Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Les papillomavirus humains (HPV) sont des petits virus non enveloppés à ADN double brin, qui infectent les épithéliums cutanés et muqueux. Très largement répandus dans la population humaine, les infections sont la plupart du temps asymptomatiques, tandis qu'une minorité provoquent des verrues cutanées, des condylomes acuminés et des carcinomes. Les cancers induits par HPV représentent 640 000 cas en 2012 dans le monde et sont localisés au niveau anogénital et au niveau des voies aérodigestives supérieures.La transformation des cellules est induite en particulier par la surexpression de deux oncoprotéines virales : E6 et E7, qui sont nécessaires à l'initiation, la promotion et la progression du phénotype cancéreux. Leur extinction provoque d'ailleurs la mort des cellules cancéreuses. E6 et E7 favorisent par exemple la dégradation de p53 et pRb, induisant ainsi une prolifération non contrôlée et accrue des cellules infectées. Mais les deux oncoprotéines virales interagissent aussi avec plus de 150 partenaires protéiques et dérégulent l'homéostasie cellulaire. Fait intéressant, E6 et E7 modulent l'expression de nombreuses protéines impliquées dans l'établissement des marques épigénétiques, impliquées dans la régulation de l'expression des gènes. D'ailleurs, la surexpression des oncoprotéines virales peut être liée à la méthylation de l'ADN d'une partie du génome viral.Puisque E6 et E7 détournent la machinerie épigénétique de la cellule, l'objectif du travail de thèse a été d'étudier les mécanismes moléculaires régulant l'expression des oncoprotéines lors du traitement des cellules de cancers du col par un agent déméthylant de l'ADN, le 5-aza-2'-déoxycytidine (5azadC).Dans des cellules issues de cancers du col de l'utérus, nous avons observé qu'un traitement déméthylant induisait une répression de la protéine E6 et une déstabilisation de ses transcrits, suggérant l'implication d'un miARN. Parmi les miARN ciblant les transcrits viraux, le miR-375 était le plus susceptible d'être impliqué dans la déstabilisation des transcrits car (i) la méthylation de son promoteur augmente avec la gravité des lésions du col de l'utérus, (ii) il cible les transcrits viraux au niveau de cinq régions différentes, et (iii) son expression est augmentée lors du traitement de cellules dérivées du cancer du col de l'utérus avec le 5azadC. Il s'est avéré que le miR-375 était effectivement impliqué dans la répression partielle des transcrits viraux lors du traitement. Les résultats de cette étude suggèrent par ailleurs qu'un autre mécanisme moléculaire pourrait être impliqué dans cette répression. C'est pourquoi, dans un deuxième temps, nous avons étudié l'implication possible d'un facteur de transcription, TBX2, dans la répression des transcrits viraux. Bien que le taux transcriptionnel de TBX2 soit augmenté lors du traitement des cellules avec des fortes concentrations de 5azadC, nos résultats suggèrent que la protéine n'est pas exprimée dans les cellules, même après traitement, et que TBX2 ne régule pas l'activité du promoteur viral intégré au génome cellulaire.Le traitement des cellules par l'agent déméthylant entraîne aussi une perte de viabilité en particulier des cellules Ca Ski. En effet, le 5azadC induit un blocage du cycle en G2/M dans ces cellules, une augmentation du pourcentage de cellules en Sub-G1 et un clivage de la PARP. Si le 5azadC est déjà utilisé en clinique pour le traitement d'autres cancers, nos données apportent un éclairage nouveau sur les mécanismes moléculaires du 5azadC dans les cancers induits par HPV, qui pourraient ainsi être traités par cette molécule. / Human papillomaviruses (HPV) are small, non-enveloped double-stranded DNA viruses and infect cutaneous and mucosal epithelia. Highly widespread in the human population, infections are mostly asymptomatic, while a minority causes cutaneous and genital warts, and carcinomas. HPV-induced cancers represent 640 000 cases in 2012 worldwide and are localized at the anogenital and head and neck sites.Cell transformation is induced by the overexpression of two viral oncoproteins E6 and E7, which are necessary for the initiation, promotion and progression of the cancerous phenotype. Indeed, their extinction causes cancer cell death. E6 and E7, for example, promote the degradation of p53 and pRb, thus inducing uncontrolled and increased proliferation of infected cells. But the two viral oncoproteins also interact with more than 150 protein partners and deregulate cell homeostasis. Interestingly, E6 and E7 modulate the expression of many proteins involved in the establishment of epigenetic marks, regulating gene expression. Furthermore, the overexpression of viral oncoproteins may be related to viral genome DNA methylation.Since E6 and E7 hijack epigenetic mechanisms, the aim of the thesis was to study the molecular mechanisms regulating the oncoprotein expression during the treatment of cervical cancer cells by a DNA demethylating agent, the 5-aza-2'-deoxycytidine (5azadC).In cervical cancer cells, we observed that a demethylating treatment induced E6 protein repression and destabilization of its transcripts, suggesting the involvement of a miRNA. Among miRNAs targeting viral transcripts, miR-375 was most likely to be involved this destabilization because (i) its promoter methylation increases with the severity of cervical lesions, (ii) it targets viral transcripts at five different regions, and (iii) its expression is increased in cervical cancer cells treated with 5azadC. In our experiences, miR-375 was indeed involved in the partial repression of viral transcripts during treatment. The results of this study further suggest that another molecular mechanism might be implicated in this repression. This is why, in a second step, we studied the possible involvement of a transcription factor, TBX2, in the viral transcript repression. Although the transcriptional rate of TBX2 is increased in cells treated with high concentrations of 5azadC, our results suggest that the protein is not expressed in cells, even after treatment, and that TBX2 does not regulate the viral promoter activity integrated into the cellular genome.Cell treatment with demethylating agent also leads to loss of viability, in particular in Ca Ski cells. Indeed, 5azadC induces a G2/M cycle arrest in these cells, an increase of Sub-G1 cells percentage and a PARP cleavage. While 5azadC is already used in clinical practice for the treatment of other cancers, our data shed new light on the molecular mechanisms of 5azadC in cancers induced by HPV, which could be treated by this molecule or analogues.

