毒感染与人端粒酶RNA基因表达的关系

    Institute of Scientific and Technical Information of China (English)

    王新帅; 陈强; 王玉峰; 高社干; 张广平; 冯笑山

    2012-01-01

    Objective To study the relationshap between human papillomavirus (HPV) DNA testing and human telomerase (hTERC) RNA gene amplification detection in esophageal cancer tissue.Methods HPV DNA was tested by polymerase chain reaction (PCR) in 82 cases of esophageal cancer tissues.The fluorescence signals of hTERC in the esophageal cancer were detected by using interphase fluorescence in situ hybridization ( FISH ).Results There was HPV infection in esophageal cancer tissue,and the infection rate was about 43.7%.Mixed type was predominant,including the HPV 16 and 18.The mutation rate of hTERC in esophageal cancer tissue was about 80.1%,and there was a positive correlation between the HPV infection and mutation of hTERC (P<0.05 ).Conclusion HPV infection and mutation of hTERC in the esophageal cancer may be important factors of pathogenesis.The mutation of hTERC gene may plav an important role in the development of sequamous cell carcinoma.%目的 探讨食管癌组织中人乳头瘤病毒(HPV)感染与人端粒酶RNA(hTERC)基因表达的关系.方法 82例经病理确诊的食管癌患者组织标本,聚合酶链反应(PCR)技术结合等离子谐振技术检测食管癌组织中HPV感染,荧光原位杂交技术检测食管癌组织中hTERC基因的表达.结果 食管癌组织中存在HPV感染,感染率为43.7%,主要以混合型为主,包括高危亚型HPV16、18;食管癌组织中hTERC 基因的表达率为80.1%;HPV感染与hTERC基因表达关系密切(x2=9.834,P<0.05).结论 食管癌组织中HPV感染与hTERC基因表达是食管癌发病的重要因素,它可能通过hTERC基因途径引起癌变结果.

  16. Impaired telomerase activity hinders proliferation and in vitro transformation of Penaeus monodon lymphoid cells.

    Science.gov (United States)

    Jayesh, P; Vrinda, S; Priyaja, P; Philip, Rosamma; Singh, I S Bright

    2016-08-01

    Retaining terminal transferase activity of telomerase, the ribonucleoprotein enzyme which add telomeric repeats on chromosome end is thought to be required to prevent cellular ageing. Additionally, telomerase considered as a marker for cell proliferation and immortalization in eukaryotes. We examined telomerase activity in tissues and lymphoid cell culture of Penaeus monodon. Along with telomerase activity, telomere repeats and an attempt on identification of telomerase reverse transcriptase (PmTERT) were made. Telomeric repeat amplification protocol revealed that telomerase-dependent telomeric lengthening has been taking place in P. monodon and the adult tissues were retaining this capacity throughout their lifespan with the highest activity in ovary, testis and lymphoid organ. However, telomerase activity could not be detected in lymphoid cells in culture. The canonical telomeric repeats added by telomerase of lymphoid tissue extract were identified as TTAGG, but pentameric repeats GGTTA and AGGTT were also added by the telomerase. PmTERT protein sequence (partial) shared 100 % identity with the TERT sequence of Daphnia pulex, 27 % sequence identity with Purple sea urchin and 24-25 % with Zebra fish. Undetectable telomerase activity in lymphoid cell culture supports the hypothesis that the inadequate telomerase activity or gene expression may be a reason that prevents neoplastic transformation and spontaneous immortalization of the cells in vitro. Thus, it is envisaged that telomerase activation in lymphoid cells may surmount cellular ageing for in vitro transformation and cell line establishment.

  17. Structure and folding of the Tetrahymena telomerase RNA pseudoknot

    Science.gov (United States)

    Cash, Darian D.; Feigon, Juli

    2017-01-01

    Telomerase maintains telomere length at the ends of linear chromosomes using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). An essential part of TER is the template/pseudoknot domain (t/PK) which includes the template, for adding telomeric repeats, template boundary element (TBE), and pseudoknot, enclosed in a circle by stem 1. The Tetrahymena telomerase holoenzyme catalytic core (p65-TER-TERT) was recently modeled in our 9 Å resolution cryo-electron microscopy map by fitting protein and TER domains, including a solution NMR structure of the Tetrahymena pseudoknot. Here, we describe in detail the structure and folding of the isolated pseudoknot, which forms a compact structure with major groove U•A-U and novel C•G-A+ base triples. Base substitutions that disrupt the base triples reduce telomerase activity in vitro. NMR studies also reveal that the pseudoknot does not form in the context of full-length TER in the absence of TERT, due to formation of a competing structure that sequesters pseudoknot residues. The residues around the TBE remain unpaired, potentially providing access by TERT to this high affinity binding site during an early step in TERT-TER assembly. A model for the assembly pathway of the catalytic core is proposed. PMID:27899638

  18. Clonal cell populations unresponsive to radiosensitization induced by telomerase inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Ju, Yeun-Jin; Shin, Hyun-Jin; Park, Jeong-Eun; Juhn, Kyoung-Mi; Woo, Seon Rang; Kim, Hee-Young; Han, Young-Hoon; Hwang, Sang-Gu; Hong, Sung-Hee; Kang, Chang-Mo [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Yoo, Young-Do [Laboratory of Molecular Cell Biology, Graduate School of Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Park, Won-Bong [Division of Natural Science, Seoul Women' s University, Seoul 139-774 (Korea, Republic of); Cho, Myung-Haing [Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul (Korea, Republic of); Park, Gil Hong, E-mail: ghpark@korea.ac.kr [Department of Biochemistry, College of Medicine, Korea University, Seoul (Korea, Republic of); Lee, Kee-Ho, E-mail: khlee@kirams.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of)

    2010-11-12

    Research highlights: {yields} In our present manuscript, we have clearly showed an interesting but problematic obstacle of a radiosensitization strategy based on telomerase inhibition by showing that: Clonal population unresponsive to this radiosensitization occasionally arise. {yields} The telomere length of unsensitized clones was reduced, as was that of most sensitized clones. {yields} The unsensitized clones did not show chromosome end fusion which was noted in all sensitized clones. {yields} P53 status is not associated with the occurrence of unsensitized clone. {yields} Telomere end capping in unsensitized clone is operative even under telomerase deficiency. -- Abstract: A combination of a radiotherapeutic regimen with telomerase inhibition is valuable when tumor cells are to be sensitized to radiation. Here, we describe cell clones unresponsive to radiosensitization after telomere shortening. After extensive division of individual transformed clones of mTERC{sup -/-} cells, about 22% of clones were unresponsive to radiosensitization even though telomerase action was inhibited. The telomere lengths of unsensitized mTERC{sup -/-} clones were reduced, as were those of most sensitized clones. However, the unsensitized clones did not exhibit chromosomal end-to-end fusion to the extent noted in all sensitized clones. Thus, a defense mechanism preventing telomere erosion is operative even when telomeres become shorter under conditions of telomerase deficiency, and results in unresponsiveness to the radiosensitization generally mediated by telomere shortening.

  19. ptsA58H质粒转染人胚腱细胞的增殖特性及端粒酶活性分析%PROLIFERATION PROPERTIES AND TELOMERASE ACTIVITY OF HUMAN EMBRYONIC TENDON CELLS TRANSFORMED BY ptsA58H PLASMID

    Institute of Scientific and Technical Information of China (English)

    解慧琪; 杨志明; 屈艺; 秦廷武; 李秀群

    2001-01-01

    Objective To analysis the proliferation properties and telomerase activity of human embryonic tendon cells transformed by ptsA58H plasmid cultured in vitro continuously. Methods The 40th, 70th, and 75th passages of transformed human embryonic tendon cells (THETC) were adopted. The collagen secretion of THETC was detected by immunohistochemical methods, the growth curve of different passages of THETC was compared, and chromosome karyotype was analyzed. Total RNA of THETC were extracted to detect human telomerase reverse transcriptase (hTERT) mRNA expression by RT-PCR technique. Results When THETC were subcultured to 70 passages, the morphological characteristics of cells changed and began replicative senescence. THETC still could secret type Ⅰcollagen normally. The chromosome of THETC was heteroploid (2n=94). There were no hTERT mRNA expression. Conclusion SV40 transfection can not make human embryonic tendon cells immortalization, on the other hand, human embryonic tendon cells transformed by ptsA58H plasmid has no tendency of malignant transformation.%目的分析体外长期传代ptsA58H质粒转染人胚腱细胞的增殖能力及端粒酶活性。方法选用连续传代培养的第40代、第70代、第75代转化人胚腱细胞,免疫组化染色观察细胞分泌胶原类型,比较细胞生长曲线,并行染色体核型分析,流式细胞术检测细胞增殖周期,提取细胞总RNA,RT-PCR二步法检测细胞人端粒酶逆转录酶mRNA表达。结果转化人胚腱细胞传代至70代以后,细胞形态发生改变,出现复制衰老现象。细胞仍具有正常分泌Ⅰ型胶原的能力,转化人胚腱细胞染色体呈异倍性(2n=94)。转化人胚腱细胞无人端粒酶逆转录酶mRNA特异扩增带,无端粒酶活性表达。结论单纯SV40转染不能使人胚腱细胞永生化,同时也说明转化人胚腱细胞无恶性转化倾向。

  20. Telomere length and telomerase activity in the context of menopause.

    Science.gov (United States)

    Pines, A

    2013-12-01

    Telomere length is a marker of cell aging, since shorter telomeres and a higher rate of telomere shortening with time are associated with poorer health status and survival. Various factors may determine telomere length and the function of the telomere maintenance system, including the hereditary load and several modifiable variables such as diet and lifestyle. Telomere length and telomerase activity were investigated extensively in a variety of diseases, such as malignancies (i.e. breast and colon cancer), cardiovascular disease and its related metabolic risk factors, cognitive, mental and psychiatric conditions, and many others. Some evidence points at an association between longer endogenous estrogen exposure (length of reproductive years of life) and greater telomere length and lower telomerase activity. However, there is probably no correlation in regard to menopause per se or the use of hormone therapy. Changing the nutrition and implementing healthy lifestyles may improve the telomere/telomerase parameters in postmenopausal women, but better understanding of this system is still needed.

  1. Telomere stability and telomerase in mesenchymal stem cells

    DEFF Research Database (Denmark)

    Serakinci, Nedime; Graakjaer, Jesper; Kølvrå, Steen

    2008-01-01

    Telomeres are repetitive genetic material that cap and thereby protect the ends of chromosomes. Each time a cell divides, telomeres get shorter. Telomere length is mainly maintained by telomerase. This enzyme is present in high concentrations in the embryonic stem cells and in fast growing...... embryonic cells, and declines with age. It is still unclear to what extent there is telomerase in adult stem cells, but since these are the founder cells of cells of all the tissues in the body, understanding the telomere dynamics and expression of telomerase in adult stem cells is very important....... In the present communication we focus on telomere expression and telomere length in stem cells, with a special focus on mesenchymal stem cells. We consider different mechanisms by which stem cells can maintain telomeres and also focus on the dynamics of telomere length in mesenchymal stem cells, both the overall...

  2. Telomere and telomerase as targets for anti-cancer and regeneration therapies

    Institute of Scientific and Technical Information of China (English)

    Yi-hsin HSU; Jing-jer LIN

    2005-01-01

    Telomerase is a ribonucleoprotein that directs the synthesis of telomeric sequence.It is detected in majority of malignant tumors, but not in most normal somatic cells.Because telomerase plays a critical role in cell immortality and tumor formation, it has been one of the targets for anti-cancer and regeneration drug development. In this review, we will discuss therapeutic approaches based mainly on small molecules that have been developed to inhibit telomerase activity, modulate telomerase expression, and telomerase directed gene therapy.

  3. Telomeres and telomerase as therapeutic targets to prevent and treat age-related diseases [version 1; referees: 4 approved

    Directory of Open Access Journals (Sweden)

    Christian Bär

    2016-01-01

    Full Text Available Telomeres, the protective ends of linear chromosomes, shorten throughout an individual’s lifetime. Telomere shortening is a hallmark of molecular aging and is associated with premature appearance of diseases associated with aging. Here, we discuss the role of telomere shortening as a direct cause for aging and age-related diseases. In particular, we draw attention to the fact that telomere length influences longevity. Furthermore, we discuss intrinsic and environmental factors that can impact on human telomere erosion. Finally, we highlight recent advances in telomerase-based therapeutic strategies for the treatment of diseases associated with extremely short telomeres owing to mutations in telomerase, as well as age-related diseases, and ultimately aging itself.

  4. A triple helix within a pseudoknot is a conserved and essential element of telomerase RNA.

    Science.gov (United States)

    Shefer, Kinneret; Brown, Yogev; Gorkovoy, Valentin; Nussbaum, Tamar; Ulyanov, Nikolai B; Tzfati, Yehuda

    2007-03-01

    Telomerase copies a short template within its integral telomerase RNA onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Telomerase action extends the proliferative potential of cells, and thus it is implicated in cancer and aging. Nontemplate regions of telomerase RNA are also crucial for telomerase function. However, they are highly divergent in sequence among species, and their roles are largely unclear. Using in silico three-dimensional modeling, constrained by mutational analysis, we propose a three-dimensional model for a pseudoknot in telomerase RNA of the budding yeast Kluyveromyces lactis. Interestingly, this structure includes a U-A.U major-groove triple helix. We confirmed the triple-helix formation in vitro using oligoribonucleotides and showed that it is essential for telomerase function in vivo. While triplex-disrupting mutations abolished telomerase function, triple compensatory mutations that formed pH-dependent G-C.C(+) triples restored the pseudoknot structure in a pH-dependent manner and partly restored telomerase function in vivo. In addition, we identified a novel type of triple helix that is formed by G-C.U triples, which also partly restored the pseudoknot structure and function. We propose that this unusual structure, so far found only in telomerase RNA, provides an essential and conserved telomerase-specific function.

  5. Two pathways recruit telomerase to Saccharomyces cerevisiae telomeres.

    Directory of Open Access Journals (Sweden)

    Angela Chan

    2008-10-01

    Full Text Available The catalytic subunit of yeast telomerase, Est2p, is a telomere associated throughout most of the cell cycle, while the Est1p subunit binds only in late S/G2 phase, the time of telomerase action. Est2p binding in G1/early S phase requires a specific interaction between telomerase RNA (TLC1 and Ku80p. Here, we show that in four telomerase-deficient strains (cdc13-2, est1A, tlc1-SD, and tlc1-BD, Est2p telomere binding was normal in G1/early S phase but reduced to about 40-50% of wild type levels in late S/G2 phase. Est1p telomere association was low in all four strains. Wild type levels of Est2p telomere binding in late S/G2 phase was Est1p-dependent and required that Est1p be both telomere-bound and associated with a stem-bulge region in TLC1 RNA. In three telomerase-deficient strains in which Est1p is not Est2p-associated (tlc1-SD, tlc1-BD, and est2A, Est1p was present at normal levels but its telomere binding was very low. When the G1/early S phase and the late S/G2 phase telomerase recruitment pathways were both disrupted, neither Est2p nor Est1p was telomere-associated. We conclude that reduced levels of Est2p and low Est1p telomere binding in late S/G2 phase correlated with an est phenotype, while a WT level of Est2p binding in G1 was not sufficient to maintain telomeres. In addition, even though Cdc13p and Est1p interact by two hybrid, biochemical and genetic criteria, this interaction did not occur unless Est1p was Est2p-associated, suggesting that Est1p comes to the telomere only as part of the holoenzyme. Finally, the G1 and late S/G2 phase pathways for telomerase recruitment are distinct and are likely the only ones that bring telomerase to telomeres in wild-type cells.

  6. 人乳头状瘤病毒L1及人端粒酶RNA基因在高危型人乳头状瘤病毒阳性子宫颈脱落细胞中的表达%Expressions of human papillomavirus L1 and human telomerase RNA component gene in high-risk types of human papillomavirus positive cervical exfoliated cells

    Institute of Scientific and Technical Information of China (English)

    孙立新; 赵宏伟; 丁进进; 韩海琼; 阎丽隽; 连翔

    2014-01-01

    目的 探讨高危型人乳头状瘤病毒(HR-HPV)阳性子宫颈脱落细胞中人乳头状瘤病毒L1(HPVL1)及人端粒酶RNA(hTERC)基因的表达及其与子宫颈病变的相关性.方法 选择有子宫颈活组织检查结果的HR-HPV阳性子宫颈脱落细胞学标本300例,其中子宫颈活组织检查结果为正常及慢性炎症46例、子宫颈上皮内瘤变(CIN)Ⅰ 95例、CINⅡ58例、CINⅢ64例、子宫颈鳞状细胞癌(SCC)37例.采用免疫细胞化学MaxVision法检测液基细胞学标本中抑癌基因HPVL1的表达,用荧光原位杂交技术(FISH)检测子宫颈脱落细胞中hTERC基因的扩增水平.结果 HPVL1阳性率在正常及慢性炎症、CIN Ⅰ、CINⅡ、CINⅢ及SCC组中分别为58.70%(27/46)、63.16%(60/95)、37.93%(22/58)、10.94%(7/64)、0(0/37),随着子宫颈病变恶性程度的增高,HPVL1阳性率逐渐降低.hTERC扩增阳性率在正常及慢性炎症、CIN Ⅰ、CINⅡ、CINⅢ、SCC组中分别为6.52%(3/46)、11.58%(11/95)、51.72%(30/58)、85.94%(55/64)、100.00%(37/37),与子宫颈病变的严重程度呈正相关(rs=0.302,P< 0.01).HPVL+/hTERC-在CIN Ⅰ中占57.89%,在CINⅢ中占4.69%;HPVL1-/hTERC+在CIN Ⅰ中占6.32%,CINⅢ中占79.69%.结论 联合检测HPVL1及hTERC在HR-HPV阳性子宫颈病变的诊断中具有重要的作用.%Objective To evaluate the significance of human papillomavirus L1 capsid protein (HPVL1) and human telomerase RNA component (hTERC) gene in the cytologic specimens of cervix which was infected by the high-risk types of human papillomavirus (HR-HPV),and to expose their relationship with cervical lesions.Methods The fluorescence signal of cytologic samples of cervix were detected by interphase FISH in chromosome enumeration double-color DNA probes TERC.The expression of HPVL1 capsid protein was detected by MaxVision immunohistochemistry method.300 samples were analyzed with HR-HPV positive from the cervical biopsy.The diagnoses as normal or chronic

  7. Friend or foe? Telomerase as a pharmacological target in cancer and cardiovascular disease.

    Science.gov (United States)

    Ait-Aissa, Karima; Ebben, Johnathan D; Kadlec, Andrew O; Beyer, Andreas M

    2016-09-01

    Aging, cancer, and chronic disease have remained at the forefront of basic biological research for decades. Within this context, significant attention has been paid to the role of telomerase, the enzyme responsible for lengthening telomeres, the nucleotide sequences located at the end of chromosomes found in the nucleus. Alterations in telomere length and telomerase activity are a common denominator to the underlying pathology of these diseases. While nuclear-specific, telomere-lengthening effects of telomerase impact cellular/organismal aging and cancer development, non-canonical, extra-nuclear, and non-telomere-lengthening contributions of telomerase have only recently been described and their exact physiological implications are ill defined. Although the mechanism remains unclear, recent reports reveal that the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), regulates levels of mitochondrial-derived reactive oxygen species (mtROS), independent of its established role in the nucleus. Telomerase inhibition has been the target of chemotherapy (directed or indirectly) for over a decade now, yet no telomerase inhibitor is FDA approved and few are currently in late-stage clinical trials, possibly due to underappreciation of the distinct extra-nuclear functions of telomerase. Moreover, evaluation of telomerase-specific therapies is largely limited to the context of chemotherapy, despite reports of the beneficial effects of telomerase activation in the cardiovascular system in relation to such processes as endothelial dysfunction and myocardial infarction. Thus, there is a need for better understanding of telomerase-focused cell and organism physiology, as well as development of telomerase-specific therapies in relation to cancer and extension of these therapies to cardiovascular pathologies. This review will detail findings related to telomerase and evaluate its potential to serve as a therapeutic target.

  8. Urine Telomerase for Diagnosis and Surveillance of Bladder Cancer

    Directory of Open Access Journals (Sweden)

    Angela Lamarca

    2012-01-01

    Full Text Available Bladder cancer has increased incidence during last decades. For those patients with nonmuscle involved tumors, noninvasive diagnosis test and surveillance methods must be designed to avoid current cystoscopies that nowadays are done regularly in a lot of patients. Novel urine biomarkers have been developed during last years. Telomerase is important in cancer biology, improving the division capacity of cancer cells. Even urinary telomerase could be a potentially useful urinary tumor marker; its use for diagnosis of asymptomatic and symptomatic patients or its impact during surveillance is still unknown. Moreover, there will need to be uniformity and standardization in the assays before it can become useful in clinical practice. It does not seem to exist a real difference between the most classical assays for the detection of urine telomerase (TRAP and hTERT. However, the new detection methods with modified TeloTAGGG telomerase or with gold nanoparticles must also be taken into consideration for the correct development of this diagnosis method. Maybe the target population would be the high-risk groups within screening programs. To date there is no enough evidence to use it alone and to eliminate cystoscopies from the diagnosis and surveillance of these patients. The combination with cytology or FISH is still preferred.

  9. Recombinant adenovirus containing PUMA gene regulated by human telomerase reverse transcriptase promoter transfected into ovarian cancer COC1 cells%hTERT启动子调控的PUMA腺病毒转染卵巢癌COC1细胞的研究

    Institute of Scientific and Technical Information of China (English)

    李雨聪; 王冬

    2014-01-01

    Objective To over-express the P53 upregulated modulator of apoptosis(PUMA) in ovarian cancer COC1 cells for playing the role to restrain the cell growth .Methods Adenovirus vector (Ad)-human telomerase reverse transcriptase promoter (hTERT)-PUMA was transfected into ovarian cancer COC1 cells .Then the growth of COC1 cells was studied by CCK-8 and the apoptosis kit and the level of PUMA was analyzed .Results After transfection of Ad-hTERT-PUMA ,PUMA was over-expressed in COC1 cells ,which restrained the cells growth and promoted the cell apoptosis .Conclusion Ad-hTERT-PUMA is successfully transfected into ovarian cancer COC1 cells and PUMA is over-expressed to play the role for restraining the cells growth .%目的:探讨卵巢癌COC1细胞中大量表达p53正向凋亡调控因子(PUMA)在抑制COC1细胞中的作用。方法采用人端粒酶逆转录酶(hTERT)启动子调控的PUMA基因腺病毒载体(Ad-hTERT-PUMA)转染卵巢癌COC1细胞,通过细胞活性检测试剂盒和凋亡试剂盒研究COC1细胞的生长情况,同时分析表达PUMA的水平。结果转染Ad-hTERT-PUMA后,PU-MA大量表达,抑制COC1细胞生长,促进细胞凋亡。结论Ad-hTERT-PUMA成功转染卵巢癌COC1细胞并大量表达,起到了抑制卵巢癌细胞增长的作用。

  10. TP53-dependent chromosome instability is associated with transient reductions in telomere length in immortal telomerase-positive cell lines

    Science.gov (United States)

    Schwartz, J. L.; Jordan, R.; Liber, H.; Murnane, J. P.; Evans, H. H.

    2001-01-01

    Telomere shortening in telomerase-negative somatic cells leads to the activation of the TP53 protein and the elimination of potentially unstable cells. We examined the effect of TP53 gene expression on both telomere metabolism and chromosome stability in immortal, telomerase-positive cell lines. Telomere length, telomerase activity, and chromosome instability were measured in multiple clones isolated from three related human B-lymphoblast cell lines that vary in TP53 expression; TK6 cells express wild-type TP53, WTK1 cells overexpress a mutant form of TP53, and NH32 cells express no TP53 protein. Clonal variations in both telomere length and chromosome stability were observed, and shorter telomeres were associated with higher levels of chromosome instability. The shortest telomeres were found in WTK1- and NH32-derived cells, and these cells had 5- to 10-fold higher levels of chromosome instability. The primary marker of instability was the presence of dicentric chromosomes. Aneuploidy and other stable chromosome alterations were also found in clones showing high levels of dicentrics. Polyploidy was found only in WTK1-derived cells. Both telomere length and chromosome instability fluctuated in the different cell populations with time in culture, presumably as unstable cells and cells with short telomeres were eliminated from the growing population. Our results suggest that transient reductions in telomere lengths may be common in immortal cell lines and that these alterations in telomere metabolism can have a profound effect on chromosome stability. Copyright 2000 Wiley-Liss, Inc.

  11. The AAA-ATPase NVL2 is a telomerase component essential for holoenzyme assembly

    Energy Technology Data Exchange (ETDEWEB)

    Her, Joonyoung [Departments of Biology and Integrated Omics for Biomedical Science, Yonsei University, Seoul 120-749 (Korea, Republic of); Chung, In Kwon, E-mail: topoviro@yonsei.ac.kr [Departments of Biology and Integrated Omics for Biomedical Science, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Identification of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. Black-Right-Pointing-Pointer NVL2 associates with catalytically active telomerase via an interaction with hTERT. Black-Right-Pointing-Pointer NVL2 is a telomerase component essential for holoenzyme assembly. Black-Right-Pointing-Pointer ATP-binding activity of NVL2 is required for hTERT binding and telomerase assembly. -- Abstract: Continued cell proliferation requires telomerase to maintain functional telomeres that are essential for chromosome integrity. Although the core enzyme includes a telomerase reverse transcriptase (TERT) and a telomerase RNA component (TERC), a number of auxiliary proteins have been identified to regulate telomerase assembly, localization, and enzymatic activity. Here we describe the characterization of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. NVL2 interacts and co-localizes with hTERT in the nucleolus. NLV2 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT. Depletion of endogenous NVL2 by small interfering RNA led to a decrease in hTERT without affecting the steady-state levels of hTERT mRNA, thereby reducing telomerase activity, suggesting that NVL2 is an essential component of the telomerase holoenzyme. We also found that ATP-binding activity of NVL2 is required for hTERT binding as well as telomerase assembly. Our findings suggest that NVL2, in addition to its role in ribosome biosynthesis, is essential for telomerase biogenesis and provides an alternative approach for inhibiting telomerase activity in cancer.

  12. Clinical significance of telomerase and its associate genes expression in the maintenance of telomere length in squamous cell carcinoma of the esophagus

    Institute of Scientific and Technical Information of China (English)

    Chung-Ping Hsu; Li-Wen Lee; Sen-Ei Shai; Chih-Yi Chen

    2005-01-01

    AIM: To observe the interaction between the expression of telomerase activity (TA) and its associate genes in regulation of the terminal restriction fragment length(TRFL) in esophageal squamous cell carcinoma (SCC).METHODS: Seventy-four specimens of esophageal SCC were examined. The TA was measured by telomeric repeat amplification protocol (TRAP) assay, and the associated genes [human telomerase-specific reverse transcriptase (hTERT), hTERC, TP1, c-Myc, TRF1,and TRF2] were detected using RT-PCR method. The TRFL was measured by Telomere Length Assay Kit and Southern blotting. The correlations between the expression of telomerase and its associated genes with the TRFL and survivals were examined.RESULTS: Expressions of the TA, hTERT, hTERC, TP1,c-Myc, TRF1, and TRF2 genes were observed in 85.1%,64.9%, 79.7%, 100.0%, 94.6%, 82.4%, and 91.9% of the tumor tissues, respectively. The TRFL of the tumor and normal esophageal tissues were 2.70±1.42 and 4.93±1.74 kb, respectively (P<0.0001). The TRFL of the telomerase positive and telomerase negative tumor tissues were 2.72±1.44 and 2.58±1.32 kb, respectively (P = 0.767).The TRFL ratios (TRFLR) of the telomerase positive and telomerase negative tumor tissues were 0.55±0.22 and 0.59±0.41, respectively (P = 0.742). The expression rates of h-TERT (P = 0.0002), hTERC (P<0.0001), and TRF1(P = 0.002) in the tumor tissues are higher than those of the normal paired tissues. Though TA is markedly activated in tumor tissues (P<0.0001), its expression is not related to clinicopathological parameters including gender, tumor differentiation, and TNM stages. The cumulative 4-year survival rates of telomerase positive and telomerase negative cases were 35.86% and 31.2%,respectively (P = 0.8442). The cumulative 4-year survival rates of patients with their TRFLR ≤85% and >85%were 38.7% and 15.7%, respectively (P = 0.1307).CONCLUSION: Though telomerase expression is not related to tumor stages and prognosis, our data support

  13. P. berghei telomerase subunit TERT is essential for parasite survival.

    Science.gov (United States)

    Religa, Agnieszka A; Ramesar, Jai; Janse, Chris J; Scherf, Artur; Waters, Andrew P

    2014-01-01

    Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA), though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO) homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR) in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF) analysis. TERT and TR were detected in blood stages and an average telomere length of ∼ 950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert- mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further investigations to

  14. P. berghei telomerase subunit TERT is essential for parasite survival.

    Directory of Open Access Journals (Sweden)

    Agnieszka A Religa

    Full Text Available Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA, though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT, is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF analysis. TERT and TR were detected in blood stages and an average telomere length of ∼ 950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert- mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further

  15. Telomerase expression abrogates rapamycin-induced irreversible growth arrest of uterine fibroid smooth muscle cells.

    Science.gov (United States)

    Suo, Guangli; Sadarangani, Anil; Tang, Wingchung; Cowan, Bryan D; Wang, Jean Y J

    2014-09-01

    Uterine fibroids are the most common solid tumors found in women of reproductive age. It has been reported that deregulation of the mammalian target of rapamycin (mTOR) pathway plays an important role in the etiology of leiomyoma. Here, we investigated the effect of rapamycin, an inhibitor of mTORC1, on the growth of primary fibroid smooth muscle cells (fSMCs) and human telomerase reverse transcriptase (hTERT)-transduced and immortalized fSMCs. With the primary fSMCs, a 24-hour treatment with rapamycin was sufficient to trigger a growth arrest that was not reversible upon drug removal. By contrast, the growth inhibitory effect of rapamycin on the hTERT-transduced fSMCs was readily reversible, as these cells resumed proliferation upon the withdrawal of the drug. These results suggest that rapamycin-induced irreversible growth arrest of fSMCs is dependent on the senescence barrier that is abrogated by the ectopic expression of telomerase.

