Mouse Telomerase Research Articles

Telomerase Reverse Transcriptase Synergizes with Calorie Restriction to Increase Health Span and Extend Mouse Longevity

Caloric restriction (CR), a reduction of food intake while avoiding malnutrition, can delay the onset of cancer and age-related diseases in several species, including mice. In addition, depending of the genetic background, CR can also increase or decrease mouse longevity. This has highlighted the importance of identifying the molecular pathways that interplay with CR in modulating longevity. Significant lifespan extension in mice has been recently achieved through over-expression of the catalytic subunit of mouse telomerase (mTERT) in a cancer protective background. Given the CR cancer-protective effects in rodents, we set to address here whether CR impacts on telomere length and synergizes with mTERT to extend mouse longevity. CR significantly decreased tumor incidence in TERT transgenic (TgTERT) mice and extended their lifespan compared to wild-type (WT) controls under the same diet, indicating a synergy between TgTERT and CR in increasing mouse longevity. In addition, longitudinal telomere length measurements in peripheral blood leukocytes from individual mice showed that CR resulted in maintenance and/or elongation telomeres in a percentage of WT mice, a situation that mimics telomere dynamics in TgTERT cohorts. These results demonstrate that CR attenuates telomere erosion associated to aging and that synergizes with TERT over-expression in increasing “health span” and extending mouse longevity.

1,25-Dihydroxyvitamin D3 Suppresses Telomerase Expression and Human Cancer Growth through MicroRNA-498

Telomerase is an essential enzyme that counteracts the telomere attrition accompanying DNA replication during cell division. Regulation of the promoter activity of the gene encoding its catalytic subunit, the telomerase reverse transcriptase, is established as the dominant mechanism conferring the high telomerase activity in proliferating cells, such as embryonic stem and cancer cells. This study reveals a new mechanism of telomerase regulation through non-coding small RNA by showing that microRNA-498 (miR-498) induced by 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) decreases the mRNA expression of the human telomerase reverse transcriptase. MiR-498 was first identified in a microarray analysis as the most induced microRNA by 1,25(OH)(2)D(3) in ovarian cancer cells and subsequently validated by quantitative polymerase chain reaction assays in multiple human cancer types. A functional vitamin D response element was defined in the 5-prime regulatory region of the miR-498 genome, which is occupied by the vitamin D receptor and its coactivators. Further studies showed that miR-498 targeted the 3-prime untranslated region of human telomerase reverse transcriptase mRNA and decreased its expression. The levels of miR-498 expression were decreased in malignant human ovarian tumors as well as human ovarian cancer cell lines. The ability of 1,25(OH)(2)D(3) to decrease human telomerase reverse transcriptase mRNA and to suppress ovarian cancer growth was compromised when miR-498 was depleted using the sponges in cell lines and mouse tumor models. Taken together, our studies define a novel mechanism of telomerase regulation by small non-coding RNAs and identify miR-498 as an important mediator for the anti-tumor activity of 1,25(OH)(2)D(3).

Overexpression of the mTERT gene by adenoviral vectors promotes the proliferation of neuronal stem cells in vitro and stimulates neurogenesis in the hippocampus of mice

We sought to construct the adenoviral vector carrying the gene encoding mouse telomerase reverse transcriptase (mTERT), as well as detect its expression and effect on the proliferation of neuronal stem cells. mTERT was amplified by RT-PCR and then the eukaryotic expression vector of pDC-EGFP-TERT was constructed. After DNA sequence analysis, we detected that there were 293 cells transfected with pDC-EGFP-TERT and helper adenovirus plasmid pBHG lox ΔE1, and three Cre using Lipofectamine 2000 mediation, named Ad-mTERT-GFP, to pack-age adenoviral particles. The Ad-mTERT-GFP was used to infect neuronal stem cells and then the expression and activity of mTERT were detected. In addition, Bromodeoxyuridine labeling test identified the impact of mTERT overexpression on proliferation of neuronal stem cells. The recombinant adenoviral vector confirmed that mTERT was successfully constructed. Overexpression of mTERT stimulated the proliferation of neuronal stem cells both in vitro and in vivo. mTERT overexpression via adenoviral vector carrying mTERT cDNA upregulated the ability of proliferation in neuronal stem cells.

