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.

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-Myc. / The 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.

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.

The acquisition and maintenance of cell division potential are important characteristics of tumorigenesis. Human telomerase reverse transcriptase (TERT) and telomerase RNA (TR) can immortalize cells through telomere maintenance, and telomerase activity is one factor that contributes to the in vitro transformation of normal cells. In vitro and in vivo evidence suggest that telomerase maintains telomeres as a functional multimer. In addition, hTERT may possess telomere maintenance-independent functions. To examine the effects of hTERT loss upon an in vitro generated tumorigenic cell line we created a tumorigenic cell line from human embryonic kidney cells through expression of the SV40 early region, H-RasG12V and a Cre-mediated excisable hTERT. These immortalized cells exhibited robust anchorage-independent colony growth and tumor formation in immuno-deficient mice. Cre recombinase expression resulted in the excision of hTERT from the tumorigenic cell lines, restoring cell mortality. A return to immortality was conferred by the re-introduction of wild-type hTERT, but not with hTERT point mutations in the N-terminus (hTEN) and reverse transcriptase (RT) domains that impair in vitro telomere elongation activity. The onset of cell mortality was not immediate, and the hTERT-excised tumorigenic cells exhibited clonal variation in the anchorage-independent colony growth assay and upon tumor formation in immuno-deficient mice. We hypothesized that tumorigenic potential was not related strictly to hTERT presence, but rather telomere length and/or integrity. To investigate this possibility we maintained the tumorigenic cell lines in the continuous presence of hTERT to permit telomere elongation prior to hTERT excision; subsequently, after hTERT excision tumor formation persisted, thus demonstrating a dependence on telomere length and not hTERT presence per se. To investigate the functional multimerization of hTERT in vivo we tested if two defective hTERT polypeptides with mutations in the hTEN and RT domains could restore telomere elongation activity in vivo. Unfortunately, during the course of these experiments an unanticipated recombination event occurred that restored a catalytically active hTERT transgene. Further in vivo analysis of hTERT multimerization should be carefully designed, accounting for the selective survival advantage bestowed by wild-type hTERT. This study provides compelling evidence that hTERT does not possess telomere maintenance-independent functions.

The acquisition and maintenance of cell division potential are important characteristics of tumorigenesis. Human telomerase reverse transcriptase (TERT) and telomerase RNA (TR) can immortalize cells through telomere maintenance, and telomerase activity is one factor that contributes to the in vitro transformation of normal cells. In vitro and in vivo evidence suggest that telomerase maintains telomeres as a functional multimer. In addition, hTERT may possess telomere maintenance-independent functions. To examine the effects of hTERT loss upon an in vitro generated tumorigenic cell line we created a tumorigenic cell line from human embryonic kidney cells through expression of the SV40 early region, H-RasG12V and a Cre-mediated excisable hTERT. These immortalized cells exhibited robust anchorage-independent colony growth and tumor formation in immuno-deficient mice. Cre recombinase expression resulted in the excision of hTERT from the tumorigenic cell lines, restoring cell mortality. A return to immortality was conferred by the re-introduction of wild-type hTERT, but not with hTERT point mutations in the N-terminus (hTEN) and reverse transcriptase (RT) domains that impair in vitro telomere elongation activity. The onset of cell mortality was not immediate, and the hTERT-excised tumorigenic cells exhibited clonal variation in the anchorage-independent colony growth assay and upon tumor formation in immuno-deficient mice. We hypothesized that tumorigenic potential was not related strictly to hTERT presence, but rather telomere length and/or integrity. To investigate this possibility we maintained the tumorigenic cell lines in the continuous presence of hTERT to permit telomere elongation prior to hTERT excision; subsequently, after hTERT excision tumor formation persisted, thus demonstrating a dependence on telomere length and not hTERT presence per se. To investigate the functional multimerization of hTERT in vivo we tested if two defective hTERT polypeptides with mutations in the hTEN and RT domains could restore telomere elongation activity in vivo. Unfortunately, during the course of these experiments an unanticipated recombination event occurred that restored a catalytically active hTERT transgene. Further in vivo analysis of hTERT multimerization should be carefully designed, accounting for the selective survival advantage bestowed by wild-type hTERT. This study provides compelling evidence that hTERT does not possess telomere maintenance-independent functions.