Background: The genes coding for human (h) chorionic somatomammotropin (CS), hCS-A and hCS-B, and placental growth hormone (GH-V), hGH-V are located at a single locus on chromosome 17q22-24. Local regulatory (5´ P and 3´ enhancer) sequences and a remote locus control region (LCR) containing a placenta-specific hypersensitive site (HS) IV, have been implicated in the efficient expression of the placental hCS/GH-V genes, in part through gene transfer studies in placental and non-placental tumor cell lines. However, low levels of endogenous expression are reported in placental tumor cells compared to normal term placenta. Thus it was hypothesized that the hCS/GH-V chromatin structure in human choriocarcinoma cells is less accessible to regulatory regions essential for efficient expression due to DNA and/or histone modifications, specifically methylation and acetylation, respectively. Approach: To assess individual hCS-A, hCS-B and hGH-V gene expression in placental and non-placental tumor cells, and assess the effect of increasing “chromatin accessibility” on hCS/GH-V RNA levels by inhibiting DNA methylation and histone deacetylation using 5-aza-2´-deoxycytidine (azadC) and trichostatin A (TSA). Principal Findings: Low levels of hCS-A, hCS-B and hGH-V RNA were detected in placental and non-placental tumor cells compared to term placenta. A significant >5-fold increase in promoter activity was seen in placental but not non-placental cells transfected with hybrid hCS promoter luciferase genes containing 3´-enhancer sequences. Placental JEG-3 cells pretreated with azadC and TSA resulted in a significant >10-fold increase in hCS-A, hCS-B and hGH-V RNA levels compared to TSA treatment alone, however, a modest ~3-fold effect was seen in non-placental MCF-7 cells. By contrast to the effect of pretreatment with azadC, post-treatment with azadC mutes the stimulatory effects of TSA on hCS/GH-V transcripts. The specificity of the response suggests that azadC treatment, and presumably hypomethylation of DNA, results in an increase in response to TSA and histone hyperacetylation at the hGH/CS locus. An assessment of histone H3/H4 hyperacetylation in JEG-3 cells treated with azadC and TSA versus TSA alone revealed significant increases consistent with a more open chromatin structure including the hCS 3´-enhancer sequences and LCR. Conclusions: These observations suggest that accessibility of remote and local regulatory regions required for efficient placental hGH/CS expression can be restricted by DNA methylation and histone acetylation status. This includes restricting access of the hCS 3´-enhancer sequences to available placental enhancer transcription factors. / May 2016

Promoter hypermethylation mediated gene silencing is a frequent epigenetic finding in many cancers that affects genes known to have important roles in several aspects of cell biology. Hematological malignancies have been reported to harbor multiple genes aberrantly silenced by promoter hypermethylation and as a result, cytosine analogs known to inhibit the DNA methylation machinery are currently being evaluated in clinical trials. As such, the general goal of this thesis was to identify genes silenced by promoter hypermethylation in human acute myeloid leukemia (AML) and to study the mechanism of promoter hypermethylation mediated gene silencing. Interestingly, the cyclin dependent kinase inhibitor p15 was found to be methylated at a high frequency in AML patients and cell lines in association with a lack of detectable p15 mRNA. Treatment with the cytosine analog 5-Aza-2-deoxycytidine (5-Aza-dC) in vitro resulted in promoter demethylation and p15 mRNA re-expression, which was associated with a release of a transcriptionally repressive complex at the p15 promoter. Importantly, 5-Aza-dC treatment also reversed specific histone amino-terminal modifications at the p15 promoter which are normally associated with transcriptionally inactive chromatin regions, implicating chromatin remodeling in promoter hypermethylation mediated gene silencing. The recently discovered DNA methylation inhibitor, zebularine considered more stable than 5-Aza-dC was also able to reconstitute p15 mRNA in vitro in association with promoter demethylation, regional enrichment of histone acetylation, and growth inhibition. To identify novel genes silenced by promoter hypermethylation in AML, cDNA microarray analysis was employed following in vitro pharmacological inhibition of DNA methylation and histone deacetylation. Of note, four genes from the metallothionein family of cysteine rich small molecules were consistently upregulated following drug treatment and further evaluation identified the gene MT1H to be hypermethylated at a high frequency in AML patients and cell lines. Taken together, the data suggests that aberrant promoter hypermethylation mediated gene silencing occurs in multiple genes from different gene families during the molecular pathogenesis of human AML. Furthermore, the mechanism of promoter methylation mediated transcriptional silencing acts in concert with specific histone modifications which, importantly, can be reversed by treatment with pharmacological inhibitors of DNA methylation.