  16. 端粒酶催化亚单位基因启动子调控Hsv-tk/GCV系统对肺癌细胞A549选择性杀伤作用的研究%A study on selective killing effect of Hsv-tk/GCV driven by human telomerase catalytic subunit promoter on human lung cancer cell A549

    Institute of Scientific and Technical Information of China (English)

    唐小军; 王艳萍; 周清华; 车国卫; 陈小禾; 朱大兴

    2007-01-01

    目的 研究人端粒酶催化亚单位(human telomerase catalytic subunit,hTERT)基因启动子调控单纯疱疹胸苷激酶/更昔洛韦(herpes simplex virus-thymidine kinase/ganciclovir, Hsv-tk/GCV)治疗系统对人肺癌细胞株A549的选择体外杀伤作用.方法 (1)用脂质体法将hTERT启动子和sv40启动子调控的tk基因表达质粒(pGL3-hTp-tk和pGL3-sv40-tk)转染端粒酶阳性的人肺腺癌细胞株A549及端粒酶阴性的人胚肺成纤维细胞株MRC-5,用逆转录-PCR方法检测转染细胞中tk基因的表达情况; (2)用MTT法检测GCV对上述转染细胞体外增殖的抑制作用; (3) 用流式细胞仪检测GCV对上述转染细胞凋亡和细胞周期的影响. 结果 (1)转染pGL3-sv40-tk的细胞A549、MRC-5和转染pGL3-hTp-tk的A549均有tk mRNA表达,转染pGL3-hTp-tk的MRC-5无tk mRNA表达; (2)GCV对转染pGL3-sv40-tk的细胞A549、MRC-5和转染pGL3-hTp-tk的A549体外增殖均有明显抑制作用,对转染pGL3-hTp-tk的MRC-5无明显抑制作用; (3) 转染pGL3-sv40-tk的A549、MRC-5和转染pGL3-hTp-tk的A549细胞,GCV处理后细胞凋亡指数(21.58%、9.35%和23.19%)均显著高于转染pGL3-hTp的A549和MRC-5细胞(0.78%和0.55%)及空白对照A549和MRC-5细胞(2.17%和0.60%),转染pGL3-hTp-tk的MRC-5细胞凋亡指数(0.88%)无明显升高.结论 hTERT启动子调控Hsv-tk基因可以在肺癌细胞中选择性表达,hTERT调控的Hsv-tk/GCV治疗系统对肺癌细胞体外增殖具有靶向性抑制作用.

  17. A Telomerase-Specific Doxorubicin-Releasing Molecular Beacon for Cancer Theranostics.

    Science.gov (United States)

    Ma, Yi; Wang, Zhaohui; Zhang, Min; Han, Zhihao; Chen, Dan; Zhu, Qiuyun; Gao, Weidong; Qian, Zhiyu; Gu, Yueqing

    2016-03-01

    A molecular beacon-based drug delivery system was designed for both detection of telomerase activity in living cells and telomerase-triggered drug release for precise cancer treatment. This system is composed of a gold nanoparticle core densely packed with FITC-labeled hairpin DNA sequences hybridized with telomerase primers. Molecules of the anticancer drug doxorubicin were intercalated into the stem region of the DNA sequence. The presence of telomerase will elongate the primers, leading to inner chain substitution followed by the release of the FITC fluorescence and the trapped doxorubicin. This molecular beacon could specifically distinguish tumor cells and normal cells based on telomerase activity, precisely release doxorubicin in response to telomerase activity in the tumor cells, and prevent toxicity to normal organs.

  18. Lack of correlation between telomere length and telomerase activity and expression in leukemic cells.

    Science.gov (United States)

    Januszkiewicz, Danuta; Wysoki, Jacek; Lewandowski, Krzysztof; Pernak, Monika; Nowicka, Karina; Rembowska, Jolanta; Nowak, Jerzy

    2003-12-01

    The expression of three components of telomerase complex (hTR, hTERT, TP1) along with telomerase activity and telomere length in leukemic cells was investigated. Cells were isolated from peripheral blood and/or bone marrow of children with acute lymphoblastic (ALL) and non-lymphoblastic (ANLL) leukemia. Expression of three components of telomerase as well as telomerase activity was found in all leukemic cells. Chemiluminescent detection of terminal restriction fragments (TRF) from DNA isolated from ALL cells showed variable patterns expressing considerable heterogeneity of telomere length. The ALL cells appeared to have both long and short telomere lengths, in contrast to normal peripheral lymphocytes, which produced limited pattern of TRF. The ANLL cells produced predominantly short telomere pattern despite high telomerase activity and expression. It can be concluded that high telomerase activity and expression in leukemic cells is not always correlated with long telomeres (TRF pattern).

  19. Telomerase inhibition abolishes the tumorigenicity of pediatric ependymoma tumor-initiating cells.

    Science.gov (United States)

    Barszczyk, Mark; Buczkowicz, Pawel; Castelo-Branco, Pedro; Mack, Stephen C; Ramaswamy, Vijay; Mangerel, Joshua; Agnihotri, Sameer; Remke, Marc; Golbourn, Brian; Pajovic, Sanja; Elizabeth, Cynthia; Yu, Man; Luu, Betty; Morrison, Andrew; Adamski, Jennifer; Nethery-Brokx, Kathleen; Li, Xiao-Nan; Van Meter, Timothy; Dirks, Peter B; Rutka, James T; Taylor, Michael D; Tabori, Uri; Hawkins, Cynthia

    2014-12-01

    Pediatric ependymomas are highly recurrent tumors resistant to conventional chemotherapy. Telomerase, a ribonucleoprotein critical in permitting limitless replication, has been found to be critically important for the maintenance of tumor-initiating cells (TICs). These TICs are chemoresistant, repopulate the tumor from which they are identified, and are drivers of recurrence in numerous cancers. In this study, telomerase enzymatic activity was directly measured and inhibited to assess the therapeutic potential of targeting telomerase. Telomerase repeat amplification protocol (TRAP) (n = 36) and C-circle assay/telomere FISH/ATRX staining (n = 76) were performed on primary ependymomas to determine the prevalence and prognostic potential of telomerase activity or alternative lengthening of telomeres (ALT) as telomere maintenance mechanisms, respectively. Imetelstat, a phase 2 telomerase inhibitor, was used to elucidate the effect of telomerase inhibition on proliferation and tumorigenicity in established cell lines (BXD-1425EPN, R254), a primary TIC line (E520) and xenograft models of pediatric ependymoma. Over 60 % of pediatric ependymomas were found to rely on telomerase activity to maintain telomeres, while no ependymomas showed evidence of ALT. Children with telomerase-active tumors had reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with tumors lacking telomerase activity. Imetelstat inhibited proliferation and self-renewal by shortening telomeres and inducing senescence in vitro. In vivo, Imetelstat significantly reduced subcutaneous xenograft growth by 40 % (p = 0.03) and completely abolished the tumorigenicity of pediatric ependymoma TICs in an orthotopic xenograft model. Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas.

  20. Detection of telomerase activity in Plasmodium falciparum using a nonradioactive method

    Directory of Open Access Journals (Sweden)

    Rubiano Claudia C

    2003-01-01

    Full Text Available A simple, quick and sensitive method was used to detect telomerase activity in Plasmodium falciparum. The telomeric repeat amplification protocol (TRAP assay was modified using electrophoresis and staining with SYBR-green I to detect telomerase activity in a range of 10² to 10(7 parasites. This might be a useful way to ascertain telomerase activity in different types of nontumor cells.

  1. Tpz1-Ccq1 and Tpz1-Poz1 interactions within fission yeast shelterin modulate Ccq1 Thr93 phosphorylation and telomerase recruitment.

    Directory of Open Access Journals (Sweden)

    Jennifer L Harland

    2014-10-01

    Full Text Available In both fission yeast and humans, the shelterin complex plays central roles in regulation of telomerase recruitment, protection of telomeres against DNA damage response factors, and formation of heterochromatin at telomeres. While shelterin is essential for limiting activation of the DNA damage checkpoint kinases ATR and ATM at telomeres, these kinases are required for stable maintenance of telomeres. In fission yeast, Rad3ATR and Tel1ATM kinases are redundantly required for telomerase recruitment, since Rad3ATR/Tel1ATM-dependent phosphorylation of the shelterin subunit Ccq1 at Thr93 promotes interaction between Ccq1 and the telomerase subunit Est1. However, it remained unclear how protein-protein interactions within the shelterin complex (consisting of Taz1, Rap1, Poz1, Tpz1, Pot1 and Ccq1 contribute to the regulation of Ccq1 Thr93 phosphorylation and telomerase recruitment. In this study, we identify domains and amino acid residues that are critical for mediating Tpz1-Ccq1 and Tpz1-Poz1 interaction within the fission yeast shelterin complex. Using separation of function Tpz1 mutants that maintain Tpz1-Pot1 interaction but specifically disrupt either Tpz1-Ccq1 or Tpz1-Poz1 interaction, we then establish that Tpz1-Ccq1 interaction promotes Ccq1 Thr93 phosphorylation, telomerase recruitment, checkpoint inhibition and telomeric heterochromatin formation. Furthermore, we demonstrate that Tpz1-Poz1 interaction promotes telomere association of Poz1, and loss of Poz1 from telomeres leads to increases in Ccq1 Thr93 phosphorylation and telomerase recruitment, and telomeric heterochromatin formation defect. In addition, our studies establish that Tpz1-Poz1 and Tpz1-Ccq1 interactions redundantly fulfill the essential telomere protection function of the shelterin complex, since simultaneous loss of both interactions caused immediate loss of cell viability for the majority of cells and generation of survivors with circular chromosomes. Based on these

  2. Modulation of yeast telomerase activity by Cdc13 and Est1 in vitro

    Science.gov (United States)

    Chen, Yu-Fan; Lu, Chia-Ying; Lin, Yi-Chien; Yu, Tai-Yuan; Chang, Chun-Ping; Li, Jing-Ru; Li, Hung-Wen; Lin, Jing-Jer

    2016-01-01

    Telomerase is the enzyme involved in extending telomeric DNA. Control of telomerase activity by modulating its access to chromosome ends is one of the most important fundamental mechanisms. This study established an in vitro yeast telomerase reconstitution system that resembles telomere replication in vivo. In this system, a tailed-duplex DNA formed by telomeric DNA was employed to mimic the structure of telomeres. The core catalytic components of telomerase Est2/Tlc1 RNA were used as the telomeric DNA extension machinery. Using the reconstituted systems, this study found that binding of Cdc13 to telomeric DNA inhibited the access of telomerase to its substrate. The result was further confirmed by a single-molecule approach using the tethered-particle motion (TPM)-based telomerase assay. The findings also showed that the inhibitory effect can be relieved by telomerase-associated protein Est1, consistent with the role of Cdc13 and Est1 in regulating telomere extension in vivo. Significantly, this study found that the DNA binding property of Cdc13 was altered by Est1, providing the first mechanistic evidence of Est1 regulating the access of telomerase to its substrate. Thus, the roles of Cdc13 and Est1 in modulating telomerase activity were clearly defined using the in vitro reconstituted system. PMID:27659693

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

    Institute of Scientific and Technical Information of China (English)

    LIU Jian-Lun; GE Lian-ying; ZHANG Gui-nian

    2005-01-01

    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 telomerase 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.

  4. Interaction between Bovine leukemia virus (BLV) infection and age on telomerase misregulation.

    Science.gov (United States)

    Hemmatzadeh, Farhid; Keyvanfar, Hadi; Hasan, Noor Haliza; Niap, Faustina; Bani Hassan, Ebrahim; Hematzade, Azar; Ebrahimie, Esmaeil; McWhorter, Andrea; Ignjatovic, Jagoda

    2015-06-01

    Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). BLV can interact with telomerase and inhibits telomere shortening, contributing in leukemogenesis and tumour induction. The role of telomerase in BLV-induced lymphosarcoma and aging has been extensively studied. To date, the interaction of both BLV and aging on telomerase mis-regulation have, however, not been investigated. In the present study, telomerase activity in BLV positive and negative cows was compared over a wide range of ages (11-85 months). Lymphocyte counts were also measured in both BLV positive and negative groups. Telomerase activity was detected in all BLV infected animals with persistent lymphocytosis (PL), especially in older individuals. This study revealed that the cells undergo the natural telomerase shortening even in the presence of an existing viral infection. We also show that viral infection, especially during the PL phase of the disease, increases telomerase activity. A statistically significant interaction between age and viral infection was observed for telomere shortening during BLV infection. Older animals with BLV infection, especially those with persistent lymphocytosis or visible tumors, exhibited a sharp increase in telomerase activity. This study demonstrates that there is a significant interaction between BLV infection and telomerase up-regulation and lymphocytosis.

  5. A telomerase em células-tronco hematopoéticas Telomerase in hematopoietic stem cells

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    Silvana Perini

    2008-02-01

    Full Text Available A proliferação das células-tronco hematopoéticas sofre a perda dos telômeros a cada divisão celular. Alguns autores discordam quanto à perda ou não do potencial proliferativo e capacidade de auto-renovação das células mais diferenciadas. Revisaremos aqui o papel da telomerase na biologia do sistema hematopoético, na diferenciação normal ou maligna, assim como no envelhecimento das células-tronco hematopoéticas. A constante renovação celular requerida pela hematopoese confere às células-tronco embrionárias, assim como à maioria das células tumorais, um aumento da capacidade proliferativa marcada pela detecção da enzima telomerase e possível manutenção dos telômeros. Estudos clínicos se farão necessários para esclarecer melhor a atividade da telomerase em células-tronco hematopoéticas, seu possível uso como marcador de diagnóstico e seu uso a fim de propósitos prognósticos.Hematopoietic stem cell proliferation leads to telomere length decreases at each cellular division. Some authors disagree about the telomere influence on the reduction of the proliferative potential and capacity of self renewal. Here we review telomerase function in the biology of the hematopoietic system, in normal or differentiation and its influence on the ageing of hematopoietic stem cells. The constant cellular renewal required to maintain the hematopoietic system, provides embryonic stem cells, as well as malignant cells, an increased proliferative capacity. This is marked by the detection of telomerase enzyme activity and possible telomere maintenance. Clinical trials will be required to clarify telomerase activity in hematopoietic stem cells, its possible use as a diagnostic marker and its use for prognostic purposes.

  6. Telomerase Inhibitor Imetelstat in Patients with Essential Thrombocythemia.

    OpenAIRE

    Baerlocher, Gabriela M.; Oppliger Leibundgut, Elisabeth; Ottmann, Oliver G; Spitzer, Gary; Odenike, Olatoyosi; McDevitt, Michael A.; Röth, Alexander; Daskalakis, Michael; Burington, Bart; Stuart, Monic; Snyder, David S.

    2015-01-01

    BACKGROUND Imetelstat, a 13-mer oligonucleotide that is covalently modified with lipid extensions, competitively inhibits telomerase enzymatic activity. It has been shown to inhibit megakaryocytic proliferation in vitro in cells obtained from patients with essential thrombocythemia. In this phase 2 study, we investigated whether imetelstat could elicit hematologic and molecular responses in patients with essential thrombocythemia who had not had a response to or who had had unacceptable s...

  7. Photosensitized damage to telomere overhang and telomerase RNA by riboflavin

    Institute of Scientific and Technical Information of China (English)

    Yuxia Liu; Fuqiang Du; Weizhen Lin; Tiecheng Tu; Wenxin Li; Nianyun Lin

    2008-01-01

    By ESR spin elimination and photodeavage assay, the mechanisms of one-electron oxidation damage of oligonucleotides by excited triplet state of riboflavin (Rb) have been elucidated. The results demonstrate that Rb, an endogenous photosensitizer, is capable of cleaving single-stranded telomeric overhang and the template region of telomerase RNA under UVA irradiation, resulting in blocking of reverse transcription of telomeric DNA which leads to the apoptosis of cancer cells ultimately.

  8. Telomere and Telomerase: From Discovery to Cancer Treatment

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    Eskandari-Nasab

    2015-07-01

    Full Text Available Context Cancer is a major cause of death worldwide. It was estimated that 7.6 million people died during 2008 due to cancer and this figure is expected to double by 2030. To conquer this disease, discovery of validated targets and new drugs is a necessity. Evidence Acquisition Telomeres are terminal structures of linear chromosomes in eukaryotes and consist of multiple repetitive sequences. Their main function is to protect and confer stability to chromosome ends and prevent their breakage, end-to-end fusion, and degeneration. Polymerases responsible for replication of DNA in eukaryotes are not able to replicate chromosome ends and, during cell division, chromosomes continuously become shorter from the telomere ends. This shortening will eventually stop cell division. In cancer cells, there is a ribonucleoprotein enzyme called telomerase that allows compensation of telomere shortening and continuation of the cell multiplication process. Results About 90% of cancers need a high level of this enzyme to continue cell multiplication. Since this enzyme set is absent in normal cells, or present at a very low level, use of telomerase inhibitors cannot have significant effects on normal cells. Conclusions Since telomerase is expressed in 90% of cancer cells, its inhibition can be considered as a goal of cancer treatment.

  9. The yeast telomerase RNA, TLC1, participates in two distinct modes of TLC1-TLC1 association processes in vivo

    OpenAIRE

    Tet Matsuguchi; Elizabeth Blackburn

    2016-01-01

    Telomerase core enzyme minimally consists of the telomerase reverse transcriptase domain-containing protein (Est2 in budding yeast S. cerevisiae) and telomerase RNA, which contains the template specifying the telomeric repeat sequence synthesized. Here we report that in vivo, a fraction of S. cerevisiae telomerase RNA (TLC1) molecules form complexes containing at least two molecules of TLC1, via two separable modes: one requiring a sequence in the 3′ region of the immature TLC1 precursor and ...

  10. Troglitazone suppresses telomerase activity independently of PPARγ in estrogen-receptor negative breast cancer cells

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    Nguyen Johnny

    2010-07-01

    Full Text Available Abstract Background Breast cancer is one the highest causes of female cancer death worldwide. Many standard chemotherapeutic agents currently used to treat breast cancer are relatively non-specific and act on all rapidly dividing cells. In recent years, more specific targeted therapies have been introduced. It is known that telomerase is active in over 90% of breast cancer tumors but inactive in adjacent normal tissues. The prevalence of active telomerase in breast cancer patients makes telomerase an attractive therapeutic target. Recent evidence suggests that telomerase activity can be suppressed by peroxisome proliferator activated receptor gamma (PPARγ. However, its effect on telomerase regulation in breast cancer has not been investigated. Methods In this study, we investigated the effect of the PPARγ ligand, troglitazone, on telomerase activity in the MDA-MB-231 breast cancer cell line. Real time RT-PCR and telomerase activity assays were used to evaluate the effect of troglitazone. MDA-MB-231 cells had PPARγ expression silenced using shRNA interference. Results We demonstrated that troglitazone reduced the mRNA expression of hTERT and telomerase activity in the MDA-MB-231 breast cancer cell line. Troglitazone reduced telomerase activity even in the absence of PPARγ. In agreement with this result, we found no correlation between PPARγ and hTERT mRNA transcript levels in breast cancer patients. Statistical significance was determined using Pearson correlation and the paired Student's t test. Conclusions To our knowledge, this is the first time that the effect of troglitazone on telomerase activity in breast cancer cells has been investigated. Our data suggest that troglitazone may be used as an anti-telomerase agent; however, the mechanism underlying this inhibitory effect remains to be determined.

  11. Telomerase activity in colorectal cancer, prognostic factor and implications in the microsatellite instability pathway

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To determine whether the telomerase activity is related to the Microsatellite instability (MSI) genetic pathway and whether it means a difference in the survival.METHODS: The population consisted of 97 colorectal cancer patients. MSI determination was performed in accordance with the NCI criteria using PCR and Genescan. Telomerase activity was determined by the TRAP-assay, an ELISA procedure based on the amplification of telomeric repeat sequences.RESULTS: 6.2% showed high MSI (MSI-H), 10.3% showed low MSI (MSI-L) and 83.5% did not show this alteration (MSS). Positive telomerase activity was detected in 92.8% of the patients. 83.3% of MSI-H tumors showed positive telomerase against 93.8% of MSS tumors. In the overall survival analysis the absence of telomerase activity conferred a better prognosis.CONCLUSION: Previous works have shown that tumors which develop via the MSI pathway present a better prognosis. No link between telomerase activity and MSI status is observed, although sample sizes are small.Patients with telomerase negative tumors had better overall survival than patients with telomerase positive tumors.

  12. Switch telomerase to ALT mechanism by inducing telomeric DNA damages and dysfunction of ATRX and DAXX

    Science.gov (United States)

    Hu, Yang; Shi, Guang; Zhang, Laichen; Li, Feng; Jiang, Yuanling; Jiang, Shuai; Ma, Wenbin; Zhao, Yong; Songyang, Zhou; Huang, Junjiu

    2016-01-01

    Activation of telomerase or alternative lengthening of telomeres (ALT) is necessary for tumours to escape from dysfunctional telomere-mediated senescence. Anti-telomerase drugs might be effective in suppressing tumour growth in approximately 85–90% of telomerase-positive cancer cells. However, there are still chances for these cells to bypass drug treatment after switching to the ALT mechanism to maintain their telomere integrity. But the mechanism underlying this switch is unknown. In this study, we used telomerase-positive cancer cells (HTC75) to discover the mechanism of the telomerase-ALT switch by inducing telomere-specific DNA damage, alpha-thalassemia X-linked syndrome protein (ATRX) knockdown and deletion of death associated protein (DAXX). Surprisingly, two important ALT hallmarks in the ALT-like HTC75 cells were observed after treatments: ALT-associated promyelocytic leukaemia bodies (APBs) and extrachromosomal circular DNA of telomeric repeats. Moreover, knocking out hTERT by utilizing the CRISPR/Cas9 technique led to telomere elongation in a telomerase-independent manner in ALT-like HTC75 cells. In summary, this is the first report to show that inducing telomeric DNA damage, disrupting the ATRX/DAXX complex and inhibiting telomerase activity in telomerase-positive cancer cells lead to the ALT switch. PMID:27578458

  13. EFFECTS OF THE HYPERTHERMIA AND HARRINGTONINE ON TELOMERASE ACTIVITY OF HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective:To understand the mechanism of the hypertherjia and harringtonine in purging of leukemia cells In vitro.telomerase activity of HL-60 cells treated by hyperthermia(42℃ for one hour) and different concentrations of Harringtonine were investigated .Methods Using telomeric Repeats Amplification Protocol(TRAP)and ELISA techniques to analyze the telomerase activity of HL-60 cells,Results:Our results showed that harringtonine inhibited the telomerase activity of HL-60 cells in a dosage related manner,Moreover,the telomerase activity of HL-60 cells was significantly decreased after the hyperthermia treatment as compared with untreated cells.Conclusion:The effect of the hyperthermia and Harringtonine on purging leukemia cellsIn vitro may be mediated by down regulation of telomerase activity of tumor cells.

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

    Institute of Scientific and Technical Information of China (English)

    Xiao Ming Zhou; Li Jia

    2008-01-01

    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 Iris-(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.

  15. TELOMERASE ACTIVITY DURING 7, 12-DIMETHYLBENZ [a] ANTHRACENE-INDUCED HAMSTER BUCCAL POUCH CARCINOGENESIS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate the roles of telomerase activity (TA) in relation to hamster buccal pouch tumor progression. Methods: male hamster were treated three times weekly with 0.5% of 7, 12-dimethyl- benzanthracene (DMBA) over a 15 weeks experimental period. Hamsters were sacrificed at 3, 6, 9, 12 and 15 weeks after treatment. Telomerase activity of hamster buccal pouch tissue were measured along with the analyses of the formation of DMBA-induced hamster buccal pouch tumors. Results: DMBA-induced squamous cell carcinomas were found at the 6th week after dosing. Telomerase activity elevation began at the 3rd week and was increasing to a plateau at the 12th week. Conclusion: Our results show that telomerase activity in the target tissue may be detected at the early stage of the DMBA-induced hamster buccal pouch tumor formation and suggests that telomerase activity may be used as a biomarker for an early clinical detection of buccal pouch cancer.

  16. Tissue formation and tissue engineering through host cell recruitment or a potential injectable cell-based biocomposite with replicative potential: Molecular mechanisms controlling cellular senescence and the involvement of controlled transient telomerase activation therapies.

    Science.gov (United States)

    Babizhayev, Mark A; Yegorov, Yegor E

    2015-12-01

    Accumulated data indicate that wound-care products should have a composition equivalent to that of the skin: a combination of particular growth factors and extracellular matrix (ECM) proteins endogenous to the skin, together with viable epithelial cells, fibroblasts, and mesenchymal stem cells (MSCs). Strategies consisting of bioengineered dressings and cell-based products have emerged for widespread clinical use; however, their performance is not optimal because chronic wounds persist as a serious unmet medical need. Telomerase, the ribonucleoprotein complex that adds telomeric repeats to the ends of chromosomes, is responsible for telomere maintenance, and its expression is associated with cell immortalization and, in certain cases, cancerogenesis. Telomerase contains a catalytic subunit, the telomerase reverse transcriptase (hTERT). Introduction of TERT into human cells extends both their lifespan and their telomeres to lengths typical of young cells. The regulation of TERT involves transcriptional and posttranscriptional molecular biology mechanisms. The manipulation, regulation of telomerase is multifactorial in mammalian cells, involving overall telomerase gene expression, post-translational protein-protein interactions, and protein phosphorylation. Reactive oxygen species (ROS) have been implicated in aging, apoptosis, and necrosis of cells in numerous diseases. Upon production of high levels of ROS from exogenous or endogenous generators, the redox balance is perturbed and cells are shifted into a state of oxidative stress, which subsequently leads to modifications of intracellular proteins and membrane lipid peroxidation and to direct DNA damage. When the oxidative stress is severe, survival of the cell is dependent on the repair or replacement of damaged molecules, which can result in induction of apoptosis in the injured with ROS cells. ROS-mediated oxidative stress induces the depletion of hTERT from the nucleus via export through the nuclear pores

  17. Decreasing Pin1 suppresses telomerase activity by NF-κB in HCT116 cells colorectal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jianwen Sun; Lijun Fan; Meining Li; Yuehong Zhang; Niuliang Cheng

    2013-01-01

    Objective: The aim of our study was to investigate the effect of Pin1 on the telomerase activity in human colorectal carcinoma HCT116 cells. Methods: Firstly, we transfected plasmid pGenesil-1-Pin1 (p-shRNA) using liposome (Lipofectamine 2000) into colorectal cancer HCT-116 cells to down-regulate the expression of Pin1. To detect the apoptotic rate of HCT116 cells was by cytometry (FCM). The expression of Pin1 and hTERT at RNA levels in human colorectal cancer HCT116 cells were determined by RT-PCR. To evaluate the activity of telomerase was by TRAP-silver staining. The subcellular localization and accumulative level of p-NF-κB/p65 protein at the nuclear was detected by Immunofluorescence and Western blotting. The DNA-binding activity of NF-κB/p65 was detected by electrophoretic mobility shift assay (EMSA). Results: Using liposome into colorectal cancer HCT-116 cells, and down-regulate the expression of Pin1 (0.392 ± 0.072-fold; P = 0.001), and the apoptotic rate was increased (11.40% ± 1.54%; P < 0.05). Compared with transfected p-CON cell group, in transfected p-shRNA cell group, the transcription of hTERT was lower (0.171 ± 0.060-fold; P = 0.001) by quantitative real-time RT-PCR, and the results of TRAP-silver staining analysis suggested that the telomerase activity was significantly declined (0.384 ± 0.015-fold; P < 0.05). Furthermore, it was demonstrated by Immunofluorescence that p-NF-κB/p65 had a nuclear localization, and the level of p-NF-κB/p65 protein at the nuclear was reduced with silencing the expression of Pin1 by Western blotting. Using EMSA, it was suggested that NF-κB/p65 was able to bind to hTERT promoter, and the direct interaction was declined with silencing the expression of Pin1. Conclusion: Taken together, silencing Pin1 may suppress activity of telomerase and the expression of hTERT by inhibiting NF-κB/p65 activity and reducing the combination of NF-κB/p65 and hTERT gene promoter.

  18. 33. The Study of Mechanism By Which The Telomerase Template Phosphorothioate Antisense Oligonucleotide(TPAO) Inhibites The Growth of Tumor Cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@The length of telomere in cells is related to the regulation of life span. The activation of telomerase is required for the maintain of telemere. In the pass several years the studies revealed that the activation of telomerase was associated with initiation and progression of tumorigenesis. There was evident that telomerase inhibitors had the inhibitory effect on tumor cells. The regulation of telo-merase activation was probably associated with cyclins. There was evident that telomerase

  19. Telomerase activation by genomic rearrangements in high-risk neuroblastoma.

    Science.gov (United States)

    Peifer, Martin; Hertwig, Falk; Roels, Frederik; Dreidax, Daniel; Gartlgruber, Moritz; Menon, Roopika; Krämer, Andrea; Roncaioli, Justin L; Sand, Frederik; Heuckmann, Johannes M; Ikram, Fakhera; Schmidt, Rene; Ackermann, Sandra; Engesser, Anne; Kahlert, Yvonne; Vogel, Wenzel; Altmüller, Janine; Nürnberg, Peter; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Mariappan, Aruljothi; Heynck, Stefanie; Mariotti, Erika; Henrich, Kai-Oliver; Gloeckner, Christian; Bosco, Graziella; Leuschner, Ivo; Schweiger, Michal R; Savelyeva, Larissa; Watkins, Simon C; Shao, Chunxuan; Bell, Emma; Höfer, Thomas; Achter, Viktor; Lang, Ulrich; Theissen, Jessica; Volland, Ruth; Saadati, Maral; Eggert, Angelika; de Wilde, Bram; Berthold, Frank; Peng, Zhiyu; Zhao, Chen; Shi, Leming; Ortmann, Monika; Büttner, Reinhard; Perner, Sven; Hero, Barbara; Schramm, Alexander; Schulte, Johannes H; Herrmann, Carl; O'Sullivan, Roderick J; Westermann, Frank; Thomas, Roman K; Fischer, Matthias

    2015-10-29

    Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.

  20. The role of chromatin-associated protein Hbsu in beta-mediated DNA recombination is to facilitate the joining of distant recombination sites.

    Science.gov (United States)

    Alonso, J C; Gutierrez, C; Rojo, F

    1995-11-01

    The beta recombinase is unable to mediate in vitro DNA recombination between two directly oriented recombination sites unless a bacterial chromatin-associated protein (Bacillus subtilis Hbsu or Escherichia [correction of Eschrichia] coli HU] is provided. By electron microscopy, we show that the role of Hbsu is to help in joining the recombination sites to form a stable synaptic complex. Some evidence supports the fact that Hbsu works by recognizing and stabilizing a DNA structure at the recombination site, rather than by serving as a bridge between beta recombinase dimers through a protein-protein interaction. We show that the mammalian HMG1 protein, which shares neither sequence nor structural homology with Hbsu, can also stimulate beta-mediated recombination. These chromatin-associated proteins share the property of binding to DNA in a relatively non-specific fashion, bending it, and having a marked preference for altered DNA structures. Hbsu, HU or HMG1 proteins probably bind specifically at the crossing-over region, since at limiting protein-DNA molar ratios they could not be outcompeted by an excess of a DNA lacking the crossing over site. Distamycin, a minor groove binder that induces local distortions in DNA, did not affect the binding of beta protein to DNA, but inhibited the formation of the synaptic complex.