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 parameters / the 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.

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.

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.

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 tumeurs. / Telomerase 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.

Les télomères sont des structures nucléoprotéiques nécessaires à la protection des extrémités des chromosomes contre les dégradations ou fusions induites par les processus de réparation de lADN. Ils sont constitués de complexes protéiques associés à des répétitions en tandem dun motif 5-(TTAGGG)-3 sous forme double brin de plusieurs kilobases, et finalisés par une extrémité 3simple brin de la même séquence de quelques centaines de bases. On observe in vitro un raccourcissement des télomères à chaque division cellulaire, ce même fait est corrélé in vivo avec le vieillissement. Lorsque les télomères se raccourcissent et atteignent une taille critique, les cellules entrent en sénescence réplicative qui se définit par un arrêt de croissance définitif et viable des cellules. La télomérase est une ADN polymerase ARN-dépendante qui allonge le télomère en lui ajoutant des séquences répétitives TTAGGG. Elle comprend une composante ARN (hTR) qui sert de matrice et une composante catalytique à activité transcriptase inverse (hTERT). Lexpression seule dhTERT suffit à immortaliser différents types cellulaires. La télomérase est fortement exprimée dans la majorité des cellules tumorales alors que son activité est difficilement détectable dans la plupart des cellules somatiques. Ces observations font de la télomérase une cible dintérêt pour des nouvelles thérapies anticancéreuses. Une de ces nouvelles stratégies consiste en lutilisation de molécules capables de stabiliser les structures en G-quadruplexe de lADN. La stabilisation des G-quadruplexes télomériques rend les télomères inaccessibles pour la télomérase et inhibe son activité par la séquestration de son substrat. Lobjectif de cette thèse est dévaluer la réponse cellulaire induite par le traitement cellulaire de deux ligands des G-quadruplexes au niveau du protéome des cellules WI38 transfectées pour exprimer hTERT. Les deux ligands, TMPyP4 et la télomestatine, inhibent la télomérase mais ont une spécificité différente pour les diverses structures G-quadruplexes. En premier lieu, nous avons étudié leffet de la transfection dhTERT sur des cellules fibroblastiques humaines (WI38). Cette première étude a été conduite afin de caractériser ladaptation cellulaire résultante de limmortalisation des cellules WI38. Par la suite, celle-ci permettra de comparer ces résultats avec ceux obtenus lors de létude protéomique de leffet des ligands des G-quadruplexes. Nous avons montré que hTERT induit une augmentation de la capacité fonctionnelle du réticulum endoplasmique ainsi quune modulation des signaux cellulaire Ca2+-dépendant. Nous proposons que cette adaptation cellulaire est responsable dune résistance accrue vis-à-vis de différents stress environnementaux. Dautres protéines impliquées dans des mécanismes doncogenèse ont été identifiées et sont différentiellement exprimées entre les cellules parentales et les cellules transfectées. Lanalyse protéomique des traitements cellulaires indique que TMPyP4 induit une altération du protéome beaucoup plus prononcée que celle induite par la télomestatine. Ceci est probablement dû au manque de spécificité de TMPyP4 pour les G-quadruplexes télomériques. TMPyP4 induit, entre autres, une sous-expression massive des hnRNPs, une modulation de la voie protéasomale, une diminution probable de la traduction et une surexpression de plusieurs chaperonnes moléculaires. La télomestatine induit notamment une surexpression de la protéine BCL2A1 qui est impliquée dans les processus de résistance aux agents anticancéreux et une probable augmentation de la traduction. Les deux ligands ont des effets communs sur la variation dexpression des chaperons CCT (sou-expression), de lHSP90 alpha (surexpression) et de lhnRNP D (sous-expression). LHSP90 est également surexprimée dans les cellules hTERT-WI38 par rapport aux cellules parentales. Cette protéine fait actuellement lobjet de nombreuses recherches visant à inhiber son activité du fait de son implication en oncogenèse ainsi que dans la modulation de lactivité de la télomérase. Enfin, nous avons montré lintérêt de ce type détude protéomique dans lévaluation dagents à vocation thérapeutique préalablement aux études cliniques.