Promoter hypermethylation mediated gene silencing is a frequent epigenetic finding in many cancers that affects genes known to have important roles in several aspects of cell biology. Hematological malignancies have been reported to harbor multiple genes aberrantly silenced by promoter hypermethylation and as a result, cytosine analogs known to inhibit the DNA methylation machinery are currently being evaluated in clinical trials. As such, the general goal of this thesis was to identify genes silenced by promoter hypermethylation in human acute myeloid leukemia (AML) and to study the mechanism of promoter hypermethylation mediated gene silencing. Interestingly, the cyclin dependent kinase inhibitor p15 was found to be methylated at a high frequency in AML patients and cell lines in association with a lack of detectable p15 mRNA. Treatment with the cytosine analog 5-Aza-2-deoxycytidine (5-Aza-dC) in vitro resulted in promoter demethylation and p15 mRNA re-expression, which was associated with a release of a transcriptionally repressive complex at the p15 promoter. Importantly, 5-Aza-dC treatment also reversed specific histone amino-terminal modifications at the p15 promoter which are normally associated with transcriptionally inactive chromatin regions, implicating chromatin remodeling in promoter hypermethylation mediated gene silencing. The recently discovered DNA methylation inhibitor, zebularine considered more stable than 5-Aza-dC was also able to reconstitute p15 mRNA in vitro in association with promoter demethylation, regional enrichment of histone acetylation, and growth inhibition. To identify novel genes silenced by promoter hypermethylation in AML, cDNA microarray analysis was employed following in vitro pharmacological inhibition of DNA methylation and histone deacetylation. Of note, four genes from the metallothionein family of cysteine rich small molecules were consistently upregulated following drug treatment and further evaluation identified the gene MT1H to be hypermethylated at a high frequency in AML patients and cell lines. Taken together, the data suggests that aberrant promoter hypermethylation mediated gene silencing occurs in multiple genes from different gene families during the molecular pathogenesis of human AML. Furthermore, the mechanism of promoter methylation mediated transcriptional silencing acts in concert with specific histone modifications which, importantly, can be reversed by treatment with pharmacological inhibitors of DNA methylation.

The full text will be available at the end of the embargo: 10th May 2024

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Les gènes suppresseurs de tumeurs (TSGs) contrôlent la prolifération cellulaire et leur inactivation joue un rôle important dans la leucémogénèse. Deux mécanismes épigénétiques majeurs sont impliqués dans la répression des TSGs: 1- la méthylation de l’ADN et 2- la déacétylation des histones des chromosomes. On les dit épigénétiques car ils n’affectent pas la séquence de l’ADN. Ces phénomènes sont réversibles, faisant donc d’eux des cibles thérapeutiques de choix. Dans le cadre de cette thèse, nous avons évalué le potentiel chimiothérapeutique de différents agents qui visent ces mécanismes épigénétiques et nous les avons administrés seuls et en combinaison dans le but d’améliorer leur efficacité. La 5-aza-2’-désoxycytidine (5-Aza-CdR) est un inhibiteur de la méthylation de l’ADN qui permet la ré-expression des TSGs. Cet agent s’est avéré efficace contre certaines maladies hématologiques et est d’ailleurs approuvé aux États-Unis dans le traitement du syndrome myélodysplasique depuis 2006. Cependant, le protocole d’administration optimal de cet agent, en termes de doses et de durée, n’est toujours pas établi. Nos recherches suggèrent que le celui-ci devrait être plus intensif que ce que rapporte la littérature. Les inhibiteurs des déacétylases des histones (HDACi) ont également montré une activité antinéoplasique intéressante. De récentes recherches ont montré que la combinaison d’agents ciblant à la fois la méthylation de l’ADN et la déacétylation des histones produit une réactivation synergique des TSGs, ce à quoi nous nous sommes intéressé. Nous avons observé que la co-administration d’un HDACi avec la 5-Aza-CdR potentialise son action anti-leucémique. Il est aussi possible d’augmenter l’activité de la 5-Aza-CdR en inhibant sa dégradation par l’enzyme cytidine (CR) désaminase. Nous avons observé que la co-administration du zebularine, un inhibiteur de la CR désaminase, avec la 5-Aza-CdR accroît son efficacité. Le zebularine est aussi un inhibiteur de la