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1
Gros, Julien. "Quadruplexes de Guanines en ADN et en ARN." Paris 6, 2009. http://www.theses.fr/2009PA066054.
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Les quadruplexes de guanines (G4) sont des structures non canoniques d’acide nucléique formées à partir de séquences ADN ou ARN riches en guanines (G) et basées sur la formation puis l’empilement de G-quartets. D’un point de vue technologique, la formation de ces structures peut être imaginée dans de nombreux domaines d’application allant de la chimie supra-moléculaire aux nanotechnologies. In vivo, ces structures pourraient être impliquées de façon transitoire dans de nombreux processus biologiques, en particulier au niveau des télomères. Les travaux de thèse présentés dans ce manuscrit relèvent de deux objectifs poursuivis. Dans le but de comprendre les mécanismes d'association et de dissociation de ces quadruplexes, nous avons étudié dans un premier temps l’association et la dissociation de G4 tétramoléculaires en ADN. L’utilisation de séquences modifiées nous a permis d’établir un certain nombre de règles régissant chacun de ces deux processus. En particulier, on peut difficilement accommoder d’autres nucléobases que des guanines au sein d’un G-quartet pour former un G4, même si certains analogues de G modifiés en positon 8 peuvent s'avérer favorables en 5' du G4. La stabilisation de G4 aux télomères à l’aide de molécules spécifiques pourrait limiter la prolifération de cellules tumorales. Dans ce cas, le(s) mécanisme(s) d’action de ces ligands demandent à être clarifié(s). Nous avons proposé une nouvelle cible G4 à l’extrémité 5’ d’hTERC, sous-unité ARN de la télomérase humaine. L’utilisation d’un système modèle nous a permis de valider la formation d’un G4 à l’extrémité 5’ d’hTERC in vitro. La formation de ce G4 est en compétition avec la formation de l’hélice P1 impliquée dans fonctionnement de la télomérase humaine. Il nous reste à valider cette hypothèse de travail sur une télomérase entière et active. Pour ce faire, nous avons modifié hTERC (mutations, délétions) et avons mis en place un test direct de l’activité télomérase reconstituée in vitro.
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Toutain, Jérôme. "Cytogénétique placentaire des retards de croissance intra-utérins : intérêts de la recherche des anomalies chromosomiques limitées au placenta et de l’estimation de la longueur télomérique placentaire." Thesis, Bordeaux 2, 2012. http://www.theses.fr/2012BOR21957/document.
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Ce travail de thèse se propose d’étudier le retard de croissance intra-utérin sous l’angle de la cytogénétique placentaire, avec deux approches distinctes et complémentaires. La première approche visera à réévaluer l’influence des anomalies chromosomiques limitées au placenta sur la croissance fœtale, car des études précédentes ont rapporté des résultats contradictoires à ce sujet. La première partie de ce travail permettra en outre d’étudier l’incidence et l’influence de la disomie uniparentale chez les fœtus issus des grossesses compliquées d’une anomalie chromosomique limitée au placenta. La deuxième approche de notre travail s’intéressera à la longueur de structures chromosomiques particulières, les télomères, au niveau placentaire. Il a récemment été décrit que la longueur des télomères des cellules placentaires était réduite au terme des grossesses compliquées d’un retard de croissance intra-utérin. La longueur télomérique placentaire n’a jamais été évaluée au cours de ces grossesses et pourrait potentiellement être utilisée comme biomarqueur placentaire du retard de croissance intra-utérin. La deuxième partie de ce travail nous permettra également d’évaluer le nombre de copies des régions chromosomiques portant les gènes codant pour les principales sous-unités du complexe enzymatique télomérase et de rechercher la présence d’agrégats télomériques au niveau placentaire en cas de retard de croissance intra-utérinThis thesis proposes to study intrauterine growth restriction in terms of cytogenetics of placenta, with two distinct and complementary approaches. The first approach will be to reassess the influence of confined placental mosaicism on fetal growth, as previous studies have reported conflicting results on this issue. The first part of this work will also study the influence of fetal uniparental disomy in case of confined placental mosaicism. The second approach of our work will focus on the length of terminal chromosomal structures, telomeres, at the placental level. It has recently been reported that telomere length was reduced in placental cells collected at term in pregnancies complicated by intrauterine growth restriction. Placental telomere length has never been evaluated in ongoing pregnancies and it could potentially be used as a placental biomarker of intrauterine growth restriction. The second part of this work will also focus on the copy number of chromosomal regions carrying genes encoding the main subunits of the telomerase enzyme complex and will look for the presence of placental telomeric aggregates in case of intrauterine growth restriction
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Silva, de Araujo Bruno Eduardo [Verfasser], Stefan [Akademischer Betreuer] Biesterfeld, and Monika [Gutachter] Hampl. "Multicolour fluorescence in situ hybridization in cervical smears: detection of amplification of hTERC, MYC, and EGFR for the diagnosis of intraepithelial neoplasia. / Bruno Eduardo Silva de Araujo ; Gutachter: Monika Hampl ; Betreuer: Stefan Biesterfeld." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2019. http://d-nb.info/1201882044/34.
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Ahmed, Shaheda Sameena. "hTERT protects mitochondria but not telomeres under oxidative stress." Thesis, University of Newcastle Upon Tyne, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435571.
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Bund, Dagmar. "hTERT, CD23 und CD229 als Tumorantigene bei der B-CLL." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-145282.
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Nimmo, Graeme A. M. "Mapping telomerase reverse transcriptase (hTERT) domains that contribute to tumorigenesis." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112547.
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Telomerase is a ribonucleoprotein that maintains telomere. It is activated in greater than 85% of human neoplasms. Traditionally, reactivation of telomerase during tumorigenesis was thought to be required solely to impart an indefinite lifespan. Recently, however, several studies have suggested that telomerase may contribute to tumorigenesis via an additional mechanism that is independent of its role in telomere lengthening. We sought to identify the region(s) of hTERT that contribute to this non-classical role of telomerase. We proposed to identify such regions by their ability to impart a tumorigenic phenotype in ALT cells transduced with activated Ras. Also, we attempted to develop methods to demonstrate that this role is not dependant of telomerase localizing to the telomere. The strategies employed and the progress gained toward each goal is presented in this thesis.
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Schaeper, Anja [Verfasser]. "Hoher prognostischer Wert der hTERT-Expression in Gastrointestinalen Stromatumoren / Anja Schaeper." Magdeburg : Universitätsbibliothek, 2012. http://d-nb.info/1052827829/34.
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Rodrigues, Katherine de Souza. "Polimorfismo rs2736100 do gene hTERT em pacientes com câncer de mama." reponame:Repositório Institucional da UnB, 2017. http://repositorio.unb.br/handle/10482/23505.
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Dissertação (mestrado)—Universidade de Brasília, Faculdade em Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2017.Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2017-05-11T18:07:41Z No. of bitstreams: 1 2017_KatherinedeSouzaRodrigues.pdf: 1435247 bytes, checksum: 20a2e26006858194d3ebf9aa5289e1d8 (MD5)
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O câncer de mama é o mais comum em mulheres e é responsável por 23% dos novos casos de câncer. O seu diagnóstico precoce assume papel decisivo para um melhor prognóstico devido à sua etiopatogenia complexa e multifatorial, já que não pode ser prevenido. Desta forma, os esforços para melhoria dos indicadores do câncer de mama são direcionados na busca por medidas que antecipem seu diagnóstico. A telomerase, uma enzima importante no processo de carcinogênese, está presente na maioria dos tumores e apresenta indícios de ser um bom marcador molecular, por isso deve ser estudada para avaliar sua relação com o câncer de mama. Este estudo analisou o polimorfismo rs2736100 da telomerase em pacientes com câncer de mama e testou a correlação de tais dados com o prognóstico e variáveis clínicas diversas. Para isso, o DNA de pacientes com câncer de mama foi extraído, submetido a PCR com os primers da região do polimorfismo e sequenciado. Ao todo, 119 pacientes com câncer de mama aceitaram participar do estudo. Foi encontrada associação em diversas características gerais do paciente (altura, idade e IMC) e características do tumor (RE, RP e Ki67), reafirmando a importância deles na clínica para a definição do melhor e mais fidedigno prognóstico. Através do sequenciamento foi identificada a região do polimorfismo rs2736100 da telomerase em 63 amostras, divididos entre aqueles com detecção do genótipo GG e AG. Foi encontrada uma deleção em 11,11% da população estudada que ainda não foi relatada na literatura. Nosso estudo demonstrou que este polimorfismo tem algumas associações com variáveis clínicas e de prognóstico do câncer de mama. Entretanto, para o polimorfismo rs2736100 da telomerase ser utilizado como marcador prognóstico no câncer de mama estudos mais detalhados devem ser realizados para confirmar seu valor clínico.
Breast cancer is the most common in women and accounts for 23% of new cases of cancer. Its early diagnosis plays a decisive role for a better prognosis because of its complex and multifactorial etiopathogenesis, which can not be prevented. Thus, efforts to improve indicators of breast cancer are targeted seeking measures that anticipate their diagnosis. Telomerase, an important enzyme in the carcinogenesis process, is present in most tumors and shows signs of being a good molecular marker, so it should be studied to evaluate its relationship with breast cancer. This study analyzed the rs2736100 polymorphism of telomerase in breast cancer patients and tested the correlation of such data with the prognosis and various clinical variables. For this, the DNA of patients with breast cancer was extracted, subjected to PCR with primers and sequenced region of the polymorphism. In all, 119 breast cancer patients agreed to participate in the study. An association was found in several general patient characteristics (height, age and BMI) and tumor characteristics (ER, PR and Ki67), reaffirming their importance in clinical settings for the definition of the best and most reliable prognosis. Through sequencing the telomerase rs2736100 polymorphism region was identified in 63 samples, divided among those with GG and AG genotype detection. A deletion was found in 11.11% of the studied population that has not yet been reported in the literature. Our study demonstrated that this polymorphism has some associations with clinical and prognostic variables of breast cancer. However, for the rs2736100 polymorphism of telomerase to be used as a prognostic marker in breast cancer more detailed studies should be performed to confirm its clinical value.
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Chebly, Alain. "L'épigénétique comme modulateur de l'expression de hTERT dans les lymphomes T cutanés." Thesis, Bordeaux, 2020. http://www.theses.fr/2020BORD0321.
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Les lymphomes T-cutanés (CTCL) sont des tumeurs télomérase-positives exprimant hTERT, dans lesquelles ni l'amplification, ni les réarrangements, ni les mutations hotspots du promoteur peuvent expliquer la ré-expression du gène. Comme le promoteur de hTERT est riche en CpG, nous avons étudié la contribution des mécanismes épigénétiques dans sa ré-expression, puisqu’aucune étude à ce jour n’a été rapporté dans les CTCL. Nous avons analysé le statut de méthylation du promoteur de hTERT dans des lignées cellulaires, des cellules de patients ainsi que dans des cellules issues de donneurs sains. Nous avons également étudié la présence sur le promoteur de hTERT des histones H3K27ac et H3K27me3. Les analyses de méthylation spécifiques des cellules CTCL ont révélé un profil de méthylation caractéristique limité aux cellules tumorales, englobant une région distale hyperméthylée de -650 pb à -150 pb et une région proximale hypométhylée de -150 pb à + 150 pb, à partir du TSS. Ce double profil de méthylation observé sur le promoteur de hTERT est identique à celui observé dans d’autres types de tumeurs. La région distale hyperméthylée identifiée dans les cellules tumorales CTCL correspond à la région nommée récemment « région TERT oncogénique hyperméthylée » (THOR) et qui est rapportée associée à la réactivation de la télomérase dans les tumeurs, mais jusqu'à présent non rapportée dans les lymphomes. Nous avons évalué l'effet sur THOR de deux inhibiteurs d’histone désacétylases (HDACi), la romidepsine et le vorinostat, tous deux approuvés pour le traitement des CTCL ainsi que d'un inhibiteur de l'ADN méthyltransférase (DNMTi) 5-azacytidine, non approuvé pour les CTCL. Nos résultats obtenus à partir d’une cohorte limitée semblent suggérer, que la 5-azacytidine ne provoque pas la déméthylation de la région hyperméthylée du promoteur de hTERT alors que ce traitement s’accompagne d’une diminution de l’expression de hTERT et, fonctionnellement d’une baisse des capacités clonogènes des cellules. La romidepsine et le vorinostat modifient peu les marques d'histones H3K27ac et H3K27me3 présentes au niveau du promoteur hTERT. En conclusion, les résultats obtenus dans les cellules CTCL comparées à ceux de cellules saines confirment que la méthylation du promoteur de hTERT dans les cellules tumorales est particulière et spécifique à ces cellules, faisant de cette méthylation un biomarqueur de la cellule tumorale. De plus, ils révèlent que la méthylation du promoteur hTERT est relativement stable même sous la pression de thérapies épigénétiques, suggérant que la régulation de hTERT par ces thérapies s’opère en priorité de manière indirecteCutaneous T-lymphomas (CTCL) are telomerase-positive tumors expressing hTERT, in which neither amplification, nor rearrangement, nor promoter hotspot mutations can explain the re-expression of the gene. As the hTERT promoter is rich in CpG, we investigated the contribution of epigenetic mechanisms in its re-expression, since no studies to date have been reported in CTCL. We analyzed the methylation status of the hTERT promoter in cell lines, patients’ cells and in cells from healthy donors. We also studied the presence, on the hTERT promoter, of histones H3K27ac and H3K27me3. Methylation analyzes in CTCL cells revealed a characteristic methylation profile specific to tumor cells, encompassing a distal hypermethylated region from -650 bp to -150 bp and a proximal hypomethylated region from -150 bp to +150 bp, relatively to the TSS. This dual methylation profile on hTERT promoter is identical to the profile seen in other types of tumors. The hypermethylated distal region identified in CTCL tumor cells corresponds to the region recently named “TERT hypermethylated oncogenic region” (THOR) and which is reported to be associated with telomerase reactivation in several tumors, but so far not reported in lymphomas. We evaluated the effect on THOR of two histone deacetylases inhibitors (HDACi), romidepsin and vorinostat, both approved for the treatment of CTCL as well as a DNA methyltransferase inhibitor (DNMTi) 5- azacytidine, not approved for CTCL. Our results, obtained from a limited cohort, seem to suggest that 5-azacytidine does not cause a demethylation of the hypermethylated region on hTERT promoter, while this treatment is accompanied by a decrease in the expression of hTERT and, functionally with a decrease in the clonogenic capacities of tumor cells. Romidepsin and vorinostat can slightly modify the H3K27ac and H3K27me3 histone marks present on hTERT promoter. In conclusion, the results obtained in CTCL cells compared with those of healthy cells confirm that hTERT promoter methylation is specific to CTCL cells, making this methylation a biomarker of tumor cells. Furthermore, they reveal that the methylation of hTERT promoter is relatively stable even under the pressure of epigenetic therapies, suggesting that the regulation of hTERT by these therapies can happen indirectly
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Wen, Victoria Wei-Yu Women's & Children's Health Faculty of Medicine UNSW. "Molecular alterations during immortalisation of human endothelial cells." Awarded by:University of New South Wales. Women's & Children's Health, 2009. http://handle.unsw.edu.au/1959.4/44743.