  1. Effects of osmotic stress on the activity of MAPKs and PDGFR-beta-mediated signal transduction in NIH-3T3 fibroblasts

    DEFF Research Database (Denmark)

    Nielsen, M-B; Christensen, Søren Tvorup; Hoffmann, E K

    2008-01-01

    in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-beta-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most...... likely via a pathway independent of PDGFR-beta and MEK1/2. Conversely, hyperosmolarity (cell shrinkage at 582 mosM) moved nuclear and phosphorylated ERK1/2 to the cytoplasm and induced the phosphorylation and nuclear translocation of p38 and phosphorylation of JNK1/2. In a series of parallel experiments......, hypoosmolarity did not affect PDGF-BB-induced activation of PDGFR-beta, whereas hyperosmolarity strongly inhibited ligand-dependent PDGFR-beta activation as well as downstream mitogenic signal components of the receptor, including Akt and the MEK1/2-ERK1/2 pathway. Based on these results, we conclude that ligand...

  2. 宫颈脱落细胞中人端粒酶RNA组分基因表达在宫颈病变中的临床价值%Clinical evaluation of the expression of human telomerase RNA component gene in cervical lesions

    Institute of Scientific and Technical Information of China (English)

    李茜; 卞美璐; 陈庆云; 马莉; 刘军

    2011-01-01

    目的:研究宫颈脱落细胞中人端粒酶RNA组分(hTERC)基因的表达,探讨其在宫颈上皮内瘤变(CIN)及鳞状上皮细胞癌(SCC)筛查与诊断中的价值.方法:采用宫颈液基细胞学(LCT)、高危型人乳头瘤病毒DNA(HC-Ⅱ)检测及组织病理学的检测,并以组织病理学为金标准,分级盲法使用荧光原位杂交(FISH)方法检测人端粒酶RNA(hTERC)基因的表达.结果:hTERC基因扩增在细胞病理学HSIL及以上病变组扩增率显著升高(P<0.05).hTERC基因的扩增率与组织病理学级别、HC-Ⅱ阳性率均显著正相关(均P<0.01).HC-Ⅱ阳性率与细胞学、组织病理学级别均显著正相关(均P<0.01).结论:在宫颈病变组织中可见hTERC基因的表达、扩增与宫颈细胞学和组织学异常以及HPV病毒感染正相关,hTERC基因的扩增与否可能作为判断有无宫颈高度病变及估计预后的指标之一.%Objective:To investigate the expression of human telomerase RNA component (hTERC)gene in the cervical shedding cells and determine its value in diagnosis of cervical intraepithelial neoplasia(CIN)and carcinoma. Methods : Two hundred and four patients were examined with liquid-based cytology (LCT),high-risk human papilomavirus (HR-HPV)test by hyrid capture Ⅱ (HC-Ⅱ),pathological classification for the gold standard and hTERC gene detection by Fluorescence in situ Hybridization (FISH).Results: (1)The expression of hTERC gene were increased significantly in HSIL and above(P<0.05).(2)The expression of hTERC gene were increased significantly in different grades of histopathology and significant rise in CIN Ⅱ and above (all P<0.01 ).(3)The more detection rate of HPV DNA,the higher grade in cytology and histology(all P<0.01).(4)The detection rate of HPV-DNA was correlated with the expression of hTERC gene positively (P<0.01).Conclusion:The hTERC gene expresses in cervical lesions were correlated with the HPV infection and the abnormality of cervical

  3. Effective gene-viral therapy for telomerase-positive cancers by selective replicative-competent adenovirus combining with endostatin gene

    Institute of Scientific and Technical Information of China (English)

    Zhang Q; Liu C; Jiang M; Fang G; Liu X; Wu M; Qian Q; Nie M; Sham J; Su C; Xue H; Chua D; Wang W; Cui Z; Liu Y

    2005-01-01

    Gene-viral therapy, which uses replication-selective transgene-expressing viruses to manage tumors, can exploit the virtues of gene therapy and virotherapy and overcome the limitations of conventional gene therapy. Using a human telomerase reverse transcriptase-targeted replicative adenovirus as an antiangiogenic gene transfer vector to target new angiogenesis and making use of its unrestrained proliferation are completely new concepts in tumor management. CNHK300-mE is a selective replication transgene-expressing adenovirus constructed to carry mouse endostatin gene therapeutically. Infection with CNHK300-mE was associated with selective replication of the adenovirus and production of mouse endostatin in telomerase-positive cancer cells. Endostatin secreted from a human gastric cell line, SGC-7901, infected with CNHK300-mE was significantly higher than that infected with nonreplicative adenovirus Ad-mE in vitro (800±94.7 ng/ml versus 132.9±9.9 ng/ml) and in vivo (610±42 ng/ml versus 126 +/- 13 ng/ml). Embryonic chorioallantoic membrane assay showed that the mouse endostatin secreted by CNHK300-mE inhibited angiogenesis efficiently and also induced distortion of pre-existing vasculature. CNHK300-mE exhibited a superior suppression of xenografts in nude mice compared with CNHK300 and Ad-mE. In summary, we provided a more efficient gene-viral therapy strategy by combining oncolysis with antiangiogenesis.

  4. Maintenance of telomeres in SV40-transformed pre-immortal and immortal human fibroblasts.

    Science.gov (United States)

    Small, M B; Hubbard, K; Pardinas, J R; Marcus, A M; Dhanaraj, S N; Sethi, K A

    1996-09-01

    Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor-derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)-transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization.

  5. Identification of Protein Components of Yeast Telomerase

    Science.gov (United States)

    2000-09-01

    rapid amplification of cDNA ends (RACE) (Frohman, 1993) (Figure 6). We were unable to extend this sequence...and one deletion were made. This sequence was extended using rapid amplification of cDNA ends (RACE) (Frohman, 1993) using human fetal thymus Marathon...the sequence deduced from the database to 374 amino acids Accession number (AF108138) using rapid amplification of cDNA ends . We were unable

  6. A novel telomerase activator suppresses lung damage in a murine model of idiopathic pulmonary fibrosis.

    Science.gov (United States)

    Le Saux, Claude Jourdan; Davy, Philip; Brampton, Christopher; Ahuja, Seema S; Fauce, Steven; Shivshankar, Pooja; Nguyen, Hieu; Ramaseshan, Mahesh; Tressler, Robert; Pirot, Zhu; Harley, Calvin B; Allsopp, Richard

    2013-01-01

    The emergence of diseases associated with telomere dysfunction, including AIDS, aplastic anemia and pulmonary fibrosis, has bolstered interest in telomerase activators. We report identification of a new small molecule activator, GRN510, with activity ex vivo and in vivo. Using a novel mouse model, we tested the potential of GRN510 to limit fibrosis induced by bleomycin in mTERT heterozygous mice. Treatment with GRN510 at 10 mg/kg/day activated telomerase 2-4 fold both in hematopoietic progenitors ex vivo and in bone marrow and lung tissue in vivo, respectively. Telomerase activation was countered by co-treatment with Imetelstat (GRN163L), a potent telomerase inhibitor. In this model of bleomycin-induced fibrosis, treatment with GRN510 suppressed the development of fibrosis and accumulation of senescent cells in the lung via a mechanism dependent upon telomerase activation. Treatment of small airway epithelial cells (SAEC) or lung fibroblasts ex vivo with GRN510 revealed telomerase activating and replicative lifespan promoting effects only in the SAEC, suggesting that the mechanism accounting for the protective effects of GRN510 against induced lung fibrosis involves specific types of lung cells. Together, these results support the use of small molecule activators of telomerase in therapies to treat idiopathic pulmonary fibrosis.

  7. A novel telomerase activator suppresses lung damage in a murine model of idiopathic pulmonary fibrosis.

    Directory of Open Access Journals (Sweden)

    Claude Jourdan Le Saux

    Full Text Available The emergence of diseases associated with telomere dysfunction, including AIDS, aplastic anemia and pulmonary fibrosis, has bolstered interest in telomerase activators. We report identification of a new small molecule activator, GRN510, with activity ex vivo and in vivo. Using a novel mouse model, we tested the potential of GRN510 to limit fibrosis induced by bleomycin in mTERT heterozygous mice. Treatment with GRN510 at 10 mg/kg/day activated telomerase 2-4 fold both in hematopoietic progenitors ex vivo and in bone marrow and lung tissue in vivo, respectively. Telomerase activation was countered by co-treatment with Imetelstat (GRN163L, a potent telomerase inhibitor. In this model of bleomycin-induced fibrosis, treatment with GRN510 suppressed the development of fibrosis and accumulation of senescent cells in the lung via a mechanism dependent upon telomerase activation. Treatment of small airway epithelial cells (SAEC or lung fibroblasts ex vivo with GRN510 revealed telomerase activating and replicative lifespan promoting effects only in the SAEC, suggesting that the mechanism accounting for the protective effects of GRN510 against induced lung fibrosis involves specific types of lung cells. Together, these results support the use of small molecule activators of telomerase in therapies to treat idiopathic pulmonary fibrosis.

  8. Stiffened yeast telomerase RNA supports RNP function in vitro and in vivo.

    Science.gov (United States)

    Lebo, Kevin J; Zappulla, David C

    2012-09-01

    The 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, in addition to providing a 16-nt template region for reverse transcription, has been proposed to act as a scaffold for protein subunits. Although accessory subunits of the telomerase ribonucleoprotein (RNP) complex function even when their binding sites are relocated on the yeast telomerase RNA, the physical nature of the RNA scaffold has not been directly analyzed. Here we explore the structure-function organization of the yeast telomerase RNP by extensively stiffening the three long arms of TLC1, which connect essential and important accessory protein subunits Ku, Est1, and Sm(7), to its central catalytic hub. This 956-nt triple-stiff-arm TLC1 (TSA-T) reconstitutes active telomerase with TERT (Est2) in vitro. Furthermore, TSA-T functions in vivo, even maintaining longer telomeres than TLC1 on a per RNA basis. We also tested functional contributions of each stiffened arm within TSA-T and found that the stiffened Est1 and Ku arms contribute to telomere lengthening, while stiffening the terminal arm reduces telomere length and telomerase RNA abundance. The fact that yeast telomerase tolerates significant stiffening of its RNA subunit in vivo advances our understanding of the architectural and functional organization of this RNP and, more broadly, our conception of the world of lncRNPs.

  9. Lack of telomerase activity in rabbit bone marrow stromal cells during differentiation along neural pathway

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhen-zhou; XU Ru-xiang; JIANG Xiao-dan; TENG Xiao-hua; LI Gui-tao; ZHOU Yü-xi

    2006-01-01

    Objective: To investigate telomerase activity in rabbit bone marrow stromal cells (BMSCs) during their committed differentiation in vitro along neural pathway and the effect of glial cell line-derived neurotrophic factor (GDNF) on the expression of telomerase.Methods: BMSCs were acquired from rabbit marrow and divided into control group, GDNF (10 ng/ml) group.No. ZL02134314. 4) supplemented with 10% fetal bovine serum (FBS) was used to induce BMSCs differentiation along neural pathway. Fluorescent immunocytochemistry was employed to identify the expressions of Nestin, neuronspecific endase (NSE), and gial fibrillary acidic protein (GFAP). The growth curves of the cells and the status of cell cycles were analyzed, respectively. During the differentiation, telomerase activitys were detected using the telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA).Results: BMSCs were successfully induced to differentiate along neural pathway and expressed specific markers of fetal neural epithelium, mature neuron and glial cells. Telomerase activities were undetectable in BMSCs during differentiation along neural pathway. Similar changes of cell growth curves, cell cycle status and telomerase expression were observed in the two groups.Conclusions: Rabbit BMSCs do not display telomerase activity during differentiation along neural pathway. GDNF shows little impact on proliferation and telomerase activity of BMSCs.

  10. Active Yeast Telomerase Shares Subunits with Ribonucleoproteins RNase P and RNase MRP.

    Science.gov (United States)

    Lemieux, Bruno; Laterreur, Nancy; Perederina, Anna; Noël, Jean-François; Dubois, Marie-Line; Krasilnikov, Andrey S; Wellinger, Raymund J

    2016-05-19

    Telomerase is the ribonucleoprotein enzyme that replenishes telomeric DNA and maintains genome integrity. Minimally, telomerase activity requires a templating RNA and a catalytic protein. Additional proteins are required for activity on telomeres in vivo. Here, we report that the Pop1, Pop6, and Pop7 proteins, known components of RNase P and RNase MRP, bind to yeast telomerase RNA and are essential constituents of the telomerase holoenzyme. Pop1/Pop6/Pop7 binding is specific and involves an RNA domain highly similar to a protein-binding domain in the RNAs of RNase P/MRP. The results also show that Pop1/Pop6/Pop7 function to maintain the essential components Est1 and Est2 on the RNA in vivo. Consistently, addition of Pop1 allows for telomerase activity reconstitution with wild-type telomerase RNA in vitro. Thus, the same chaperoning module has allowed the evolution of functionally and, remarkably, structurally distinct RNPs, telomerase, and RNases P/MRP from unrelated progenitor RNAs.

  11. Telomerase Activity Detected by Quantitative Assay in Bladder Carcinoma and Exfoliated Cells in Urine

    Directory of Open Access Journals (Sweden)

    Roberta Fedriga

    2001-01-01

    Full Text Available Early diagnosis is one of the most determining factors for patient survival. The detection of telomerase activity is a potentially promising tool in the diagnosis of bladder and other types of cancer due to the high expression of this enzyme in tumor cells. We carried out a quantitative evaluation of telomerase activity in urine samples in an attempt to determine a cut-off capable of identifying cancer patients. Telomerase activity was quantified by fluorescence TRAP assay in urine from 50 healthy volunteers and in urine and bioptic tumor samples from 56 previously untreated bladder cancer patients and expressed in arbitrary enzymatic units (AEU. Telomerase activity in urine ranged from 0 to 106 AEU (median 0 in healthy donors and from 0 to 282 AEU (median 87 in patients with cancer. A telomerase expression higher than the cut off value determined by receiver operating characteristic (ROC analysis was observed in 78% of cases, regardless of tumor grade and in 71% (15/21 of cases of nonassessable or negative cytology. The quantitative analysis of telomerase activity in urine enabled us to define cut-off values characterized by different sensitivity and specificity. Cytologic and telomerase determination, used sequentially, enabled us to detect about 90% of tumors.

  12. Colorimetry and SERS dual-mode detection of telomerase activity: combining rapid screening with high sensitivity.

    Science.gov (United States)

    Zong, Shenfei; Wang, Zhuyuan; Chen, Hui; Hu, Guohua; Liu, Min; Chen, Peng; Cui, Yiping

    2014-01-01

    As an important biomarker and therapeutic target, telomerase has attracted considerable attention concerning its detection and monitoring. Here, we present a colorimetry and surface enhanced Raman scattering (SERS) dual-mode telomerase activity detection method, which has several distinctive advantages. First, colorimetric functionality allows rapid preliminary discrimination of telomerase activity by the naked eye. Second, the employment of SERS technique results in greatly improved detection sensitivity. Third, the combination of colorimetry and SERS into one detection system can ensure highly efficacious and sensitive screening of numerous samples. Besides, the avoidance of polymerase chain reaction (PCR) procedures further guarantees fine reliability and simplicity. Generally, the presented method is realized by an "elongate and capture" procedure. To be specific, gold nanoparticles modified with Raman molecules and telomeric repeat complementary oligonucleotide are employed as the colorimetric-SERS bifunctional reporting nanotag, while magnetic nanoparticles functionalized with telomerase substrate oligonucleotide are used as the capturing substrate. Telomerase can synthesize and elongate telomeric repeats onto the capturing substrate. The elongated telomeric repeats subsequently facilitate capturing of the reporting nanotag via hybridization between telomeric repeat and its complementary strand. The captured nanotags can cause a significant difference in the color and SERS intensity of the magnetically separated sediments. Thus both the color and SERS can be used as indicators of the telomerase activity. With fast screening ability and outstanding sensitivity, we anticipate that this method would greatly promote practical application of telomerase-based early-stage cancer diagnosis.

  13. TELOMERASE ACTIVITY OF FIBROBRONCHOSCOPIC BRUSHING CELLS IN NON-SMALL CELL LUNG CANCER

    Institute of Scientific and Technical Information of China (English)

    吴晓红; 应可净; 张行

    2003-01-01

    Objective: To evaluate the clinical significance of telomerase activity particularly in terms of prognostic impact in non-small cell lung cancer (NSCLC). Methods: The exfoliated cells from fibrobronchoscopic brushing were studied using polymerase chain reaction based on a telomerase repeat amplification protocal assay. Samples were taken from 60 NSCLC and 20 pulmonary infection cases. Results: Telomerase activity was detected in 53 of 60(88.3%) NSCLC specimens from the lesion side and in 5 of 25(20.0%) from the contralateral side but only in 2 of 20 pulmonary infection samples (P<0.05). The telomerase activity levels in NSCLC (medium 0.109) were significantly higher than those in pulmonary infection (medium 0.018, U=4.95, P<0.05). The telomerase activity levels in tumor staged IIIb-IV (medium 0.173) were higher than those in staged I-IIIa (medium 0.132, U=1.899, P<0.05). Conclusion: Telomerase activity is one of the most important marker in patients with NSCLC. Telomerase activity increases with the advance of tumor stage and can be used as a prognostic indicator of advanced NSCLC.

  14. Lysine-specific demethylase 1 (LSD1 Is required for the transcriptional repression of the telomerase reverse transcriptase (hTERT gene.

    Directory of Open Access Journals (Sweden)

    Qingjun Zhu

    Full Text Available BACKGROUND: Lysine-specific demethylase 1 (LSD1, catalysing demethylation of mono- and di-methylated histone H3-K4 or K9, exhibits diverse transcriptional activities by mediating chromatin reconfiguration. The telomerase reverse transcriptase (hTERT gene, encoding an essential component for telomerase activity that is involved in cellular immortalization and transformation, is silent in most normal human cells while activated in up to 90% of human cancers. It remains to be defined how exactly the transcriptional activation of the hTERT gene occurs during the oncogenic process. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we determined the effect of LSD1 on hTERT transcription. In normal human fibroblasts with a tight hTERT repression, a pharmacological inhibition of LSD1 led to a weak hTERT expression, and a robust induction of hTERT mRNA was observed when LSD1 and histone deacetylases (HDACs were both inhibited. Small interference RNA-mediated depletion of both LSD1 and CoREST, a co-repressor in HDAC-containing complexes, synergistically activated hTERT transcription. In cancer cells, inhibition of LSD1 activity or knocking-down of its expression led to significant increases in levels of hTERT mRNA and telomerase activity. Chromatin immunoprecipitation assay showed that LSD1 occupied the hTERT proximal promoter, and its depletion resulted in elevated di-methylation of histone H3-K4 accompanied by increased H3 acetylation locally in cancer cells. Moreover, during the differentiation of leukemic HL60 cells, the decreased hTERT expression was accompanied by the LSD1 recruitment to the hTERT promoter. CONCLUSIONS/SIGNIFICANCE: LSD1 represses hTERT transcription via demethylating H3-K4 in normal and cancerous cells, and together with HDACs, participates in the establishment of a stable repression state of the hTERT gene in normal or differentiated malignant cells. The findings contribute to better understandings of hTERT/telomerase

  15. Telomerase as an emerging target to fight cancer--opportunities and challenges for nanomedicine.

    Science.gov (United States)

    Philippi, C; Loretz, B; Schaefer, U F; Lehr, C M

    2010-09-01

    Telomerase as an enzyme is responsible for the renewal of the chromosomal ends, the so-called telomeres. By preventing them from shortening with each cell cycle, telomerase is able to inhibit cellular senescence and apoptosis. Telomerase activity, which is detectable in the majority of cancer cells, allows them to maintain their proliferative capacity. The thus obtained immortality of those cells again is a key to their malignancy. Based on these discoveries, it is obvious that telomerase inhibitors would represent an innovative approach to fight cancer, and a variety of such candidate molecules are currently in the pipeline. Telomerase inhibitors largely fall in two classes of compounds: small synthetic molecules and nucleotide-based biologicals. For several candidates, some proof of concept studies have been demonstrated, either on cell cultures or in animal models. But the same studies also revealed that inefficient delivery is largely limiting the translational step into the clinic. The most appealing feature of telomerase inhibitors, which distinguishes them from conventional anticancer drugs, is probably seen in their intrinsic non-toxicity to normal cells. Nevertheless, efficient delivery to the target cells, i.e. to the tumor, is still required. Here, some well-known biopharmaceutical problems such as insufficient solubility, permeability or even metabolic stability are frequently encountered. To address these challenges, there is a clear need for adequate delivery technologies, for example by using nanomedicines, that would allow to overcome their biopharmaceutical shortcomings and to warrant a sufficient bioavailability at the target side. This review first briefly explains the concept of telomerase and telomerase inhibition in cancer therapy. It secondly aims to provide an overview of the different currently known telomerase inhibitors. Finally, the biopharmaceutical limitations of these molecules are discussed as well as the possibilities to overcome

  16. Clinical significance of human telomerase reverse transcriptase gene expression in giant cell tumor of bone%人端粒酶逆转录酶基因在骨巨细胞瘤组织的表达及其临床意义

    Institute of Scientific and Technical Information of China (English)

    朱宇; 马希峰; 来秋山; 张明生; 巴玉峰; 黄涛; 陈清汉

    2012-01-01

    Objective To study the clinical significance of human telomerase reverse transcriptase (hTERT) gene expression in giant cell tumor of bone.Methods By using in situ hybridization (ISH) techniques,the clinical significance of hTERT mRNA expression was tested in 84 cases of giant cell tumor of bone,32 cases of hyperostosis,28 cases of heterotopic ossification and 20 cases of normal bone tissues.These cases were collected from Jan.2008 to Dec.2011 in our department.The expression level and the location of hTERT gene were observed by using the image analyzing system.Results hTERT was expressed in 46.4% (39/84) of giant cell tumors of bone and it had an obvious correlation with the differentiation level of tumor cells ( P < 0.05 ).There was a close correlation between the expression of hTERT and the location of tumor cells.hTERT gene was not detected in 32 cases of hyperostosis,28 cases of heterotopic ossification or 20 cases of normal bone tissues.Conclusion hTERT gene may play an important role in the pathogenesis of giant cell tumors of bone.%目的 观察骨巨细胞瘤组织中人端粒酶逆转录酶(hTERT)基因的表达,并探讨其临床意义.方法 选择我院2008年1月至2011年12月病理科保存的标本,标本中骨巨细胞瘤84例、骨质增生骨组织32例、异位骨化组织28例,对照组为正常骨组织20例;采用原位分子杂交技术检测和定位标本中的端粒酶hTERT基因,并用图像分析系统分析其表达水平.、结果 端粒酶hTERT基因在骨巨细胞瘤组织中的表达阳性率为46.4%(39/84),表达强度与骨巨细胞瘤的分化程度明显相关(P<0.05),表达水平与肿瘤细胞的分布定位一致;端粒酶hTERT基因在骨质增生、异位骨化和正常骨组织中的表达均为阴性.结论 骨巨细胞瘤组织的恶化可能与端粒酶hTERT基因相关.

  17. Telomerase expression in the glial scar of rats with spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Mingkun Yang; Weibin Sheng; Tao Xu; Kai Huang; Yanjiao Wang

    2012-01-01

    A rat model of spinal cord injury was established using the weight drop method. A cavity formed 14 days following spinal cord injury, and compact scar tissue formed by 56 days. Enzyme-linked immunosorbent assay and polymerase chain reaction enzyme-linked immunosorbent assay results demonstrated that glial fibrillary acidic protein and telomerase expression increased gradually after injury, peaked at 28 days, and then gradually decreased. Spearman rank correlation showed a positive correlation between glial fibrillary acidic protein expression and telomerase expression in the glial scar. These results suggest that telomerase promotes glial scar formation.

  18. 端粒酶活性在肿瘤诊治中的价值%Value of Telomerase Activity on the Diagnosis and Treatment in Tumor

    Institute of Scientific and Technical Information of China (English)

    易铁男; 周云峰

    2001-01-01

    Telomerase activity highly increases in almost all huma n malignant tumors,but in benign lesions or normal tissues it dosent express e xcept germ cell and stem cell.Besides its diagnostic value,telomerase activity a ppears to be associated with tumor therapeutic effect,monitoring recurrence of t umor,predicting tumor aggressiveness.So it could be of values as a marker of tum or and a therapeutic target.This review gives a detailed presentation of value o f telomerase activity on the diagnosis and treatment in human tumor.%端粒酶几乎在所有的恶性肿瘤进程中表达,但除生殖细胞和干细 胞外良性病变和正常组织均不表达。因为它的广泛表达可作为肿瘤的标志物及癌症治疗的靶 点,使端粒酶成为目前全世界研究的热点,本文就端粒酶活性的检测与肿瘤临床的诊断、治 疗、对残存或复发癌症的监测以及病人的预后方面的研究进展作一综述。

  19. Insights into the evolution of mammalian telomerase: Platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes

    Directory of Open Access Journals (Sweden)

    Hrdličková Radmila

    2012-06-01

    Full Text Available Abstract Background The TERT gene encodes the catalytic subunit of the telomerase complex and is responsible for maintaining telomere length. Vertebrate telomerase has been studied in eutherian mammals, fish, and the chicken, but less attention has been paid to other vertebrates. The platypus occupies an important evolutionary position, providing unique insight into the evolution of mammalian genes. We report the cloning of a platypus TERT (OanTERT ortholog, and provide a comparison with genes of other vertebrates. Results The OanTERT encodes a protein with a high sequence similarity to marsupial TERT and avian TERT. Like the TERT of sauropsids and marsupials, as well as that of sharks and echinoderms, OanTERT contains extended variable linkers in the N-terminal region suggesting that they were present already in basal vertebrates and lost independently in ray-finned fish and eutherian mammals. Several alternatively spliced OanTERT variants structurally similar to avian TERT variants were identified. Telomerase activity is expressed in all platypus tissues like that of cold-blooded animals and murine rodents. OanTERT was localized on pseudoautosomal regions of sex chromosomes X3/Y2, expanding the homology between human chromosome 5 and platypus sex chromosomes. Synteny analysis suggests that TERT co-localized with sex-linked genes in the last common mammalian ancestor. Interestingly, female platypuses express higher levels of telomerase in heart and liver tissues than do males. Conclusions OanTERT shares many features with TERT of the reptilian outgroup, suggesting that OanTERT represents the ancestral mammalian TERT. Features specific to TERT of eutherian mammals have, therefore, evolved more recently after the divergence of monotremes.

  20. Telomerase and HER-2/neu as targets of genetic cancer vaccines in dogs.

    Science.gov (United States)

    Peruzzi, Daniela; Mesiti, Giuseppe; Ciliberto, Gennaro; La Monica, Nicola; Aurisicchio, Luigi

    2010-02-03

    Pet dogs represent a valuable pre-clinical model to assess the efficacy of oncology drugs. Additionally, canine cancers occur with an incidence similar to that of humans and share many features with human malignancies including histological appearance, tumor genetics, biological behavior and response to conventional therapies. The telomerase reverse transcriptase (TERT) is reactivated in most of human and dog tumors. Similarly, HER-2/neu oncoprotein is overexpressed in a proportion of canine breast cancers. Therefore, TERT and HER-2/neu can constitute valid tumor associated antigens (TAA), suitable targets for translational cancer immunotherapy in dogs. In this study, we have evaluated the ability of DNA electroporation (DNA-EP) and Adenovirus serotype 6 (Ad6) to induce immune responses against dog TERT (dTERT) and HER-2/neu in healthy dogs. Vaccination was effective in all treated animals and the adaptive immune response remained detectable and long-lasting in the absence of autoimmunity or other side-effects. Our results show that DNA-EP/Ad6-based cancer vaccine induces adaptive immune responses against TAA in canine subjects and support further evaluation of this approach in cancer dog patients.

  1. 端粒、端粒酶与肿瘤%Telomere, Telomerase and Tumor

    Institute of Scientific and Technical Information of China (English)

    张岭; 李龙芸

    1999-01-01

    Telomere is the end structure of chromosome and it will be shortened during replication. Telomerase is a reverse transcripatse consisting of both RNA and protein components and synthesizes telomeric DNA by copying the template sequence of its own RNA components to maintain telomere length for function.Telomerase activity in germline cells,immortal and neoplastic cells was detected,but not in mostly normal cells.The telomere-telomerase hypothesis was brung out to explain this phenomenon.According to this hypothesis,re-actived telomerase will maintain telomere's length for protecting chromosome to make the cell immortal.The aging procedure will be explained by this hypothesis too.This hypothesis provides a new concepts of cancer and cancer's treatment.

  2. Effect of Mifepristone on the Telomerase Activity in Chorion and Decidua during Early Pregnancy

    Institute of Scientific and Technical Information of China (English)

    Ge-qing XIA; Ya-li XIONG; Yong-hong SUN

    2004-01-01

    Objective To investigate telomerase activity in chorion and decidua from abortion induced by mifepristone incorporated with misoprostol at early pregnancy Methods TRAP-SYBR Green assay was used to detect the expression of telomerase. Forty specimen were obtained from medicinal abortion (experiment group) and forty were from normal induced abortion (control group).Results Positive expression, of chorion telomerase was significantly different between the experimental group (28%, 11/40) and the control group (73%, 29/40) (P<0. 05).While in decidua, the positive rate was 28% (11/40) in the experimental group and 20% (9/40) in the control group, there was no significant difference (P>0. 05).Conclusion It is suggested that miferistone may significantly decrease the telomerase activity in chorion but not in decidua.

  3. POT1a and components of CST engage telomerase and regulate its activity in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Kyle B Renfrew

    2014-10-01

    Full Text Available Protection of Telomeres 1 (POT1 is a conserved nucleic acid binding protein implicated in both telomere replication and chromosome end protection. We previously showed that Arabidopsis thaliana POT1a associates with the TER1 telomerase RNP, and is required for telomere length maintenance in vivo. Here we further dissect the function of POT1a and explore its interplay with the CST (CTC1/STN1/TEN1 telomere complex. Analysis of pot1a null mutants revealed that POT1a is not required for telomerase recruitment to telomeres, but is required for telomerase to maintain telomere tracts. We show that POT1a stimulates the synthesis of long telomere repeat arrays by telomerase, likely by enhancing repeat addition processivity. We demonstrate that POT1a binds STN1 and CTC1 in vitro, and further STN1 and CTC1, like POT1a, associate with enzymatically active telomerase in vivo. Unexpectedly, the in vitro interaction of STN1 with TEN1 and POT1a was mutually exclusive, indicating that POT1a and TEN1 may compete for the same binding site on STN1 in vivo. Finally, unlike CTC1 and STN1, TEN1 was not associated with active telomerase in vivo, consistent with our previous data showing that TEN1 negatively regulates telomerase enzyme activity. Altogether, our data support a two-state model in which POT1a promotes an extendable telomere state via contacts with the telomerase RNP as well as STN1 and CTC1, while TEN1 opposes these functions.