Statement of Retraction

Withdrawn: Dong X, Shi W, Zeng Q, Xie L. Roles of Adherence and Matrix Metalloproteinases in Growth Patterns of Fungal Pathogens in Cornea. Current Eye Research 2005; 30(8):613–620doi:10.1080/02713680590968196Withdrawn: Ma L, Xie L, Dong X, Shi W. Role of extracellular phospholipase B of Canddida albicans as a virulent factor in experimental keratomycosis. Current Eye Research 2009; 34(9):761–768doi:10.1080/02713680903056391Withdrawn: Min X, Zhou Q, Dong X, Wang Y, Xie L. Expression Profile and Regulation of Telomerase Reverse Transcriptase on Oxygen-Induced Retinal Neovascularization. Current Eye Research 2011; 36(2):135–142doi:10.3109/02713683.2010.525679The above articles duplicate previously published work and are hereby retracted in both the print and online editions of Current Eye Research. These articles appeared earlier in Chinese-language journals (referenced below). As far as can be determined, no permission was sought for the translation and re-publication of the articles. The English-language versions do not cite or refer to the prior publications, and are therefore redundant to scientific literature. The articles submitted in English to Current Eye Research were peer-reviewed and published in good faith.Roles of Adherence and Matrix Metalloproteinases in Growth Patterns of Fungal Pathogens in Cornea in by Xiaoguang Dong, Weiyun Shi, Qingyan Zeng and Lixin Xie of the Shandong Eye Institute, Qingdao People’s Republic of China, was previously published by Dong, X., Shi,W., Zeng, Q., and Xie, L. (2004) in the Chinese Journal of Ophthalmology, Volume 40, pages: 774–776.Role of extracellular phospholipase B of Canddida albicans as a virulent factor in experimental keratomycosis by Lin Ma, Lixin Xie, Xiaoguang Dong and Weiyun Shi of The State Key Lab Cultivation Base, Shandong Provincial Key Lab of Ophthalmology and Shandong Eye Institute, Qingdao, China was previously published with the same authors as Virulence of extracellular phospholipase B of Candida albicans in rabbit experimental keratomycosis in the Chinese Journal of Ophthalmology, Volume 44, pages: 237-43.Expression Profile and Regulation of Telomerase Reverse Transcriptase on Oxygen-Induced Retinal Neovascularization by Xiaojie Min, Qingjun Zhou, Xiaoguang Dong, Yiqiang Wang, Lixin Xie of the State Key Lab Cultivation Base, Shandong Provincial Key Lab of Ophthalmology, Shandong Eye Institute, Qingdao, China (Xiaojie Min now works at the Department of Ophthalmology, Fourth Affiliated Hospital, China Medical University, Shenyang, China) was previously published as: Min X, Zhou Q, Liu T, Yin H, Dong X, Xie L. Expression of mouse telomerase reverse transcription in a mouse model of oxygen-induced retinopathy. Chinese Journal of Ophthalmology 2009;45:199–205, 2009; and: Min X, Dong X, Zhou Q, Liu T, Yin H, Xie L. TERT-siRNA inhibits oxygen-induced retinal neovascularization in mice. Chinese Journal of Ophthalmology 2009;45:1111–1117.The journal’s policy in this respect is clear: Current Eye Research considers all manuscripts on the strict condition that they have been submitted only to Current Eye Research, that they neither contain material that has been published already, nor are they under consideration for publication or in press elsewhere.Current Eye Research published these articles in good faith, and on the basis of legal warranties made by the corresponding authors regarding the originality of their work.The PublishersNovember 2011

Cloning of mouse telomerase reverse transcriptase gene promoter and identification of proximal core promoter sequences essential for the expression of transgenes in cancer cells