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Replicative exhaustion of endothelial cells (ECs) contributes to the pathogenesis of age-related vascular disorders, including atherosclerosis and impaired wound healing. Conversely, abnormal proliferation of ECs underlies the development of EC-derived malignancies, such as haemangioblastoma and angiosarcoma. The central objective of this thesis was to delineate mechanisms that regulate the replicative lifespan of human ECs and molecular alterations that occur during immortalisation of ECs. The gradual shortening of telomeres (chromosome-end structures) is one mechanism that restricts the replicative lifespan of human ECs. Telomere shortening initiates an irreversible growth arrest or senescence through activation of a TP53-mediated DNA damage response. Expression of the cyclin-dependent kinase inhibitor, p16INK4a, is also increased and reinforces senescence via the retinoblastoma pathway. Overexpression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity and extends the lifespan of human ECs, but is not sufficient for immortalisation. The current study demonstrated that p16INK4a repression by promoter methylation was a frequent event during immortalisation of hTERT-transduced bone marrow ECs (BMECs), occurring in 5 of 12 clones. Repression of p16INK4a concurred with the development of recurring chromosomal aberrations, which appeared to be a consequence of telomere dysfunction and chromosome fusions. Loss of p16INK4a and the development of a complex karyotype were associated with a more transformed phenotype in hTERT-immortalised BMECs. The investigations described in this thesis were the first to associate loss of p16INK4a expression with the accumulation of chromosome aberrations. Repression of p16INK4a in only a subset of immortal BMECs provided impetus for investigating whether there was a functionally analogous defect in the hTERT-immortalised BMECs that retained p16INK4a expression. In normal human cells, oncogenic Ras upregulates p16INK4a and induces senescence independently of telomere shortening. This thesis demonstrates that the immortal BMECs that retained p16INK4a expression had a defective response to oncogenic Ras, which may have contributed to the immortalisation of these cells. Whole genome and proteome analyses identified additional alterations in gene copy number and protein expression specific to p16INK4a-positive or -negative immortal BMECs. Overall, these investigations provide new insight to the potential consequences of p16INK4a repression during carcinogenesis and describe novel molecular alterations that occur during immortalisation of human ECs.
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Liu, Qingyuan. "Epigenetic Regulation of hTERT in Human Acute Promyelocytic Leukemia Cell Line NB4 and Role of c-Myc." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA11T103.
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La régulation de la télomérase s’effectue à de nombreux niveaux dont la transcription de la sous-Unité catalytique (hTERT). Les travaux du laboratoire effectués sur les cellules NB4, modèle de Leucémie Aiguë Promyélocytaire (LAP), ont montré que l'acide rétinoïque tout-Trans (ATRA) réprime la transcription de hTERT. Cette répression peut être associée à la différenciation (cas des cellules NB4) ou en être dissociée conduisant à la mort des cellules (cas des cellules NB4-LR1 résistantes à la maturation induite par l’ATRA). A partir de la lignée NB4-LR1 a été sélectionnée la lignée NB4-LR1SFD résistante à cette mort cellulaire du fait de la ré-Expression de hTERT même en présence d’ATRA. Cependant cette résistance à la répression de hTERT peut être levée par le co-Traitement ATRA et trioxide d’Arsenic (As2O3) qui conduit à la mort des cellules. Il s'agit donc d'une propriété nouvelle de cette lignée dont le mécanisme reste à élucider.Les résultats obtenus par le laboratoire suggèrent l'importance du statut de méthylation de l’ADN du promoteur de hTERT jusque là peu explorée qui pourrait rendre compte de la résistance à la répression de hTERT. Mon projet a pour objectif de valider cette hypothèse en tirant profit de la diversité des réponses biologiques (différenciation, prolifération, mort cellulaire et expression de hTERT) des variants cellulaires du modèle NB4. Une coopération entre le statut épigénétique (méthylation de l’ADN et modification des histones) du promoteur de hTERT et la fixation de facteurs activateurs et/ou répresseurs sera étudiée. Le statut de méthylation du promoteur de hTERT sur une région allant de -2500pb à +1000pb par rapport au site d’initiation de la transcription a été étudié par la technique de séquençage (Illumina) après traitement des cellules NB4-LR1SFD par l’ATRA seul ou en combinaison avec As2O3. Le résultat obtenu à ce jour montre une hypométhylation d’une région limitée du domaine distal (de -1300pb à -800pb) du promoteur de hTERT associée à la répression de hTERT dans les cellules traitées par la combinaison ATRA+ As2O3 par rapport aux traitements seuls par ATRA ou As2O3. Ceci renforce l’importance du statut de méthylation de cette région du promoteur dans la régulation de l’expression de hTERT. Ce co-Traitement induit également une diminution de l’expression protéique de cMyc et WT1, et aussi de l’ADN methyltransférase 1 (DNMT1) suggérant un rôle de cette enzyme dans le maintien de la méthylation de cette région du promoteur de hTERT. Dans le but d’évaluer le rôle de c-Myc dans la régulation de hTERT, nous avons montré qu’un analogue de l’AMPc, le 8-CPT-CAMP, induisait une dégradation (en partie protéasome dépendant) de la protéine c-Myc dès 6h de traitement dans la lignée résistante NB4-LR1SFD et non la lignée parentale NB4. La lignée NB4-LR1SFD est caractérisée par un déficit en sous unité régulatrice PKA RII. Spécifique knock-Down de PKA RII et l’utilisation d’agonistes et d’antagonistes spécifiques de PKAI a montré : 1) PKAI et PKAII ont des rôles différents sur la stabilité de la protéine c-Myc; 2) le rapport PKAI/PKAII déterminait la stabilité de c-Myc suite à l’activation de la signalisation PKA. Ces résultats suggèrent un rôle possible de PKA comme régulatrice de expression de hTERT via son implication dans le maintien de la stabilité de la protéine c-MycThe regulation of telomerase occurs at various levels, including the transcriptional regulation of hTERT. Previous results in our laboratory from acute promyolocytic leukemia cell model NB4, have shown that all-Trans retinoid acid (ATRA) repress the transcription of hTERT. This repression can be associated with differentiation (in the case of NB4 cells), or be dissociated with differentiation and triggers cellular death (the case of maturation resistant NB4-LR1 cells). Another variant NB4-LR1SFD cells were isolated from NB4-LR1 cells with continuous presence of ATRA and were resistant to the cellular death induced by ATRA. In fact, this resistance is related to the re-Expression of hTERT in presence of ATRA. However, this resistance can be overcome by combination of ATRA and AS2O3 and triggers cellular death.The results obtained in our laboratory suggested the importance of the DNA methylation status in the promoter region of hTERT and could be the one mechanism of the resistance to the repression of hTERT induced by ATRA. My project is by taking the diversity of biological response of the NB4 cells variants to validate the hypothesis. And the cooperation between epigenetic modifications and the binding of transcriptional factors will be equally studied.The DNA methylation status in the promoter region of hTERT from -2500bp to +1000bp has been analyzed with the sequencing technique (illumina) in NB4-LR1SFD treated by ATRA alone or in combination with AS2O3. The results showed a distal hypomethylated region from -1300bp to -800bp associated with the repression of hTERT by the co-Treatment of ATRA and AS2O3 compared with the treatment by ATRA or AS2O3 alone. This result strengthens the importance of methylation status in this region in the regulation of hTERT. The co-Treatment induces also a diminution in protein expression of cMyc, WT1 and DNA methyltransferase 1 (DNMT 1), suggesting this enzyme may play a role in the maintenance of methylation level in this region.In order to evaluate the role of cMyc in the regulation of hTERT, we have shown that an analog de cAMP, 8-CPT-CAMP, induces degradation (partly proteasome-Dependent) of c-Myc protein since 6h in NB4-LR1SFD cells but not in NB4 cells. NB4-LR1SFD cells are characterized by a defect of the PKA regulatory subunit II. Specific knockdown of PKA RII and utilizations of agonists and antagonists of PKA I have shown that: 1) PKA I and PKA II have distinct functional roles on the steady-State of c-Myc protein. 2) The ratio of PKA I/PKA II determines the stability of c-Myc protein with the activation of PKA signalization. These results suggest a possible role of PKA in the regulation of hTERT expression through its modulation on the stability of c-Myc
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Wellnitz, Dominique [Verfasser]. "Aktivierung hTERT-spezifischer T-Lymphozyten bei Patienten mit Nicht-Kleinzelligem Lungenkarzinom / Dominique Wellnitz." Kiel : Universitätsbibliothek Kiel, 2013. http://d-nb.info/1033548596/34.
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Klingenfeld, Catina [Verfasser], and Guido [Akademischer Betreuer] Sauter. "hTERT Promoter-Mutationen in humanen Tumoren der Leber / Catina Klingenfeld. Betreuer: Guido Sauter." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2015. http://d-nb.info/1069986216/34.
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Dale, Tina Patricia. "Investigating the chondrogenic phenotype in clinically relevant cells : the effect of hTERT expression." Thesis, Keele University, 2016. http://eprints.keele.ac.uk/2440/.
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Damaged or diseased mature articular cartilage cannot undergo effective tissue repair and due to its avascular, hypocellular nature defects become widespread and painful. No ‘gold standard’ treatment exists for this indication and the ultimate recourse is prosthetic joint replacement. Cartilage is therefore an ideal target for regenerative medicine therapies aiming to recapitulate native cartilage. Despite over fifty years of research and encouraging outcomes, re-creation of the hyaline tissue has yet to be consistently achieved, possibly as a result of the application of a sub-optimal cell type. Chondrocytes and bone marrow mesenchymal stem cells (MSCs) have been used clinically, with future prospects for other alternative MSC sources and human embryonic stem cell (hESC)-derived cells. Further in vitro study of cellular chondrogenic capacity is desirable but hampered by cell changes and senescence. This work examines the hypothesis that the re-introduction of the catalytic sub-unit human telomerase reverse transcriptase (hTERT) can extend the proliferative cell capacity of cells whilst concomitantly bypassing changes associated with cell aging and senescence. The utility of umbilical cord blood (UCB) as a possible alternative source of more naive MSCs was also investigated. Human bone marrow MSCs, chondrocytes, and hESC-derived cells were transduced with hTERT and their resulting chondrogenic capacity, assessed principally by extracellular matrix (ECM) production and gene expression, examined and compared to that of the three non-transduced, parental cell sources. UCB was not found to be a viable alternative MSC source due to a very low cell number and colony recovery; however, foetal bovine serum (FBS) batch and atmospheric oxygen tension were identified as key to influencing recovery outcomes. Of the three parental cell types examined for chondrogenic potential MSCs and chondrocytes produced similar amounts of sGAG but chondrocytes produced a more homogeneous ECM with persistent chondrogenesis, whereas MSCs became hypertrophic. hESC derived cells had a more muted chondrogenic response with similarities to both chondrocytes and MSCs. TERT extended the proliferative capacity of all three cell types, two extensively but was also associated with changes in cell phenotype and a reduction, although not complete ablation, in the subsequent chondrogenic capacity. Taken together the results demonstrate that with current differentiation techniques primary articular chondrocytes provide the most optimal result, supporting their continued use for clinical therapies, and this capacity may not be preserved by the application of hTERT transduction strategies.
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Masserot, Caroline. "WT1 et régulation de hTERT : cas du neuroblastome et de la leucémie aiguë promyélocytaire." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA11T091.