  4. A sensitive, label-free electrochemical detection of telomerase activity without modification or immobilization.

    Science.gov (United States)

    Liu, Xu; Wei, Min; Xu, Ensheng; Yang, Haitang; Wei, Wei; Zhang, Yuanjian; Liu, Songqin

    2017-05-15

    Telomerase has become one of the most typical tumor marker because it is closely related to cancers. In this paper, a simple label-free electrochemical detection of telomerase activity by using methylene blue (MB) as a G-quadruplex binding probe was proposed, avoiding commonly used complex label procedures, nano-probe synthesis, complicated electrode modification, probe immobilization or signal amplification. In the presence of telomerase substrate (TS) primer, the binding of MB on primer was weak. When repeats of (TTAGGG) were extended on the TS primer under the action of telomerase, they formed multiple G-quadruplexes with the help of K(+). As a result, a large amount of MB bounded to multiple G-quadruplexes because they have more strong interaction with G-quadruplexes than TS primer. As a result, the diffusion current of MB decreased sharply, which was strongly dependent on the telomerase activity. The DPV current change has a linear correlation with the logarithm of HeLa cell number in the range of 10-10,000 cells, with the detection limit of 3 cells. The high sensitivity was due to the formed multiple G-quadruplexes. Using indium tin oxide (ITO) as working electrode without modification ensured the good reproducibility of the method. The method was also simple, rapid, and has been successfully applied in the telomerase activity detection in urine with good selectivity and reproducibility, which is significant for cancer diagnosis, anticancer drugs screening, and cancer therapy evaluation.

  5. Structural Features of the Telomerase RNA Gene in the Naked Mole Rat Heterocephalus glaber

    Science.gov (United States)

    Evfratov, S. A.; Smekalova, E. M.; Golovin, A. V.; Logvina, N. A.; Zvereva, M. I.; Dontsova, O. A.

    2014-01-01

    Telomere length, an important feature of life span control, is dependent on the activity of telomerase (a key enzyme of the telomere-length-maintaining system). Telomerase RNA is a component of telomerase and, thus, is crucial for its activity. The structures of telomerase RNA genes and their promoter regions were compared for the long-living naked mole rat and different organisms. Two rare polymorphisms in Heterocephalus glaber telomerase RNA (hgTER) were identified: A→G in the first loop of pseudoknot P2b-p3 (an equivalent of 111nt in hTR) and G→A in the scaRNA domain CR7-p8b (an equivalent of 421nt in hTR). Analysis of TER promoter regions allowed us to identify two new transcription factor binding sites. The first one is the ETS family site, which was found to be a conserved element for all the analyzed TER promoters. The second site is unique for the promoter region of TER of the naked mole rat and is a binding site for the SOX17 transcription factor. The absence of one Sp1 site in the TER promoter region of the naked small rat is an additional specific feature of the promoter area of hgTER. Such variation in the hgTER transcription regulation region and hgTER itself could provide increased telomerase activity in stem cells and an extended lifespan to H. glaber. PMID:25093110

  6. Telomerase inhibitory effects of medicinal mushrooms and lichens, and their anticancer activity.

    Science.gov (United States)

    Xu, Baojun; Li, Chantian; Sung, Changkeun

    2014-01-01

    Telomerase has been widely accepted as a cancer marker and a promising therapeutic target for novel anticancer drugs. The aim of this study was to investigate the in vitro telomerase inhibitory effects of mushrooms and their anticancer properties. The inhibitory effects of mushrooms and lichens against telomerase activity of HL-60 cells were systematically assessed using polymerase chain reaction based on assay of telomeric repeat amplification protocol. Telomerase inhibitory samples were further tested for antiproliferation effects against the gastric cell line SNU-1 using the MTT method. Ethyl acetate extract of Pleurotus ostreatus, ethyl acetate and water extracts of Lasiosphaera fenzlii, hexane extract of Strobilomyces floccopus, water extract of Sarcodon aspratus, and hexane, ethyl acetate, and water extracts from Umbilicaria esculenta showed strong positive telomerase inhibitory activity. Hexane extract of S. floccopus and water extracts from the edible lichen U. esculenta exhibited strong anticancer effects against SNU-1 cells through antiproliferation assay. The water extract of U. esculenta has a great potential to be developed into an anticancer agent that targets telomerase.

  7. Analysis of telomerase activity based on a spired DNA tetrahedron TS primer.

    Science.gov (United States)

    Li, Yan; Wen, Yanli; Wang, Lele; Liang, Wen; Xu, Li; Ren, Shuzhen; Zou, Ziying; Zuo, Xiaolei; Fan, Chunhai; Huang, Qing; Liu, Gang; Jia, Nengqin

    2015-05-15

    The development of sensitive telomerase biosensors is hindered by the restricted accessibility of telomere strand (TS) primer and the limited enzyme reaction space, which is mainly confined by the vertical distance. In this work, we designed an electrochemical telomerase biosensor based on a spired DNA tetrahedron TS primer (STTS). By adding a rigid dsDNA spire onto the top of the DNA tetrahedron, we successfully regulated the distance between the TS primer and the surface, and thus greatly facilitated the telomerase elongation on surface. The signal-to-noise ratio was 2 times higher than TSP without the spire structure. The limit of detection was calculated to be lower than 10 HeLa cells, which is at least 2 magnitudes lower than other surface extension-based electrochemical telomerase sensors without amplification. The practicability of STTS sensor was also demonstrated by analysing various other cell lines including cancer cells, stem cells of high telomerase activity and somatic cells of low telomerase activity.

  8. Induction of endogenous telomerase (hTERT) by c-Myc in WI-38 fibroblasts transformed with specific genetic elements.

    Science.gov (United States)

    Casillas, Mark A; Brotherton, Scott L; Andrews, Lucy G; Ruppert, J Michael; Tollefsbol, Trygve O

    2003-10-16

    Elucidation of the mechanisms governing expression of the human telomerase reverse transcriptase (hTERT) is important for understanding cancer pathogenesis. Approximately 90% of tumors express hTERT, the major catalytic component of telomerase. Activation of telomerase is an early event, and high levels of this activity correlate with poor prognosis. Recent studies have shown that the transcription factors c-Myc and Mad1 activate and repress hTERT, respectively. It is not clear how these transcription factors compete for the same recognition sequence in the hTERT core promoter region. Studies have shown that the combined expression of SV40 large T antigen (T-Ag), hTERT, and H-Ras is able to transform human cells. In this study, we used a distinct human cell type, WI-38 fetal lung fibroblasts used extensively for senescence studies. We transduced cells with amphotropic retroviral constructs containing SV40 T antigen, hTERT, and activated H-ras. Transduced cells exhibited anchorage independence in soft agar and expressed increased levels of c-Myc and endogenous hTERT. These effects were observed by 25 population doublings (PDs) following the establishment of the neoplastic cell line. During the process of transformation, we observed a switch from Mad1/Max to c-Myc/Max binding to oligonucleotide sequences containing the hTERT promoter distal and proximal E-boxes. c-Myc can bind specifically to the hTERT promoter in vitro, indicating that c-Myc expression in tumors may account for the increased expression of hTERT observed in vivo. These findings indicate that the widely used model system of WI-38 fibroblasts can be employed for transformation studies using defined genetic elements and that the endogenous hTERT and c-Myc are induced in these cells during early tumorigenesis. Such studies should have important implications in the mechanisms of hTERT and c-Myc induction in the beginning stages of tumorigenesis and facilitate extension of these studies to novel models of

  9. Completion of cell division is associated with maximum telomerase activity in naturally synchronized cultures of the green alga Desmodesmus quadricauda.

    Science.gov (United States)

    Ševčíková, Tereza; Bišová, Kateřina; Fojtová, Miloslava; Lukešová, Alena; Hrčková, Kristýna; Sýkorová, Eva

    2013-03-18

    Telomerase maintains the ends of eukaryotic chromosomes, and its activity is an important parameter correlating with the proliferative capacity of cells. We have investigated cell cycle-specific changes in telomerase activity using cultures of Desmodesmus quadricauda, a model alga naturally synchronized by light/dark entrainment. A quantitative telomerase assay revealed high activity in algal cultures, with slight changes during the light period. Significantly increased telomerase activity was observed at the end of the dark phase, when cell division was complete. In contrast to other models, a natural separation between nuclear and cellular division typical for the cell cycle in D. quadricauda made this observation possible.

  10. A common variant in the telomerase RNA component is associated with short telomere length.

    Directory of Open Access Journals (Sweden)

    Omer T Njajou

    Full Text Available BACKGROUND: Telomeres shorten as cells divide. This shortening is compensated by the enzyme telomerase. We evaluated the effect of common variants in the telomerase RNA component (TERC gene on telomere length (TL in the population-based Health Aging and Body Composition (Health ABC Study and in two replication samples (the TwinsUK Study and the Amish Family Osteoporosis Study, AFOS. METHODOLOGY: Five variants were identified in the TERC region by sequence analysis and only one SNP was common (rs2293607, G/A. The frequency of the G allele was 0.26 and 0.07 in white and black, respectively. Testing for association between TL and rs2293607 was performed using linear regression models or variance component analysis conditioning on relatedness among subjects. RESULTS: The adjusted mean TL was significantly shorter in 665 white carriers of the G allele compared to 887 non-carriers from the Health ABC Study (4.69±0.05 kbp vs. 4.86±0.04 kbp, measured by quantitative PCR, p = 0.005. This association was replicated in another white sample from the TwinsUK Study (6.90±0.03 kbp in 301 carriers compared to 7.06±0.03 kbp in 395 non-carriers, measured by Southern blots, p = 0.009. A similar pattern of association was observed in whites from the family-based AFOS and blacks from the Health ABC cohort, although not statistically significant, possibly due to the lower allele frequency in these populations. Combined analysis using 2,953 white subjects from 3 studies showed a significant association between TL and rs2293607 (β = -0.19±0.04 kbp, p = 0.001. CONCLUSION: Our study shows a significant association between a common variant in TERC and TL in humans, suggesting that TERC may play a role in telomere homeostasis.

  11. Breast Field Cancerization: Isolation and Comparison of Telomerase-Expressing Cells in Tumor and Tumor Adjacent, Histologically Normal Breast Tissue

    Science.gov (United States)

    Trujillo, Kristina A.; Hines, William C.; Vargas, Keith M.; Jones, Anna C.; Joste, Nancy E.; Bisoffi, Marco; Griffith, Jeffrey K.

    2011-01-01

    Telomerase stabilizes chromosomes by maintaining telomere length, immortalizes mammalian cells, and is expressed in more than 90% of human tumors. However, the expression of human telomerase reverse transcriptase (hTERT) is not restricted to tumor cells. We have previously shown that a subpopulation of human mammary epithelial cells (HMEC) in tumor-adjacent, histologically normal (TAHN) breast tissues expresses hTERT mRNA at levels comparable with levels in breast tumors. In the current study, we first validated a reporter for measuring levels of hTERT promoter activity in early-passage HMECs and then used this reporter to compare hTERT promoter activity in HMECs derived from tumor and paired TAHN tissues 1, 3, and 5 cm from the tumor (TAHN-1, TAHN-3, and TAHN-5, respectively). Cell sorting, quantitative real-time PCR, and microarray analyses showed that the 10% of HMECs with the highest hTERT promoter activity in both tumor and TAHN-1 tissues contain more than 95% of hTERT mRNA and overexpress many genes involved in cell cycle and mitosis. The percentage of HMECs within this subpopulation showing high hTERT promoter activity was significantly reduced or absent in TAHN-3 and TAHN-5 tissues. We conclude that the field of normal tissue proximal to the breast tumors contains a population of HMECs similar in hTERT expression levels and in gene expression to the HMECs within the tumor mass and that this population is significantly reduced in tissues more distal to the tumor. PMID:21775421

  12. Aberrant gene expression profiles, during in vitro osteoblast differentiation, of telomerase deficient mouse bone marrow stromal stem cells (mBMSCs)

    DEFF Research Database (Denmark)

    Saeed, H.; Iqtedar, M.

    2015-01-01

    Background: Telomerase deficiency has been associated with inadequate differentiation of mesenchymal stem cells. However, the effect of telomerase deficiency on differential regulation of osteoblast specific genes, based on functional gene grouping, during in vitro osteoblast differentiation has ...

  13. Colorimetry and SERS dual-mode detection of telomerase activity: combining rapid screening with high sensitivity

    Science.gov (United States)

    Zong, Shenfei; Wang, Zhuyuan; Chen, Hui; Hu, Guohua; Liu, Min; Chen, Peng; Cui, Yiping

    2014-01-01

    As an important biomarker and therapeutic target, telomerase has attracted considerable attention concerning its detection and monitoring. Here, we present a colorimetry and surface enhanced Raman scattering (SERS) dual-mode telomerase activity detection method, which has several distinctive advantages. First, colorimetric functionality allows rapid preliminary discrimination of telomerase activity by the naked eye. Second, the employment of SERS technique results in greatly improved detection sensitivity. Third, the combination of colorimetry and SERS into one detection system can ensure highly efficacious and sensitive screening of numerous samples. Besides, the avoidance of polymerase chain reaction (PCR) procedures further guarantees fine reliability and simplicity. Generally, the presented method is realized by an ``elongate and capture'' procedure. To be specific, gold nanoparticles modified with Raman molecules and telomeric repeat complementary oligonucleotide are employed as the colorimetric-SERS bifunctional reporting nanotag, while magnetic nanoparticles functionalized with telomerase substrate oligonucleotide are used as the capturing substrate. Telomerase can synthesize and elongate telomeric repeats onto the capturing substrate. The elongated telomeric repeats subsequently facilitate capturing of the reporting nanotag via hybridization between telomeric repeat and its complementary strand. The captured nanotags can cause a significant difference in the color and SERS intensity of the magnetically separated sediments. Thus both the color and SERS can be used as indicators of the telomerase activity. With fast screening ability and outstanding sensitivity, we anticipate that this method would greatly promote practical application of telomerase-based early-stage cancer diagnosis.As an important biomarker and therapeutic target, telomerase has attracted considerable attention concerning its detection and monitoring. Here, we present a colorimetry and

  14. Human telomerase reverse transcriptase transfection of bone marrow mesenchymal stem cells in the treatment of diabetic rats%人端粒酶反转录酶转染的骨髓间充质干细胞移植治疗大鼠糖尿病

    Institute of Scientific and Technical Information of China (English)

    赵海莲

    2015-01-01

    BACKGROUND:Human telomerase reverse transcriptase (hTERT) is the first choice for regulating the proliferation and directional differentiation, with multiple biological effects. OBJECTIVE:To observe the therapeutic effect of hTERT-transfected bone marrow mesenchymal stem cels transplantation in diabetic rats. METHODS: Bone marrow mesenchymal stem cels from Sprague-Dawley rats were culturedin vitro and transfected with retrovirus PLXSN carrying hTERT. RT-PCR was used to detect the hTERT expression in the bone marrow mesenchymal stem cels before and after transfection. Sixty male Sprague-Dawley rats were selected and equaly randomized into four groups: normal control group, transfection group, cel transplantation group, and model group. In the latter three groups, rats were injected with 45 mg/kg chain urea to establish diabetes models, and then injectedvia the tail vein with 0.2 mL hTERT-transfected bone marrow mesenchymal stem cels, 0.2 mL bone marrow mesenchymal stem cels, and 0.2 mL normal saline, respectively. RESULTS AND CONCLUSION:At 48 hours after hTERT transfection, the expression of hTERT mRNA was detected in the bone marrow mesenchymal stem cels, and mainly concentrated in the nuclei. At 14 days after transfection, the fasting glucose level in the model group was higher than that in the normal control group (P 0.05). These findings indicate that the transplantation of hTERT-transfected bone marrow mesenchymal stem cels is effective in the treatment of diabetic rats.%背景:人端粒酶反转录酶是调控增殖及定向分化的首选生长因子之一,具有多重生物学效应。目的:观察人端粒酶反转录酶表达的骨髓间充质干细胞移植治疗大鼠糖尿病的效果。方法:体外培养 SD 大鼠骨髓间充质干细胞,经反转录病毒 PLXSN 为载体介导人端粒酶反转录酶基因转染骨髓间充质干细胞,在转染前后用RT-PCR检测骨髓间充质干细胞人端粒酶反转录酶基因的表达。60

  15. 人端粒酶RNA基因荧光原位杂交检测在宫颈细胞学检查中的应用%Detection of human telomerase RNA component gene by fluorescent in situ hybridization for screening of cervical lesions

    Institute of Scientific and Technical Information of China (English)

    高莉; 朱炎; 刘素萍; 高妍; 朱明华

    2011-01-01

    目的 探讨人类染色体端粒酶RNA基因(hTERC)扩增的荧光原位杂交(FISH)检测在宫颈脱落细胞防癌筛查中的意义.方法 收集2008年2-10月上海长海医院门诊就诊146例患者宫颈液基细胞学样本,应用间期双色FISH技术检测hTERC基因的扩增情况,并与细胞学和组织学结果进行对照.结果 杂交成功120例(细胞学阴性20例、细胞学阳性100例).hTERC基因扩增的阳性率与细胞学等级成正相关(r=0.465,P<0.01),与组织学等级成正相关(r=0.610,P<0.01),各级的阳性率分别为:炎性病变0(0/13)、宫颈上皮内瘤样病变(CIN)Ⅰ级2 8.6%(6/21)、CINⅡ级11/18、CINⅢ级75.0%(18/24)、鳞状细胞癌91.7%(22/24);FISH方法检出高级别(CINⅡ/Ⅲ级)以上病变的敏感性为77.3%(51/66),特异性为82.4%(28/34).阳性预测值89.5%,阴性预测值65.1%,低级别(CIN Ⅰ级)以下病变的hTERC阳性率与高级别以上病变相比差异有统计学意义(x2=32.550,P<0.01).结合hTERC高倍扩增(信号数>4)的出现,检出高级别以上病变的敏感性提高到81.2%.结论 hTERC基因扩增的FISH检测有助于辅助细胞学诊断,提高高危病变的检出率.实验结果判断除参照阈值之外,还应结合hTERC基因的扩增类型,出现高倍扩增亦提示高级别以上病变.%Objective To investigate the value of fluorescence in situ hybridization (FISH)detection of human telomerase RNA component ( hTERC ) gene amplification in screening of cervical lesions.Methods A total of 146 post-thinPrep cytology test (TCT) samples were analyzed using FISH by two-color interphase probe targeting hTERC gene at chromosome 3q26 and the data were compared with the cytological and histological results. Results FISH analysis was successful in 120 cases (20 cases of normal and 100 abnormal cases by TCT). Gene amplification of hTERC by FISH had a positive correlation with the cytological (r = 0. 465, P < 0. 01 ) and histological grade results ( r = 0. 610, P < 0

  16. Super-telomeres in transformed human fibroblasts.

    Science.gov (United States)

    Chiodi, Ilaria; Belgiovine, Cristina; Zongaro, Samantha; Ricotti, Roberta; Horard, Beatrice; Lossani, Andrea; Focher, Federico; Gilson, Eric; Giulotto, Elena; Mondello, Chiara

    2013-08-01

    Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process. Just after telomerase catalytic subunit (hTERT) expression, cen3tel telomeres shortened despite the presence of telomerase activity. At a later stage and concomitantly with transformation, cells started elongating telomeres, which reached a mean length greater than 100kb in about 900 population doublings. Super-telomeres were stable and compatible with cell growth and tumorigenesis. Telomere extension was associated with increasing levels of telomerase activity that were linked to the deregulation of endogenous telomerase RNA (hTERC) and exogenous telomerase reverse transcriptase (hTERT) expression. Notably, the increase in hTERC levels paralleled the increase in telomerase activity, suggesting that this subunit plays a role in regulating enzyme activity. Telomeres ranging in length between 10 and more than 100kb were maintained in an extendible state although TRF1 and TRF2 binding increased with telomere length. Super-telomeres neither influenced subtelomeric region global methylation nor the expression of the subtelomeric gene FRG1, attesting the lack of a clear-cut relationship between telomere length, subtelomeric DNA methylation and expression in human cells. The cellular levels of the telomeric proteins hTERT, TRF1, TRF2 and Hsp90 rose with transformation and were independent of telomere length, pointing to a role of these proteins in tumorigenesis.

  17. A spectrum of severe familial liver disorders associate with telomerase mutations.

    Directory of Open Access Journals (Sweden)

    Rodrigo T Calado

    Full Text Available BACKGROUND: Telomerase is an enzyme specialized in maintaining telomere lengths in highly proliferative cells. Loss-of-function mutations cause critical telomere shortening and are associated with the bone marrow failure syndromes dyskeratosis congenita and aplastic anemia and with idiopathic pulmonary fibrosis. Here, we sought to determine the spectrum of clinical manifestations associated with telomerase loss-of-function mutations. METHODOLOGY/PRINCIPAL FINDINGS: Sixty-nine individuals from five unrelated families with a variety of hematologic, hepatic, and autoimmune disorders were screened for telomerase complex gene mutations; leukocyte telomere length was measured by flow fluorescence in situ hybridization in mutation carriers and some non-carriers; the effects of the identified mutations on telomerase activity were determined; and genetic and clinical data were correlated. In six generations of a large family, a loss-of-function mutation in the telomerase enzyme gene TERT associated with severe telomere shortening and a range of hematologic manifestations, from macrocytosis to acute myeloid leukemia, with severe liver diseases marked by fibrosis and inflammation, and one case of idiopathic pulmonary fibrosis but not with autoimmune disorders. Additionally, we identified four unrelated families in which loss-of-function TERC or TERT gene mutations tracked with marrow failure, pulmonary fibrosis, and a spectrum of liver disorders. CONCLUSIONS/SIGNIFICANCE: These results indicate that heterozygous telomerase loss-of-function mutations associate with but are not determinant of a large spectrum of hematologic and liver abnormalities, with the latter sometimes occurring in the absence of marrow failure. Our findings, along with the link between pulmonary fibrosis and telomerase mutations, also suggest a common pathogenic mechanism for fibrotic diseases in which defective telomere repair plays important role.

  18. A transposable element within the Non-canonical telomerase RNA of Arabidopsis thaliana modulates telomerase in response to DNA damage [corrected].

    Directory of Open Access Journals (Sweden)

    Hengyi Xu

    2015-06-01

    Full Text Available Long noncoding RNAs (lncRNAs have emerged as critical factors in many biological processes, but little is known about how their regulatory functions evolved. One of the best-studied lncRNAs is TER, the essential RNA template for telomerase reverse transcriptase. We previously showed that Arabidopsis thaliana harbors three TER isoforms: TER1, TER2 and TER2S. TER1 serves as a canonical telomere template, while TER2 is a novel negative regulator of telomerase activity, induced in response to double-strand breaks (DSBs. TER2 contains a 529 nt intervening sequence that is removed along with 36 nt at the RNA 3' terminus to generate TER2S, an RNA of unknown function. Here we investigate how A. thaliana TER2 acquired its regulatory function. Using data from the 1,001 Arabidopsis genomes project, we report that the intervening sequence within TER2 is derived from a transposable element termed DSB responsive element (DRE. DRE is found in the TER2 loci of most but not all A. thaliana accessions. By analyzing accessions with (TER2 and without DRE (TER2Δ we demonstrate that this element is responsible for many of the unique properties of TER2, including its enhanced binding to TERT and telomerase inhibitory function. We show that DRE destabilizes TER2, and further that TER2 induction by DNA damage reflects increased RNA stability and not increased transcription. DRE-mediated changes in TER2 stability thus provide a rapid and sensitive switch to fine-tune telomerase enzyme activity. Altogether, our data shows that invasion of the TER2 locus by a small transposon converted this lncRNA into a DNA damage sensor that modulates telomerase enzyme activity in response to genome assault.

  19. Immunocalization of telomerase in cells of lizard tail after amputation suggests cell activation for tail regeneration.

    Science.gov (United States)

    Alibardi, L

    2016-02-01

    Tail amputation (autotomy) in most lizards elicits a remarkable regenerative response leading to a new although simplified tail. No information on the trigger mechanism following wounding is known but cells from the stump initiate to proliferate and form a regenerative blastema. The present study shows that telomerases are mainly activated in the nuclei of various connective and muscle satellite cells of the stump, and in other tissues, probably responding to the wound signals. Western blotting detection also indicates that telomerase positive bands increases in the regenerating blastema in comparison to the normal tail. Light and ultrastructural immunocytochemistry localization of telomerase shows that 4-14 days post-amputation in lizards immunopositive nuclei of sparse cells located among the wounded tissues are accumulating into the forming blastema. These cells mainly include fibroblasts and fat cells of the connective tissue and satellite cells of muscles. Also some immature basophilic and polychromatophilic erytroblasts, lymphoblasts and myelocytes present within the Bone Marrow of the vertebrae show telomerase localization in their nuclei, but their contribution to the formation of the regenerative blastema remains undetermined. The study proposes that one of the initial mechanisms triggering cell proliferation for the formation of the blastema in lizards involve gene activation for the production of telomerase that stimulates the following signaling pathways for cell division and migration.

  20. The telomerase inhibitor imetelstat depletes cancer stem cells in breast and pancreatic cancer cell lines.

    Science.gov (United States)

    Joseph, Immanual; Tressler, Robert; Bassett, Ekaterina; Harley, Calvin; Buseman, Christen M; Pattamatta, Preeti; Wright, Woodring E; Shay, Jerry W; Go, Ning F

    2010-11-15

    Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus, inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study, we investigated the effects of imetelstat (GRN163L), a potent telomerase inhibitor, on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro, telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally, imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat, but not control oligonucleotides, also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after imetelstat, suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy.

  1. Effect of telomerase inhibition on preclinical models of malignant rhabdoid tumor.

    Science.gov (United States)

    Hu, Yafang; Bobb, Daniel; Lu, Yunbiao; He, Jianping; Dome, Jeffrey S

    2014-09-01

    Novel treatment approaches are desperately needed for malignant rhabdoid tumor (MRT). Telomerase is an attractive therapeutic target because it is specific to cancer and critical for cancer cell immortality. We evaluated the effect of the telomerase inhibitor imetelstat in preclinical models of MRT. Three MRT cell lines, BT-12, G401, and RT-peri, were treated with the telomerase inhibitor imetelstat. The effects of imetelstat on telomere length, DNA damage response, and cell proliferation were assessed. The efficacy of imetelstat in vivo was evaluated in subcutaneous xenografts derived from each of the cell lines. Treatment with imetelstat resulted in inhibition of telomerase activity, marked telomere shortening, and activation of the DNA damage response pathway, as measured by formation of γ-H2AX nuclear foci, phosphorylation of ATM, and phosphorylation of TP53. Imetelstat-treated G401 cells underwent complete growth arrest after 16 passages. The other two cell lines exhibited growth inhibition. Imetelstat resulted in 40-50% growth inhibition compared to placebo-treated controls in all three xenograft models. The activity of imetelstat as a single agent suggests that further studies of telomerase inhibitors in combination with other agents may be warranted.

  2. Comparison of telomerase activity in prostate cancer, prostatic intraepithelial neoplasia and benign prostatic hyperplasia

    Directory of Open Access Journals (Sweden)

    Soleiman Mahjoub

    2006-11-01

    Full Text Available BACKGROUND: Telomerase is a reverse transcriptase enzyme that synthesizes telomeric DNA on chromosome ends. The enzyme is important for the immortalization of cancer cells because it maintains the telomeres. METHODS: Telomerase activity (TA was measured by fluorescence-based telomeric repeat amplification protocol (FTRAP assay in prostate carcinoma and benign prostatic hyperplasia (BPH. RESULTS: TA was present in 91.4% of 70 prostate cancers, 68.8% of 16 prostatic intraepithelial neoplasia (PIN, 43.3% of 30 BPH*, 21.4% of 14 atrophy and 20% of 15 normal samples adjacent to tumor. There was not any significant correlation between TA, histopathological tumor stage or gleason score. In contrast to high TA in the BPH* tissue from the cancer-bearing gland, only 6.3% of 32 BPH specimens from patients only diagnosed with BPH were telomerase activity-positive. CONCLUSIONS: These results indicate that TA is present in most prostate cancers. The high rate of TA in tissue adjacent to tumor may be attributed either to early molecular alteration of cancer that was histologically unapparent, or to the presence of occult cancer cells. Our findings suggest that the re-expression of telomerase activity could be one step in the transformation of BPH to PIN. KEY WORDS: Telomerase activity, prostate cancer, prostatic intraepithelial neoplasia, benign prostatic hyperplasia.

  3. Effects of Curcuma longa Extract on Telomerase Activity in Lung and Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Nosratollah Zarghami

    2014-10-01

    Full Text Available Background: The purpose of this study is to evaluate the effect of Curcuma longa extract on the telomerase gene expression in QU-DB lung cancer and T47D breast cancer cell lines. Materials and Methods: The present study is an experimental research. Using 3 different phases n-hexane, dichloromethane and methanol, total extract of Curcuma longa in a serial dilution was prepared and three phases was analyzed for determining which phase has more curcuminoids. Then the extract cytotoxicity effect was tested on breast cancer cell line (T47D, and lung cancer cell line (QU-DB by 24, 48 and 72 h MTT (Dimethyl thiazolyl diphenyl tetrazolium assay. Then, the cells were treated with serial concentrations of the extract. Finally, total protein was extracted from the control and test groups, its quantity was determined and telomeric repeat amplification protocol (TRAP assay was performed for measurement of possible inhibition of the telomerase activity. Results: Cell viability and MTT-based cytotoxicity assay show that the total extract of Curcuma longa has cytotoxic effect with different IC50s in breast and lung cancer cell lines. Analysis of TRAP assay also shows a significant reduction in telomerase activity on both cancer cells with different levels. Conclusion: Curcuma longa extract has anti-proliferation and telomerase inhibitory effects on QU-DB lung cancer and T47D breast cancer cells with differences in levels of telomerase inhibition.