méthylation de l’ADN, ce qui pourrait contribuer à la potentialisation de la réponse anti-leucémique observée lors de la co-administration de ces deux agents. En résumé, il est possible d’augmenter l’efficacité anti-leucémique de la 5-Aza-CdR en : 1- intensifiant son protocole d’administration, en termes de doses et de durée, 2- la combinant avec un HDACi, et 3- diminuant sa dégradation par la CR désaminase. L’utilisation de ces résultats précliniques dans l’élaboration de protocoles cliniques pourrait être bénéfique à beaucoup de patients. / The silencing of tumor suppressor genes (TSG) that normally regulate cells proliferation plays an important role in leukemogenesis. Two major mechanisms are involved in TSG’s silencing: DNA methylation and histones deacetylation. Because those phenomenons are reversible, it makes them interesting therapeutic targets for chemotherapeutic agents. We evaluated the antineoplastic potential of different agents that target those events and we administered them alone or in combination with the goal of improving their efficiency. 5-aza-2’-deoxycytidine (5-Aza-CdR) is a DNA methylation inhibitor that can re-express TSGs that are silenced by methylations. This agent demonstrated its efficacy against hematological malignancies. Therefore, 5-Aza-CdR is used since 2006 in United States of America against myelodysplastic syndrome; but its optimal dose-schedule still needs to be established. Our researches suggest that the dose-schedule of 5-Aza-CdR should be more intensive than what is reported from the literature. Inhibitors of histones deacetylation (HDACi) also demonstrated some interesting antineoplastic activity. Recently, observations showed that combination of chemotherapeutic agent that targets both DNA methylation and histones deacetylation lead to a synergic reactivation of silenced TSG. This finding allowed us to observe that the co-administration of an HDACi with 5-Aza-CdR improve its antileukemic potential. Moreover, it is possible to increase the activity of 5-Aza-CdR by preventing its degradation by cytidine (CR) deaminase. We demonstrated that the co-administration of zebularine, an inhibitor of CR deaminase, with 5-Aza-CdR increases its activity. Zebularine is also an inhibitor of DNA methylation, which may contribute to the enhancement of the antileukemic action of this combination. In summary, our preclinical data indicate that the antileukemic activity of 5-Aza-CdR can be enhanced by: 1- increasing his dosage, 2- combining it with HDACi, and 3- preventing its inactivation by CR deaminase. The translation of those preclinical observations into clinical protocols may be effective in patients with advanced leukemia.

Embora a transferência nuclear de células somáticas (TNCS) seja uma ferramenta promissora, seu amplo uso é impedido devido às altas taxas de mortalidade durante o desenvolvimento dos animais clonados. Acredita-se que a reprogramação epigenética anormal seja a principal causa desta baixa eficiência. Nós hipotetizamos que agentes modificadores de cromatina (AMCs) atingindo a acetilação das histonas e a metilação do DNA poderiam alterar a configuração da cromatina e torná-la mais facilmente reprogramável. Deste modo, fibroblastos bovinos foram tratados com 5-aza-2\'-deoxicitidina (AZA) mais tricostatina A (TSA) ou hidralazina (HH) mais ácido valpróico (VPA) enquanto, em outro experimento, zigotos bovinos clonados foram tratados com TSA. O tratamento dos fibroblastos com AZA+TSA ou HH+VPA aumentou a acetilação das histonas, mas não afetou o nível de metilação do DNA. Entretanto, o tratamento com HH+VPA diminuiu a viabilidade/proliferação celular. O uso destas células como doadoras de núcleo não mostrou efeitos positivos sobre o desenvolvimento pré- e pós-implantação. Em relação ao tratamento dos zigotos clonados com TSA, o tratamento destes mostrou um aumento nos padrões de acetilação das histonas, mas não reduziu o nível de metilação do DNA. Além disso, este tratamento não resultou em efeito positivo sobre o desenvolvimento pré- e pósimplantação. Este trabalho fornece evidências de que o tratamento de células doadoras de núcleo ou zigotos clonados com AMCs não tem efeito positive sobre o desenvolvimento pré- e pós-implantação de bovinos clonados. / Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatinmodifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2\'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and post-implantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and post-implantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and post-implantation development of cloned cattle.