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La télomérase, enzyme qui permet le maintien de la longueur des télomères est activée dans la majorité des cellules cancéreuses. Du fait de son rôle dans l’immortalité cellulaire, elle a été proposée comme cible de nouvelles stratégies anticancéreuses; il est donc fondamental d’identifier les mécanismes de sa régulation. Des travaux récents du laboratoire ont démontré, en utilisant une lignée de leucémie aiguë promyélocytaire résistante à la différenciation par les rétinoïdes (NB4-LR1), que l’acide rétinoïque tout trans (ATRA) induit une répression transcriptionnelle de hTERT, sous-unité catalytique de la télomérase, indépendamment de la différenciation. Cette répression est également associée à la mort des clones résistants. Il a également été montré lors du traitement par l’ATRA de cellules résistantes à cette répression, NB4-LR1SFD, qu’il existait une hyperméthylation de la région distale du promoteur de hTERT associée à la persistance de sa transcription et à la prolifération cellulaire. Ceci nous a conduit à proposer un modèle de régulation de hTERT dans lequel l'induction de modifications épigénétiques de la région distale de son promoteur empêcherait la fixation d'éventuels répresseurs. Des résultats récents suggèrent que WT1, facteur connu pour contribuer à la répression de hTERT, pourrait être un de ces facteurs. WT1 code pour un facteur de transcription à doigt de zinc et a été décrit pour la première fois comme délété dans les tumeurs de Wilms (tumeurs rénales de l’enfant). Cependant le rôle de WT1 dans développement tumoral varie en fonction des modèles cellulaires ; il peut avoir un rôle d’oncogène ou au contraire agir comme un gène suppresseur de tumeur. Dans le but d’élucider les mécanismes de régulation de la télomérase, nous avons donc voulu étudier le rôle de WT1 dans la répression de hTERT. Pour ce travail, nous avons utilisé deux modèles cellulaires :• La Leucémie aigue promyélocytaire (LAP) : leucémie aigue myéloblastique caractérisée par un transcrit de fusion impliquant le récepteur aux rétinoïdes.• Le neuroblastome : tumeur solide extra crânienne la plus fréquente chez l’enfant. Cette maladie est très hétérogène aussi bien au niveau biologique que clinique ; en effet certaines formes peuvent régresser spontanément alors que d’autres, très agressives, sont résistantes à toutes les thérapeutiques actuelles. Cette hétérogénéité clinique est notamment liée à la différenciation de la tumeur et également à la présence ou non de l’amplification de l’oncogène N-Myc qui a un rôle pronostic majeur dans cette maladie. Les voies de signalisations impliquées dans le fort pouvoir tumorigène des neuroblastomes ne sont pas encore clairement identifiées.Nous avons pu montrer dans ces modèles que WT1 réprime hTERT et que cette répression semble fonction de l'état de méthylation de la région distale du promoteur de hTERTLa 2ème partie de mon travail a été d’étudier le rôle de WT1 dans les neuroblastomes. Les résultats de nos travaux montrent que WT1 est plus exprimé dans les tumeurs sans amplification de N-Myc et dont la différenciation est stromale. Cependant la surexpression de WT1 dans des tumeurs sans amplification de N-Myc semble associée à un moins bon pronostic. L’étude de l’expression de WT1 pourrait constituer un outil pronostic intéressant dans ces tumeursTelomerase is expressed and active in most immortalized cells. Whereas telomerase becomes activated during neoplastic transformation, its activity decreases during differentiation of various immortal cells in response to pharmacological agents, including retinoids. We showed using both an Acute Promyelocytic Leukemia (APL) cellular model (NB4 cell model) and Neuroblastoma cells that all-trans-retinoic acid (ATRA) induced a transcriptional repression of the catalytic subunit of telomerase, hTERT, associated with differentiation. This repression also occurred independently of differentiation, as demonstrated during long term treatment of ATRA-induced maturation resistant NB4-LR1, leading to telomere shortening, growth arrest and cell death. Changes in chromatin environment of hTERT promoter and binding of transcriptional factors have been demonstrated in differentiating cells when hTERT is repressed. However, it is not clear whether these changes are directly involved in hTERT repression or only linked to differentiation. A variant cell line was isolated from NB4-LR1 cells, (NB4-LR1SFD), which bypassed the death step because of a re-activated telomerase and resisted to hTERT repression despite the continuous presence of ATRA. Using both NB4-LR1 and NB4-LR1SFD cells, it was shown that the mechanisms of ATRA-induced hTERT repression involved: 1) a switch from c-myc to mad1 binding at the proximal domain of hTERT promoter, 2) epigenetic modifications of the distal domain of hTERT promoter (-600 -250 nucleotides). Indeed, the persistence of hTERT transcription was associated with an hypermethylation of the distal domain in ATRA-treated NB4-LR1SFD. Interestingly, the binding of a known hTERT repressor, WT1, to this distal domain, was defective in NB4-LR1SFD. The first part of my thesis was to investigate the role in hTERT transcriptionnal regulation of both c-myc and WT1, two transcription factors, which bind to the proximal and the distal region of hTERT promoter, respectively. The second part was to determine the role of WT1 in neuroblastoma. Neuroblastoma is the most common extra cranial solid tumor in childhood and the most frequently diagnosed neoplasm during the infancy. A striking feature of this tumor is its clinical heterogeneity. Among tumor progression markers delineated so far, MYCN amplification occurs in about 25% of total neuroblastoma cases and this percentage increases at 30% in advanced stage NEUROBLASTOMA. Despite the fact that MYCN amplification is strongly correlated with neuroblastoma of poor outcome, MYCN status cannot predict all cases of poor survival in neuroblastoma. In addition, neuroblastoma without MYCN amplification (about 70-80% of neuroblastoma) are far to display favorable behavior. WT1 was initially identified as a tumor suppressor gene involved in the development of a pediatric renal tumor (Wilm’s tumor). Here, we describe an inverse correlation between WT1 expression and MYCN amplification and expression. However and most notably, our results show that WT1 gene expression is associated with a poor outcome for patients showing non-MYCN-amplified tumors. Thus WT1 expression may be a prognostic marker for a better risk-stratification and for an optimized therapeutic management of neuroblastoma
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Salvatico, Jose. "The Expression of MKRN1, an E3 Ubiquitin Ligase for Telomerase Reverse Transcriptase, Is Induced with Differentiation Therapy in Leukemia." Master's thesis, University of Central Florida, 2009. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/3744.
Full textAbstract:
Telomeres are important structural and functional components of chromosomes, serving to provide stability and enabling full replication of the chromosomes. However, a shortening of the telomeres occurs with each cell division that can be fixed by a polymerase activity provided by telomerase, preventing this loss which would otherwise eventually lead to chromosome end-to-end fusions, senescence and cell death. The telomerase activity is present in stem cells and germ line cells, but absent or barely noticeable in adult somatic cells. However, in approximately 80-90% of transformed somatic cells the telomerase activity is recovered, resulting in a "telomerase positive phenotype". This phenotype has been a prime target in cancer research, and recently a novel mechanism for regulating telomerase levels has been uncovered. Makorin 1 RING finger protein (MKRN1) was found to be an E3 ubiquitin ligase for hTERT, the rate-limiting catalytic component of telomerase, leading to the ubiqutin-mediated 26s proteasomal degradation of hTERT and reduced telomerase activity. So, MKRN1 plays a role in telomere homeostasis. In this study we looked at the expression of MKRN1 in numerous tumor cell lines (Hela, HCT116, HL60) and the normal diploid fibroblasts (WI-38). In the latter cell line, basal levels of MKRN1 were found to increase 6-fold when the cells were serum starved and arrested in G1/G0. In contrast, the cancer cell lines expressed MKRN1 at low levels or undetectable. This would indicate that MKRN1 is up-regulated in resting or G1 arrested cells.In one cell line the promyelocytic leukemia, HL-60, showed no protein levels of MKRN1. This cell line is able to be terminally differentiated upon ATRA treatment, when cells are arrested at G1. In this model system of cellular differentiation hTERT mRNA levels and telomerase activity decrease drastically and quickly. We hypothesized that the differentiation of HL-60 induced by ATRA would be accompanied by an increase in MKRN1 levels. MKRN1 mRNA and protein levels were strongly up-regulated during the ATRA-mediated differentiation of HL-60 cells. Although, a decrease in hTERT mRNA is a contributor to telomerase inhibition during cellular differentiation; our data indicate that the up-regulation of MKRN1 ensures the effective removal of residual telomerase activity by the ubiquitin-mediated degradation pathway at the proteasome.M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular and Microbiology MS
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張綺雲 and Yee-wan Cheung. "Quantitative analysis of hTERT mRNA expression in gestational trophoblastic disease by real-time PCR." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B31970436.
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Cheung, Yee-wan. "Quantitative analysis of hTERT mRNA expression in gestational trophoblastic disease by real-time PCR." Hong Kong : University of Hong Kong, 2002. http://sunzi.lib.hku.hk/hkuto/record.jsp?B25139198.
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Bund, Dagmar [Verfasser], and Michael [Akademischer Betreuer] Hallek. "hTERT, CD23 und CD229 als Tumorantigene bei der B-CLL / Dagmar Bund. Betreuer: Michael Hallek." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1024243443/34.
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Yin, Zhanhai. "Establishment of a clonal immortalized human mesenchymal stem cell line expressing hTERT using lentiviral gene transfer." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-145290.
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Hasan, Rana. "Functional identification and mapping of a gene that represses telomerase hTERT transcription in prostate cancer cells." Thesis, Brunel University, 2010. http://bura.brunel.ac.uk/handle/2438/5683.
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Telomerase is present in over 90% of tumour tissues and immortalized cells and is tightly regulated in most normal somatic cells. This suggests the existence of regulatory mechanisms repressing telomerase in normal cells that somehow have become inactive during cancer development. In this project, I used genetic complementation in the form of microcell-mediated monochromosome transfer (MMCT) to search for chromosomes that repress telomerase activity in a prostate cancer cell line, PC-3. Microcell hybrids generated by introducing normal human chromosome 11 strongly inhibited telomerase. Telomerase is regulated primarily at the level of hTERT transcription, its catalytic subunit. Consequently, endogenous hTERT mRNA levels were measured by quantitative RT-PCR in microcell hybrids generated by transferring normal human chromosomes into a PC-3 sub-clone (PC- 3/hTERT) ectopically expressing hTERT cDNA to prevent senescence. Only hybrids constructed with transferred chromosome 11 showed strong transcriptional repression of hTERT. Next, hybrids were constructed by the MMCT transfer of chromosome 11 fragments (X-ray-induced). FISH analysis of clones with completely silenced endogenous hTERT transcription revealed in all cases a discrete chromosome 11 fragment with both the p-arm and q-arm material. A randomly selected hTERT-repressed clone was treated with ganciclovir to select against the HyTK marker and reverse the phenotype. hTERT expression in majority of GCV-resistant clones returned to levels comparable to the parent PC-3/hTERT cells. Collectively, these results provide strong functional evidence for the presence of a powerful telomerase repressor sequence on the fragment. Transfer of one repressive fragment back into mouse A9 cells was then carried out to facilitate fine-structure mapping of its sequence content. High density STS mapping of the fragment in each of the clones revealed a considerable DNA content heterogeneity across the panel. These content maps, together with a further round of MMCT to confirm hTERTrepressive activity, enabled me to identify three candidate regions on the q-arm of chromosome 11 where the repressor sequence may be located: the first region lies between map positions 64.70Mb to 65.42Mb and the other two regions each flank a single positive STS marker at 69.71Mb and 127.32Mb. KAT5, a histone modifying gene has been identified as a potential candidate for repressing hTERT.
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Suppiah, Aravind. "Humoral immunity in colorectal cancer : evaluation of the anti-p53 and anti-hTERT auto-antibody responses." Thesis, University of Hull, 2010. http://hydra.hull.ac.uk/resources/hull:5741.