  4. Clinical Relevance of Telomere Status and Telomerase Activity in Colorectal Cancer.

    Science.gov (United States)

    Fernández-Marcelo, Tamara; Sánchez-Pernaute, Andrés; Pascua, Irene; De Juan, Carmen; Head, Jacqueline; Torres-García, Antonio-José; Iniesta, Pilar

    2016-01-01

    The role of telomeres and telomerase in colorectal cancer (CRC) is well established as the major driving force in generating chromosomal instability. However, their potential as prognostic markers remains unclear. We investigated the outcome implications of telomeres and telomerase in this tumour type. We considered telomere length (TL), ratio of telomere length in cancer to non-cancer tissue (T/N ratio), telomerase activity and TERT levels; their relation with clinical variables and their role as prognostic markers. We analyzed 132 CRCs and paired non-cancer tissues. Kaplan-Meier curves for disease-free survival were calculated for TL, T/N ratio, telomerase activity and TERT levels. Overall, tumours had shorter telomeres than non-tumour tissues (P Telomere lengths of non-tumour tissues and CRCs were positively correlated (P telomere status and clinical variables, the lowest degree of telomere shortening was shown by tumours located in the rectum (P = 0.021). Regarding prognosis studies, patients with tumours showing a mean TL telomere shortening relapsed during the follow-up period (P = 0.043). The mean TL in CRCs emerged as an independent prognostic factor in the Cox analysis (P = 0.017). Telomerase-positive activity was identified as a marker that confers a trend toward a poor prognosis. In CRC, our results support the use of telomere status as an independent prognostic factor. Telomere status may contribute to explaining the different molecular identities of this tumour type.

  5. Telomerase in relation to expression of p53, c-Myc and estrogen receptor in ovarian tumours

    NARCIS (Netherlands)

    Wisman, GBA; Hollema, H; Helder, MN; Knol, AJ; Van Der Meer, GT; Krans, M; De Jong, S; De Vries, EGE; Van Der Zee, AGJ

    2003-01-01

    Telomerase activity and its subunits (hTERC, hTERT mRNA) were evaluated in ovarian tumours in relation to the expression of p53, c-Myc and estrogen receptor (ER). Furthermore, relations between telomerase activity, hTERC and hTERT with known clinicopathologic prognostic factors and survival in patie

  6. Telomerase activity is spontaneously increased in lymphocytes from patients with atopic dermatitis and correlates with cellular proliferation

    DEFF Research Database (Denmark)

    Wu, Kehuai; Volke, Anne Rehné; Lund, Marianne;

    1999-01-01

    , and staphylococcal enterotoxin A (SEA) (0.1 microg/ml). Telomerase activity was measured by the telomeric repeat amplification protocol-based telomerase polymerase chain reaction enzyme-linked immunosorbent assay at 0 and 72 h of incubation. In addition, DNA synthesis of the cells was assayed using 3H...

  7. The effects of erythropoietin signaling on telomerase regulation in non-erythroid malignant and non-malignant cells

    Energy Technology Data Exchange (ETDEWEB)

    Uziel, Orit, E-mail: Oritu@clalit.org.il [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Kanfer, Gil [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Dep. of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Beery, Einat [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Yelin, Dana; Shepshelovich, Daniel [Medicine A, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Bakhanashvili, Mary [Unit of Infectious Diseases, Sheba Medical Center, Tel-Hashomer (Israel); Nordenberg, Jardena [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Dep. of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Endocrinology Laboratory, Beilinson Medical Center, Petah-Tikva (Israel); Lahav, Meir [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Medicine A, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel)

    2014-07-18

    Highlights: • We assumed that some of erythropoietin adverse effects may be mediated by telomerase activity. • EPO administration increased telomerase activity, cells proliferation and migration. • The inhibition of telomerase modestly repressed the proliferative effect of erythropoietin. • Telomere shortening caused by long term inhibition of the enzyme totally abolished that effect. • This effect was mediated via the Lyn–AKT axis and not by the canonical JAK2–STAT pathway. - Abstract: Treatment with erythropoietin (EPO) in several cancers is associated with decreased survival due to cancer progression. Due to the major importance of telomerase in cancer biology we hypothesized that some of these effects may be mediated through EPO effect on telomerase. For this aim we explored the possible effects of EPO on telomerase regulation, cell migration and chemosensitivity in non-erythroid malignant and non-malignant cells. Cell proliferation, telomerase activity (TA) and cell migration increased in response to EPO. EPO had no effect on cancer cells sensitivity to cisplatinum and on the cell cycle status. The inhibition of telomerase modestly repressed the proliferative effect of EPO. Telomere shortening caused by long term inhibition of the enzyme abolished the effect of EPO, suggesting that EPO effects on cancer cells are related to telomere dynamics. TA was correlated with the levels of Epo-R. The increase in TA was mediated post-translationally through the Lyn-Src and not the canonical JAK2 pathway.

  8. Feasibility of Telomerase-Specific Adoptive T-cell Therapy for B-cell Chronic Lymphocytic Leukemia and Solid Malignancies.

    Science.gov (United States)

    Sandri, Sara; Bobisse, Sara; Moxley, Kelly; Lamolinara, Alessia; De Sanctis, Francesco; Boschi, Federico; Sbarbati, Andrea; Fracasso, Giulio; Ferrarini, Giovanna; Hendriks, Rudi W; Cavallini, Chiara; Scupoli, Maria Teresa; Sartoris, Silvia; Iezzi, Manuela; Nishimura, Michael I; Bronte, Vincenzo; Ugel, Stefano

    2016-05-01

    Telomerase (TERT) is overexpressed in 80% to 90% of primary tumors and contributes to sustaining the transformed phenotype. The identification of several TERT epitopes in tumor cells has elevated the status of TERT as a potential universal target for selective and broad adoptive immunotherapy. TERT-specific cytotoxic T lymphocytes (CTL) have been detected in the peripheral blood of B-cell chronic lymphocytic leukemia (B-CLL) patients, but display low functional avidity, which limits their clinical utility in adoptive cell transfer approaches. To overcome this key obstacle hindering effective immunotherapy, we isolated an HLA-A2-restricted T-cell receptor (TCR) with high avidity for human TERT from vaccinated HLA-A*0201 transgenic mice. Using several relevant humanized mouse models, we demonstrate that TCR-transduced T cells were able to control human B-CLL progression in vivo and limited tumor growth in several human, solid transplantable cancers. TERT-based adoptive immunotherapy selectively eliminated tumor cells, failed to trigger a self-MHC-restricted fratricide of T cells, and was associated with toxicity against mature granulocytes, but not toward human hematopoietic progenitors in humanized immune reconstituted mice. These data support the feasibility of TERT-based adoptive immunotherapy in clinical oncology, highlighting, for the first time, the possibility of utilizing a high-avidity TCR specific for human TERT. Cancer Res; 76(9); 2540-51. ©2016 AACR.

  9. A platinum(II) complex of liriodenine from traditional Chinese medicine (TCM): Cell cycle arrest, cell apoptosis induction and telomerase inhibition activity via G-quadruplex DNA stabilization.

    Science.gov (United States)

    Li, Yu-Lan; Qin, Qi-Pin; Liu, Yan-Cheng; Chen, Zhen-Feng; Liang, Hong

    2014-08-01

    Liriodenine (L), an antitumor active ingredient from the traditional Chinese medicine (TCM), Zanthoxylum nitidum, afforded a platinum(II) complex (1) of L, cis-[PtCl2(L)(DMSO)], which previously reported for its in vitro antitumor activity and intercalative binding with DNA. In this study, complex 1 was further discussed for its antitumor mechanism and structure-activity relationship, comparing with L and cisplatin. Towards the most sensitive BEL-7404 human hepatoma cells, complex 1 significantly induced cell cycle arrest at both G2/M phase and S phase. It suggests that double helix DNA is not the simplex intracellular target for 1. On the other hand, the BEL-7404 cells incubated with 1 and stained by Hoechst 33258 and AO/EB showed typical cell apoptosis in dose-dependent manner. The BEL-7404 cells incubated with 1 and stained by JC-1 were also characteristic for cell apoptosis on the loss of mitochondrial membrane potential. Furthermore, the G-quadruplex DNA binding property of complex 1 was also investigated by spectroscopic analyses, fluorescent indicator displacement (FID) assay and fluorescence resonance energy transfer (FRET) assay. The results indicated that 1 stabilized the human telomeric G4-HTG21 DNA better than L. The telomerase inhibition ratio of 1 ((62.50±0.03)%), which was examined by telomerase polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), was much higher than L ((21.77±0.01)%). It can be ascribed to the better G4-HTG21 DNA stabilization of 1 than L. The results suggested that the nuclei, mitochondria and telomerase via G-quadruplex DNA stabilization all should be key targets for the antitumor mechanism of 1, in which the central platinum(II) played a key role.

  10. 碱性成纤维细胞生长因子和环磷酸腺苷对神经前体细胞内源性端粒酶反转录酶基因转录水平的调控%Regulation of transcription level of endogenous human telomerase reverse transcriptase gene in neural progenitor cells by bFGF and cAMP

    Institute of Scientific and Technical Information of China (English)

    张小燕; 王亚军; 刘胜勇; 王建伟; 陆爱丽; 王珂; 张卫光; 沈丽

    2011-01-01

    目的 探讨人类神经前体细胞中内源性人端粒酶反转录酶(hTERT)基因的转录活性及增殖与分化相关调控机制.方法 采用系列hTERT基因启动子的荧光素酶报告载体和β-半乳苷酶表达载体,瞬时共转染HeLa细胞和hNPCs-G3细胞,检测不同长度启动子的活性.分析碱性成纤维细胞生长因子(bFGF)促进增殖过程中hTERT基因启动子活性,分析cAMP诱导分化过程中hTERT基因启动子活性.结果 hNPCs-G3神经前体细胞内源性hTERT基因的转录活性较低,主要依赖近端启动子区.bFGF上调hNPCs-G3神经前体细胞内源性hTERT基因的表达,其调控主要作用在-2 098~-1 099bp区域和-3 216bp~-2 098bp区域内;cAMP 抑制hNPCs-G3神经前体细胞hTERT基因的转录,其调控主要作用在近端启动子区.结论 神经前体细胞内源性hTERT基因的转录活性较低,bFGF可上调神经前体细胞内源性hTERT基因的表达,cAMP则可抑制hTERT基因的转录.%Objective Using hTERT-immortalized human neural progenitor cell line, hNPCs-G3 , it was studied on the transcription activity of endogenous human telomerase reverse transcriptase( hTERT ) gene and the mechanisms of proliferation and differentiation in the human neural progenitor cells. Methods Transient co-transf'ection of hTERT promoter-luciferase constructs and pSV-β-Gal control vector into HeLa cells and hNPCs-G3 cells, and detection of the transcription activities of different hTERT promoter-luciferase constructs. After co-tansfection as above, hNPCs-G3 cells were induced by bFGF, and the transcription activities of different length of hTERT promoters responsive to proliferation were analyzed. After the co-tansfection as ahove, hNPCs-G3 cells were induced by cAMP, and the transcription activities of different length of hTERT promoters responsive to differentiation were analyzed. Results The transcription activity of endogenous hTERT gene was low in human neural progenitor cells , and activity of

  11. TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines

    Directory of Open Access Journals (Sweden)

    C.A. Dalla Torre

    2002-01-01

    Full Text Available Measurement of telomerase activity in clinically obtained tumor samples may provide important information for use as both a diagnostic marker and a prognostic indicator for patient outcome. In order to evaluate telomerase activity in tumor tissue without radiolabeling the product, we developed a simple telomeric repeat amplification protocol-silver-staining assay that is less time-consuming, is safe and requires minimal equipment. In addition, we determined the sensitivity of the silver-staining method by using extracts of telomerase-positive thyroid carcinoma cell lines which were serially diluted from 5,000 to 10 cells. Telomerase activity was also assayed in 19 thyroid tumors, 2 normal controls and 27 bone marrow aspirates. The results indicate that the technique permits the detection of telomerase activity from 5000 to as few as 10 cells. We propose that it could be immediately applicable in many laboratories due to the minimal amount of equipment required.

  12. Imetelstat, a telomerase inhibitor, differentially affects normal and malignant megakaryopoiesis.

    Science.gov (United States)

    Mosoyan, G; Kraus, T; Ye, F; Eng, K; Crispino, J D; Hoffman, R; Iancu-Rubin, C

    2017-03-08

    Imetelstat (GRN163L) is a specific telomerase inhibitor which has demonstrated clinical activity in patients with myeloproliferative neoplasms (MPN) and in patients with solid tumors. The antitumor effects were associated with the development of thrombocytopenia, one of the common side effects observed in patients treated with imetelstat. The events underlying these adverse effects are not apparent. In this report we investigated the potential mechanisms that account for imetelstat's beneficial effects in MPN patients and the manner by which imetelstat treatment leads to a reduction in platelet numbers. Using a well-established system of ex vivo megakaryopoiesis, we demonstrated that imetelestat treatment affects normal megakaryocyte (MK) development by exclusively delaying maturation of MK precursor cells. By contrast, additional stages along MPN MK development were affected by imetelstat resulting in reduced numbers of assayable colony-forming unit (CFU)-MK and impaired MK maturation. In addition, treatment with imetelstat inhibited the secretion of fibrogenic growth factors by malignant but not by normal MK. Our results indicate that the delay observed in normal MK maturation may account for imetelstat-induced thrombocytopenia, while the more global effects of imetelstat on several stages along the hierarchy of MPN megakaryopoiesis may be responsible for the favorable clinical outcomes reported in MPN patients.Leukemia accepted article preview online, 08 March 2017. doi:10.1038/leu.2017.78.

  13. Amplification of hTERT and hTERC genes in leukemic cells with high expression and activity of telomerase.

    Science.gov (United States)

    Nowak, Tomasz; Januszkiewicz, Danuta; Zawada, Mariola; Pernak, Monika; Lewandowski, Krzysztof; Rembowska, Jolanta; Nowicka, Karina; Mankowski, Przemyslaw; Nowak, Jerzy

    2006-08-01

    Reactivation of telomerase plays an important role in carcinogenesis. Malignant cells almost always possess high activity and expression of telomerase. The aim of this study was to see whether there is any relationship between telomerase activity and expression and hTERT and hTERC gene amplification in acute lymphoblastic leukemia (ALL) and non-lymphoblastic leukemia (ANLL) cells. In addition telomere length was tested in leukemic cells at the time of diagnosis and during remission. Expression of the three components of telomerase (hTERT, hTERC and TP1) as well as telomerase activity was found both in ALL and ANLL cells. Telomerase activity was diminished in patients in remission. The leukemic cells showed considerable heterogeneity of terminal restriction fragments, that is telomere length. ALL cells showed a variable pattern of telomere length in contrast to ANLL cells which produced a predominantly short telomere pattern. Telomere length in the lymphocytes of leukemia patients was shorter in remission as compared to the time of diagnosis. FISH analysis revealed amplification of hTERT and hTERC genes in ALL and ANLL cells. Quantitative analysis showed that leukemic cells possess higher number of hTERT and hTERC copies than the normal PBL. Our results suggest that the activation of telomerase in leukemic cells is connected with amplification of hTERT and hTERC genes. The high expression and activity of telomerase found in leukemic cells may be partially explained by amplified hTERT and hTERC genes. Amplification of the telomerase genes seems to be a common event in carcinogenesis and may play a role in telomerase reactivation leading to cell immortalization.

  14. The telomerase reverse transcriptase subunit from the dimorphic fungus Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Dolores Bautista-España

    Full Text Available In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1 contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.

  15. The telomerase reverse transcriptase subunit from the dimorphic fungus Ustilago maydis.

    Science.gov (United States)

    Bautista-España, Dolores; Anastacio-Marcelino, Estela; Horta-Valerdi, Guillermo; Celestino-Montes, Antonio; Kojic, Milorad; Negrete-Abascal, Erasmo; Reyes-Cervantes, Hortensia; Vázquez-Cruz, Candelario; Guzmán, Plinio; Sánchez-Alonso, Patricia

    2014-01-01

    In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.

  16. Peroxiredoxin 1 Protects Telomeres from Oxidative Damage and Preserves Telomeric DNA for Extension by Telomerase

    Directory of Open Access Journals (Sweden)

    Eric Aeby

    2016-12-01

    Full Text Available Oxidative damage of telomeres can promote cancer, cardiac failure, and muscular dystrophy. Specific mechanisms protecting telomeres from oxidative damage have not been described. We analyzed telomeric chromatin composition during the cell cycle and show that the antioxidant enzyme peroxiredoxin 1 (PRDX1 is enriched at telomeres during S phase. Deletion of the PRDX1 gene leads to damage of telomeric DNA upon oxidative stress, revealing a protective function of PRDX1 against oxidative damage at telomeres. We also show that the oxidized nucleotide 8-oxo-2′deoxyguanosine-5′-triphosphate (8oxodGTP causes premature chain termination when incorporated by telomerase and that some DNA substrates terminating in 8oxoG prevent extension by telomerase. Thus, PRDX1 safeguards telomeres from oxygen radicals to counteract telomere damage and preserve telomeric DNA for elongation by telomerase.

  17. Increased fibroblast telomerase expression precedes myofibroblast α-smooth muscle actin expression in idiopathic pulmonary fibrosis

    Directory of Open Access Journals (Sweden)

    Daniel Reis Waisberg

    2012-09-01

    Full Text Available OBJECTIVE: This study sought to identify the relationship between fibroblast telomerase expression, myofibroblasts, and telomerase-mediated regulatory signals in idiopathic pulmonary fibrosis. METHODS: Thirty-four surgical lung biopsies, which had been obtained from patients with idiopathic pulmonary fibrosis and histologically classified as usual interstitial pneumonia, were examined. Immunohistochemistry was used to evaluate fibroblast telomerase expression, myofibroblast α-smooth muscle actin expression and the tissue expression of inter leu kin-4, transforming growth factor-β, and basic fibroblast growth factor. The point-counting technique was used to quantify the expression of these markers in unaffected, collapsed, mural fibrosis, and honeycombing areas. The results were correlated to patient survival. RESULTS: Fibroblast telomerase expression and basic fibroblast growth factor tissue expression were higher in collapsed areas, whereas myofibroblast expression and interleukine-4 tissue expression were higher in areas of mural fibrosis. Transforming growth factor-β expression was higher in collapsed, mural fibrosis and honeycombing areas in comparison to unaffected areas. Positive correlations were found between basic fibroblast growth factor tissue expression and fibroblast telomerase expression and between interleukin-4 tissue expression and myofibroblast α-smooth muscle actin expression. Negative correlations were observed between interleukin-4 expression and basic fibroblast growth factor tissue expression in areas of mural fibrosis. Myofibroblast α-smooth muscle actin expression and interleukin-4 tissue expression in areas of mural fibrosis were negatively associated with patient survival. CONCLUSION: Fibroblast telomerase expression is higher in areas of early remodeling in lung tissues demonstrating typical interstitial pneumonia, whereas myofibroblast α-smooth muscle actin expression predominates in areas of late remodeling

  18. A trans-spliced telomerase RNA dictates telomere synthesis in Trypanosoma brucei

    Institute of Scientific and Technical Information of China (English)

    Ranjodh Sandhu; Samantha Sanford; Shrabani Basu; MinA Park; Unnati M Pandya; Bibo Li; Kausik Chakrabarti

    2013-01-01

    Telomerase is a ribonucleoprotein enzyme typically required for sustained cell proliferation.Although both telomerase activity and the telomerase catalytic protein component,TbTERT,have been identified in the eukaryotic pathogen Trypanosoma brucei,the RNA molecule that dictates telomere synthesis remains unknown.Here,we identify the RNA component of Trypanosoma brucei telomerase,TbTR,and provide phylogenetic and in vivo evidence for TbTR's native folding and activity.We show that TbTR is processed through trans-splicing,and is a capped transcript that interacts and copurifies with TbTERT in vivo.Deletion of TbTR caused progressive shortening of telomeres at a rate of 3-5 bp/population doubling (PD),which can be rescued by ectopic expression of a wild-type allele of TbTR in an apparent dose-dependent manner.Remarkably,introduction of mutations in the TbTR template domain resulted in corresponding mutant telomere sequences,demonstrating that telomere synthesis in T.brucei is dependent on TbTR.We also propose a secondary structure model for TbTR based on phylogenetic analysis and chemical probing experiments,thus defining TbTR domains that may have important functional implications in telomere synthesis.Identification and characterization of TbTR not only provide important insights into T.brucei telomere functions,which have been shown to play important roles in T.brucei pathogenesis,but also offer T.brucei as an attractive model system for studying telomerase biology in pathogenic protozoa and for comparative analysis of telomerase function with higher eukaryotes.

  19. Telomere shortening and cell senescence induced by perylene derivatives in A549 human lung cancer cells.

    Science.gov (United States)

    Taka, Thanachai; Huang, Liming; Wongnoppavich, Ariyaphong; Tam-Chang, Suk-Wah; Lee, T Randall; Tuntiwechapikul, Wirote

    2013-02-15

    Cancer cells evade replicative senescence by re-expressing telomerase, which maintains telomere length and hence chromosomal integrity. Telomerase inhibition would lead cancer cells to senesce and therefore prevent cancer cells from growing indefinitely. G-quadruplex ligands can attenuate telomerase activity by inducing G-quadruplex formation at the 3'-overhang of telomere and at the human telomerase reverse transcriptase (hTERT) promoter; the former prevents telomerase from accessing the telomere, and the latter acts as a transcriptional silencer. The present investigation found that perylene derivatives PM2 and PIPER induced G-quadruplex formation from both telomeric DNA and the hTERT promoter region in vitro. Further, TRAP assay showed that these compounds inhibited telomerase in a dose-dependent manner. When A549 human lung cancer cells were treated with these compounds, hTERT expression was down-regulated. Moreover, the crude protein extract from these treated cells exhibited less telomerase activity. In the long-term treatment of A549 lung cancer cells with sub-cytotoxic dose of these perylenes, telomere shortening, reduction of cell proliferation and tumorigenicity, and cell senescence were observed. The results of this study indicate that perylene derivatives warrant further consideration as effective agents for cancer therapy.

  20. Improved Inhibition of Telomerase by Short Twisted Intercalating Nucleic Acids under Molecular Crowding Conditions

    DEFF Research Database (Denmark)

    Agarwal, Tani; Pradhan, Devranjan; Géci, Imrich;

    2012-01-01

    crowding conditions mimicking physiological milieu, stabilization of the telomeric G-quadruplex is often lost. We attempted to demonstrate the enhanced G-quadruplex stabilizing ability under molecular conditions by using twisted intercalating nucleic acids (TINA)-modified oligonucleotides. We have shown......-based telomerase repeat amplification assay (TRAP) assay as well as nondenaturing polyacrylamide gel electrophoresis-based TRAP, we demonstrate remarkable enhancement in their anti-telomerase activity even under molecular crowding conditions. This is the first time in which a G-quadruplex stabilizing agent has...... demonstrated enhanced activity even under molecular crowding conditions....

  1. Telomere attrition and restoration in the normal teleost Oryzias latipes are linked to growth rate and telomerase activity at each life stage.

    Science.gov (United States)

    Hatakeyama, Hitoshi; Yamazaki, Hiromi; Nakamura, Ken-Ichi; Izumiyama-Shimomura, Naotaka; Aida, Junko; Suzuki, Hiroetsu; Tsuchida, Shuichi; Matsuura, Masaaki; Takubo, Kaiyo; Ishikawa, Naoshi

    2016-01-01

    Telomere shortening occurs when cells divide, both in vitro and in vivo. On the other hand, telomerase is able to maintain telomere length in cells by adding TTAGGG repeats to the ends of telomeres. However, the interrelationships existing among telomere length, telomerase activity and growth in vertebrates remain to be clarified. In the present study we measured telomere length (terminal restriction fragment length), telomerase activity and body growth of Oryzias latipes from the embryo stage until senescence. During the rapid growth stage (age 0-7 months), telomeres shortened in parallel with decreasing telomerase activity. Then, during adolescence (age 7 months - 1 year), telomeres lengthened quickly as growth slowed and telomerase activity increased. In the adult stage (age 1-4 years) characterized by little growth, telomerase activity decreased gradually and telomeres shortened. Our data indicate that telomere attrition and restoration are linked to growth and telomerase activity, and suggest that critical loss of telomere homeostasis is associated with mortality in this animal.

  2. Telomeres and telomerase of structure and regulation%端粒与端粒酶的结构及调节

    Institute of Scientific and Technical Information of China (English)

    李新萍; 李娟; 欧阳建

    2014-01-01

    Telomeres ,as a major factor of maintaining chromosomal integrity ,is an essential activity of regulatory factors of aging and tumor sup-pression mechanism ,and are highly associated with cance ;in normal somatic cells ,when telomeres shorten to a certain limit (ie after about 50 -70 times a cell division)cell gene becomes unstable causing human tissue and cell senescence until death ;but some escape apoptotic cells almost universally express a ribonucleoprotein ,telomerase(a cellular reversal transcriptase ,adding new DNA to telomerestheends of chromosomes ) ,in order to maintain steady but short telomeres ;while in most cancer cells ,hematopoietic stem cells ,germ cells ,telomeres ,telomerase are major factors in maintaining chromosome stability ,not only with cellular senescence direct relationship is also closely related to tumor development . Telomerase is a major regula-tor factors of the telomere length ,which provide new targets for anti -aging and cancer treatment . Here we review mechanisms of maintain telomere length and the latest developments of telomerase composition 、regulation.%端粒作为维持染色体完整性的主要因素,在衰老与肿瘤抑制机制中是一个必不可少的活性调节因素,是与癌症高度相关的;在正常体细胞中,当端粒缩短至一定极限时(即经过大约50-70次的细胞分裂)细胞基因变的极不稳定导致人类组织和细胞衰老直至凋亡;但是,一些逃脱凋亡的细胞几乎普遍表达一种核糖核蛋白,端粒酶(一种细胞逆转录酶,增加新的 DNA 到位于染色体末端的端粒上面),从而维护稳定但短的端粒;而在大多数癌症细胞、造血干细胞、生殖细胞中,端粒、端粒酶是维持染色体稳定的主要因素,因此二者不仅与细胞衰老有直接关系,还与肿瘤的发生发展有密切的关系。端粒酶做为端粒长度维持的主要调节因素,为抗衰老、肿瘤治疗提供了新的靶

  3. Telomerase-pulsed dendritic cells: preclinical results and outcome of a clinical phase I/II trial in patients with metastatic renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Schmiedel, Alexandra

    2006-02-01

    Full Text Available Objective: Therapeutic vaccination with dendritic cells (DC showed promising results in first clinical trials in cases of metastatic renal cell carcinoma (RCC. Human telomerase reverse transcriptase (hTERT could be a potential target because it is detectable in more than 85% of human tumors including RCC. Design: 10 patients with progressive metastatic RCC were enrolled in a clinical phase I/II trial using DC pulsed with hTERT-peptide. Beside toxicity and feasibility aspects, a complex immune monitoring including in vitro data were evaluated. In addition to detection of tumor-specific effector cells we investigated their functionality like IFN-γ secretion and cytotoxic activity against tumor cells. Results: The vaccine was well tolerated. Two patients showed a mixed response (MR and one patient a stable disease (SD. Interestingly, responders showed cytotoxic activity already before start of therapy and there was a significant increase in cytotoxic activity of effector cells from all responders (SD and MR patients after the first vaccination. In contrast non-responders showed no cytotoxic activity before and during treatment. Therefore, cytotoxic activity might be used as a predictive marker in the future. Tetramer staining detected higher amounts of tumor-specific cytotoxic cells in responding patients compared to non-responders. Also, responders possessed increasing amounts of IFN-γ producing immunological effector cells. Conclusion: Telomerase-pulsed DC could enhance a tumor-specific immune response against RCC.

  4. Telomerase reverse transcriptase (TERT) promoter mutations in Korean melanoma patients.

    Science.gov (United States)

    Roh, Mi Ryung; Park, Kyu-Hyun; Chung, Kee Yang; Shin, Sang Joon; Rha, Sun Young; Tsao, Hensin

    2017-01-01

    Telomerase reverse transcriptase (TERT) is the reverse transcriptase component of the telomeric complex, which synthesizes terminal DNA to protect chromosomal ends and to maintain genomic integrity. In melanoma, mutation in TERT promoter region is a common event and theses promoter variants have been shown to be associated with increased gene expression, decreased telomere length and poorer outcome. In this study, we determined the frequency of TERT promoter mutation in 88 Korean primary melanoma patients and aimed to see the association of TERT promoter mutation status to other major molecular features, such as BRAF, NRAS, KIT mutations and correlate with clinicopathological features. In our study, acral melanoma (n=46, 52.3%) was the most common type. Overall, TERT promoter mutation was observed in 15 cases (17%) with ten c. -124C>T altertions and five c. -146C>T alterations. None of our samples showed CC>TT mutation which is considered pathognomonic of UV induction. Among the 46 acral melanoma patients, 5 patients (10.9%) harbored TERT promoter mutation. Tumors with TERT promoter mutation showed significantly greater Breslow thickness compared to WT tumors (P=0.039). A combined analysis for the presence of TERT promoter and BRAF mutations showed that patients with both TERT promoter and BRAF mutation showed decreased survival compared with those with only TERT promoter mutation, only BRAF mutation, or without mutations in either TERT promoter or BRAF (P=0.035). Our data provides additional evidence that UV-induced TERT promoter mutation frequencies vary depending on melanoma subtype, but preserves its prognostic value.

  5. Coordinate increase of telomerase activity and c-Myc expression in Helicobacter pylori-associated gastric diseases

    Institute of Scientific and Technical Information of China (English)

    Guo-Xin Zhang; Yan-Hong Gu; Zhi-Quan Zhao; Shun-Fu Xu; Hong-Ji Zhang; Hong-Di Wang; Bo Hao

    2004-01-01

    AIM: To detect the telomerase activity and c-Myc expression in gastric diseases and to examine the relation between these values and Helicobacter pylori (H pylori) as a risk factor for gastric cancer.METHODS: One hundred and seventy-one gastric samples were studied to detect telomerase activity using a telomerase polymerase chain reaction enzyme linked immunosorbent assay (PCR-ELTSA), and c-Myc expression using immunohistochemistry.RESULTS: The telomerase activity and c-Myc expression were higher in cancers (87.69% and 61.54%) than in noncancerous tissues. They were higher in chronic atrophic gastritis with severe intestinal metaplasia (52.38% and 47.62%) than in chronic atrophic gastritis with mild intestinal metaplasia (13.33% and 16.67%). Tn chronic atrophic gastritis with severe intestinal metaplasia, the telomerase activity and c-Myc expression were higher in cases with -H pylori infection (67.86% and 67.86%) than in those without infection (21.43%and 7.14%). c-Myc expression was higher in gastric cancer with H pylori infection (77.27%) than in that without infection (28.57%). The telomerase activity and c-Nyc expression were coordinately up-regulated in H pylori infected gastric cancer and chronic atrophic gastritis with severe intestinal metaplasia.CONCLUSION: H pylori infection may influence both telomerase activity and c-Myc expression in gastric diseases,especially in chronic atrophic gastritis.