Telomerase is a ribonucleoprotein complex, whose function is to add motif-specific nucleotides to the end of chromosomes. Telomerase consists of three major subunits, the telomerase RNA template (hTR), the telomerase-associated protein (TEP1) and telomerase reverse transcriptase (TERT). TERT is the most important component responsible for the catalytic activity of telomerase and a rate-limiting determinant of the activity. Telomerase activities were at high levels in approximately 90% of mouse cancers or tumor-derived cell lines through TERT transcriptional up-regulation. Unlike human telomerase, telomerase activity exists in colon, liver, ovary and testis but not in brain, heart, stomach and muscle in normal mouse tissues. In this study, we prepared 5' truncations of 1086 bp fragments upstream of the initiating ATG codon of the mTERT gene to construct luciferase reporter gene plasmids, and transfected these plasmids into a normal mouse cell line and several cancer lines to identify the core promoter region essential for transcriptional activation in cancer cells by a luciferase assay. We constructed a eukaryotic expression vector of membrane-expressing staphylococcal endotoxin A (SEA) gene driven by the core promoter region of the mTERT gene and observed if the core promoter region could express the SEA gene in these cancer cells, but not in normal cells following transfection with the construct. The results showed that the transcriptional activities of each fragment of the mTERT gene promoter in the cancer cell lines Hepa1-6, B16 and CT26 were higher than those in NIH3T3 cells, and the proximal 333-bp fragment was the core promoter of the mTERT gene in the cancer cells. The proximal 333-bp fragment was able to make the SEA express on the surface of the cancer cells, but not in NIH3T3 cells. It provides a foundation for cancer targeting gene therapy by using the mTERT gene promoter.

The telomerase activator TA‐65 elongates short telomeres and increases health span of adult/old mice without increasing cancer incidence

Here, we show that a small-molecule activator of telomerase (TA-65) purified from the root of Astragalus membranaceus is capable of increasing average telomere length and decreasing the percentage of critically short telomeres and of DNA damage in haploinsufficient mouse embryonic fibroblasts (MEFs) that harbor critically short telomeres and a single copy of the telomerase RNA Terc gene (G3 Terc(+/-) MEFs). Importantly, TA-65 does not cause telomere elongation or rescue DNA damage in similarly treated telomerase-deficient G3 Terc(-/-) littermate MEFs. These results indicate that TA-65 treatment results in telomerase-dependent elongation of short telomeres and rescue of associated DNA damage, thus demonstrating that TA-65 mechanism of action is through the telomerase pathway. In addition, we demonstrate that TA-65 is capable of increasing mouse telomerase reverse transcriptase levels in some mouse tissues and elongating critically short telomeres when supplemented as part of a standard diet in mice. Finally, TA-65 dietary supplementation in female mice leads to an improvement of certain health-span indicators including glucose tolerance, osteoporosis and skin fitness, without significantly increasing global cancer incidence.

TRAPping telomerase within the intestinal stem cell niche

Adult intestinal stem cells (ISCs) reside at the crypt base, where they continuously proliferate to maintain homeostasis of the intestinal epithelium. As a result of a lifetime of cell division, these LGR5+ ISCs face the combined risk of genomic mutations and telomere attrition. In this issue of EMBOJ , [Schepers et al (2011)][1] examine whether ISCs employ telomerase expression and asymmetric chromosome segregation to protect their genome. [1]: #ref-9

Telomerase Inhibitor PinX1 Provides a Link between TRF1 and Telomerase to Prevent Telomere Elongation

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.

Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells

The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5(+)) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and to allow for a response to tissue injury. Here, we identify a subpopulation of slowly cycling ISCs marked by mouse telomerase reverse transcriptase (mTert) expression that can give rise to Lgr5(+) cells. mTert-expressing cells distribute in a pattern along the crypt-villus axis similar to long-term label-retaining cells (LRCs) and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert(+) cells give rise to all differentiated intestinal cell types, persist long term, and contribute to the regenerative response following injury. Consistent with other highly regenerative tissues, our results demonstrate that a slowly cycling stem cell population exists within the intestine.