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Carcinogenesis is a multi-factorial and multi-aetiological process involving suppressions, alterations and re-activation of key biomolecular markers. Some of these changes are recognised by the humoral system and are known as Tumour Associated Antigens (TAA) against which the humoral system is able to mount an auto-antibody response. Cancer cells are subject to two key mortality barriers (M1 & M2) as described in the "2-hit" hypothesis which are overcome by p53 dysfunction (M1) and hTERT re-expression (M2). These two molecular events generate TAA which are recognised by the humoral immune system with a corresponding auto-antibody response. The aim of this thesis was to investigate the significance of the humoral anti-p53 auto-antibody and anti-hTERT auto-antibody responses in colorectal cancer (CRC). This was performed by evaluating all published literature (1979-2009) on anti-p53 auto-antibody response and its association with p53 mutation to provide the largest cumulative sample size to date spanning 30 years. A critical review was performed of all anti-p53 auto-antibody studies in CRC, followed by an investigation into the long-term prognostic significance (minimum 5 years follow-up) of anti-p53 auto-antibody in CRC. The second aim of this thesis was to optimise a method of detecting anti-hTERT in CRC patients and correlate this with anti-p53 auto-antibody in order to investigate the significance of a joint humoral response against the two key TAA responsible for CRC immortality. The overall prevalence of anti-p53 auto-antibody was 18.4% (3292/17,859) in all cancers and 2.2% (88/3,946) in normal/benign disease controls. The anti-p53 autoantibody presence in all published cancers reports was plotted against the reported p53 mutational rates in individual cancers and showed partial correlation (R2=0.5, correlation=0.7) between anti-p53 auto-antibody presence and p53 mutation. Anti-p53 was present in 21.5% (786/3,653) in all CRC only studies, and 19.9% (479/2,409) in CRC studies using ELISA. Anti-p53 was not associated with clinico-pathological factors or prognosis in majority of the studies. Only 4 studies associate anti-53 autoantibody with adverse clinico-pathological parameters, mostly in selective groups. The weaknesses of these studies are discussed. This association leads to anti-p53 association with adverse prognosis but only in selective analysis. The prognostic significance is observed in univariate analysis but lost in multivariate analysis when stronger traditional prognostic factors are incorporated. This thesis initially compared serum with plasma anti-p53 auto-antibody titres and excluded plasma titres from further analysis due to the potential contamination by non-specific binding leading to falsely elevated levels (17-73% variation) of anti-p53 auto-antibody. Serum anti-p53 auto-antibody was present in 21.7% (20/92) CRC patients and 0% (0/20) controls. There was no association with age (p=0.750), sex (p-0.468), Dukes' / TNM stage (p=1.000), T- (p=0.900), N- (p=0.912), M-stage (p=0.632), location (p=0.175), differentiation (p=0.117) or mucinous component (p=0.699). The median follow-up was 97 months with median DPS and OS of 73 months and 62 months respectively. Dukes' / TNM stage, T-, N-, M-stage were prognostic indicators in univariate DFS and OS analysis. Only Dukes'/TNM stage remained an independent prognostic indicators in multivariate analysis (p=0.001). Anti-p53 auto-antibody did not display prognostic significance in univariate or multivariate analysis of OS or DFS. Anti-hTERT auto-antibody has only been reported once in the literature, using molecular recombination to develop hTERT antigen. This thesis optimisation processes aimed develop a method of detecting anti-hTERT using less restrictive technology, and further development of a WB or ELISA to allow mass detection of serum anti-hTERT. The first step aimed to isolate hTERT using a streptavidin immune-affinity column with biotinylated anti-hTERT to capture hTERT from cancer cell lysates. This was unsuccessful and further attempts were made at identifying hTERT using Western blot (WB). Five different anti-hTERT antibodies and a multitude of WB conditions were trialled in duplicate (>100 WB), each with multiple ECL exposures. hTERT was not identified. The reason for this was narrowed down in the final experiments to the lack of specificity of the primary antibodies available. The raising of a sufficiently specific anti-hTERT antibody is required to isolate hTERT antigen. Early CRC detection is vital in improving outcomes. The humoral response to TAA incarcinogenesis could enable earlier identification of CRC and impact prognosis.
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Izgi, Ahu. "Investigation Of Telomerase Activity And Gene Expression In Colorectal Cancer." Master's thesis, METU, 2012. http://etd.lib.metu.edu.tr/upload/12614409/index.pdf.
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Human telomerase is a reverse transcriptase which synthesizes telomeric repeat sequences at the ends of chromosomes. The telomerase enzyme has two essential subunits to be functional which are called telomerase reverse transcriptase (hTERT) and human telomerase RNA (hTR). Telomerase uses its RNA subunit as a template for the addition of hexameric repeats at the ends of chromosomes. The activity of telomerase has been detected in immortal cells but not in most normal somatic cells. Therefore, its activity could serve as diagnostic or prognostic marker in malignancies.
Telomeres are heterochromatic DNA sequences bound by a number of telomere binding proteins in order to maintain the stability of chromosomes. Protection of telomere 1(POT1) is a single stranded telomere binding protein which is thought to have significant role in the recruitment of telomerase to telomeres.
The objective of the current study to investigate telomerase activity and gene expression of hTERT and hPOT1 in human colorectal cancer tissues. The activity of telomerase was examined in colorectal tumors and normal adjacent specimens by and improved telomeric repeat amplification protocol (TRAP)-Silver Staining Assay. The expression levels of hTERT and hPOT1 genes was analysed by qPCR.
The results showed that colorectal cancer tumors showed significantly high telomerase activity whereas normal adjacent tissues were found to be telomerase negative. Among clinicopathological parametersthe stage, histological type, distant metastasis and lymph node metastasis status of tumors were found to show significant differences in terms of telomerase activity. Moreover, the expression of human telomerase reverse transcriptase (hTERT) was found to be overexpressed in tumor tissues compared to normal adjacent tissues. Likewise, colorectal tumors expressed high level of hPOT1 compared to normal tissues. Both the expression of hTERT and hPOT1 correlated with telomerase activity. It can be concluded from the results of the current study that high telomerase activity and overexpression of hTERT and hPOT1, may indicate that they could serve as diagnostic or prognostic indicators in colorectal cancer.
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Palumbo, SunMi Lee. "Characterization of Secondary DNA Structures Formed in the c-myb and hTERT Promoters and Their Potential Role in the Regulation of Transcription." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/194266.
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In this dissertation, the formation of unusual G-quadruplexes in the critical regions of the c-myb and hTERT promoters for control of promoter activity was investigated.The c-myb promoter contains three copies of an almost perfect (GGA)4 sequence. We demonstrate that the each (GGA)4 repeat forms a tetrad:heptad G-quadruplex and any two of the three can intramolecularly dimerize to form T:H:H:T G-quadruplexes. The three T:H:H:T G-quadruplex combinations are of differing degrees of stability and can be further stabilized by G-quadruplex interactive compounds. We also demonstrate that the c-myb G-quadruplex forming region is a critical transcriptional regulatory element and interacts with various nuclear proteins including MAZ (Myc Associated Zinc finger protein). The data from luciferase reporter assay show that the c-myb GGA repeat region plays dual roles as a transcriptional activator and an inhibitor by serving as binding sites for the activators and by forming G-quadruplex structures in the region, respectively. Furthermore, we show that MAZ is a transcriptional repressor of the c-myb promoter and binds to both the double-stranded and T:H:H:T G-quadruplex-folded conformations of the GGA repeat region of the c-myb promoter.The hTERT core promoter contains a G-rich region of 12 consecutive G-tracts, which includes three critical Sp1 binding sites. Although this G-rich region has the potential to form multiple G-quadruplexes, our investigation on the full-length G-rich sequence demonstrate that the G-rich region forms a unique G-quadruplex structure in which two tandem intramolecular G-quadruplex structures are present, consisted of one G-quadruplex formed by the G-tracts 1-4 and the other formed by the G-tracts 5, 6, 11, and 12. We also demonstrate that the latter unusual structure contains a 26-base middle loop that likely forms a hairpin structure and is more stable than the other conventional G-quadruplex. Significantly, the formation of this unusual tandem G-quadruplex structure in the full-length will disable all three critical Sp1 binding sites, which will dramatically downregulate hTERT expression. G-quadruplex formation in the hTERT promoter suggests that the effect of G-quadruplex interactive ligands on telomerase inhibition and telomere shortening may be exerted by the direct interaction between the hTERT G-quadruplex structure and the ligands.
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Kang, Hyun-Jin, Yunxi Cui, Holly Yin, Amy Scheid, William P. D. Hendricks, Jessica Schmidt, Aleksandar Sekulic, et al. "A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters." AMER CHEMICAL SOC, 2016. http://hdl.handle.net/10150/621444.
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Activation of human telomerase reverse transcriptase (hTERT) is necessary for limitless replication in tumorigenesis. Whereas hTERT is transcriptionally silenced in normal cells, most tumor cells reactivate hTERT expression by alleviating transcriptional repression through diverse genetic and epigenetic mechanisms. Transcription-activating hTERT promoter mutations have been found to occur at high frequencies in multiple cancer types. These mutations have been shown to form new transcription factor binding-sites that drive hTERT expression, but this model cannot fully account for differences in wild-type (WT) and mutant promoter activation and has not yet enabled a selective therapeutic strategy. Here, we demonstrate a novel mechanism by which promoter mutations activate hTERT transcription, which also sheds light on a unique therapeutic opportunity. Promoter mutations occur in a core promoter region that forms tertiary structures consisting of a pair of G-quadruplexes involved in transcriptional silencing. We show that promoter mutations exert a detrimental effect on the folding of one of these G-quadruplexes, resulting in a nonfunctional silencer element that alleviates transcriptional repression. We have also identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, GTC365, that acts at an early step in the G-quadruplex folding pathway to redirect mutant promoter G-quadruplex misfolding, partially reinstate the correct folding pathway, and reduce hTERT activity through transcriptional repression. This transcription-mediated repression produces cancer cell death through multiple routes including both induction of apoptosis through inhibition of hTERT's role in regulating apoptosis-related proteins and induction of senescence by decreasing telomerase activity and telomere length. We demonstrate the selective therapeutic potential of this strategy in melanoma cells that overexpress, hTERT.
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Linne, Hannah Louise. "Investigating telomerase regulation in human breast cancer cells : a search for telomerase repressor sequences localised to chromosome 3P." Thesis, Brunel University, 2015. http://bura.brunel.ac.uk/handle/2438/11620.
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Cellular immortality is one of the ten hallmarks of human cancer and has been shown to be an essential prerequisite for malignant progression (Hanahan and Weinberg., 2011, Newbold et al., 1982, Newbold and Overell., 1983). In contrast, normal human somatic cells proliferate for a limited number of population doublings before entering permanent growth arrest known as replicative senescence. This is thought to be due to the progressive shortening of telomeric sequences with each round of cell division. Over 90% of human tumours, but not the majority of human somatic cells, have been found to express telomerase activity (Kim et al., 1994). The rate-limiting component of the human telomerase enzyme is the telomerase reverse transcriptase subunit, which is encoded by the hTERT gene. Transfection of hTERT cDNA into normal human fibroblasts and epithelial cells may sometimes be sufficient to confer cellular immortality (Newbold., 2005, Stampfer and Yaswen., 2002). Therefore, de-repression of hTERT and telomerase re-activation are thought to be critical events in human carcinogenesis and is the predominant mechanism by which cancer cells maintain their proliferative capacity. Previously, our group has shown that introduction of a normal, intact copy of human chromosome 3 into the 21NT primary breast cancer cell line by microcell-mediated monochromosome transfer (MMCT), is associated with strong telomerase repression and induction of cell growth arrest within the majority of hybrid clones (Cuthbert et al., 1999). Structural mapping of chromosome 3 within telomerase-positive revertent clones revealed two regions of deletion: 3p21.3-p22 and 3p12-p21.1, thought to harbour the putative telomerase repressor sequence(s). Subsequent studies showed that the chromosome 3p-encoded telomerase repressor sequence(s) mediates its function by means of transcriptional silencing of hTERT, in part, through chromatin remodelling of two sites within intron 2 of the hTERT gene (Ducrest et al., 2001, Szutorisz et al., 2003). Attempts to achieve positional cloning of hTERT repressor sequences on chromosome 3p identified two interesting candidates; the histone methyltransferase SETD2 and an adjacent long non-coding RNA (lncRNA) sequence known as FLJ/KIF9-AS1 (Dr. T. Roberts, unpublished data). Through MMCT-mediated introduction of intact chromosomes 3 and 17 into the 21NT cell line, I have demonstrated that at least two as yet unidentified telomerase repressor sequences (one located on each of these two normal chromosomes) may function to repress telomerase activity within the same breast cancer cell line, which suggests that multiple, independent telomerase regulatory pathways may be inactivated within the same cancer type. Furthermore, by examining the consequences of forced SETD2 and FLJ expression within the 21NT cell line, together with siRNA-mediated knockdown of SETD2 within a single telomerase-repressed 21NT-chromosome 3 hybrid, I have provided evidence to show that neither of these two candidate genes may function as a regulator of hTERT transcription. Through interrogation of relevant literature, a set of four candidate 3 telomerase regulatory genes (BAP1, RASSF1A, PBRM1 and PARP-3) were selected for further investigation based on their location within the 3p21.1-p21.3 region together with their documented role in the epigenetic regulation of target gene expression. Using mammalian expression vectors containing candidate gene cDNA sequences, my colleague Dr. T. Roberts and I demonstrated that forced overexpression of BAP1 and PARP-3 within the 21NT cell line is associated with consistent, but not always sustained, repression of hTERT transcriptional activity and telomerase activity. It is therefore possible that at least two sequences may exist on chromosome 3p that function collectively to regulate hTERT expression within breast cancer cells. Finally, using an in vitro model of human mammary epithelial cell (HMEC) immortalization, involving the targeted abrogation of two pathologically relevant genes, p16 and p53 to generate a series of variant clones at different stages of immortal transformation (developed by my colleague Dr. H. Yasaei), I have shown that single copy deletions on chromosome 3p are a frequent, clonal event, specifically associated with hTERT de-repression and immortal transformation. Subsequent high-density single nucleotide polymorphism (SNP) array analysis of immortal variants carried out by Dr. H. Yasaei, identified a minimal common region of deletion localized to 3p14.2-p22. Together, these findings provide additional evidence to show that chromosome 3p may harbour critical hTERT repressor sequences, that are lost as an early event during breast carcinogenesis.
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Singhapol, Chatchawan. "Mitochondrial localisation of hTERT protects against nuclear DNA damage and mitochondrial ROS production after endogenous and exogenous stress." Thesis, University of Newcastle upon Tyne, 2013. http://hdl.handle.net/10443/2216.