  6. Label-free molecular beacons-based cascade amplification DNA machine for sensitive detection of telomerase activity.

    Science.gov (United States)

    Li, Kan; Wang, Lei; Xu, Xiaowen; Jiang, Wei

    2017-05-15

    Sensitive detection of telomerase activity is critical to cancer diagnosis, screening of anticancer drugs and evaluation of cancer therapy. Herein, a label-free molecular beacons-based DNA machine was developed for sensitive detection of telomerase activity. The DNA machine consisted of T7 exonuclease (T7 Exo), label-free recognition molecular beacon (RMB) and signal molecular beacon (SMB) with projecting 5'-terminuses, which can protect RMB and SMB from being digested by T7 Exo. Firstly, telomerase elongated telomerase substrate (TS) primer, generating a telomerase elongation production (TEP) with tandem repeats (TTAGGG)n. Next, TEP activated the DNA machine by hybridizing with RMB, unfolding RMB with a recessed 5'-terminus, making RMB deprotection from T7 Exo. Then T7 Exo-assisted cycling cleavage was incurred, releasing intact TEP and numerous DNA fragments (trigger DNA), which got recycling I. Subsequently, trigger DNA specifically opened SMB and was recycled by T7 Exo, liberating multiple G-quadruplex (G4) structures, which got recycling II. Finally, TEP and the liberative G4 structures strongly interacted with N-methyl-mesoporphyrin IX (NMM), yielding a significantly enhanced fluorescence together. In this way, per telomerase-mediated elongation event was efficiently converted into the greatly amplified fluorescence signals. Telomerase activity in crude HeLa cells extracts equivalent to 50 cells/mL was successfully measured with a linear range from 50 cells/mL to 2000 cells/mL. Besides, the strategy was also successfully used to assay the inhibition effect of a telomerase-inhibiting drug, demonstrating the strategy holds the potential to screen telomerase inhibitors.

  7. Detection of K-ras point mutation and telomerase activity during endoscopic retrograde cholangiopancreatography in diagnosis of pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Guo-Xiong Zhou; Jie-Fei Huang; Zhao-Shen Li; Guo-Ming Xu; Feng Liu; Hong Zhang

    2004-01-01

    AIM: To study the value of monitoring K-ras point mutation at codon 12 and telomerase activity in exfoliated cells obtained from pancreatic duct brushings during endoscopic retrograde cholangiopancreatography (ERCP) in the diagnosis of pancreatic cancer.METHODS: Exfoliated cells obtained from pancreatic duct brushings during ERCP were examined in 27 patients: 23with pancreatic cancers, 4 with chronic pancreatitis. K-fas point mutation was detected with the polymerase chain reaction and restriction fragment-length polymorphism (PCR-RFLP). Telomerase activity was detected by PCR and telomeric repeat amplification protocol assay (PCR-TRAPELISA).RESULTS: The telomerase activities in 27 patients were measured in 21 exfoliated cell samples obtained from pancreatic duct brushings. D450 value of telomerase activities in pancreatic cancer and chronic pancreatitis were 0.446±0.27and 0.041±0.0111, respectively. Seventy-seven point eight percent (14/18) of patients with pancreatic cancer and none of the patients with chronic pancreatitis showed telomerase activity in cells collected from pancreatic duct brushings when cutoff value of telomerase activity was set at 2.0. The K-ras gene mutation rate (72.2%) in pancreatic cancer was higher than that in chronic pancreatitis (33.3%)(P<0.05). In considering of both telomerase activities and K-ras point mutation, the total positive rate was 83.3%(15/18), and the specificity was 100%.CONCLUSION: Changes of telomerase activities and K-ras point mutation at codon 12 may be an early event of malignant progression in pancreatic cancer. Detection of telomerase activity and K-ras point mutation at codon 12may be complementary to each other, and is useful in diagnosis of pancreatic cancer.

  8. Telomerase is essential to alleviate pif1-induced replication stress at telomeres

    NARCIS (Netherlands)

    Chang, Michael; Luke, Brian; Kraft, Claudine; Li, Zhijian; Peter, Matthias; Lingner, Joachim; Rothstein, Rodney

    2009-01-01

    Pif1, an evolutionarily conserved helicase, negatively regulates telomere length by removing telomerase from chromosome ends. Pif1 has also been implicated in DNA replication processes such as Okazaki fragment maturation and replication fork pausing. We find that overexpression of Saccharomyces cerv

  9. Regulative Function of Telomerase and Extracelluar Regulated Protein Kinases to Leukemic Cell Apoptosis

    Institute of Scientific and Technical Information of China (English)

    李登举; 张瑶珍; 曹文静; 孙岚; 徐慧珍; 路武

    2002-01-01

    Summary: In order to investigate the regulative function of telomerase and phosphorylated (acti-vated) extracelluar regulated protein kinase (ERK) i and 2 in the leukemic cell lines HL-60 andK562 proliferation inhibition and apoptosis, three chemotherapeutic drugs Harringtonine (HRT),Vincristine(VCR)and Etoposide(Vp16)were selected as inducers. The proliferation inhibition ratewas detected by MTT method, the cell cycle and cell apoptosis was analyzed by flow cytometryand the telomerase activity was detected by the telomeric repeat amplification protocol (TRAP)assay and bioluminescence analysis method. The phosphorylated ERK1/2 protein expression wasdetected by western blot method. The results showed that HRT, VCR and Vp16 could inhibit cellproliferation, induce apoptosis, inhibit telomerase activity and down-regulate the protein expres-sion of phosphorylated ERK. It was suggested that ERK signal transduction pathway was involvedin the down-regulation of telomerase activity and the onset of apoptosis in the leukemic cells treat-ed by HRT, VCR and Vp16.

  10. Telomerase gene therapy rescues telomere length, bone marrow aplasia, and survival in mice with aplastic anemia.

    Science.gov (United States)

    Bär, Christian; Povedano, Juan Manuel; Serrano, Rosa; Benitez-Buelga, Carlos; Popkes, Miriam; Formentini, Ivan; Bobadilla, Maria; Bosch, Fatima; Blasco, Maria A

    2016-04-07

    Aplastic anemia is a fatal bone marrow disorder characterized by peripheral pancytopenia and marrow hypoplasia. The disease can be hereditary or acquired and develops at any stage of life. A subgroup of the inherited form is caused by replicative impairment of hematopoietic stem and progenitor cells due to very short telomeres as a result of mutations in telomerase and other telomere components. Abnormal telomere shortening is also described in cases of acquired aplastic anemia, most likely secondary to increased turnover of bone marrow stem and progenitor cells. Here, we test the therapeutic efficacy of telomerase activation by using adeno-associated virus (AAV)9 gene therapy vectors carrying the telomerase Tert gene in 2 independent mouse models of aplastic anemia due to short telomeres (Trf1- and Tert-deficient mice). We find that a high dose of AAV9-Tert targets the bone marrow compartment, including hematopoietic stem cells. AAV9-Tert treatment after telomere attrition in bone marrow cells rescues aplastic anemia and mouse survival compared with mice treated with the empty vector. Improved survival is associated with a significant increase in telomere length in peripheral blood and bone marrow cells, as well as improved blood counts. These findings indicate that telomerase gene therapy represents a novel therapeutic strategy to treat aplastic anemia provoked or associated with short telomeres.

  11. Telomere-independent functions of telomerase in nuclei, cytoplasm, and mitochondria.

    Science.gov (United States)

    Chiodi, Ilaria; Mondello, Chiara

    2012-01-01

    Telomerase canonical activity at telomeres prevents telomere shortening, allowing chromosome stability and cellular proliferation. To perform this task, the catalytic subunit (telomerase reverse transcriptase, TERT) of the enzyme works as a reverse transcriptase together with the telomerase RNA component (TERC), adding telomeric repeats to DNA molecule ends. Growing evidence indicates that, besides the telomeric-DNA synthesis activity, TERT has additional functions in tumor development and is involved in many different biological processes, among which cellular proliferation, gene expression regulation, and mitochondrial functionality. TERT has been shown to act independently of TERC in the Wnt-β-catenin signaling pathway, regulating the expression of Wnt target genes, which play a role in development and tumorigenesis. Moreover, TERT RNA-dependent RNA polymerase activity has been found, leading to the genesis of double-stranded RNAs that act as precursor of silencing RNAs. In mitochondria, a TERT TERC-independent reverse transcriptase activity has been described that could play a role in the protection of mitochondrial integrity. In this review, we will discuss some of the extra-telomeric functions of telomerase.

  12. Telomerase activity in needle biopsies from prostate cancer and benign prostates

    NARCIS (Netherlands)

    Wymenga, LFA; Wisman, GBA; Ruiters, MHJ; Mensink, HJA; Veenstra, R.

    2000-01-01

    Background Telomerase activation is thought to be essential for the immortality of cancer cells. It may be a prognostic factor in small volume well differentiated prostate cancers and hence a guide for the aggressiveness of the approach. The length of the chromosome tips (telomeres) are maintained b

  13. HOT1 is a mammalian direct telomere repeat-binding protein contributing to telomerase recruitment

    NARCIS (Netherlands)

    Kappei, D.; Butter, F.; Benda, C.; Scheibe, M.; Draskovic, Irena; Stevense, M.; Novo, C.L.; Basquin, C.; Araki, M.; Araki, K.; Krastev, D.B.; Kittler, R.; Jessberger, R.; Londono-Vallejo, J.A.; Mann, M.; Buchholz, F.

    2013-01-01

    Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the

  14. Determination of telomerase activity in stem cells and non-stem cells of breast cancer

    Institute of Scientific and Technical Information of China (English)

    LI Zhi; HE Yanli; ZHANG Jiahua; ZHANG Jinghui; HUANG Tao

    2007-01-01

    Although all normal tissue cells,including stem cells,are genetically homologous,variation in gene expression patterns has already determined the distinct roles for individual cells in the physiological process due to the occurrence of epigenetic modification.This is of special importance for the existenee of tissue stem cells because they are exclusively immortal within the body,capable of selfreplicating and differentiating by which tissues renew and repair itself and the total tissue cell population maintains a steady-state.Impairment of tissue stem cells is usually accompanied by a reduction in cell number,slows down the repair process and causes hypofunction.For instance,chemotherapy usually leads to depression of bone marrow and hair loss.Cellular aging is closely associated with the continuous erosion of the telomere while activation of telomerase repairs and maintains telomeres,thus slowing the aging process and prolonging cell life.In normal adults,telomerase activation mainly presents in tissue stem cells and progenitor cells giving them unlimited growth potential.Despite the extensive demonstration of telomerase activation in malignancy(>80%),scientists found that heterogeneity also exists among the tumor cells and only minorities of cells,designated as cancer stem cells,andergo processes analogous to the self-renewal and differentiation of normal stem ceils while the rest have limited lifespans.In this study,telomerase activity was measured and compared in breast cancer stem cells and non-stem cells that were phenotypically sorted by examining surface marker expression.The results indicated that cancer stem cells show a higher level of enzyme activity than non-stem cells.In addition,associated with the repair of cancer tissue(or relapse)after chemotherapy,telomerase activity in stem cells was markedly increased.

  15. The pif1 helicase, a negative regulator of telomerase, acts preferentially at long telomeres.

    Directory of Open Access Journals (Sweden)

    Jane A Phillips

    2015-04-01

    Full Text Available Telomerase, the enzyme that maintains telomeres, preferentially lengthens short telomeres. The S. cerevisiae Pif1 DNA helicase inhibits both telomerase-mediated telomere lengthening and de novo telomere addition at double strand breaks (DSB. Here, we report that the association of the telomerase subunits Est2 and Est1 at a DSB was increased in the absence of Pif1, as it is at telomeres, suggesting that Pif1 suppresses de novo telomere addition by removing telomerase from the break. To determine how the absence of Pif1 results in telomere lengthening, we used the single telomere extension assay (STEX, which monitors lengthening of individual telomeres in a single cell cycle. In the absence of Pif1, telomerase added significantly more telomeric DNA, an average of 72 nucleotides per telomere compared to the 45 nucleotides in wild type cells, and the fraction of telomeres lengthened increased almost four-fold. Using an inducible short telomere assay, Est2 and Est1 no longer bound preferentially to a short telomere in pif1 mutant cells while binding of Yku80, a telomere structural protein, was unaffected by the status of the PIF1 locus. Two experiments demonstrate that Pif1 binding is affected by telomere length: Pif1 (but not Yku80 -associated telomeres were 70 bps longer than bulk telomeres, and in the inducible short telomere assay, Pif1 bound better to wild type length telomeres than to short telomeres. Thus, preferential lengthening of short yeast telomeres is achieved in part by targeting the negative regulator Pif1 to long telomeres.

  16. Dynamic telomerase gene suppression via network effects of GSK3 inhibition.

    Directory of Open Access Journals (Sweden)

    Alan E Bilsland

    Full Text Available BACKGROUND: Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression. METHODOLOGY/PRINCIPAL FINDINGS: In a focused promoter screen, several GSK3 inhibitors suppressed hTERT reporter activity. GSK3 inhibition using 6-bromoindirubin-3'-oxime suppressed hTERT expression, telomerase activity and telomere length in several cancer cell lines and growth and hTERT expression in ovarian cancer xenografts. Microarray analysis, network modelling and oligonucleotide binding assays suggested that multiple transcription factors were affected. Extensive remodelling involving Sp1, STAT3, c-Myc, NFkappaB, and p53 occurred at the endogenous hTERT promoter. RNAi screening of the hTERT promoter revealed multiple kinase genes which affect the hTERT promoter, potentially acting through these factors. Prolonged inhibitor treatments caused dynamic expression both of hTERT and of c-Jun, p53, STAT3, AR and c-Myc. CONCLUSIONS/SIGNIFICANCE: Our results indicate that GSK3 activates hTERT expression in cancer cells and contributes to telomere length homeostasis. GSK3 inhibition is a clinical strategy for several chronic diseases. These results imply that it may also be useful in cancer therapy. However, the complex network effects we show here have implications for either setting.

  17. Epidermal growth factor activates telomerase activity by direct binding of Ets-2 to hTERT promoter in lung cancer cells.

    Science.gov (United States)

    Hsu, Chung-Ping; Lee, Li-Wen; Tang, Sheau-Chung; Hsin, I-Lun; Lin, Yu-Wen; Ko, Jiunn-Liang

    2015-07-01

    Growth signals are directly or indirectly involved in telomerase regulation. In this study, we investigated molecular mechanisms of the effect of EGF (epidermal growth factor) on regulating hTERT (human telomerase reverse transcriptase) expression. To elucidate specific transcription factors involved in EGF-stimulated hTERT transcription in A549 and H1299 lung cancer cells, transcription factors drives hTERT promoter activity, such as Myc, Mad, and Ets-2, was evaluated on luciferase reporter assay. The upregulation of hTERT promoter by Ets-2 and Myc were abolished by Mad. Using DAPA (DNA affinity precipitation assay), Ets-2 binding to SNP (T) was stronger than Ets-2 binding to SNP (C) at -245 bp upstream of the transcription start site within the core promoter of hTERT. Ets-2 silence by siRNA decreased hTERT expression at mRNA and protein levels. The regulation of hTERT promoter by EGF/Ets-2 was diminished via the EGFR kinase signal pathway-specific inhibitors AG1478 and Iressa. Inhibitors of Erk and Akt inhibited Ets-2-activated hTERT promoter activity. These data suggested that Ets-2, a genuine cancer-specific transcription factor, is actively involved in EGFR kinase-induced hTERT overexpression pathway in lung cancer cells. Blockage of this pathway may contribute to targeted gene therapy in lung cancer.

  18. Formation and stabilization of the telomeric antiparallel G-quadruplex and inhibition of telomerase by novel benzothioxanthene derivatives with anti-tumor activity

    Science.gov (United States)

    Zhang, Wen; Chen, Min; Ling Wu, Yan; Tanaka, Yoshimasa; Juan Ji, Yan; Lin Zhang, Su; He Wei, Chuan; Xu, Yan

    2015-09-01

    G-quadruplexes formed in telomeric DNA sequences at human chromosome ends can be a novel target for the development of therapeutics for the treatment of cancer patients. Herein, we examined the ability of six novel benzothioxanthene derivatives S1-S6 to induce the formation of and stabilize an antiparallel G-quadruplex by EMSA, UV-melting and CD techniques and the influence of S1-S6 on A549 and SGC7901 cells through real-time cell analysis, wound healing, trap assay methods. Results show that six compounds could differentially induce 26 nt G-rich oligonucleotides to form the G-quadruplex with high selectivity vs C-rich DNA, mutated DNA and double-stranded DNA, stabilize it with high affinity, promote apoptosis and inhibit mobility and telomerase activity of A549 cells and SGC7901 cells. Especially, S1, S3, S4 displayed stronger abilities, of which S3 was the most optimal with the maximum ΔTm value being up to 29.8 °C for G-quadruplex, the minimum IC50 value being 0.53 μM and the maximum cell inhibitory rate being up to 97.2%. This study suggests that this type of compounds that induce the formation of and stabilize the telomeric antiparallel G-quadruplex, and consequently inhibit telomerase activity, leading to cell apoptosis, can be screened for the discovery of novel antitumor therapeutics.

  19. Risk of progression of early cervical lesions is associated with integration and persistence of HPV-16 and expression of E6, Ki-67, and telomerase

    Directory of Open Access Journals (Sweden)

    Arianna Vega-Peña

    2013-01-01

    Full Text Available Background: Low-grade squamous intraepithelial lesions (LSIL are the earliest lesions of the uterine cervix, the persistence and integration of high-risk human papillomavirus (HR-HPV as type 16, which promotes the development of more aggressive lesions. Aim: To select more aggressive lesions with tendency to progress to invasive cervical cancer. Materials and Methods: A total of 75 cytological specimens in liquid base (Liqui-PREP were analyzed: 25 specimens were with no signs of SIL (NSIL and without HPV; 25 NSIL with HPV-16, and 25 with both LSIL and HPV-16. The expression of Ki-67, telomerase, and viral E6 was evaluated by immunocytochemistry; and the detection of viral DNA was done by polymerase chain reaction (PCR and restriction fragment length polymorphism (RFLPs for genotyping or sequencing of HPV-16. The physical state of HPV-16 was evaluated by in situ hybridization with amplification with tyramide. Results: Of the total group, 58.6% had LSIL associated with persistence and of these 59.3% was associated with integrated state of HPV as intense expression of E6, Ki-67 (P = 0.013, P = 0.055 has except for the expression of telomerase present a non-significant association (P<0.341. Conclusions: Overexpression of E6 and Ki-67 is associated with the integration of HPV-16, favoring viral persistence, and increasing the risk of progression in women with NSIL and LSIL.

  20. The role of telomerase protein TERT in Alzheimer's disease and in tau-related pathology in vitro.

    Science.gov (United States)

    Spilsbury, Alison; Miwa, Satomi; Attems, Johannes; Saretzki, Gabriele

    2015-01-28

    The telomerase reverse transcriptase protein TERT has recently been demonstrated to have a variety of functions both in vitro and in vivo, which are distinct from its canonical role in telomere extension. In different cellular systems, TERT protein has been shown to be protective through its interaction with mitochondria. TERT has previously been found in rodent neurons, and we hypothesize that it might have a protective function in adult human brain. Here, we investigated the expression of TERT at different stages of Alzheimer's disease pathology (Braak Stages I-VI) in situ and the ability of TERT to protect against oxidative damage in an in vitro model of tau pathology. Our data reveal that TERT is expressed in vitro in mouse neurons and microglia, and in vivo in the cytoplasm of mature human hippocampal neurons and activated microglia, but is absent from astrocytes. Intriguingly, hippocampal neurons expressing TERT did not contain hyperphosphorylated tau. Vice versa, neurons that expressed high levels of pathological tau did not appear to express TERT protein. TERT protein colocalized with mitochondria in the hippocampus of Alzheimer's disease brains (Braak Stage VI), as well as in cultured neurons under conditions of oxidative stress. Our in vitro data suggest that the absence of TERT increases ROS generation and oxidative damage in neurons induced by pathological tau. Together, our findings suggest that TERT protein persists in neurons of the adult human brain, where it may have a protective role against tau pathology.

  1. The changes in telomerase activity and telomere length in HeLa cells undergoing apop- tosis induced by sodium butyrate

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The changes in telomerase activity and telomere length during apoptosis in HeLa cells as induced by sodium butyrate (SB) have been studied. After a 48 h SB treatment, HeLa cells demonstrated characteristic apoptotic hallmarks including chromatin condensation, formation of apoptotic bodies and DNA Laddering which were caused by the cleavage and degradation of DNA between nucleosomes. There were no significant changes in telomerase activity of apoptotic cells, while the telomere length shortened markedly. In the meanwhile, cells became more susceptible to apoptotic stimuli and telomere became more vulnerable to degradation after telomerase activity was inhibited. All the results suggest that the apoptosis induced by SB is closely related to telomere shortening, while telomerase enhances resistance of HeLa cells to apoptotic stimuli by protecting telomere.

  2. Clinical significance of telomerase activity in peritoneal lavage fluid from patients with gastric cancer and its relationship with cellular proliferation

    Institute of Scientific and Technical Information of China (English)

    Ming-Xu Da; Xiao-Ting Wu; Tian-Kang Guo; Zi-Guang Zhao; Ting Luo; Kun Qian; Ming-Ming Zhang; Jie Wang

    2007-01-01

    AIM: To evaluate the efficacy of telomerase activity assay and peritoneal lavage cytology (PLC) examination in peritoneal lavage fluid for the prediction of peritoneal metastasis in gastric cancer patients, and to explore the relationship between telomerase activity and proliferating cell nuclear antigen expression.METHODS: Telomeric repeated amplification protocol (TRAP)-enzyme-linked immunosorbent assay (ELISA) was performed to measure the telomerase activity in 60 patients with gastric cancer and 50 with peptic ulcer. PLC analysis of the 60 patients with gastric cancer was used for comparison. The proliferating cell nuclear antigen (PCNA) in gastric carcinoma was immunohistochemically examined.RESULTS: The telomerase activity and PLC positive rate in peritoneal lavage fluid from patients with gastric cancer was 41.7% (25/60), and 25.0% (15/60), respectively. The positive rate of telomerase activity was significantly higher than that of PLC in the group Of pT4 (15/16 vs 9/16, P < 0.05), P1-3 (13/13 vs 9/13, P < 0.05) and diffuse type (22/42 vs 13/42, P < 0.05). The patients with positive telomerase activity, peritoneal metastasis, and serosal invasion had significantly higher levels of average PCNA proliferation index (PI), (55.00 ± 6.59 vs 27.43 ± 7.72, 57.26 ± 10.18 vs 29.15 ± 8.31, and 49.82 ± 6.74 vs 24;65 ± 7.33, respectively, P < 0.05).CONCLUSION: The TRAP assay for telomerase activity is a useful adjunct for cytologic method in the diagnosis of peritoneal micrometastasis and well related to higher proliferating activity of gastric cancer. The results of this study also suggest a promising future therapeutic strategy for treating peritoneal dissemination based on telomerase inhibition.

  3. Effects of PCB126 and PCB153 on telomerase activity and telomere length in undifferentiated and differentiated HL-60 cells.

    Science.gov (United States)

    Xin, Xing; Senthilkumar, P K; Schnoor, Jerald L; Ludewig, Gabriele

    2016-02-01

    PCBs are persistent organic pollutants that are carcinogenic and immunotoxic and have developmental toxicity. This suggests that they may interfere with normal cell maturation. Cancer and stem/progenitor cells have telomerase activity to maintain and protect the chromosome ends, but lose this activity during differentiation. We hypothesized that PCBs interfere with telomerase activity and the telomere complex, thereby disturbing cell differentiation and stem/progenitor cell function. HL-60 cells are cancer cells that can differentiated into granulocytes and monocytes. We exposed HL-60 cells to PCB126 (dioxin-like) and PCB153 (nondioxin-like) 6 days before and during 3 days of differentiation. The differentiated cells showed G0/G1 phase arrest and very low telomerase activity. hTERT and hTR, two telomerase-related genes, were downregulated. The telomere shelterins TRF1, TRF2, and POT1 were upregulated in granulocytes, and TRF2 was upregulated and POT1 downregulated in monocytes. Both PCBs further reduced telomerase activity in differentiated cells, but had only small effects on the differentiation and telomere-related genes. Treatment of undifferentiated HL-60 cells for 30 days with PCB126 produced a downregulation of telomerase activity and a decrease of hTERT, hTR, TRF1, and POT1 gene expression. With PCB153, the effects were less pronounced and some shelterin genes were increased after 30 days of exposure. With each PCB, no differentiation of cells was observed and cells continued to proliferate despite reduced telomerase activity, resulting in shortened telomeres after 30 days of exposure. These results indicate cell-type and PCB congener-specific effects on telomere/telomerase-related genes. Although PCBs do not seem to strongly affect differentiation, they may influence stem or progenitor cells through telomere attrition with potential long-term consequences for health.

  4. Inhibitory effects of antisense phosphorothioate oligodeoxynucleotides on pancreatic cancer cell Bxpc-3 telomerase activity and cell growth in vitro

    Institute of Scientific and Technical Information of China (English)

    Yun-Feng Wang; Ke-Jian Guo; Bei-Ting Huang; Yong Liu; Xiao-Yun Tang; Jian-Jun Zhang; Qiang Xia

    2006-01-01

    AIM: To investigate the effect of telomerase hTERT gene antisense oligonucleotide (hTERT-ASO) on proliferation and telomerase activity of pancreatic cancer cell line Bxpc-3.METHODS: MTT assay was used to detect the effect of different doses of hTERT-ASO on proliferation of Bxpc-3 cell for different times. To study the anti-tumor activity,the cells were divided into there groups: Control group (pancreatic cancer cell Bxpc-3); antisense oligonucleotide (hTERT-ASO) group; and nosense oligonucleotide group decorated with phosphorothioate. Telomerase activity was detected using TRAP-PCR-ELISA. Cell DNA distribution was examined using flow cytometry assay.Cell apoptosis was observed by transmission electron microscope in each group.RESULTS: After treatment with 6 mmol/L hTERTASO, cell proliferation was inhibited in dose- and timedependent manner. The telomerase activity decreased after treatment with hTERT-ASO for 72 h. Flow cytometry showed the cell number of G0/G1 phase increased from 2.7% to 14.7%, the cell number of S phase decreased from 72.7% to 51.0%, and a sub-G1 stage cell apoptosis peak appeared in front of G1 stage.CONCLUSION: Telomerase antisense oligodeoxynucleotide can inhibit the proliferation of pancreatic cancer cell line Bxpc-3 and decrease the telomerase activity and increase cell apoptosis rate in vitro.

  5. The green tea polyphenol, epigallocatechin-3-gallate inhibits telomerase and induces apoptosis in drug-resistant lung cancer cells.

    Science.gov (United States)

    Sadava, David; Whitlock, Elizabeth; Kane, Susan E

    2007-08-17

    Epidemiological studies on humans and investigations in animal models suggest that consumption of green tea has anti-cancer effects. Small-cell lung carcinoma (SCLC) has a poor prognosis, particularly due to the development of drug resistance. We investigated the effects of the green tea polyphenol, epigallocatechin-3-gallate (EGCG) on human SCLC cells. EGCG had similar effects (IC(50) of approximately 70 microM) on drug-sensitive (H69) and drug-resistant (H69VP) SCLC cells, indicating that it is not part of the drug resistance phenotype expressed in these cells. In both cell lines, incubation in EGCG at 1 x IC(50) for 24h resulted in 50-60% reduced telomerase activity as measured by a PCR-based assay for telomeric repeats. Colorimetric assays of cells treated for 36 h with EGCG demonstrated a reduction in activities of caspases 3 (50%) and 9 (70%) but not caspase 8, indicating initiation of apoptosis. DNA fragmentation as measured by ELISA occurred within cells treated with EGCG and this was confirmed by TUNEL staining. Flow cytometric analysis of SCLC cells incubated for 36 h in EGCG indicated a cell-cycle block in S phase. These data indicate the potential use of EGCG, and possibly green tea, in treating SCLC.

  6. Using of Telomerase Enzyme in Urine as a Non invasive Marker for Cancer Bladder Detection

    Directory of Open Access Journals (Sweden)

    Azza A Hassan*, Fawzia A . El- Sheshtawey** , Seliem A. Seliem

    2008-12-01

    Full Text Available Background: Urinary bladder cancer is one of the major health problem all over the world. Cystoscopy remains the gold standard for identifying bladder cancer but it is invasive and expensive, therefore, a simple, non invasive test for detecting bladder cancer would be helpful. Several biomarkers for bladder cancer have been used, but no single marker has been accurate and conclusive. Aim: The current study aimed to measure telomerase enzyme in urine as a useful non invasive marker for detection of bladder cancer. Methods : Forty eight patients ( 39 males and 9 females were included, They are complaining of urinary symptoms and undergo cystoscopy with biopsy of bladder lesions and histopathological examination. They were divided into groups: Group I: 16 patients ( 11 males and 5 females have benign urologic conditions. Group II: 32 patients (28 males and 4 females have proven bladder cancer patients underwent transurethral resection of bladder tumor or cystoscopy with biopsy of bladder lesions. Also, 15 apparently healthy volunteers with matched age and sex with patients were served as a control group. All subjects were submitted to laboratory estimation of the following in urine: urinary creatinine, urine cytology, telomerase enzyme in urine by telomerase PCR and complete urine examination. Results : The results of this study revealed that a highly significant increase in the frequency of cytolological positive cases for tumor cells in malignant group than each of benign group and healthy subjects, while no significant difference was detected between benign group and healthy subjects. The frequency of telomerase in urine was significantly higher in malignant group than each of benign group and healthy subjects, while no significant difference was detected between benign group and healthy subjects. The telomerase activity has sensitivity of 90.6% for diagnosis of cancer bladder with 93.7% for specificity and PPV was 96.6%, NPV was 83.3% and

  7. 人端粒酶逆转录酶肿瘤相关反馈调节机制的研究进展%Research progress in the feedback regulation of telomerase reverse transcriptase related in cancer

    Institute of Scientific and Technical Information of China (English)

    刘莹; 董成永; 崔晓楠

    2015-01-01

    人端粒酶逆转录酶( human telomerase reverse transcriptase,hTERT)是端粒酶的催化亚单位,尤其是端粒酶活性的限速因子,大量转录因子参与hTERT的调节。 hTERT在超过90%的肿瘤中表达,对肿瘤细胞的持续增殖发挥着重要作用。此外, hTERT能调节诸如细胞周期调控、细胞信号转导等不同细胞生物学过程中众多基因的表达。因此,hTERT在肿瘤中既是效应因子又是调节因子。然而,hTERT与其靶基因之间的相互作用机制还尚未完全明确。本综述重点关注不同的信号转导通路和基因参与hTERT的反馈调节及其机制,进一步认识端粒酶的非端粒延长功能,从而可能成为肿瘤治疗潜在的新靶点。%Telomerase reverse transcriptase ( TERT) is the catalytic component of telomerase, especially the rate limiting determinant of telomerase activity.A comprehensive network of transcription factors has been shown to be involved in the regulation of TERT.TERT has been reported to be over-expressed in more than 90%of cancers, thereby playing a criti-cal role in sustained proliferation and survival potentials of various cancer cells.Furthermore, accumulating evidence has suggested that TERT could modulate the expression of numerous genes involved in diverse group of cellular processes, in-cluding cell cycle regulation and cellular signaling.Therefore, it indicates that TERT is both an effector and a regulator in carcinoma.However, the mechanisms of the interaction between TERT and its target genes are still not fully understood.In this review, we focus on various signaling pathways and genes that participate in the feedback regulation of TERT and the underlying feedback regulation mechanism of TERT, to further provide new insights into non-telomeric functions of telom-erase and potentially novel therapeutic target for cancer.