NFAT5 regulates transcription of the mouse telomerase reverse transcriptase gene

We aimed to clarify the transcription-regulation mechanisms of the mouse telomerase reverse transcriptase gene ( mTERT). First, we searched for the promoter region required for transcriptional activation of mTERT and identified an enhancer cis-element (named mTERT-EE) located between − 200 and − 179 bp of the mouse TERT gene ( mTERT). EMSA results suggested that nuclear factor of activated T cells (NFAT) member proteins bind to mTERT-EE. We then identified NFAT5 as the factor binding to mTERT-EE and found that it activates the transcription of the mTERT core promoter. The results that siRNA directed against NFAT5 significantly reduced mTERT expression and mTERT core promoter activity and that the expressions of NFAT5 and mTERT were well correlated in various mouse tissues except liver suggest that NFAT5 dominantly and directly regulates mTERT expression. To clarify their functionality further, we investigated the effect of hypertonic stress, a known stimulus affecting the expression and transcriptional activity of NFAT5, on mTERT expression. The result indicated that hypertonic stress activates mTERT transcription via the activation and recruitment of NFAT5 to the mTERT promoter. These results provide useful information about the transcription-regulation mechanisms of mTERT.

A Cajal body-independent pathway for telomerase trafficking in mice

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.

Regulatory mechanisms of human and mouse telomerase reverse transcriptase gene transcription: distinct dependency on c-Myc

Telomerase-a complex ribonucleoprotein enzyme-synthesizes telomeric repeats to avoid telomere loss that accompanies cell division and chromosomal replication. Expression of telomerase is detectable in embryonic cells and cancer cells, but not in normal human cells. On the other hand, in mice, substantial expression of telomerase is detected in normal cells and tissues as well as in immortalized cells. These results suggest that the regulatory mechanisms of telomerase activity in humans and mice differ. Considering these results along with the fact that the expression of the telomerase reverse transcriptase (TERT) gene is a rate-limiting step for telomerase activity, we compared transcriptional regulatory mechanisms of both the species. A series of luciferase assays and RT-PCR analyses demonstrated that c-Myc, a dominant transactivator for human TERT (hTERT), is not involved in the regulation of mouse TERT (mTERT). These results suggest that distinct molecules and pathways are involved in the process of immortalization and tumorigenesis in human and mouse cells.

In vivo administration of artificial Antigen Presenting Cells licenses low avidity T cells for treatment of cancer (131.13)

Abstract The development of effective anti-tumor immune responses is normally constrained by low avidity, tumor-specific cytotoxic T lymphocytes (CTL) which are unable to eradicate the tumor. Therefore, new strategies to rescue anti-tumor activity of low avidity melanoma-specific CTL in vivo may improve immunotherapy efficacy. To boost the in vivo effectiveness of low avidity CTL we immunized mice bearing metastatic lung melanoma with artificial Antigen Presenting Cells (aAPC), made by covalently coupling pepMHC-Ig dimers and B7.1-Ig molecules to magnetic beads. aAPC treatment induced significant tumor reduction in a mouse telomerase antigen system and complete tumor eradication in a mouse TRP-2 antigen system, when low avidity CTL specific for these antigens were adoptively transferred. In addition, in an in vivo treatment model of subcutaneous melanoma, aAPC injection also augmented the activity of adoptively transferred CTL and significantly delayed tumor growth. In vivo tumor clearance due to aAPC administration correlated with in situ proliferation of the transferred CTL. In vitro studies showed that aAPC effectively stimulated cytokine release and TCR down-regulation in low avidity CTL. Therefore, in vivo aAPC administration represents a potentially novel approach to improve adoptive immunotherapy of cancer.

Robust activation of the human but not mouse telomerase gene during the induction of pluripotency