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Under oxidative stress condition, telomerase catalytic subunit can shuttle from the nucleus and localises within mitochondria. hTERT can improve mitochondrial functions and contribute to a decreased oxidative stress suggesting an entirely new function of telomerase in protecting mitochondria and cells under stress. However, there are still many questions about the mechanism and what factors influence the protective function of telomerase. In this study we investigated the kinetic exclusion of hTERT, the catalytic subunit of telomerase, in various cell lines under different oxidative stress conditions. We also used organelle specific hTERT localisation vectors to model hTERT localisation and investigated a correlation between hTERT location, nuclear DNA damage and ROS production. We found that cells excluded endogenous hTERT from the nucleus in a heterogeneous fashion independently of the cell types. Importantly, nuclear DNA damage showed a significant correlation with the localisation of hTERT. Cells where hTERT remained in the nucleus displayed high DNA damage while cells which excluded hTERT from the nucleus displayed no or very low DNA damage. Our results from specific hTERT localisation vectors specified that mitochondrial localisation of hTERT protects the nucleus from DNA damage and did not showed any sign of apoptosis induction while nuclear localisation of hTERT correlated with higher amounts of DNA damage and apoptosis. Moreover, mitochondrial localisation of hTERT decreased mitochondrial ROS generation levels directly after both endogenous and exogenous stress which we interpret as the reason for the prevention of nuclear DNA damage. Additionally, we analysed whether p53 status might influence the protective function of telomerase. Our results in an isogenic cell pair of glioblastoma cells showed that p53 status does not prominently influence the protective function of mitochondrial hTERT under low stress condition. However, nuclear hTERT of cells which contained inactive p53 displayed a significantly higher nuclear DNA damage than cells which contained an active p53 and this became more pronounced when stress levels were increased. We hypothesise that telomerase localisation might possibly interact with p53 when a cancer cell is under stress condition. However, the molecular mechanism for that is unknown. Our results demonstrate a novel link between mitochondrial localisation of hTERT, decrease of mitochondrial ROS generation and the protective capacity of hTERT to nuclear DNA from damage after stress treatments.
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Krämer, Kai. "Hemmung der humanen Telomerase Reverse Transkriptase-Expression mittels synthetischer Nukleinsäuren in Harnblasenkarzinomzellen." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2006. http://nbn-resolving.de/urn:nbn:de:swb:14-1142361278611-72521.
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Das Harnblasenkarzinom (BCa) ist die zweithäufigste bösartige urologische Tumorerkrankung sowie die siebthäufigste tumorbedingte Todesursache bei Männern. Zur Senkung des erheblichen Rezidiv- und Progressionsrisikos oberflächlicher BCa kommen lokale Immun- oder Chemotherapeutika zum Einsatz, die jedoch starke Nebenwirkungen verursachen können bzw. ungenügende langfristige Effekte bewirken. Eine neuartige Therapieoption besteht in der gezielten Expressionshemmung von Genen, die den Tumorzellen einen Wachstumsvorteil vermitteln. Hierfür eignen sich besonders synthetische Nukleinsäuren wie Antisense-Oligodesoxynukleotide (AS-ODN) und small interfering RNAs (siRNAs). In der vorliegenden Arbeit wurde die Expressionshemmung des potenziellen Targetgens hTERT (humane Telomerase Reverse Transkriptase) mit AS-ODN und siRNAs in BCa-Zellen untersucht. Die Tumorspezifität der hTERT-mRNA-Expression konnte zunächst an tumor- und tumorfreien Gewebeproben von BCa-Patienten gezeigt werden. Die verwendeten AS-ODN reduzierten die hTERT-mRNA-Expression auf bis zu 40%, womit eine Verringerung der Telomeraseaktivität einherging. Die AS-ODN-Behandlung bewirkte des Weiteren eine konzentrationsabhängige Viabilitätsreduktion verschiedener BCa-Zelllinien sowie eine verminderte Zellkoloniebildungsrate. Diese antiproliferativen Effekte waren auf eine Apoptoseinduktion zurückzuführen. Durch eine Vorbehandlung von vier BCa-Zelllinien mit hTERT-AS-ODN konnten die zytotoxischen Effekte der für das BCa relevanten Chemotherapeutika Cisplatin, Mitomycin C und Gemcitabin signifikant verstärkt werden. Nach Untersuchung der AS-ODN-Wirkung in vitro erfolgte die Etablierung eines subkutanen Xenotransplantantmodells der Nacktmaus. Die Eignung einer intraperitonealen Applikation wurde mit fluoreszenzmarkierten AS-ODN belegt. In weiteren Zellkulturexperimenten kamen hTERT-siRNAs, als alternative Methode der Geninhibition, zum Einsatz. Die Reduktion der hTERT-mRNA-Expression auf 50% war mit der durch AS-ODN bewirkten Inhibition vergleichbar. Im Gegensatz zur AS-ODN-Behandlung induzierten siRNAs keine unmittelbare Apoptose. Eine Kombination der siRNAs mit Cisplatin und Mitomycin C bewirkte jedoch eine Verdopplung der Apoptoserate. Um die molekularen Mechanismen der Wirkung der nukleinsäurebasierten hTERT-Inhibitoren und den Einfluss targetunabhängiger Effekte zu untersuchen, wurden transkriptomweite Expressionsanalysen mittels Oligonukleotid-Microarrays durchgeführt. Hierbei zeigte sich, dass die AS-ODN-Behandlung vorwiegend zu einer gesteigerten Expression von Genen führte, die mit einer zellulären Stressantwort assoziiert sind (u.a. ATF3, EGR1, GADD45). Diese Expressionsmuster stimmten in hohem Maße mit denen überein, die durch Transfektion mit AS-ODN gegen andere Targets erhalten wurden. Diese Ergebnisse deuten auf eine, zumindest teilweise, durch off-Targeteffekte ausgelöste Wachstumshemmung hin. Die siRNA-Behandlungen gegen unterschiedliche Targets zeigten relativ geringe Übereinstimmungen in den Expressionsmustern und somit eine höhere Spezifität. Außerdem wurde erstmalig gezeigt, dass eine hTERT-Inhibition mit siRNAs zur trankriptionellen Hemmung der Onkogene EGFR und FOSL1 führt. Diese Daten sowie die Ergebnisse anderer Arbeitsgruppen deuten auf einen wechselseitigen Zusammenhang zwischen hTERT und EGFR in der Regulation der EGFR-stimulierten Proliferation von BCa-Zellen hin. Zusammenfassend lässt sich feststellen, dass hTERT als tumorspezifisch exprimierter und funktionell relevanter Faktor ein hervorragendes Target für eine nukleinsäurebasierte BCa-Therapieoption darstellt. Im Vergleich zu AS-ODN wirken siRNAs grundsätzlich targetspezifischer. Die therapeutische Wertigkeit der lokal applizierten Inhibitoren, insbesondere in Kombination mit herkömmlichen Chemotherapeutika, sollte in nachfolgenden Experimenten im Rahmen eines orthotopen BCa-Xenotransplantatmodells untersucht werden.
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Bikkul, Mehmet Ural. "Using drug treatments to control genome behaviour in normal and Hutchinson-Gilford Progeria Syndrome fibroblasts, with and without hTERT immortalisation." Thesis, Brunel University, 2016. http://bura.brunel.ac.uk/handle/2438/12774.
Full textAbstract:
Hutchinson-Gilford Progeria Syndrome (HGPS) is an exceedingly rare genetic condition with striking features reminiscent of marked premature ageing. HGPS is commonly caused by a ‘classic’ mutation in the A-type lamin gene, LMNA (G608G). This leads to the expression of an aberrant truncated lamin A protein, progerin. The nuclear lamina is known to anchor chromosomes, stabilising and regulating the genome. Interphase chromosomes are non-randomly positioned in the nuclei of cells and they occupy specific locations with respect to a radial distribution, gene-poor chromosomes are positioned at the nuclear periphery and gene-rich chromosomes are positioned towards the nuclear interior. The findings indicated that FTI-277, pravastatin, Zoledronic acid, N-acetyl-L-cysteine and all three combination treatments; FG, PZ, FPZ, have a positive effect on anchoring the genome to the nuclear matrix in AG01972 cell line. Furthermore, it was shown that in terms of positiong of chromosomes 18 and X, treatment of AG01972 HGPS cells with FTI + GGTI and FTI-277 + pravastatin + zoledronic acid drug combinations greatly restored the chromosomal organisation as well as the chromosome repositioning. The data in this thesis indicated that HP1α was found affected in T08 cells and upon lovastatin treatment T08 cells exhibited increased HP1α staining which is the good indication of rescue of heterochromatin organization. Whole exome sequencing data obtained from AG08466 atypical HGPS cells revealed that 2457589 position of promoter region is missing on LMNB2 gene located on chromosome 19. Intriguinly, the radial positions of chromosomes 10, 13, 18, and X results revealed that first time ever in our lab chromosome X found occupying the nuclear interior in atypical T08 cells. Colocalisation analysis of chromosome and fibrillarin findings confirmed chromosome positioning results since chromosomes X colocalised with fibrillarin with higher percentages in T08 cells than other cells suggesting the situation appears to be due to elongated telomeres with more chromosome territories being in the nuclear interior associated with nucleoli. M-FISH karyotyping analysis results confirmed unequivocally metaphase chromosome findings that immortalised T08 cell line has aneuoploidy including deletions and translocations. Histone modification marks H3K9me3, H3K27me3, H4K20me3 and HP1α results indicated that proteins were severely affected in T06 cells, suggesting that expression of truncuated progerin protein alters chromatin organization in T06 cells and also the structure of histone marks are severely affected in atypical T08 cells. The structure of nucleolus is affected in both typical and atypical immortalised HGPS cells, suggesting epigenetic regulation to have a crucial role in HGPS. Subsequently, the telomere lengths of immortalised normal and atypical T08 HGPS cell lines were assessed using IQ-FISH. The results indicated that both immortalised cells had chromosomes with relatively longer telomeric repeats in comparison to the control NB1 cells. The small non-peptidic, non-nucleosidic synthetic compounds (BIBR1532) was utilised to target the telomerase/telomere complex of our immortalised cell lines to shorten telomeres in order to test the hypothesis that elongated telomeres had mislocalised chromosomes. Furthermore, the effect of BIBR1532 on telomeres position in cells was assessed. Remarkably, results demonstrated that BIBR1532 is capable of shortening telomere length of immortalised cells to the level of normal NB1 fibroblasts and immortalised cell lines were capable of proliferating after drug treatment. Furher, results revealed that BIBR1532 treatment can restore the position of chromosome X towards the nuclear periphery within the NB1T and T08 nucleus. Furthermore, although BIBR1532 treatment can restore the position of chromosome 18 towards the nuclear periphery in NB1T cells, treatment did not alter the position of chromosome 18 in T08 cells. Finally, the telomere dysfunction-induced foci (TIF) assay was performed to detect DNA damage in NB1T and T08 cells using an antibody against DNA damage marker γ-H2AX and a synthetic PNA probe for telomeres. Surprisingly, results indicated that BIBR1532 treatment of cells did not give rise to high DNA damage response in both NB1T and T08 cells.
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Kuhlmann, Anne-Sophie. "Rôle de la protéine HBZ du virus HTLV-1 dans l'activation de la télomérase au cours du processus leucémogène." Lyon, Ecole normale supérieure, 2010. http://www.theses.fr/2010ENSL0562.
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Le rétrovirus HTLV-1 infecte 10 à 15 millions de personnes à travers le monde dont 5% développeront la leucémie ATL. La protéine virale Tax, exprimée au cours des phases précoces de l'infection, serait impliquée dans l'initiation du processus leucémogène conduisant à la leucémie ATL. La protéine HBZ, seul produit viral détecté dans les cellules leucémiques de patients, serait impoliquée dans les phases plus tardives du processus. Les travaux réalisés au cours de ma thèse s'attachent à comprendre l'évolution du processus leucémogène de la cellule infectée vers la cellule leucémique ATL et permettent de définir HBZ comme une protéine virale essentielle à la progression de la leucémie associée à l'infection par le virus HTLV-1HTLV-1 retrovirus infects 10 to 15 millions of people worldwide and among them 5% will develop ATL leukemia. The viral protein Tax is expressed during the early steps of the infection and would be involved in the initiation of the leukemogenic process leading to ATL leukemia. The viral protein HBZ is the only viral product detected in the leukemic cells from the patients and could be involved in the later steps of the leukemogenesis. The work performed during my phD aims at understanding the evolution of the leukemogenic process from the infected cell toward the ATL leukemic cell and defines HBZ as a protein which is essential to the leukemogenesis associated to HTLV-1 infection
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Krämer, Kai. "Hemmung der humanen Telomerase Reverse Transkriptase-Expression mittels synthetischer Nukleinsäuren in Harnblasenkarzinomzellen." Doctoral thesis, Technische Universität Dresden, 2005. https://tud.qucosa.de/id/qucosa%3A24678.