  8. EFFECTS OF THE HYPERTHERMIA AND HARRINGTONINE ON TELOMERASE ACTIVITY OF HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    王宝燕; 张海涛; 李静

    2002-01-01

    Objective To understand the mechanism of the hype rthermia and harringtonine in purging of leukemia cells In Vitro. telomerase activity of HL-60 cells treated by hyperthermia (42℃ for one hour) and di fferent concentrations of Harringtonine were investigated.Methods Using Telomeric Repeats Amplification Protocol (TRAP) a nd ELISA techniques to analyze the telomerase activity of HL-60 cells. Results Our results showed that harringtonine inhibited the tel omerase activity of HL-60 cells in a dosage related manner. Moreover, the telom erase activity of HL-60 cells was significantly decreased after the hyperthermi a treatment as compared with untreated cells.Conclusion The effect of the hyperthermia and Harringtonine on purging leukemia cells In Vitro may be mediated by down regulation of telome rase activity of tumor cells.

  9. Structural Basis for Telomerase Catalytic Subunit TERT Binding to RNA Template and Telomeric DNA

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, M.; Gillis, A; Futahashi, M; Fujiwara, H; Skordalakes, E

    2010-01-01

    Telomerase is a specialized DNA polymerase that extends the 3{prime} ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA template and motifs 2 and B{prime} position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT-RNA template and TERT-telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases, suggesting common mechanistic aspects of DNA replication between the two families of enzymes.

  10. Telomeric repeat-containing RNA (TERRA) and telomerase are components of telomeres during mammalian gametogenesis.

    Science.gov (United States)

    Reig-Viader, Rita; Vila-Cejudo, Marta; Vitelli, Valerio; Buscà, Rafael; Sabaté, Montserrat; Giulotto, Elena; Caldés, Montserrat Garcia; Ruiz-Herrera, Aurora

    2014-05-01

    Telomeres are ribonucleoprotein structures at the end of chromosomes composed of telomeric DNA, specific-binding proteins, and noncoding RNA (TERRA). Despite their importance in preventing chromosome instability, little is known about the cross talk between these three elements during the formation of the germ line. Here, we provide evidence that both TERRA and the telomerase enzymatic subunit (TERT) are components of telomeres in mammalian germ cells. We found that TERRA colocalizes with telomeres during mammalian meiosis and that its expression progressively increases during spermatogenesis until the beginning of spermiogenesis. While both TERRA levels and distribution would be regulated in a gender-specific manner, telomere-TERT colocalization appears to be regulated based on species-specific characteristics of the telomeric structure. Moreover, we found that TERT localization at telomeres is maintained throughout spermatogenesis as a structural component without affecting telomere elongation. Our results represent the first evidence of colocalization between telomerase and telomeres during mammalian gametogenesis.

  11. Telomerase Activity in Peripheral Blood Mononuclear Cells from Senile Patients with Pneumonia

    Institute of Scientific and Technical Information of China (English)

    LIU Jian; ZHOU Zhen; LIU Xiaoqing

    2006-01-01

    To investigate the changes of the activity of telomerase in peripheral blood mononuclear cells (PBMCs) from senile patients with pneumonia, the telomerase activity was examined before and after the stimulation of phytohemagglutinin-M (PHA-M) in PBMCs from 10 control subjects (group A), 12 non-senile patients with pneumonia (group B) and 9 senile patients with pneumonia (group C). Also observed was the proliferative response of these PBMCs to PHA-M. The results showed that, both with or without the stimulation of PHA-M, the values of telomerase activity in PBMCs from group C patients (A values: pre-stimulation, 0.43±0.04; post-stimulation, 0.63±0.03) were significantly lower than those in PBMCs from both group A patients (A values: prestimulation, 0.65±0.05;post-stimulation, 1.26±0.13;P<0.001, respectively) and group B patients (A values: pre-stimulation, 0.63±0.03; post-stimulation, 0.93±0.03;P<0.05, respectively). The results of MTT test showed that the proliferative activity of PBMCs in group C patients (A value: 0.35±0.03) was also significantly lower than that in group A patients (A value:0. 55±0.04; P<0.05) and group B patients (A value: 0.46±0.03;P<0.05). These results indicate that the telomerase activity decreases in senile patients with pneumonia, which may be one of the mechanisms for the weakened immune function in those patients.

  12. [Suppression of telomerase activity leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase].

    Science.gov (United States)

    Pokrovskaya, M V; Zhdanov, D D; Eldarov, M A; Aleksandrova, S S; Veselovskiy, A V; Pokrovskiy, V S; Grishin, D V; Gladilina, Ju A; Sokolov, N N

    2017-01-01

    The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrАE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.

  13. Effects of Curcuma longa Extract on Telomerase Activity in Lung and Breast Cancer Cells

    OpenAIRE

    Nosratollah Zarghami; Javad Ranjbari; Abbas Alibakhshi; Roghayeh Arezumand; Mohammad Pourhassan-Moghaddam; Mohammad Rahmati; Mohammad Mehdi Namvaran

    2014-01-01

    Background: The purpose of this study is to evaluate the effect of Curcuma longa extract on the telomerase gene expression in QU-DB lung cancer and T47D breast cancer cell lines. Materials and Methods: The present study is an experimental research. Using 3 different phases n-hexane, dichloromethane and methanol, total extract of Curcuma longa in a serial dilution was prepared and three phases was analyzed for determining which phase has more curcuminoids. Then the extract cytotoxicity effe...

  14. Combined Telomerase Inhibition and Immunotherapy in the Prevention and Treatment of Mammary Carcinomas

    Science.gov (United States)

    2009-02-01

    deficiency on organs with high cellular proliferation. To address this question, we examined the spermatogenesis in the telomerase-null mice. MMT-92d G0MMT...revealed impairment of spermatogenesis in mTerc–/–MMT mice, especially in the late generations of the mice. Testes from MMT and G0 mTerc+/–MMT mice showed...normal structure with well-developed seminiferous tubules, germ cells and supporting cells, whereas impairment of spermatogenesis was observed in

  15. The effects of telomerase inhibition on prostate tumor-initiating cells.

    Science.gov (United States)

    Marian, Calin O; Wright, Woodring E; Shay, Jerry W

    2010-07-15

    Prostate cancer is the most common malignancy in men, and patients with metastatic disease have poor outcome even with the most advanced therapeutic approaches. Most cancer therapies target the bulk tumor cells, but may leave intact a small population of tumor-initiating cells (TICs), which are believed to be responsible for the subsequent relapse and metastasis. Using specific surface markers (CD44, integrin alpha(2)beta(1) and CD133), Hoechst 33342 dye exclusion, and holoclone formation, we isolated TICs from a panel of prostate cancer cell lines (DU145, C4-2 and LNCaP). We have found that prostate TICs have significant telomerase activity which is inhibited by imetelstat sodium (GRN163L), a new telomerase antagonist that is currently in Phase I/II clinical trials for several hematological and solid tumor malignancies. Prostate TICs telomeres were of similar average length to the telomeres of the main population of cells and significant telomere shortening was detected in prostate TICs as a result of imetelstat treatment. These findings suggest that telomerase inhibition therapy may be able to efficiently target the prostate TICs in addition to the bulk tumor cells, providing new opportunities for combination therapies.

  16. Heroin abuse accelerates biological aging: a novel insight from telomerase and brain imaging interaction.

    Science.gov (United States)

    Cheng, G L F; Zeng, H; Leung, M-K; Zhang, H-J; Lau, B W M; Liu, Y-P; Liu, G-X; Sham, P C; Chan, C C H; So, K-F; Lee, T M C

    2013-05-21

    Heroin abuse and natural aging exert common influences on immunological cell functioning. This observation led to a recent and untested idea that aging may be accelerated in abusers of heroin. We examined this claim by testing whether heroin use is associated with premature aging at both cellular and brain system levels. A group of abstinent heroin users (n=33) and matched healthy controls (n=30) were recruited and measured on various biological indicators of aging. These measures included peripheral blood telomerase activity, which reflects cellular aging, and both structural and functional measures of brain magnetic resonance imaging. We found that heroin users were characterized by significantly low telomerase activity (0.21 vs 1.78; 88% reduction; t(61)=6.96, Pbrain region implicated in aging. Using the PFC location identified from the structural analyses as a 'seed' region, it was further revealed that telomerase activity interacted with heroin use to impact age-sensitive brain functional networks (AlphaSim corrected Pbrain system and behavioral measures in the context of substance abuse. The present finding that heroin abuse is associated with accelerated aging at both cellular and brain system levels is novel and forms a unique contribution to our knowledge in how the biological processes of drug abusers may be disrupted.

  17. The Anti-Proliferative Effect of Inhibitor of Telomerase on Cultured Retinal Pigment Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to provide a new method for treating proliferative vitreoretinopathy (PVR), the effects of anti-proliferation and apoptosis induction of inhibitors of telomerase and heat shock protein 90 (Hsp90) on the cultured retinal pigment epithelial (RPE) cells were investigated. The rate of apoptosis cells was measured by using TUNEL on the cultured RPE cells, the co-cultured RPE cells with inhibitor of telomerase (camptothecin) or the co-cultured RPE cells with inhibitor of Hsp90 (geldanamycin). The cell proliferation status was measured in the above three groups by using MTT method. The rate of apoptosis in the RPE cells co-cultured with camptothecin or geldanamycin was increased remarkably (P<0.05). MTT showed the rate of growth inhibition was 8.4 %, 32.3 % and 72.3 % at the concentrations of camptothecin 1 μmol/L, 5 μmol/L, 10 μmol/L, respectively, and 6.5 %, 30.9 %, 71.9 % at the concentrations of geldanamycin 1 μmol/L, 5 μmol/L, 10 μmol/L, respectively. It was concluded that telomerase and Hsp90 can promote the proliferation of the cultured RPE cells, while the inhibitor of them can induce apoptosis and inhibit the growth of the RPE cells.

  18. Mixed Integer Linear Programming based machine learning approach identifies regulators of telomerase in yeast.

    Science.gov (United States)

    Poos, Alexandra M; Maicher, André; Dieckmann, Anna K; Oswald, Marcus; Eils, Roland; Kupiec, Martin; Luke, Brian; König, Rainer

    2016-06-02

    Understanding telomere length maintenance mechanisms is central in cancer biology as their dysregulation is one of the hallmarks for immortalization of cancer cells. Important for this well-balanced control is the transcriptional regulation of the telomerase genes. We integrated Mixed Integer Linear Programming models into a comparative machine learning based approach to identify regulatory interactions that best explain the discrepancy of telomerase transcript levels in yeast mutants with deleted regulators showing aberrant telomere length, when compared to mutants with normal telomere length. We uncover novel regulators of telomerase expression, several of which affect histone levels or modifications. In particular, our results point to the transcription factors Sum1, Hst1 and Srb2 as being important for the regulation of EST1 transcription, and we validated the effect of Sum1 experimentally. We compiled our machine learning method leading to a user friendly package for R which can straightforwardly be applied to similar problems integrating gene regulator binding information and expression profiles of samples of e.g. different phenotypes, diseases or treatments.

  19. Mixed Integer Linear Programming based machine learning approach identifies regulators of telomerase in yeast

    Science.gov (United States)

    Poos, Alexandra M.; Maicher, André; Dieckmann, Anna K.; Oswald, Marcus; Eils, Roland; Kupiec, Martin; Luke, Brian; König, Rainer

    2016-01-01

    Understanding telomere length maintenance mechanisms is central in cancer biology as their dysregulation is one of the hallmarks for immortalization of cancer cells. Important for this well-balanced control is the transcriptional regulation of the telomerase genes. We integrated Mixed Integer Linear Programming models into a comparative machine learning based approach to identify regulatory interactions that best explain the discrepancy of telomerase transcript levels in yeast mutants with deleted regulators showing aberrant telomere length, when compared to mutants with normal telomere length. We uncover novel regulators of telomerase expression, several of which affect histone levels or modifications. In particular, our results point to the transcription factors Sum1, Hst1 and Srb2 as being important for the regulation of EST1 transcription, and we validated the effect of Sum1 experimentally. We compiled our machine learning method leading to a user friendly package for R which can straightforwardly be applied to similar problems integrating gene regulator binding information and expression profiles of samples of e.g. different phenotypes, diseases or treatments. PMID:26908654

  20. NBS1 plays a synergistic role with telomerase in the maintenance of telomeres in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Najdekrova Lucie

    2012-09-01

    Full Text Available Abstract Background Telomeres, as elaborate nucleo-protein complexes, ensure chromosomal stability. When impaired, the ends of linear chromosomes can be recognised by cellular repair mechanisms as double-strand DNA breaks and can be healed by non-homologous-end-joining activities to produce dicentric chromosomes. During cell divisions, particularly during anaphase, dicentrics can break, thus producing naked chromosome tips susceptible to additional unwanted chromosome fusion. Many telomere-building protein complexes are associated with telomeres to ensure their proper capping function. It has been found however, that a number of repair complexes also contribute to telomere stability. Results We used Arabidopsis thaliana to study the possible functions of the DNA repair subunit, NBS1, in telomere homeostasis using knockout nbs1 mutants. The results showed that although NBS1-deficient plants were viable, lacked any sign of developmental aberration and produced fertile seeds through many generations upon self-fertilisation, plants also missing the functional telomerase (double mutants, rapidly, within three generations, displayed severe developmental defects. Cytogenetic inspection of cycling somatic cells revealed a very early onset of massive genome instability. Molecular methods used for examining the length of telomeres in double homozygous mutants detected much faster telomere shortening than in plants deficient in telomerase gene alone. Conclusions Our findings suggest that NBS1 acts in concert with telomerase and plays a profound role in plant telomere renewal.

  1. Study of the expressions of p53 and bcl-2 genes, the telomerase activity and apoptosis in GIST patients

    Institute of Scientific and Technical Information of China (English)

    Qiang Wang; You-Wei Kou

    2007-01-01

    AIM: To explore the relationship between clinicobiological behavior and the expression levels of telomerase activity,apoptosis, p53 gene and bcl-2 gene in gastrointestinal stromal tumors (GISTs).METHODS: The intensity of telomerase activity,apoptosis, p53 and bcl-2 expression in GISTs were detected by telomeric repeat amplification protocol, in situ end-labeling technique, and immunohistochemistry,respectively.RESULTS: The positive rates of telomerase activity of malignant GIST, potential malignant GIST and benign GIST were 85% (17/20), 22.8% (2/9) and 0 (0/9),respectively. The apoptosis indices of malignant GIST,potential malignant GIST, and benign GIST were 11.7 ± 5.4, 30.2 ± 5.6 and 45.2 ± 7.2, respectively. The intensity of telomerase activity and apoptosis were related to the biological characteristics of GISTs (85% vs 22.8%, 0, 0; P < 0.01 or 11.7±5.4 vs 30.2 ± 5.6, 45.2 ± 7.2, 72.1 ± 9.3; P < 0.05). The intensity of telomerase activity was negatively correlated with cellular apoptosis (22.9 ± 8.4 vs 9.5 ± 5.7, P < 0.01). The intensity of telomerase activity was positively correlated with p53,bcl-2 expression (40.0% vs 78.9%, 40.0% vs 84.2%;P < 0.05).CONCLUSION: The detection of telomerase activity,apoptosis and its control genes in GIST will be helpful for the discrimination of the malignant and benign GIST and evaluation of the prognosis.

  2. Mesenchymal stem cells with high telomerase expression do not actively restore their chromosome arm specific telomere length pattern after exposure to ionizing radiation

    DEFF Research Database (Denmark)

    Graakjaer, Jesper; Christensen, Rikke; Kolvraa, Steen;

    2007-01-01

    BACKGROUND: Previous studies have demonstrated that telomeres in somatic cells are not randomly distributed at the end of the chromosomes. We hypothesize that these chromosome arm specific differences in telomere length (the telomere length pattern) may be actively maintained. In this study we...... investigate the existence and maintenance of the telomere length pattern in stem cells. For this aim we studied telomere length in primary human mesenchymal stem cells (hMSC) and their telomerase-immortalised counterpart (hMSC-telo1) during extended proliferation as well as after irradiation. Telomere lengths...... were measured using Fluorescence In Situ Hybridization (Q-FISH). RESULTS: A telomere length pattern was found to exist in primary hMSC's as well as in hMSC-telo1. This pattern is similar to what was previously found in lymphocytes and fibroblasts. The cells were then exposed to a high dose of ionizing...

  3. Silencing effects of telomerase Cajal body protein 1-shRNA on human oral squamous cells%shRNA慢病毒对人口腔鳞状细胞癌细胞端粒酶卡侯体蛋白1基因的沉默效应

    Institute of Scientific and Technical Information of China (English)

    胡玲; 孙崇奎; 苟雅萍; 李燕; 肖丽英

    2013-01-01

    目的:探讨shRNA慢病毒对人口腔鳞状细胞癌Cal27细胞系端粒酶新蛋白,即端粒酶卡侯体蛋白1(TCAB1)基因的沉默效应,以期探索肿瘤基因治疗的新途径。方法:根据TCAB1基因,构建慢病毒shTCAB1-A~D,测定病毒滴度;用重组慢病毒体外感染Cal27细胞,荧光显微镜观察绿色荧光蛋白表达推断感染效率;半定量逆转录聚合酶链反应(RT-PCR)检测TCAB1 mRNA水平;Western blot检测TCAB1蛋白质的表达水平;四甲基偶氮唑盐比色(MTT)法检测细胞体外增殖能力;Transwell小室法检测细胞体外侵袭能力改变。结果:经PCR与测序鉴定证实成功构建靶向TCAB1的慢病毒RNAi载体,病毒滴度为1.5×108~3×108 U·mL-1。荧光显微镜观察显示,绝大部分细胞表达绿色荧光。与空白细胞对照组相比,4种不同的shTCAB1-A~D慢病毒感染组TCAB1 mRNA表达下调48.7%~62.0%;蛋白抑制率达45.6%~77.5%,shTCAB1-C组最明显。shTCAB1-C慢病毒能明显抑制Cal27细胞增殖能力,也能降低其侵袭能力,穿过人工基底膜的平均细胞数明显低于空白对照组及非特异性对照组,侵袭抑制率高达67.5%(P<0.05)。结论:成功构建并包装了端粒酶新蛋白TCAB1靶向的RNAi慢病毒,该病毒可高效感染人口腔鳞状细胞癌细胞,产生特异性的基因沉默效应。%Objective To investigate the inhibitory effects of telomerase Cajal body protein 1(TCAB1)-shRNA on oral squamous cell carcinoma Cal27 and to explore a novel cancer gene therapy approach by depletion of TCAB1 using RNA interference. Methods Four recombinant lentiviruses, called shTCAB1-A-D, which contain oligonucleotide(67 bp) targeting TCAB1 and green fluorescent protein EGFP Puro, were constructed based on different sites in the TCAB1 gene. A series of experiments were then carried out to demonstrate the silencing effects of the shTCAB1 lentiviruses on Cal27 cells. Semiquantitative

  4. Telomere Length in Peripheral Blood Mononuclear Cells of Patients on Chronic Hemodialysis Is Related With Telomerase Activity and Treatment Duration.

    Science.gov (United States)

    Stefanidis, Ioannis; Voliotis, Georgios; Papanikolaou, Vassilios; Chronopoulou, Ioanna; Eleftheriadis, Theodoros; Kowald, Axel; Zintzaras, Elias; Tsezou, Aspasia

    2015-09-01

    Telomere shortening to a critical limit is associated with replicative senescence. This process is prevented by the enzyme telomerase. Oxidative stress and chronic inflammation are factors accelerating telomere loss. Chronic hemodialysis, typically accompanied by oxidative stress and inflammation, may be also associated with replicative senescence. To test this hypothesis, we determined telomere length and telomerase activity in peripheral blood mononuclear cells (PBMCs) in a cross-sectional study. Hemodialysis patients at the University Hospital Larissa and healthy controls were studied. Telomere length was determined by the TeloTAGGG Telomere Length Assay and telomerase activity by Telomerase PCR-ELISA (Roche Diagnostics GmbH, Mannheim, Germany). We enrolled 43 hemodialysis patients (17 females; age 65.0 ± 12.7 years) and 23 controls (six females; age 62.1 ± 15.7 years). Between the two groups, there was no difference in telomere length (6.95 ± 3.25 vs. 7.31 ± 1.96 kb; P = 0.244) or in telomerase activity (1.82 ± 2.91 vs. 2.71 ± 3.0; P = 0.085). Telomere length correlated inversely with vintage of hemodialysis (r = -0.332, P = 0.030). In hemodialysis patients, positive telomerase activity correlated with telomere length (r = 0.443, P = 0.030). Only age, and neither telomere length nor telomerase activity, was an independent survival predictor (hazard ratio 1.116, 95% confidence interval 1.009-1.234, P = 0.033). In this study, telomere length and telomerase activity in PBMCs are not altered in hemodialysis patients compared with healthy controls. Long duration of hemodialysis treatment is associated with telomere shortening and positive telomerase activity with an increased telomere length in PBMCs of hemodialysis patients. The underlying mechanism and clinical implications of our findings require further investigation.

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

    Institute of Scientific and Technical Information of China (English)

    CAO Sun-qiong; REN Chang-shan

    2008-01-01

    Objective:To research the silence of NBS1 after transfection microRNA expressing eukaryotic recombinants and the changes of telomerase activation in teiomerase-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 NBS1 gene and telomerase activity.

  6. 48. The value of CT scan and detection of telomerase activity in biopsy specimens for early diagnosis of lung carcinoma

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To evaluate the diagnostic value of telomerase activity in the specimens of biopsy with bronchoscopy or cutting needle. Methods: Telomerase activity was measured in the biopsy apecimens taken from 52 patients suspected of having early lung cancer by CT scan. The PCR based silver staining telomeric repeat amplification protocol (TRAP) was used for detection of telomerase activity in 22 patients with early lung cancer (T1N0M0). Control study was made on the specimens taken from 24 patients with benign disease (cyst 3, TB 6, pseudtumor 5, pneumonjia 10). Results: The positive rates of telomerase activity were 86.45% (19/22) and 4.2% (1/24) in early lung cancers and benign lesions respectively (P<0.01). It was significantly higher in early lung cancers than in benign disease. All cases were diagnosed with surgical pathology and following for 2 years. Conclusion: Detecting telomerase activity in preoperative bronchoscope and cutting needle biopsy specimens may contribute to diagnosis of early lung cancer.

  7. Anti-tumor effect of 5-aza-2'-deoxycytidine by inhibiting telomerase activity in hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Shuang-Fen Tao; Chang-Song Zhang; Xian-Ling Guo; Yun Xu; Shan-Shan Zhang; Jian-Rui Song; Rong Li

    2012-01-01

    AIM:To investigate the effect of the demethylating reagent 5-aza-2'-deoxycitidine (DAC) on telomerase activity in hepatocellular carcinoma (HCC) cell lines,SMMC-7721 and HepG2.METHODS:The related gene expression in cell lines was examined by real-time reverse transcription-polymerase chain reaction and Western blotting analysis.The telomerase activity was examined by telomeric repeat amplification protocol-enzyme-linked immunosorbent assay and DNA methylation was determined by methylation-specific polymerase chain reaction.RESULTS:The telomerase activity was significantly reduced in both cell lines treated with DAC,accompanied by downregulation of telomerase reverse transcriptase (hTERT).We also observed the effect of DAC on the methylation status of hTERT promoter and the expression of regulatory genes,such as c-myc,p15,p16,p21,E2F1,and WT1.The methylation status of hTERT promoter could be reversed in SMMC-7721 by DAC,but not in HepG2 cells.However,p16 expression could be reactivated by demethylation of its promoter,and c-Myc expression was repressed in both cell lines.Moreover,DAC could enhance the sensitivity to the chemotherapeutic agents,such as cisplatin,by induction of apoptosis of HCC cells.CONCLUSION:The DAC exerts its anti-tumor effects in HCC cells by inhibiting the telomerase activity.

  8. A novel DNA tetrahedron-hairpin probe for in situ"off-on" fluorescence imaging of intracellular telomerase activity.

    Science.gov (United States)

    Feng, Qiu-Mei; Zhu, Meng-Jiao; Zhang, Ting-Ting; Xu, Jing-Juan; Chen, Hong-Yuan

    2016-04-21

    A novel three-dimensionally structured DNA probe is reported to realize in situ"off-on" imaging of intracellular telomerase activity. The probe consists of a DNA tetrahedron and a hairpin DNA on one of the vertices of the DNA tetrahedron. It is composed of four modified DNA segments: S1-Au nanoparticle (NP) inserting a telomerase strand primer (TSP) and S2-S4, three Cy5 dye modified DNA segments. Fluorescence of Cy5 at three vertices of the DNA tetrahedron is quenched by the Au NP at the other vertex due to the effective fluorescence resonance energy transfer (FRET) ("off" state). When the probe meets telomerase, the hairpin structure changes to rod-like through complementary hybridization with the telomerase-triggered stem elongation product, resulting in a large distance between the Au NP and Cy5 and the recovery of Cy5 fluorescence ("on" state). The molar ratio of 3 : 1 between the reporter (Cy5) and the target related TSP makes the probe show high sensitivity and recovery efficiency of Cy5 in the presence of telomerase extracted from HeLa cells. Given the functional and compact nanostructure, the mechanically stable and noncytotoxic nature of the DNA tetrahedron, this FRET-based probe provides more opportunities for biosensing, molecular imaging and drug delivery.

  9. Structural Basis for Telomerase RNA Recognition and RNP Assembly by the Holoenzyme La Family Protein p65

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Mahavir; Wang, Zhonghua; Koo, Bon-Kyung; Patel, Anooj; Cascio, Duilio; Collins, Kathleen; Feigon, Juli (UCLA); (UCB)

    2012-07-01

    Telomerase is a ribonucleoprotein complex essential for maintenance of telomere DNA at linear chromosome ends. The catalytic core of Tetrahymena telomerase comprises a ternary complex of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and the essential La family protein p65. NMR and crystal structures of p65 C-terminal domain and its complex with stem IV of TER reveal that RNA recognition is achieved by a combination of single- and double-stranded RNA binding, which induces a 105{sup o} bend in TER. The domain is a cryptic, atypical RNA recognition motif with a disordered C-terminal extension that forms an {alpha} helix in the complex necessary for hierarchical assembly of TERT with p65-TER. This work provides the first structural insight into biogenesis and assembly of TER with a telomerase-specific protein. Additionally, our studies define a structurally homologous domain (xRRM) in genuine La and LARP7 proteins and suggest a general mode of RNA binding for biogenesis of their diverse RNA targets.

  10. A randomized phase II study of the telomerase inhibitor imetelstat as maintenance therapy for advanced non-small-cell lung cancer

    OpenAIRE

    Chiappori, A. A.; Kolevska, T.; Spigel, D. R.; Hager, S.; Rarick, M; Gadgeel, S; Blais, N.; von Pawel, J.; Hart, L; Reck, M.; Bassett, E.; Burington, B.; Schiller, J H

    2014-01-01

    Imetelstat, a novel telomerase inhibitor, failed to improve significantly median PFS and OS as maintenance therapy (±bevacizumab) in advanced NSCLC. Telomere length (TL) biomarker results were consistent with the hypothesis that telomerase inhibition is of greater benefit to patients with tumors possessing shorter telomeres; the patients with shorter TL had a trend toward longer median PFS and OS.

  11. Label-free ultrasensitive detection of telomerase activity via multiple telomeric hemin/G-quadruplex triggered polyaniline deposition and a DNA tetrahedron-structure regulated signal.

    Science.gov (United States)

    Liu, Yuanjian; Wei, Min; Liu, Xu; Wei, Wei; Zhao, Hongyu; Zhang, Yuanjian; Liu, Songqin

    2016-01-31

    Label-free detection of telomerase activity was done by using telomeric hemin/G-quadruplex triggered polyaniline deposition, not only on themselves but also on the DNA tetrahedron-structure (DTS). DTS size has a great impact on telomerase accessibility, reactivity and detection sensitivity. The method has been used to evaluate bladder cancer development.

  12. The correlation between telomerase activity and Bax/Bcl-2 ratio in valproic acid-treated MCF-7 breast cancer cell line

    Directory of Open Access Journals (Sweden)

    Zahra Vafaiyan

    2015-07-01

    Conclusion: Our study demonstrated that cell viability of MCF-7 cells was decreased after treatment with VPA, probably through a reduction of telomerase activity and an increase in Bax/bcl-2 ratio. Therefore, it could be concluded that VPA is a potent anti-cancer agent for breast cancer cells through inhibition of telomerase activity and induction of apoptosis.

  13. Telomerase reverse transcriptase promoter-driven expression of iodine pump genes for targeted radioiodine therapy of malignant glioma cells

    Institute of Scientific and Technical Information of China (English)

    Jian Tan; Wei Li; Peng Wang

    2011-01-01

    Radioiodine is a routine therapy for differentiated thyroid cancers. Non-thyroid cancers can intake radioiodine after transfection of the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter, an excellent tumor-specific promoter, has potential value for targeted gene therapy of glioma. We used the hTERT promoter to drive the expression of the hNIS and human thyroid peroxidase (hTPO) gene as a primary step for testing the effects of radioiodine therapy on malignant glioma. The U87 and U251 cells were co-transfected with two adenoviral vectors, in which the hNIS gene had been coupled to the hTERT promoter and the hTPO gene had been coupled to the CMV promoter, respectively. Then, we performed Western blot, 135l intake and efflux assays, and clonogenic assay with cancer cells. We also did 99mTc tumor imaging of nude mice models. After co-transfection with Ad-hTERT-hNIS and Ad-CMV-hTPO, glioma cells showed the 125l intake almost 1.5 times higher than cells transfected with Ad-hTERT-hNIS alone. Western blots revealed bands of approximately 70 kDa and 110 kDa, consistent with the hNIS and hTPO proteins. In clonogenic assay, approximately 90% of co transfected cells were killed, compared to 50% of control cells after incubated with 37 MBq of 131I. These results demonstrated that radioiodine therapy was effective in treating malignant glioma cell lines following induction of tumor-specific iodide intake by the hTERT promoter-directed hNIS expression in vitro. Co transfected hNIS and hTPO genes can result in increased intake and longer retention of radioiodine. Nude mice harboring xenografts transfected with Ad-hTERT-NIS can take 99mTc scans.