Pluripotent stem cells (PSCs) express telomerase and have unlimited proliferative potential. To study telomerase activation during reprogramming, 3 classes of embryonic stem cell (ESC)-like clones were isolated from mouse fibroblasts containing a transgenic hTERT reporter. Class I expressed few pluripotency markers, whereas class II contained many, but not Oct4, Nanog, and Sox2. Neither class of cells differentiated efficiently. Class III cells, the fully reprogrammed induced PSCs (iPSCs), expressed all pluripotency markers, formed teratomas indistinguishable from those of mESCs, and underwent efficient osteogenic differentiation in vitro. Interestingly, whereas the endogenous mTERT gene expression was only moderately increased during reprogramming, the hTERT promoter was strongly activated in class II cells and was further elevated in class III cells. Treatment of class II cells with chemical inhibitors of MEKs and glycogen synthase kinase 3 resulted in their further reprogramming into class III cells, accompanied by a strong activation of hTERT promoter. In reprogrammed human cells, the endogenous telomerase level, although variable among different clones, was dramatically elevated. Only in cells with the highest telomerase were telomeres restored to the lengths in hESCs. Our data, for the first time, demonstrated that the hTERT promoter was strongly activated in discrete steps, revealing a critical difference in human and mouse cell reprogramming. Because telomere elongation is crucial for self-renewal of hPSCs and replicative aging of their differentiated progeny, these findings have important implications in the generation and applications of iPSCs.

Functional interaction between telomere protein TPP1 and telomerase

Human chromosome end-capping and telomerase regulation require POT1 (Protection of Telomeres 1) and TPP1 proteins, which bind to the 3' ssDNA extension of human telomeres. POT1-TPP1 binding to telomeric DNA activates telomerase repeat addition processivity. We now provide evidence that this POT1-TPP1 activation requires specific interactions with telomerase, rather than it being a DNA substrate-specific effect. First, telomerase from the fish medaka, which extends the same telomeric DNA primer as human telomerase, was not activated by human POT1-TPP1. Second, mutation of a conserved glycine, Gly100 in the TEN (telomerase essential N-terminal) domain of TERT, abolished the enhancement of telomerase processivity by POT1-TPP1, in contrast to other single amino acid mutations. Chimeric human-fish telomerases that contained the human TEN domain were active but not stimulated by POT1-TPP1, showing that additional determinants of processivity lie outside the TEN domain. Finally, primers bound to mouse POT1A and human TPP1 were activated for extension by human telomerase, whereas mPOT1A-mTPP1 was most active with mouse telomerase, indicating that these mammalian telomerases have specificity for their respective TPP1 proteins. We suggest that a sequence-specific interaction between TPP1 in the TPP1-POT1-telomeric DNA complex and the G100 region of the TEN domain of TERT is necessary for high-processivity telomerase action.

Reprogramming Murine Telomerase Rapidly Inhibits the Growth of Mouse Cancer Cells In vitro and In vivo

Telomerase plays a critical role in cancer, prompting the pursuit of various telomerase-based therapeutic strategies. One such strategy, telomerase interference, exploits the high telomerase activity in cancer cells and reprograms telomerase to encode "toxic" telomeres. To date, telomerase interference has been tested in human cancer cells xenografted into mice, an approach that does not recapitulate spontaneous malignancy and offers few insights about host toxicities, because human telomerase is targeted in a mouse host. To address these limitations, we designed and validated two new gene constructs specifically targeting mouse telomerase: mutant template mouse telomerase RNA (MT-mTer) and small interfering RNA against wild-type mouse telomerase RNA (α-mTer-siRNA). Using lentiviral delivery in mouse prostate cancer cells, we achieved α-mTer-siRNA-mediated knockdown of wild-type mTer (80% depletion) and concurrent overexpression of MT-mTer (50-fold). We showed that the two constructs effectively synergize to reprogram murine telomerase to add mutant instead of wild-type telomeric repeats, resulting in rapid telomeric uncapping (5-fold increase in DNA damage foci). This, in turn, led to rapid and significant apoptosis (>90% of cells) and growth inhibition in vitro (90% reduction in viable cell mass) and in vivo (75% reduction in tumor allograft wet weight). In summary, we have shown that mouse cancer cells are vulnerable to direct telomerase interference using novel murine telomerase-targeting constructs; this approach can now be used to study the true therapeutic potential of telomerase interference in mouse spontaneous cancer models.