Full textAbstract:
Das Harnblasenkarzinom (BCa) ist die zweithäufigste bösartige urologische Tumorerkrankung sowie die siebthäufigste tumorbedingte Todesursache bei Männern. Zur Senkung des erheblichen Rezidiv- und Progressionsrisikos oberflächlicher BCa kommen lokale Immun- oder Chemotherapeutika zum Einsatz, die jedoch starke Nebenwirkungen verursachen können bzw. ungenügende langfristige Effekte bewirken. Eine neuartige Therapieoption besteht in der gezielten Expressionshemmung von Genen, die den Tumorzellen einen Wachstumsvorteil vermitteln. Hierfür eignen sich besonders synthetische Nukleinsäuren wie Antisense-Oligodesoxynukleotide (AS-ODN) und small interfering RNAs (siRNAs). In der vorliegenden Arbeit wurde die Expressionshemmung des potenziellen Targetgens hTERT (humane Telomerase Reverse Transkriptase) mit AS-ODN und siRNAs in BCa-Zellen untersucht. Die Tumorspezifität der hTERT-mRNA-Expression konnte zunächst an tumor- und tumorfreien Gewebeproben von BCa-Patienten gezeigt werden. Die verwendeten AS-ODN reduzierten die hTERT-mRNA-Expression auf bis zu 40%, womit eine Verringerung der Telomeraseaktivität einherging. Die AS-ODN-Behandlung bewirkte des Weiteren eine konzentrationsabhängige Viabilitätsreduktion verschiedener BCa-Zelllinien sowie eine verminderte Zellkoloniebildungsrate. Diese antiproliferativen Effekte waren auf eine Apoptoseinduktion zurückzuführen. Durch eine Vorbehandlung von vier BCa-Zelllinien mit hTERT-AS-ODN konnten die zytotoxischen Effekte der für das BCa relevanten Chemotherapeutika Cisplatin, Mitomycin C und Gemcitabin signifikant verstärkt werden. Nach Untersuchung der AS-ODN-Wirkung in vitro erfolgte die Etablierung eines subkutanen Xenotransplantantmodells der Nacktmaus. Die Eignung einer intraperitonealen Applikation wurde mit fluoreszenzmarkierten AS-ODN belegt. In weiteren Zellkulturexperimenten kamen hTERT-siRNAs, als alternative Methode der Geninhibition, zum Einsatz. Die Reduktion der hTERT-mRNA-Expression auf 50% war mit der durch AS-ODN bewirkten Inhibition vergleichbar. Im Gegensatz zur AS-ODN-Behandlung induzierten siRNAs keine unmittelbare Apoptose. Eine Kombination der siRNAs mit Cisplatin und Mitomycin C bewirkte jedoch eine Verdopplung der Apoptoserate. Um die molekularen Mechanismen der Wirkung der nukleinsäurebasierten hTERT-Inhibitoren und den Einfluss targetunabhängiger Effekte zu untersuchen, wurden transkriptomweite Expressionsanalysen mittels Oligonukleotid-Microarrays durchgeführt. Hierbei zeigte sich, dass die AS-ODN-Behandlung vorwiegend zu einer gesteigerten Expression von Genen führte, die mit einer zellulären Stressantwort assoziiert sind (u.a. ATF3, EGR1, GADD45). Diese Expressionsmuster stimmten in hohem Maße mit denen überein, die durch Transfektion mit AS-ODN gegen andere Targets erhalten wurden. Diese Ergebnisse deuten auf eine, zumindest teilweise, durch off-Targeteffekte ausgelöste Wachstumshemmung hin. Die siRNA-Behandlungen gegen unterschiedliche Targets zeigten relativ geringe Übereinstimmungen in den Expressionsmustern und somit eine höhere Spezifität. Außerdem wurde erstmalig gezeigt, dass eine hTERT-Inhibition mit siRNAs zur trankriptionellen Hemmung der Onkogene EGFR und FOSL1 führt. Diese Daten sowie die Ergebnisse anderer Arbeitsgruppen deuten auf einen wechselseitigen Zusammenhang zwischen hTERT und EGFR in der Regulation der EGFR-stimulierten Proliferation von BCa-Zellen hin. Zusammenfassend lässt sich feststellen, dass hTERT als tumorspezifisch exprimierter und funktionell relevanter Faktor ein hervorragendes Target für eine nukleinsäurebasierte BCa-Therapieoption darstellt. Im Vergleich zu AS-ODN wirken siRNAs grundsätzlich targetspezifischer. Die therapeutische Wertigkeit der lokal applizierten Inhibitoren, insbesondere in Kombination mit herkömmlichen Chemotherapeutika, sollte in nachfolgenden Experimenten im Rahmen eines orthotopen BCa-Xenotransplantatmodells untersucht werden.
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Borowiak, Malgorzata. "HBZ-induced functional deregulation of menin - new insights into the mechanism of telomerase activation during HTLV-1-mediated leukemogenesis." Phd thesis, Ecole normale supérieure de lyon - ENS LYON, 2013. http://tel.archives-ouvertes.fr/tel-00860179.
Full textAbstract:
Adult T-cell leukemia (ATL) is an aggressive lymphoproliferative disorder associated with human T-cell leukemia virus type 1 (HTLV-1) infection. Reactivation of telomerase, a critical event in tumor progression observed in late phases of ATL development, has been shown to be caused by HBZ (HTLV-1 bZIP factor), a regulatory protein encoded by the negative strand of the HTLV-1 genome. The HBZ-mediated up-regulation of the telomerase catalytic subunit is dependent on JunD, which in the cellular context occurs in the complex with menin, the product of the MEN-1 tumor suppressor gene. Interaction with menin represses JunD-dependent transcription and converts JunD into a growth suppressor, whereas it acts as a growth promoter in the absence of menin. My results demonstrate that the viral protein HBZ abrogates tumor suppressor function of menin, resulting in the activation of JunD transcriptional activity and finally in the up-regulation of its target gene, the human telomerase reverse transcriptase (hTERT). I showed that HBZ, JunD and menin can coexist in the same protein complex and that HBZ and menin exert opposite effects on JunD transcriptional activity. Moreover menin inhibits the JunD-mediated activation of the hTERT proximal promoter and HBZ is able to counteract this effect. Finally, I proposed that HBZ, by recruiting p300 histone acetyltransferase, reverses the histone deacetylation conducted by menin-recruited HDACs and therefore up-regulates the expression of the hTERT gene. Altogether, my work led to the identification of the molecular mechanism leading to the functional impairment of the menin tumor suppressor, which results in the deregulation of AP-1 signaling in HTLV-1 infected cells. Finally this work gave new insights into the mechanism of the transcriptional up-regulation of the hTERT gene upon HTLV-1 infection, being a key event during the development of Adult T-cell leukemia and a necessary step towards the progression into more aggressive courses.
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Amarnath, Shoba Maria Prescilla. "Specific cytotoxic T lymphocyte immunity against hTERT in breast cancer and optimisation of dendritic cell maturation for presenting tumour associated antigens." Thesis, University of Hull, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411929.
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Tumkur, Sitaram Raviprakash. "Signalling pathways in renal cell carcinoma with a focus on telomerase regulation." Doctoral thesis, Umeå universitet, Patologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-38121.
Full textAbstract:
Telomerase is a ribonucleoprotein complex that catalyses telomeric repeat addition at the ends of chromosomes. The catalytic subunit, hTERT, acts as a key determinant for telomerase activity control; the induction of hTERT expression is required for telomerase activity. hTERT participates in cellular immortalization and is elevated in certain malignant tissues. Several tumours exhibit telomerase activity, which contributes to the infinite proliferation capacity that promotes tumour progression. Renal cell carcinoma (RCC) represents 2% of all adult malignancies and has a high mortality rate. The WHO classifies RCC into several sub-types based on cytogenetic aberrations and morphological features; the most prevalent sub-types are clear cell (ccRCC), papillary (pRCC), and chromophobe RCC (chRCC). The aims of this thesis were to study the expression patterns of various signalling molecules, to elucidate the functional links among them, and to define the roles of these signalling molecules in the regulation of hTERT gene expression and telomerase activity in RCC. The first paper included in this thesis revealed mRNA overexpression of DJ-1 (a PTEN inhibitor), cMyc, and hTERT in clinical ccRCC samples compared to tumour-free kidney cortex tissues. Significant, positive correlations were detected for DJ-1, cMyc, and hTERT mRNA levels in ccRCC, but not in pRCC. In vitro knockdown of DJ-1 by siRNA in ccRCC cells induced downregulation of p-Akt, cMyc, hTERT, and telomerase activity. Forced overexpression of DJ-1 in an ovarian carcinoma cell line was followed by increased hTERT promoter activity, which appeared to be dependent on cMYC binding to the promoter. Collectively, the in vitro studies verified a functional link among DJ-1, cMyc, and hTERT as implied in the clinical ccRCC samples. The second paper included in this thesis demonstrated overexpression of NBS1 mRNA levels in ccRCC compared to the kidney cortex. NBS1 mRNA levels exhibited significant, positive correlations with DJ-1, cMyc, and S phase, but not with hTERT. In vitro experiments suggested that DJ-1 could regulate NBS1 gene expression. The role of the hTERT transcriptional repressor WT1 in RCC was evaluated in the third paper included in this thesis. ccRCC samples displayed low WT1 mRNA levels compared to kidney cortex samples. Interestingly, WT1 expression was negatively associated with hTERT and cMyc both of which were elevated in ccRCC. Forced overexpression of WT1 isoforms in a ccRCC cell line increased the expression of several negative transcriptional regulators of hTERT and diminished the expression of hTERT positive regulators. In consequence, hTERT mRNA levels and telomerase activity were reduced. Chromatin immunoprecipitation verified direct binding of WT1 to the cMyc, Smad3, and hTERT promoters. Taken together, these data suggested that in ccRCC, WT1 affects hTERT at the transcriptional level via a combined effect on both positive and negative regulators. In conclusion, DJ-1 can regulate hTERT and telomerase activity through the PI3K pathway encompassing PTEN, NBS1, p-Akt, and cMyc in ccRCC, but not in pRCC. WT1 negatively regulates hTERT and telomerase activity directly and indirectly through multiple pathways in ccRCC.
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Slusher, Aaron L. "COUNTERREGULATORY EFFECTS OF PTX3 ON INFLAMMATION AND CELLULAR AGING." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5287.
Full textAbstract:
Pentraxin 3 (PTX3) is a vital regulator of innate immune function that has been shown to counterregulate pro-inflammatory signaling and protect against the development of cardiovascular disease (CVD). Less is known about how PTX3 may mitigate against CVD risk by regulating the pro-inflammatory response at the cellular level. Therefore, this dissertation details four manuscripts which aimed to examine the capacity of PTX3 to regulate the innate immune response of peripheral blood mononuclear cells (PBMCs) isolated from healthy adults. Manuscript 1 examined the capacity of PTX3 to alter the inflammatory milieu following in vitro stimulation of isolated PBMCs with the pro-inflammatory lipid palmitate. In addition, Manuscript 2 sought to examine how participation in acute exercise, a powerful anti-inflammatory behavior that reduces CVD risk, alters the inflammatory phenotype and response of mononuclear cells following ex vivo stimulation with lipopolysaccharide (LPS). Manuscript 3 aimed to further elucidate the potential impact of cardiorespiratory fitness on the capacity of PTX3 to stimulate an innate immune response prior to and immediately following acute exercise in aerobically trained and untrained individuals. Finally, Manuscript 4 investigated the impact of healthy aging on plasma PTX3 concentrations and its relationship with telomere length in middle-aged compared to young adults. The capacity of isolated PBMCs to express a key cellular mechanism involved in maintaining longer telomere lengths, human telomerase reverse transcriptase (hTERT), following cellular stimulation with LPS, PTX3, and PTX3+LPS was also examined to address a mechanism that might explain how persistent exposure of circulating immune cells to the age-related pro-inflammatory milieu contributes to the shortening of telomere lengths.
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Yin, Zhanhai [Verfasser], and Wolf [Akademischer Betreuer] Mutschler. "Establishment of a clonal immortalized human mesenchymal stem cell line expressing hTERT using lentiviral gene transfer : no / Zhanhai Yin. Betreuer: Wolf Mutschler." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1024658546/34.
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Wöster, Katrin [Verfasser]. "Aktivierung hTERT-spezifischer T-Lymphozyten mit zytokin- oder ligandengereiften Dendritischen Zellen bei gesunden Probanden und Patienten mit Nicht-kleinzelligem Lungenkarzinom / Katrin Wöster." Kiel : Universitätsbibliothek Kiel, 2013. http://d-nb.info/1036872084/34.
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Goulart, Ana Paula Szezepaniak. "Avalia??o da express?o de telomerase (hTert), Ki-67 e P16iNK4a em les?es intraepiteliais cervicais de baixo e alto grau." Pontif?cia Universidade Cat?lica do Rio Grande do Sul, 2016. http://tede2.pucrs.br/tede2/handle/tede/7035.
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Objective: To evaluate association between histological grade of low (CIN I) and high-grade intraepithelial cervical lesions (CIN II and III) and the immunohistochemical expression of p16INK4a, hTert and ki67 in order to establish the behavior of these lesions and recurrence during follow-up of two years. Patients and Methods: a historical cohort study of 94 women to analyze 3 groups of patients with low (CIN I) and high-grade (CIN II and III) cervical intraepithelial lesions underwent to knife, to determine the recurrence of disease associated to expression of immunohistochemical markers p16INK4a, Ki67 and hTERT. Results: Mean age of the patients was 38.2 years. Patients with CIN I, there was a lower p16INK4a expression, while in patients CIN II or I / II a higher frequency of p16INK4a ?10% was observed. In patients with CIN III there was a higher frequency of p16INK4a expression (> 50%). There were more patients with Ki67 ?10% of the cell population and lower frequency of Ki67 expression (> 50%) in CIN I. In CIN III there were fewer patients in the category Ki67 ?10% and CIN II and III group there were more patients with absent expression of Ki67. There wasn't association between hTert and histological grade. When comparing the markers between subjects with and without recurrence there was no statistically significant difference. Conclusion: There was a statistically significant association between p16INK4a, P16iNK4aM and Ki67 and histological grade, however there was no statistical difference in the expression of these markers in relation to recurrence of disease during the study period.