  14. Frequency of TERT promoter mutations in human cancers.

    Science.gov (United States)

    Vinagre, João; Almeida, Ana; Pópulo, Helena; Batista, Rui; Lyra, Joana; Pinto, Vasco; Coelho, Ricardo; Celestino, Ricardo; Prazeres, Hugo; Lima, Luis; Melo, Miguel; da Rocha, Adriana Gaspar; Preto, Ana; Castro, Patrícia; Castro, Ligia; Pardal, Fernando; Lopes, José Manuel; Santos, Lúcio Lara; Reis, Rui Manuel; Cameselle-Teijeiro, José; Sobrinho-Simões, Manuel; Lima, Jorge; Máximo, Valdemar; Soares, Paula

    2013-01-01

    Reactivation of telomerase has been implicated in human tumorigenesis, but the underlying mechanisms remain poorly understood. Here we report the presence of recurrent somatic mutations in the TERT promoter in cancers of the central nervous system (43%), bladder (59%), thyroid (follicular cell-derived, 10%) and skin (melanoma, 29%). In thyroid cancers, the presence of TERT promoter mutations (when occurring together with BRAF mutations) is significantly associated with higher TERT mRNA expression, and in glioblastoma we find a trend for increased telomerase expression in cases harbouring TERT promoter mutations. Both in thyroid cancers and glioblastoma, TERT promoter mutations are significantly associated with older age of the patients. Our results show that TERT promoter mutations are relatively frequent in specific types of human cancers, where they lead to enhanced expression of telomerase.

  15. Reptin is required for the transcription of telomerase reverse transcriptase and over-expressed in gastric cancer

    Directory of Open Access Journals (Sweden)

    Liu Tiantian

    2010-05-01

    Full Text Available Abstract Background Telomerase is activated in oncogenesis, which confers an immortal phenotype to cancer cells. The AAA + ATPase Reptin is required for telomerase biogenesis by maintaining telomerase RNA (hTER stability and is aberrantly expressed in certain cancers. Given its role in chromatin remodeling and transcription regulation, we determined the effect of Reptin on the transcription of the telomerase reverse transcriptase (hTERT gene, a key component of the telomerase complex and its expression in gastric cancer. Results Knocking down Reptin or its partner Pontin using small interfering RNA in gastric and cervical cancer cells led to significant decreases in hTERT mRNA, but hTERT promoter activity was inhibited in only Reptin-depleted cells. Reptin interacted with the c-MYC oncoprotein and its stimulatory effect on the hTERTpromoter was significantly dependent on functional E-boxes in the promoter. Moreover, Reptin bound to the hTERT proximal promoter and the loss of the Reptin occupancy led to dissociation of c-MYC from the hTERT promoter in Reptin-depleted cells. Reptin inhibition dramatically impaired clonogenic potential of gastric cancer cells by inducing cell growtharrest and over-expression of Reptin was observed in primary gastric cancer specimens. Conclusions The hTERT gene is a direct target of Reptin, and hTERT transcription requires constitutive expression of Reptin and its cooperation with c-MYC. Thus, Reptin regulates telomerase at two different levels. This finding, together with the requirementof Reptin for the clonogenic potential of cancer cells and its over-expression in gastriccancer and other solid tumors, suggests that Reptin may be a putative therapeutic target.

  16. Mathematical model of a telomerase transcriptional regulatory network developed by cell-based screening: analysis of inhibitor effects and telomerase expression mechanisms.

    Directory of Open Access Journals (Sweden)

    Alan E Bilsland

    2014-02-01

    Full Text Available Cancer cells depend on transcription of telomerase reverse transcriptase (TERT. Many transcription factors affect TERT, though regulation occurs in context of a broader network. Network effects on telomerase regulation have not been investigated, though deeper understanding of TERT transcription requires a systems view. However, control over individual interactions in complex networks is not easily achievable. Mathematical modelling provides an attractive approach for analysis of complex systems and some models may prove useful in systems pharmacology approaches to drug discovery. In this report, we used transfection screening to test interactions among 14 TERT regulatory transcription factors and their respective promoters in ovarian cancer cells. The results were used to generate a network model of TERT transcription and to implement a dynamic Boolean model whose steady states were analysed. Modelled effects of signal transduction inhibitors successfully predicted TERT repression by Src-family inhibitor SU6656 and lack of repression by ERK inhibitor FR180204, results confirmed by RT-QPCR analysis of endogenous TERT expression in treated cells. Modelled effects of GSK3 inhibitor 6-bromoindirubin-3'-oxime (BIO predicted unstable TERT repression dependent on noise and expression of JUN, corresponding with observations from a previous study. MYC expression is critical in TERT activation in the model, consistent with its well known function in endogenous TERT regulation. Loss of MYC caused complete TERT suppression in our model, substantially rescued only by co-suppression of AR. Interestingly expression was easily rescued under modelled Ets-factor gain of function, as occurs in TERT promoter mutation. RNAi targeting AR, JUN, MXD1, SP3, or TP53, showed that AR suppression does rescue endogenous TERT expression following MYC knockdown in these cells and SP3 or TP53 siRNA also cause partial recovery. The model therefore successfully predicted several

  17. Effect of antisense oligonucleotides targeting telomerase catalytic subunit on tumor cell proliferationin vitro

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To screen specific antitumor drugs targeting telomerase catalytic subunit (hEST2), 12 different hEST2 antisense oligonucleotides were designed based on hEST2 mRNA second structure and transfected into tumor cell lines by the lipofectin-mediated method. Cell growth activity was evaluated by MTT assay. hEST212 was picked out and its specificity, antitumor tree and continuous effect were analyzed. The results showed that hEST212 had promising antitumor activity in vitro, hEST2 can be used as a pratical target and an antisense drug candidate for cancer.

  18. Telomerase regulation and the intimate relationship with aging

    OpenAIRE

    Morgan G

    2013-01-01

    Garrett Morgan1–31Department of Physiology, University of Utah School of Medicine, University of Utah, 2George E. Wahlen Department of Veterans Affairs Medical Center, 3Geriatric Research, Education and Clinical Center, VA Salt Lake City Health Care System, Salt Lake City, UT, USAAbstract: Advancing age in humans is associated with a decline in function of physiological systems, as well as the onset and progression of many chronic diseases. An ongoing focus of basic aging research i...

  19. Telomerase antagonist imetelstat increases radiation sensitivity in esophageal squamous cell carcinoma.

    Science.gov (United States)

    Wu, Xuping; Zhang, Jing; Yang, Sijun; Kuang, Zhihui; Tan, Guolei; Yang, Gang; Wei, Qichun; Guo, Zhigang

    2017-01-13

    The morbidity and mortality of esophageal cancer is one of the highest around the world and the principal therapeutic method is radiation. Thus, searching for sensitizers with lower toxicity and higher efficiency to improve the efficacy of radiation therapy is critical essential. Our research group has previously reported that imetelstat, the thio-phosphoramidate oligonucleotide inhibitor of telomerase, can decrease cell proliferation and colony formation ability as well as increase DNA breaks induced by radiation in esophageal cancer cells. Further study in this project showed that imetelstat significantly sensitized esophageal cancer cells to radiation in vitro. Later study showed that imetelstat leads to increased cell apoptosis. We also measured the expression level of several DNA repair and apoptosis signaling proteins. pS345 CHK1, γ-H2AX, p53 and caspase3 expression were up-regulated in imetelstat treated cells, identifying these factors as molecular markers. Mouse in vivo model using imetelstat at clinically achievable concentrations and fractionated irradiation scheme yielded results demonstrating radiosensitization effect. Finally, TUNEL assay, caspase 3 and Ki67 staining in tumor tissue proved that imetelstat sensitized esophageal cancer to radiation in vivo through promoting cell apoptosis and inhibiting cell proliferation. Our study supported imetelstat increase radiation sensitivity of esophageal squamous cell carcinoma through inducing cell apoptosis and the specific inhibitor of telomerase might serve as a potential novel therapeutic tool for esophageal squamous cell carcinoma therapy.

  20. Allium telomeres unmasked: the unusual telomeric sequence (CTCGGTTATGGG)n is synthesized by telomerase.

    Science.gov (United States)

    Fajkus, Petr; Peška, Vratislav; Sitová, Zdeňka; Fulnečková, Jana; Dvořáčková, Martina; Gogela, Roman; Sýkorová, Eva; Hapala, Jan; Fajkus, Jiří

    2016-02-01

    Phylogenetic divergence in Asparagales plants is associated with switches in telomere sequences. The last switch occurred with divergence of the genus Allium (Amaryllidaceae) from the other Allioideae (formerly Alliaceae) genera, resulting in uncharacterized telomeres maintained by an unknown mechanism. To characterize the unknown Allium telomeres, we applied a combination of bioinformatic processing of transcriptomic and genomic data with standard approaches in telomere biology such as BAL31 sensitivity tests, terminal restriction fragment analysis, the telomere repeat amplification protocol (TRAP), and fluorescence in situ hybridization (FISH). Using these methods, we characterize the unusual telomeric sequence (CTCGGTTATGGG)n present in Allium species, demonstrate its synthesis by telomerase, and characterize the telomerase reverse transcriptase (TERT) subunit of Allium cepa. Our findings open up the possibility of studying the molecular details of the evolutionary genetic change in Allium telomeres and its possible role in speciation. Experimental studies addressing the implications of this change in terms of the interplay of telomere components may now be designed to shed more light on telomere functions and evolution in general.

  1. Design and study of telomerase inhibitors based on G-quadruplex ligands

    Directory of Open Access Journals (Sweden)

    Negrutska V. V.

    2013-05-01

    Full Text Available In this review we have summarized the results of our recent research on telomerase inhibitors and G-quadruplex DNA ligands. A series of potential enzyme inhibitors were synthesized and studied. These compounds were based on tricyclic heteroaromatic systems (thiazolobenzimidazoles phenazines, acridones, amino-substituted cyanines and natural and synthetic porphyrins and their metalocomplexes. A number of compounds, including cyanines and especially porphyrin derivatives and conjugates, were found to efficiently inhibit telomerase at low micromolar concentrations in the in vitro TRAP assay. Porphyrins demonstrated antiproliferative activity in tumor cell cultures at micro- and nanomolar concentrations. Spectral-fluorescent and electrophoretic experiments were performed to investigate the interaction of ligands with duplex and quadruplex DNA, and in many cases binding mode was established. Convenient G-octet model of G-quadruplex was developed to study the ligand-target binding using quantum-chemical methods. QM/MM hybrid approach ONIUM2 was employed to model the interaction of small molecules with Tel22 quadruplex DNA.

  2. Analysis of the age of Panax ginseng based on telomere length and telomerase activity.

    Science.gov (United States)

    Liang, Jiabei; Jiang, Chao; Peng, Huasheng; Shi, Qinghua; Guo, Xiang; Yuan, Yuan; Huang, Luqi

    2015-01-23

    Ginseng, which is the root of Panax ginseng (Araliaceae), has been used in Oriental medicine as a stimulant and dietary supplement for more than 7,000 years. Older ginseng plants are substantially more medically potent, but ginseng age can be simulated using unscrupulous cultivation practices. Telomeres progressively shorten with each cell division until they reach a critical length, at which point cells enter replicative senescence. However, in some cells, telomerase maintains telomere length. In this study, to determine whether telomere length reflects ginseng age and which tissue is best for such an analysis, we examined telomerase activity in the main roots, leaves, stems, secondary roots and seeds of ginseng plants of known age. Telomere length in the main root (approximately 1 cm below the rhizome) was found to be the best indicator of age. Telomeric terminal restriction fragment (TRF) lengths, which are indicators of telomere length, were determined for the main roots of plants of different ages through Southern hybridization analysis. Telomere length was shown to be positively correlated with plant age, and a simple mathematical model was formulated to describe the relationship between telomere length and age for P. ginseng.

  3. Evaluation of Manisa propolis effect on leukemia cell line by telomerase activity.

    Science.gov (United States)

    Gunduz, Cumhur; Biray, Cigir; Kosova, Buket; Yilmaz, Berna; Eroglu, Zuhal; Sahin, Fahri; Omay, Serdar Bedii; Cogulu, Ozgur

    2005-11-01

    Propolis is a resinous substance which is used by bees to repair and maintain their hives. It has more than 180 compounds including flavonoids, phenolic acids and its esters which have anti-inflammatory, antibacterial, antiviral, immunomodulatory, antioxidant and antiproliferative effects. Propolis is shown to inhibit cell division and protein synthesis. However the exact mechanism underlying antitumor effect is not clearly described. On the other hand progressive telomere shortening to a critical level results with senescence of normal cells by inducing apoptosis and telomerase prevents erosion of telomeres. In this study we aimed to evaluate hTERT ratios in propolis-treated T-cell acute lymphoblastic leukemia (CCFR-CEM) cell line. Cell counts and cell viability of propolis-treated and propolis-free T-cell acute lymphoblastic leukemia (CCFR-CEM) cell line were assessed by trypan blue dye exclusion test and MTT assay. The LightCycler instrument was used (online real-time PCR) for the quantification of hTERT in CCFR-CEM cell line. The hTERT ratio significantly decreased 60 and 93% after 24 and 72 h respectively compared to the initial value of the cells incubated with propolis. It had almost no cytotoxic effect and caused 30, 30, 22 and 12% decrease in cell counts after 24, 48, 72 and 96 h respectively which is statistically significant. In conclusion propolis may show antitumor and apoptotic effect via inhibiting telomerase expression besides the mechanisms which have been described previously.

  4. Telomerase: a target for therapeutic effects of curcumin and a curcumin derivative in Aβ1-42 insult in vitro.

    Directory of Open Access Journals (Sweden)

    Zijian Xiao

    Full Text Available This study was designed to investigate whether telomerase was involved in the neuroprotective effect of curcumin and Cur1. Alzheimer's disease is a consequence of an imbalance between the generation and clearance of amyloid-beta peptide in the brain. In this study, we used Aβ1-42 (10 µg/ml to establish a damaged cell model, and curcumin and Cur1 were used in treatment groups. We measured cell survival and cell growth, intracellular oxidative stress and hTERT expression. After RNA interference, the effects of curcumin and Cur1 on cells were verified. Exposure to Aβ1-42 resulted in significant oxidative stress and cell toxicity, and the expression of hTERT was significantly decreased. Curcumin and Cur1 both protected SK-N-SH cells from Aβ1-42 and up-regulated the expression of hTERT. Furthermore, Cur1 demonstrated stronger protective effects than curcumin. However, when telomerase was inhibited by TERT siRNA, the neuroprotection by curcumin and Cur1 were ceased. Our study indicated that the neuroprotective effects of curcumin and Cur1 depend on telomerase, and thus telomerase may be a target for therapeutic effects of curcumin and Cur1.

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective 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 μmol/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 mad1 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 mad1 mRNA expression in cadmium-transformed 16HBE cells and selenium concentration is significantly correlated with these changes.

  6. Estrogen deficiency leads to telomerase inhibition,telomere shortening and reduced cell proliferation in the adrenal gland of mice

    Institute of Scientific and Technical Information of China (English)

    Sharyn Bayne; Margaret EE Jones; He Li; Alex R Pinto; Evan R Simpson; Jun-Ping Liu

    2008-01-01

    Estrogen deficiency mediates aging, but the underlying mechanism remains to be fully determined. We report here that estrogen deficiency caused by targeted disruption of aromatase in mice results in significant inhibition oftelomerase activity in the adrenal gland in vivo. Gene expression analysis showed that, in the absence of estrogen, telomerase reverse transcriptase (TERT) gene expression is reduced in association with compromised cell proliferation in the adrenal gland cortex and adrenal atrophy. Stem cells positive in c-kit are identified to populate in the parenchyma of adrenal cortex. Analysis of telomeres revealed that estrogen deficiency results in significantly shorter telomeres in the adrenal cortex than that in wild-type (WT) control mice. To further establish the causal effects of estrogen, we conducted an estrogen replacement therapy in these estrogen-deficient animals. Administration of estrogen for 3 weeks restores TERT gene expression, telomerase activity and cell proliferation in estrogen-deficient mice. Thus, our data show for the first time that estrogen deficiency causes inhibitions of TERT gene expression, telomerase activity, telomere maintenance, and cell proliferation in the adrenal gland of mice in vivo, suggesting that telomerase inhibition and telomere shortening may mediate cell proliferation arrest in the adrenal gland, thus contributing to estrogen deficiency-induced aging under physiological conditions.

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

    Energy Technology Data Exchange (ETDEWEB)

    Parra, E.R.; Pincelli, M.S. [Departamento de Patologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Teodoro, W.R.; Velosa, A.P.P. [Disciplina de Reumatologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Martins, V.; Rangel, M.P.; Barbas-Filho, J.V.; Capelozzi, V.L. [Departamento de Patologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil)

    2014-06-04

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

  8. Expression of telomerase gene and apoptosis related genes in benign and malignant breast lesion%乳腺良恶性疾病端粒酶hTERT及调亡相关蛋白的表达

    Institute of Scientific and Technical Information of China (English)

    Mengquan Li; Jingruo Li; Jing Su; Jianzhang Li; Jiangtao Li

    2007-01-01

    Objective: To analyse the expression of telomerase and apoptosis related protein, and explore the possible mechanism of breast cancer development. Methods: Immunohistochemistry method (SP) was used to detect the expression of hTERT, p53 and bcl-2 in the tissues of 48 cases of human breast cancer and 42 cases of benign lesions in breast. Results:The positive rates of expression of hTERT, p53 and bcl-2 in breast cancer were 87.50%, 56.25% and 54.17%, respectively.Compared with the groups of adjacent noncancerous and benign lesions, there was a significant difference among three types of tissues (P < 0.05). The positive rates of expression of p53 and bcl-2 in the group with positive expression of hTERT were 64.28% and 61.90%, respectively, and their difference was significant compared with the negative group (P < 0.05).Conclusion: There is a correlation between the activation of telomerases and p53 gene mutation in the development of breast cancer, and they are perhaps relation to the down regulation of bcl-2.

  9. Value of endoscopic methylene blue and Lugol's iodine double staining and detection of GST-Π and telomerase in the early diagnosis of esophageal carcinoma

    OpenAIRE

    Zhu, Xuan; Zhang, Shuang-Hong; ZHANG, KUN-HE; Li, Bi-Ming; Chen, Jiang

    2005-01-01

    AIM: To explore the expressions of GST-Π and telomerase activity in esophageal carcinoma and premalignant lesions and to investigate the value of endoscopic methylene blue (MB) and Lugol's iodine double staining.

  10. Telomerase inhibitor imetelstat has preclinical activity across the spectrum of non-small cell lung cancer oncogenotypes in a telomere length dependent manner

    OpenAIRE

    Frink, Robin E.; Peyton, Michael; Schiller, Joan H.; Gazdar, Adi F.; Shay, Jerry W.; Minna, John D.

    2016-01-01

    Telomerase was evaluated as a therapeutic oncotarget by studying the efficacy of the telomerase inhibitor imetelstat in non-small cell lung cancer (NSCLC) cell lines to determine the range of response phenotypes and identify potential biomarkers of response. A panel of 63 NSCLC cell lines was studied for telomere length and imetelstat efficacy in inhibiting colony formation and no correlation was found with patient characteristics, tumor histology, and oncogenotypes. While there was no overal...

  11. Modulation of telomere shelterin by TRF1 [corrected] and TRF2 interacts with telomerase to maintain the telomere length in non-small cell lung cancer.

    Science.gov (United States)

    Hsu, Chung-Ping; Ko, Jiunn-Liang; Shai, Sen-Ei; Lee, Li-Wen

    2007-12-01

    Our previous report demonstrated good correlations between the expressions of h-TERT and its associated genes, such as c-Myc, TRF1 and TRF2. To observe the interaction between telomerase activity and expression of its associated genes in regulation of the telomere restriction fragment length (TRFL) in non-small cell lung cancer (NSCLC), 79 NSCLC specimens were examined. Telomerase activity, h-TERT, TRF1 and TRF2 genes expression were observed in 60.8, 66.7, 74.7, and 83.5% of the tumour tissues, respectively. The TRFL were shorter in both tumour tissues and telomerase positive tissues, as compared to their counterparts. The t/n-TRFLR (tumour-to-normal TRFL ratio) was also lower in telomerase positive tissues. When telomerase was negative, the t/n-TRFLR was lower in both TRF1 positive and TFR2 positive. However, when telomerase was positive, the t/n-TRFLR was only lower in the TFR2 positive group. When t/n-TRFLR level was equal to or less than 75%, the majority of the specimens became TRF1 and TRF2 positive. To explain these findings, our hypothesis is that when the TRF length becomes shorter during tumour progression, the tumour cells can sustain a better tolerance to shorter telomere with the help of both TRF1 and TRF2, but without immediate activation of the telomerase. However, when the TRF length reaches a critical level, changing the telomere shelterin by persistent expression of the TRF2, which in combination with telomerase activation reverses the telomere shortening.

  12. Effects of Ganoderma lucidum (Higher Basidiomycetes) Extracts on the miRNA Profile and Telomerase Activity of the MCF-7 Breast Cancer Cell Line.

    Science.gov (United States)

    Gonul, Oyku; Aydin, Hikmet Hakan; Kalmis, Erbil; Kayalar, Husniye; Ozkaya, Ali Burak; Atay, Sevcan; Ak, Handan

    2015-01-01

    Ganoderma lucidum is a medicinal higher Basidiomycetes mushroom that exerts anticancer effects through several different mechanisms. This study investigated the effects of G. lucidum on the telomerase activity and microRNA (miRNA) profiles of MCF-7 cells. According to the cytotoxicity results, the G. lucidum ether extract exhibits the highest cytotoxic potency; therefore it was chosen for the subsequent telomerase activity assay and miRNA profiling. The telomerase activity observed in the cells treated with a half-maximal inhibitory concentration of G. lucidum ether extract (100 µg/mL in dimethyl sulfoxide) was 32.2% lower than that of the control cells treated with 1% dimethyl sulfoxide. Among 1066 miRNAs, the most downregulated miRNA was hsa-miR-27a* (4.469-fold), and the most upregulated miRNA was hsa-miR-1285 (10.462-fold). A database search revealed the predicted miRNAs that target the catalytic subunit of the telomerase enzyme telomerase reverse transcriptase, and only miR-3687 (upregulated 2.153-fold) and miR-1207-5p (upregulated 2.895-fold) were changed by at least 2-fold. The miRNA profile changes demonstrated in this study provide a data set regarding their effects on the pathways that regulate telomerase activity in MCF-7 breast cancer cells treated with G. lucidum. These data should aid the development of novel cancer treatment strategies.

  13. Topological effects of charge transfer in telomere G-quadruplex: Mechanism on telomerase activation and inhibition

    CERN Document Server

    Wang, Xin

    2015-01-01

    We explore charge transfer in the telomere G-Quadruplex (TG4) DNA theoretically by the nonequilibrium Green's function method, and reveal the topological effect of charge transport in TG4 DNA. The consecutive TG4(CTG4) is semiconducting with 0.2 ~ 0.3eV energy gap. Charges transfers favorably in the consecutive TG4, but are trapped in the non-consecutive TG4 (NCTG4). The global conductance is inversely proportional to the local conductance for NCTG4. The topological structure transition from NCTG4 to CTG4 induces abruptly ~ 3nA charge current, which provide a microscopic clue to understand the telomerase activated or inhibited by TG4. Our findings reveal the fundamental property of charge transfer in TG4 and its relationship with the topological structure of TG4.

  14. Risk of multiple myeloma is associated with polymorphisms within telomerase genes and telomere length

    DEFF Research Database (Denmark)

    Campa, Daniele; Martino, Alessandro; Varkonyi, Judit;

    2015-01-01

    that a TERT variant, rs2242652, is associated with reduced MM susceptibility (OR = 0.81; 95% CI: 0.72-0.92; p = 0.001). In addition we measured the leukocyte telomere length (LTL) in a subgroup of 140 cases who were chemotherapy-free at the time of blood donation and 468 controls, and found that MM patients...... had longer telomeres compared to controls (OR = 1.19; 95% CI: 0.63-2.24; ptrend  = 0.01 comparing the quartile with the longest LTL versus the shortest LTL). Our data suggest the hypothesis of decreased disease risk by genetic variants that reduce the efficiency of the telomerase complex. This reduced...... efficiency leads to shorter telomere ends, which in turn may also be a marker of decreased MM risk....

  15. Topological Effects of Charge Transfer in Telomere G-Quadruplex Mechanism on Telomerase Activation and Inhibition

    Science.gov (United States)

    Wang, Xin; Liang, Shi-Dong

    2013-02-01

    We explore the charge transfer in the telomere G-Quadruplex (TG4) DNA theoretically by the nonequilibrium Green's function method, and reveal the topological effect of the charge transport in TG4 DNA. The consecutive TG4 (CTG4) is semiconducting with 0.2 0.3 eV energy gap. Charges transfer favorably in the CTG4, but are trapped in the nonconsecutive TG4 (NCTG4). The global conductance is inversely proportional to the local conductance for NCTG4. The topological structure transition from NCTG4 to CTG4 induces abruptly 3nA charge current, which provide a microscopic clue to understand the telomerase activated or inhibited by TG4. Our findings reveal the fundamental property of charge transfer in TG4 and its relationship with the topological structure of TG4.

  16. The interaction between the yeast telomerase RNA and the Est1 protein requires three structural elements.

    Science.gov (United States)

    Lubin, Johnathan W; Tucey, Timothy M; Lundblad, Victoria

    2012-09-01

    In the budding yeast Saccharomyces cerevisiae, the telomerase enzyme is composed of a 1.3-kb TLC1 RNA that forms a complex with Est2 (the catalytic subunit) and two regulatory proteins, Est1 and Est3. Previous work has identified a conserved 5-nt bulge, present in a long helical arm of TLC1, which mediates binding of Est1 to TLC1. However, increased expression of Est1 can bypass the consequences of removal of this RNA bulge, indicating that there are additional binding site(s) for Est1 on TLC1. We report here that a conserved single-stranded internal loop immediately adjacent to the bulge is also required for the Est1-RNA interaction; furthermore, a TLC1 variant that lacks this internal loop but retains the bulge cannot be suppressed by Est1 overexpression, arguing that the internal loop may be a more critical element for Est1 binding. An additional structural feature consisting of a single-stranded region at the base of the helix containing the bulge and internal loop also contributes to recognition of TLC1 by Est1, potentially by providing flexibility to this helical arm. Association of Est1 with each of these TLC1 motifs was assessed using a highly sensitive biochemical assay that simultaneously monitors the relative levels of the Est1 and Est2 proteins in the telomerase complex. The identification of three elements of TLC1 that are required for Est1 association provides a detailed view of this particular protein-RNA interaction.

  17. Telomerase activity is increased and telomere length shortened in T cells from blood of patients with atopic dermatitis and psoriasis

    DEFF Research Database (Denmark)

    Wu, Kehuai; Higashi, N; Hansen, E R;

    2000-01-01

    We studied telomerase activity and telomere length in PBMC and purified CD4(+) and CD8(+) T cells from blood obtained from a total of 32 patients with atopic dermatitis, 16 patients with psoriasis, and 30 normal controls. The telomerase activity was significantly increased in PBMC from the patients...... compared with PBMC from normal donors. This increase was most pronounced in the subpopulation of CD4(+) T cells, which were significantly above the activity of the CD8(+) T cells in atopic dermatitis, psoriasis patients, and control persons. The telomere length was significantly reduced in all T cell...... subsets from both atopic dermatitis and psoriasis patients compared with normal individuals. Furthermore, the telomere length was found to be significantly shorter in CD4(+) memory T cells compared with the CD4(+) naive T cells, and both of the cell subsets from diseases were shown to be of significantly...

  18. Telomerase inhibition effectively targets mouse and human AML stem cells and delays relapse following chemotherapy

    DEFF Research Database (Denmark)

    Bruedigam, Claudia; Bagger, Frederik O; Heidel, Florian H;

    2014-01-01

    Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer maintained by rare populations of leukemia stem cells (LSCs). Selective targeting of LSCs is a promising approach for treating AML and preventing relapse following chemotherapy, and developing such therapeutic modalities is a key...

  19. Immunohistochemical detection of human telomerase reverse transcriptase in oral cancer and pre-cancer

    Directory of Open Access Journals (Sweden)

    Jayanthi Palani

    2011-01-01

    Conclusion: There was increased expression of hTERT protein in OSCC and leukoplakia samples when compared to normal oral mucosa. The cellular localization, LI and LS in OSF were significantly different from OSCC and leukoplakia.

  20. Design of a ribozyme targeting human telomerase reverse transcriptase and cloning of it's gene

    Institute of Scientific and Technical Information of China (English)

    Zhi-Ming Hap; Jin-Yan Luo; Jin Cheng; Quan-Yin Wang; Guang-Xiao Yang

    2003-01-01

    AIM: To design a hammerhead ribozyme targeting humantelomerase reverse transcriptase (hTERT) and clone it's genefor future use in the study of tumor gene therapy.METHODS: Using the software RNAstructure, the secondarystructure of hTERT mRNA was predicted and the cleavagesite of ribozyme was selected. A hammerhead ribozymetargeting this site was designed and bimolecular fold betweenthe ribozyme and hTERT was predicted. The DNA encodingthe ribozyme was synthesized and cloned into pGEMEX-1and the sequence of the ribozyme gene was confirmed byDNA sequencing.RESULTS: Triplet GUC at 1742 of hTERT mRNA was chosenas the cleavage site of the ribozyme. The designed ribozymewas comprised of 22nt catalytic core and 17nt flankingsequence. Computer-aided prediction suggested that theribozyme and hTERT mRNA could cofold into a properconformation. Endonuclease restriction and DNA sequencingconfirmed the correct insertion of the ribozyme gene intothe vector pGEMEX-1.CONCLUSION: This fundamental work of successfuldesigning and cloning of an anti-hTERT hammerheadribozyme has paved the way for further study of inhibitingtumor cell growth by cleaving hTERT mRNA with ribozyme.