In vivo Administration of Artificial Antigen-Presenting Cells Activates Low-Avidity T Cells for Treatment of Cancer

The development of effective antitumor immune responses is normally constrained by low-avidity, tumor-specific CTLs that are unable to eradicate the tumor. Strategies to rescue antitumor activity of low-avidity melanoma-specific CTLs in vivo may improve immunotherapy efficacy. To boost the in vivo effectiveness of low-avidity CTLs, we immunized mice bearing lung melanoma metastases with artificial antigen-presenting cells (aAPC), made by covalently coupling (pep)MHC-Ig dimers and B7.1-Ig molecules to magnetic beads. aAPC treatment induced significant tumor reduction in a mouse telomerase antigen system, and complete tumor eradication in a mouse TRP-2 antigen system, when low-avidity CTLs specific for these antigens were adoptively transferred. In addition, in an in vivo treatment model of subcutaneous melanoma, aAPC injection also augmented the activity of adoptively transferred CTLs and significantly delayed tumor growth. In vivo tumor clearance due to aAPC administration correlated with in situ proliferation of the transferred CTL. In vitro studies showed that aAPC effectively stimulated cytokine release, enhanced CTL-mediated lysis, and TCR downregulation in low-avidity CTLs. Therefore, in vivo aAPC administration represents a potentially novel approach to improve cancer immunotherapy.

Induction of specific CTL elicited by dendritic cells transfected with recombinant adenovirus vector expressing the mouse telomerase reverse transcriptase in vitro

Objective To investigate the mouse telomerase reverse transcriptase (mTERT) spe-cific CTL elicited by dendritic cells (DC) transfected with adenovirus vector expressing the mouse telom-erase reverse transcriptase (AdmTERT) and its anti-tumor effect in vitro. Methods Flow cytometry and electron microscopy were used to observe DC phenotype and morphology. DC were transfected with Ad-mTERT. T cells were isolated from mouse spleen. After third stimulation, cytotoxicity of mTERT specific CTL was determined by using 51Cr release assay. H22 and CT26 cells were used as stimulators, and CD8 +T cells as responders. IFN-γ positive blots were developed and calculated following the kit's instruction. IFN-γ ELISPOT assays for antigen specific IFN-γ production by T cells. Results Phenotype and mor-phous observe mouse bone marrow from DC was full-blown and express mTERT fusion protein. IFN-γsecre-ted (208.6 μ/L) and IFN-γ secreting cells (341/106 spleen cell)elicited by the AdmTERT infected DCs were much stronger and higher than that by Ad-GFP group (14.2 μg/L,33/106 spleen cell) and DC group (12.1 μg/L,19/106 spleen cell). High cytotoxicity of mTERT specific CTL could be elicited by AdmTERT transfected DC groups (54.2%), in comparison, no specific lysis was observed for T cells eocultured with control groups. Conclusion DC transfected with adenovirus encoding mTERT can induce strong and functional mTERT antigen specific CTL and remarkable anti-minor effect in vitro. Key words: Carcinoma; hepatocellular; Dendritic cell; Telomerase; Cytotoxicity T lympho-cyte

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Antitumor Activity of Small Double-Stranded Oligodeoxynucleotides Targeting Telomerase RNA in Malignant Melanoma Cells

Human telomerase RNA (hTR) is an intrinsic component of telomerase enzyme. Small interfering RNAs (siRNAs) and single-stranded antisense oligonucleotides have been used previously for silencing of the hTR. The objective of this study was to investigate the effect of partially double-stranded oligodeoxynucleotides (ODNs), in vitro and in vivo in comparison to single-stranded antisense ODNs and siRNAs. ODNs were designed on the basis of structural properties of an ODN from previous studies on HIV, to target the hTR in the human cervical carcinoma HeLa cell line and mouse telomerase RNA (mTR) in the murine metastatic melanoma B16-F10 cell line, respectively. Our results indicate that ODNs were able to inhibit the hTR by 68% and the mTR by 81% in the respective cell lines. This correlated with ODN-mediated rapid inhibition of cell proliferation and induction of apoptosis excluding slow effects on telomerase function. The inhibition of the hTR was decreased by knock-down of the cellular RNases H suggesting their contribution. Furthermore, we showed a reduction in numbers of metastases by 70% after intravenous administration of ODN-transfected B16-F10 cells in C57BL/6 mice. Our study demonstrates the potential utility of these hairpin-loop-structured ODNs as a different group of nucleic acids for telomerase-based antiproliferative strategies.