Objetivo: Avaliar a associa??o entre gradua??o histol?gica e a express?o imunoistoqu?mica para P16iNK4a, hTert e ki67, a fim de estabelecer o comportamento dessas les?es quanto ? recorr?ncia durante o seguimento de dois anos. Pacientes e M?todos: Estudo de coorte hist?rica incluindo 94 mulheres, em que foram analisados 3 grupos de pacientes com les?es intraepiteliais cervicais de baixo (NIC I) e alto grau , classificadas em NIC II e III, submetidas ? coniza??o, a fim de determinar a recorr?ncia da doen?a conforme a express?o dos marcadores imunoistoqu?micos P16iNK4a, Ki67 e hTert. Resultados: A idade m?dia das pacientes foi de 38,2 anos. Nas pacientes com NIC I, houve maior frequ?ncia de P16iNK4a ausente, nas pacientes NIC II ou I/II observou-se maior frequ?ncia com P16iNK4a ?10%. Nas pacientes NIC III observou-se maior frequ?ncia de express?o de P16iNK4a (>50%). Na categoria NIC I houve mais pacientes com Ki67 ?10% e menor frequ?ncia de Ki67 (>50%). Na categoria NIC III houve menos pacientes na categoria Ki67 ?10% e no grupo NIC II e III houve mais pacientes com Ki67 ausente. N?o houve associa??o entre a express?o do marcador imunoistoqu?mico hTert e gradua??o histol?gica. Quando comparadas as express?es dos marcadores entre sujeitos com e sem recorr?ncia, n?o houve diferen?as estatisticamente significativas. Conclus?o: Houve uma associa??o estatisticamente significativa entre P16iNK4a, P16iNK4aM e Ki67 e a gradua??o histol?gica, entretanto n?o houve diferen?a estat?stica na express?o desses marcadores em rela??o ? recorr?ncia da doen?a no per?odo avaliado.
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Deville, Laure. "Régulation de la Télomérase dans dans la leucémie Myéloïde chronique : Etude de l'expression et de la fonction des variants d'épissage de hTERT." Paris 11, 2010. http://www.theses.fr/2010PA11T050.
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Chen, Yan. "Characterization of Bacillus Spore Membrane Proteomes and Investigation of Their Roles in the Spore Germination Process." Diss., Virginia Tech, 2014. http://hdl.handle.net/10919/64934.
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Components of the bacterial spore germination apparatus are crucial for survival and for initiation of infection by some pathogens. While some components of the germination apparatus are well conserved in spore-forming species, such as the spoVA operon, each species may possess a different and possibly unique germinant recognition mechanism. The significance of several individual proteins in the germination process has been characterized. However, the mechanisms of how these proteins perform their functions and the network connecting these proteins in the complete germination process are still a mystery.
In this study, we characterized a Bacillus subtilis superdormant spore population and investigated the abundance of 11 germination-related proteins. The relative quantities of these proteins in dormant, germinating and superdormant spores suggested that variation in the levels of proteins, other than germinant receptor proteins may result in superdormancy. Specifically, variation in the abundance of the GerD lipoprotein may contribute to heterogeneity of spore germination rates.
Spore membrane proteomes of Bacillus anthracis and B. subtilis were characterized to generate a candidate protein list that can be further investigated. Proteins that were not
previously known to be spore-associated were identified, and many of these proteins shared great similarity in both Bacillus species. A significant number of these proteins are implicated in functions that play major roles in spore formation and germination, such as amino acid or inorganic ion transport and protein fate determination.
By analyzing the in vivo and in vitro activity of HtrC, we proved that the protease is responsible for YpeB proteolytic processing at specific sites during germination. However, without HtrC present in the spore, other proteases appear to degrade YpeB at a reduced rate. The activity of purified HtrC in vitro was stimulated by a relatively high concentration of Mn2+ or Ca2+ ions, but the mechanism behind the stimulation is not clear. We also demonstrated that YpeB and SleB, in the absence of their partner protein, were degraded by unknown proteases other than HtrC during spore formation. Identification and characterization of these unknown proteases would be a future direction for revealing the roles of proteases in spore germination.Ph. D.
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Ripoll, Martín Roberto. "Cuantificación en tiempo real de la subunidad hTERT (Telomerase Reverse Transcriptase) del gen de la telomerasa en plasma de pacientes con cáncer colorrectal." Doctoral thesis, Universitat de València, 2007. http://hdl.handle.net/10803/9600.
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INTRODUCCIÓN: Los telómeros son estructuras compuestas por secuencias de DNA repetitivo y de proteínas, ubicadas en los extremos de los cromosomas que intervienen en su funcionamiento y mantienen su estabilidad. El DNA telomérico se acorta con cada división celular. La telomerasa es una enzima con actividad reverso-transcriptasa, que permite elongar el DNA de los telómeros durante la división celular, habiendo correlación entre la expresión del gen que sintetiza la subunidad hTERT y la actividad enzimática. La actividad telomerasa, es prácticamente indetectable en tejidos sanos y se encuentra aumentada en más del 85% de los cánceres (en el cáncer colorrectal, en el 90-100% de los casos).HIPÓTESIS DE TRABAJO: La determinación de la subunidad hTERT del gen de la telomerasa en plasma de pacientes con cáncer colorrectal, puede utilizarse como marcador tumoral. OBJETIVOS: Cuantificar en plasma de pacientes con cáncer colorrectal, la subunidad hTERT del gen de la telomerasa, mediante transcripción inversa-reacción en cadena de la polimerasa. Analizar el poder de discriminación de este marcador comparándolo con un grupo control. Valorar la correlación de la expresión hTERT con la edad y el sexo, los valores del CEA preoperatorio de los pacientes, el estadio tumoral y el grado de diferenciación tumoral. Determinar la actividad telomerasa en células tumorales, comparándola con células de mucosa sana de colon adyacente al tumor en un subgrupo de pacientes.MATERIAL Y MÉTODO: Se han incluido 81 pacientes intervenidos de cáncer colorrectal en el Servicio de Cirugía del Hospital Clínico Universitario de Valencia, la mediana de edad fue de 69 años, 51 eran varones. Como grupo control se seleccionaron 50 voluntarios con ausencia de patología neoplásica, la mediana de edad fue de 40 años, 25 eran varones. Se extrajeron 5-8ml de sangre venosa del antebrazo. Se ha cuantificado mediante espectrofotometría el RNA del plasma de la muestra, con transcripción inversa del mismo a DNA y posterior amplificación por Reacción en Cadena de la Polimerasa, obteniéndose la expresión del gen hTERT. Se determinó la actividad telomerasa en tejidos en 10 de los pacientes estudiados por método TRAP. En el estudio estadístico, los resultados se abordaron desde un enfoque no paramétrico, utilizándose el test de Spearman como prueba de correlación. Se determinó la sensibilidad y la especificidad del estudio confeccionando la curva ROC.RESULTADOS: El grupo con CCR ha obtenido un hTERT de 10.94, más elevado que en el grupo control que fue de 0.29 (p<0.001). Con respecto al estadio tumoral, el hTERT ha sido de 5.36, 8.91, 10.24 y 20.18 para los estadios I, II, III y IV respectivamente (p=0.032). 10 pacientes presentaban metástasis hepáticas, estos pacientes han obtenido un hTERT de 20.18, superior al obtenido en el resto de pacientes sin metástasis; 8.64 (p=0.005). El hTERT de los pacientes con adenocarcinomas pobremente diferenciados fue significativamente superior (15.15) con respecto al de los pacientes con AC bien diferenciados (5.65) (p=0.048). La actividad telomerasa ha sido significativamente más elevada en células tumorales de CCR (17.46) que en células de mucosa sana de colon (3.88) (p=0.005). Según la curva ROC confeccionada, hemos obtenido una sensibilidad del hTERT en el estudio del 91.4%, una especificidad del 96%, un valor predictivo positivo del 97.4% y un valor predictivo negativo del 87.3% (área bajo la curva: 0.985). CONCLUSIONES: La cuantificación en el RNA plasmático de la subunidad hTERT del gen de la telomerasa, ha sido mucho mayor en pacientes con cáncer colorrectal, en comparación con el grupo control, encontrándose significación estadística entre los niveles hTERT y el estadio de la enfermedad. La actividad telomerasa, es más elevada en tejidos tumorales de CCR con respecto a tejidos de mucosa sana de colon de forma significativa.BLACKGROUND: Telomerase is the enzime responsible for synthesizing DNA from chomosome ends during cellular division. Increased telomerase activity can be found in almost 90% of colorectal tumours.WORKING HYPOTHESIS: The determination of the subunit hTERT of the gene of telomerase in plasma of patients with colorrectal cancer, can be in use as scoreboard tumour.OBJECTIVES: To quantify in plasma the hTERT gene in a prospective series of patients operated on for colorectal cancer and to compare these values to a control group. To value the correlation of expression hTERT with the age and the sex, pre-operative CEA levels, stages and differentiation tumour. To determine the telomerase activity in tumour cells, and cells of mucous colon in a subgroup patients. MATERIALS AND METHODS: Eighty one patients undergoing surgery for colorectal cancer and a control group of fifty healthy volunteers were prospectively studied. Pre-operative venous blood samples were taken from all cancer patients and volunteers. Plasma hTERT expression was determined from peripheal blood based on real-time quantitative for transcription inverse and chain reaction of polimerase (RT-PCR), method normalized to the amount of RNA input using GAPDH gene expression. The telomerase activity was determinated in ten patients by method TRAP.RESULTS: Median values normalized hTERT (hTERTN) gene expression were higher in colorectal cancer patients (10.94, range 0.93-54.72) than healthy volunteers (0.29, range 0.00-4.63) (P<0.001). No significant differences in hTERTN expression between sex or with age (>0.05). No significant correlation was found between hTERTN expression and CEA values (P=0.218). We found significant differences between hTERTN expression and tumour stage (P=0.104). Median values of activity telomerase were higher in tumour cells of colorectal cancer (17.27, range 4.74-58.71), than cells of mucous colon (3.88, range 1.36-12.56). Sensitivity and specificity of the assay for colorectal cancer detection were 91.4% and 96%, respectively. CONCLUSIONS: These results show that detection of mRNA based on the qRT-PCR of the telomerase hTERTN gene in plasma clearly differentiates between healthy and colorectal cancer patients and that hTERTN can be detected and quantified in plasma. This opens up a new field as a non-invasive blood test for colorectal cancer diagnosis.
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Peich, Marco Polo [Verfasser], J. [Akademischer Betreuer] Buchmann, I. [Akademischer Betreuer] Classen-Linke, and B. [Akademischer Betreuer] Fischer. "Einflüsse ubiquitär vorkommender Xenobiotika auf humane immortalisierte endometriale Epithelzellen (hTERT-EEC B37) / Marco Polo Peich. Betreuer: J. Buchmann ; I. Classen-Linke ; B. Fischer." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2011. http://d-nb.info/102520302X/34.
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Oliveira, Sabrina Rocha Luna de. "Efeito do tabagismo no perfil de metilação de DNA no promotor de genes MHL1, hTERT e TP53 em células epiteliais da mucosa bucal." Universidade Federal da Paraíba, 2014. http://tede.biblioteca.ufpb.br:8080/handle/tede/6652.
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DNA methylation, characterized by the addition of a methyl group in cytosines within CpG dinucelotides can modified gene transcription, leading to decrease or even silence a gene. The ability of the environmental factors to induce epigenetic changes has been investigated and many studies have shown a relationship between them. Studies show that pesticides, metal ions, drugs, diet, alcohol dependence and smoking are associated with epigenetic changes. Smoking is often associated with the risk of cancer in various tissues and cardiovascular diseases, being considered the leading cause of preventable death. The MLH1 gene is related to the repair of badly paired bases of DNA (DNA mismatch repair (MMR)). The hTERT gene comprises the catalytic subunit of telomerase enzyme, which is considered a biological clock, a marker indicating that the cellular senescence can be installed inevitably form. The TP53 is a tumor suppressor gene and its hypermethylation is related to the development of various cancers. The aim of this work was to investigate the smoking habit influence on DNA methylation status in the promoter of cancer-related genes, MLH1, hTERT and TP53 in oral epithelial cells of healthy subjects. Samples of oral epithelium of smokers, nonsmokers and former smokers were collected by rinsing and DNA was extracted. After, DNA Methylation analysis was performed by Methylation Sensitive Restriction Enzymes, using two restriction enzymes, the HpaII and HhaI, which cleave different sites. Following the enzymatic digestion, DNA was amplified by PCR, subjected to electrophoresis on a 6% polyacrylamide gel and stained with silver nitrate. Statistical analysis was performed by Chi-square test at a significance level of 5%. The investigated CpG dinucleotides located at HhaI and HpaII sites in the MLH1 gene promoter were observed to be fully methylated in DNA majority samples from the smoker group and statistical differences were found between nonsmokers and smokers and between smokers and former smokers (p<0.05). The same was observed in the hTERT gene promoter at HhaI site (p<0.05) and for HpaII site the unmethylated condition was more frequent in smoker in comparison to nonsmokers (p<0.05). For TP53 no differences were found among groups (p>0.05) which the fully methylated condition was found to be an usual event in healthy oral epithelial cells. We conclude that smoking may induce changes in DNA methylation status in cancer-related genes, such as MLH1 and hTERT of healthy oral epithelial cells and the cessation of smoking reversed the process.
A metilação de DNA é uma modificação química na molécula de DNA, e consiste na presença de um radical metil em dinucleotídeos CpG, presente principalmente em regiões promotoras do gene. Uma das principais funções da metilação de DNA é regular a transcrição gênica, sendo que a presença do radical metil pode suprimir por completo a expressão gênica. Estudos mostram que o meio ambiente pode modular a metilação de DNA. Como exemplo de fatores ambientais temos: a radiação ultravioleta, agrotóxicos, dieta, fármacos, uso crônico do álcool e o hábito de fumar. O fumo é frequentemente associado ao risco de câncer em diversos tecidos e doenças cardiovasculares, sendo considerado a maior causa de morte evitável. O gene MLH1 está relacionado ao reparo de bases mal pareadas do DNA (DNA mismatch repair (MMR)). O gene hTERT compõe a subunidade catalítica da enzima telomerase, a qual é considerada um relógio biológico, um marcador que indica que a senescência celular poderá se instalar de forma inevitável. O TP53 é um gene supressor tumoral e sua hipermetilação está relacionada ao desenvolvimento de diversos tipos de câncer. Assim, o objetivo deste estudo foi investigar o efeito do tabagismo no perfil de metilação de DNA em genes relacionados ao câncer, MLH1, hTERT e TP53 em células da mucosa bucal de indivíduos saudáveis. Para tanto, amostras de epitélio da mucosa bucal de indivíduos fumantes, não fumantes e ex-fumantes foram coletadas por bochecho e o DNA dessas células foi extraído. Após esse processo, a análise de metilação de DNA foi feita utilizando o método de Digestão Enzimática Sensível à Metilação, utilizando-se de duas enzimas de restrição, a HhaI e a HpaII, as quais clivam sítios diferentes. Em seguida à digestão enzimática, DNA foi amplificado por PCR, submetido à eletroforese em gel de poliacrilamida a 6% e corado pelo nitrato de prata. A análise estatística foi realizada pelo Teste de Qui-Quadrado ao nível de significância de 5%. Os dinucleotídeos CpG localizados nos sítios HhaI e HpaII no promotor do gene MLH1 mostraram-se totalmente metilados na maioria dos indivíduos do grupo fumante e diferenças significativas foram observadas entre fumantes e não fumantes e entre fumantes e ex-fumantes (p<0,05). O mesmo foi observado para o sítio HhaI no promotor do gene hTERT (p<0,05) e para o sítio HpaII a condição não metilada foi mais frequente em fumantes em comparação com não fumantes (p<0,05). Para o gene TP53 não foram encontradas diferenças entre os grupos (p>0,05), sendo a condição totalmente metilada um evento usual das células saudáveis da mucosa bucal. Assim, concluímos que o fumo está associado a alterações no perfil de metilação de DNA em genes relacionados ao câncer, como MLH1 e hTERT em células epiteliais saudáveis da mucosa bucal e a cessação do hábito de fumar reverteu o processo.
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Lebel, Réjean. "Visualisation de la liaison du complexe hétérodimérique des b-HLH-LZS de c-Myc et de Max aux séquences E-box du promoteur HTERT." Mémoire, Université de Sherbrooke, 2006. http://savoirs.usherbrooke.ca/handle/11143/3884.
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Les protéines Myc et Max appartiennent à la famille de facteurs de transcription de type b-HLH-LZ. L'hétérodimérisation entre Myc et Max, ou l'homodimérisation de Max permet à ces protéines de lier des séquences spécifiques au niveau de l'ADN (E-box,"CACGTG"). Des résultats récents suggèrent que l'hétérodimère c-Myc/Max pourrait interagir de manière tête-à-queue, composant ainsi un dimère de dimères, et former une boucle dans les promoteurs contenant plus d'une séquence E-box. Afin d'améliorer les connaissances actuelles du mécanisme transcriptionnel de l'hétérodimère c-Myc/Max, les complexes formés entre ce dernier et un fragment du promoteur hTERT (comprenant 2 E-box) ont été visualisés par AFM. Bien que la spécificité de la liaison de l'hétérodimère ait été observée au niveau de chaque site, aucune boucle ne fut détectée. Ce résultat a aussi été confirmé par des essais sur gel dans lesquels l'hétérodimère c-Myc/Max est capable de retarder une sonde E-box avec la masse moléculaire apparente d'un dimère. Un contrôle positif, effectuer avec un tétramère artificiel et obligatoire, a permis d'observer par AFM des boucles et croisements spécifiques parmi les brins d'ADN.Les résultats obtenus indiquent que si le dimère de dimères c-Myc/Max existe en solution, les interactions impliquées dans son oligomérisation sont probablement trop faibles pour permettre la formation de structures complexes dans un promoteur.
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Dimitrova, Lora. "Entwicklung eines neuen Assays zum Nachweis der humanen Telomerase." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15875.
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Die Telomere sind spezialisierte DNA-Protein-Komplexe, die sich an den Enden der Chromosomen der eukaryotischen Zellen befinden. Die Telomerase ist ein Ribonukleoprotein, welches für die vollständige Replikation der Telomere bei den meisten Eukaryoten verantwortlich ist. Die katalytische Untereinheit des Enzyms (hTERT beim Menschen) besitzt Reverse-Transkriptase-Aktivität, und nutzt eine integrierte RNA (hTR beim Menschen) als Template, um Telomer-Wiederholungssequenzen an den Enden der Chromosomen zu synthetisieren. Die Telomerase ist in den meisten normalen humanen somatischen Zellen unterdrückt. In den meisten Krebszellen jedoch, stellt die Reaktivierung der Telomerase zur Beibehaltung der Telomerlänge eine Voraussetzung für deren unbegrenztes Wachstumspotential dar. Im Rahmen dieser Arbeit sollte ein neuer, einfacher und selektiver Assay für den Nachweis der humanen Telomerase entwickelt werden. In dem neuen Assay sollten die beiden Kernkomponenten des Enzyms, die Protein-Untereinheit und die RNA, die Targets sein. Der Test ist in seiner Grundstruktur wie folgt aufgebaut : 1. Immobilisierung der Telomerase über die hTERT an eine Festphase, beschichtet mit Phosphorothioat-modifizierten (PS) Oligonukleotiden oder Heparin. Zusammen mit der Telomerase werden bei diesem Schritt die Heparin-bindenden Proteine, die in der Probe enthalten sind, an die Festphase gebunden. 2. Spezifischer Nachweis der hTR. Zur Detektion der hTR wird ein Oligonukleotid-Ligations-Assay (OLA) oder eine Reverse-Transkriptase-PCR (RT-PCR) eingesetzt. In der optimierten Endversion wurde zur Immobilisierung des Enzyms eine Festphase, beschichtet mit PS-Oligonukleotiden, verwendet. Die hTR wurde mittels RT-PCR nachgewiesen. Mit dem neuen Assay wurden erfolgreich 75 Tumorzellen detektiert.Telomeres are specialized DNA-Protein structures located at the ends of linear eukaryotic chromosomes. Telomerase is a ribonucleoprotein, which is responsible for the complete replication of the telomeres in most eukaryotes. The catalytic reverse transcriptase protein subunit (hTERT in humans) of the nucleoprotein uses an integral RNA (hTR in humans) as a template for the addition of telomeric repeat sequences to the ends of chromosomes. Telomerase is repressed in most normal human somatic cells, while the reactivation of telomerase to maintain telomere length is necessary for the unlimited growth potential of most human cancer cells. The aim of this work was the development of a new, simple and selective assay for the detection of human telomerase. The targets of the new assay were the two core subunits of the enzyme : hTERT and hTR. The test comprises two principal steps : 1. Immobilization of the telomerase via the hTERT subunit on a solid phase, coated with heparin or phosphorothioate-modified (PS) oligonucleotides. In this step telomerase is bound together with the heparin-binding proteins of the analysed sample to the surface. 2. Specific detection of the hTR. For the detection of the hTR an oligonucleotide ligation assay (OLA) or a reverse transcriptase PCR (RT-PCR) was used. In the optimized final version of the assay a PS-coated solid phase was used for the immobilization of the enzyme. Reverse transcriptase PCR was applied for detection of the hTR. 75 tumor cells were successfully detected with the new assay.
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Sachs, Patrick. "REGULATION OF TELOMERASE EXPRESSION IN STEM CELL REPROGRAMMING." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/40.
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A great need exists for an abundant, easily accessible source of patient-specific cells that will function for use in regenerative medicine. One promising source is the adult stem cell derived from adipose tissue (ASCs). Isolated from waste lipoaspiration, these cells could serve as a readily available source for the regeneration of damaged tissues. To further define the biology of ASCs, we have isolated multiple cell strains from different adipose tissue sources, indicating wide-spread distribution in the body. We find that a widely used set of cell surface markers fail to distinguish ASCs from normal fibroblasts. However, our ASC isolations are multipotent while fibroblasts show no differentiation potential. In further contrast to fibroblasts, these cells also show expression of genes associated with pluripotent cells, Oct-4, SOX2, and NANOG. Together, our data suggest that while the cell surface profile of ASCs do not distinguish them from normal fibroblasts and their lack of telomerase shows their limited proliferation capacity, the expression of genes closely linked to pluripotency and their differentiation capacity clearly define ASCs as multipotent stem cells. iPS cells are another promising cell type for tissue regeneration, due to their expression of hTERT and their capacity to differentiate into all three germ layers. Interestingly, telomerase is activated during the induction process, accomplished by the exogenous expression of four genes in normal, non-hTERT-expressing fibroblasts. To elucidate the mechanisms behind this activation, we examined the overexpression of these four factors in BJ fibroblasts and ASCs, which resulted in undetectable hTERT expression. We then demonstrated a lack of an acetylated histone H3K9 with the opposing di-methylation, indicative of a closed chromatin state at the hTERT promoter. Subsequent treatment of cells with TSA alone showed an upregulation of hTERT mRNA without telomerase activity. However, telomerase activity was found when ASCs, but not BJs were treated with TSA and all four factors, indicating differential regulation of hTERT in cells of similar mesenchymal origins. Our data suggest that while hTERT’s expression is universally dependent on the presence of a relaxed chromatin state and sufficient transactivating factors, other cell to cell differences can prevent its expression.
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Chebel, Amel. "Influence de la stimulation et de la sénescence réplicative des lymphocytes T sur le métabolisme des télomères." Thesis, Lyon 1, 2010. http://www.theses.fr/2010LYO10008.
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Les lymphocytes constituent un modèle original de cellules somatiques puisqu’ils sont capables de réactiver la télomérase lorsqu’ils sont stimulés. Nous avons montré que les lymphocytes, en culture prolongée et soumis à des stimulations itératives par la PHA, présentent une diminution progressive de l’activité télomérasique interrompue à chaque stimulation par une augmentation transitoire. Ces variations sont corrélées positivement aux variations de hTERT et de la longueur des télomères. Les foyers γ-H2AX et 53BP1 et leur localisation au niveau des télomères augmentent lors du vieillissement cellulaire. Nous montrons un dysfonctionnement des télomères au cours de la sénescence lymphocytaire in vitro résultant d’une érosion accrue des télomères et d’une diminution de l’expression des protéines qui les coiffent. Le mécanisme des variations précoces de l’expression de hTERT lors de l’activation lymphocytaire restaient à comprendre. Les conséquences du traitement des lymphocytes par différents immunosuppresseurs agissant tous de façon directe ou indirecte sur l’activation de NFAT suggéraient le rôle de NFAT dans la régulation transcriptionnelle de hTERT. Nous avons montré i) 5 éléments de réponse potentiels pour NFAT au niveau du promoteur de hTERT, ii) l’activation in vitro du promoteur de hTERT par NFAT essentiellement via un site consensus localisé dans le coeur du promoteur de hTERT en position -40 et une synergie fonctionnelle entre NFAT et SP1, iii) la liaison directe de NFAT sur le promoteur de hTERT via ce site consensus in vivo. Ainsi, NFAT1 régule la transcription de hTERT et est impliqué dans l’activation de la télomérase lors de la stimulation lymphocytaireLymphocytes are an example of somatic cells capable to induce telomerase activity when stimulated. We showed that lymphocytes, during long-term culture and repeated PHA stimulations, present a progressive drop in telomerase activity interrupted at each stimulation by a transitory increase. These variations are positively correlated with hTERT and telomere length variations. γ-H2AX and 53BP1 foci and their localization on telomeres increase with cell aging. We show a telomere dysfunction during in vitro lymphocyte senescence resulting from an excessive telomere shortening and a decrease in shelterin content. The mechanism involved in early variations of hTERT expression during lymphocyte activation remained to be understood. Consequences of lymphocyte treatment with different immunosuppressors, all acting directly or indirectly on NFAT activation, suggested a role for NFAT in the regulation of hTERT transcription. Five putative responsive elements for NFAT were identified in the hTERT promoter. We showed that NFAT activates in vitro the hTERT promoter mainly via a consensus site localized in the promoter core at position -40 and a functional synergy between NFAT and SP1. Furthermore, NFAT1 binds directly to the endogenous hTERT promoter via this consensus site in vivo. Thus, NFAT positively regulates the hTERT transcription and we propose its implication in telomerase activation during lymphocyte stimulation
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Jensen, Keith Douglas Ostergaard. "Dual Regulation of Telomerase Activity By HSF1 And Its Role in Prostate Cancer Progression." VCU Scholars Compass, 2006. http://hdl.handle.net/10156/1630.
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Saliter, Julia [Verfasser], and Matthias [Akademischer Betreuer] Folwaczny. "Einfluss von quervernetzten und nicht quervernetzten xenogenen Barrieremembranen auf die Proliferation von humanen mesenchymalen Stammzellen (hMSC) und immortalisierten multipotenten parodontalen Ligamentzellen (PDL-hTERT) / Julia Saliter ; Betreuer: Matthias Folwaczny." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1199816566/34.
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Aloysius, Mark. "Anti-cancer vaccination with dendritic cells pulsed with class I and II peptides of human telomerase reverse transcriptase (hTERT) : optimization of anti-cancer cytotoxic T killer cell activity in patients with advanced malignancy." Thesis, University of Nottingham, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.546266.
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