69 research outputs found

    Non-destructive, label free identification of cell cycle phase in cancer cells by multispectral microscopy of autofluorescence

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    Background: Cell cycle analysis is important for cancer research. However, available methodologies have drawbacks including limited categorisation and reliance on fixation, staining or transformation. Multispectral analysis of endogenous cell autofluorescence has been shown to be sensitive to changes in cell status and could be applied to the discrimination of cell cycle without these steps. Methods: Cells from the MIA-PaCa-2, PANC-1, and HeLa cell lines were plated on gridded dishes and imaged using a multispectral fluorescence microscope. They were then stained for proliferating cell nuclear antigen (PCNA) and DNA intensity as a reference standard for their cell cycle position (G1, S, G2, M). The multispectral data was split into training and testing datasets and models were generated to discriminate between G1, S, and G2 + M phase cells. A standard decision tree classification approach was taken, and a two-step system was generated for each line. Results: Across cancer cell lines accuracy ranged from 68.3% (MIA-PaCa-2) to 73.3% (HeLa) for distinguishing G1 from S and G2 + M, and 69.0% (MIA-PaCa-2) to 78.0% (PANC1) for distinguishing S from G2 + M. Unmixing the multispectral data showed that the autofluorophores NADH, FAD, and PPIX had significant differences between phases. Similarly, the redox ratio and the ratio of protein bound to free NADH were significantly affected. Conclusions: These results demonstrate that multispectral microscopy could be used for the non-destructive, label free discrimination of cell cycle phase in cancer cells. They provide novel information on the mechanisms of cell-cycle progression and control, and have practical implications for oncology research.Jared M. Campbell, Abbas Habibalahi, Saabah Mahbub, Martin Gosnell, Ayad G. Anwer, Sharon Paton, Stan Gronthos and Ewa Goldy

    The Potential of Raman Spectroscopy in the Diagnosis of Premalignant Oral Lesions

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    Thesis submitted in partial fulfilment for the degree of Doctor of Philosophy (PhD) Technological University Dublin, School of Physics December 2017

    Inducible photoreceptor degeneration model in goldfish

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    2011 Summer.Includes bibliographical references.Photoreceptor degenerative diseases are among the leading causes of vision loss and there is presently no known cure. The future success of biological and prosthetic vision rescue approaches following photoreceptor loss remains questionable, due to the morphological and functional changes occurring in the remaining retinal circuitry. In the current study we sought to establish a chemically-induced photoreceptor degenerative model in goldfish, based on the ability of teleost to regenerate their retina following damage. N-methyl-N-nitrosourea (MNU) was chosen to chemically induce the photoreceptor degeneration, because it has been found to be potent, and selective in mammalian studies. We hypothesized that MNU would induce selective and complete photoreceptor loss in the goldfish retina as well as the consequent morphological changes observed in mammalian retinas. Under anesthesia, fish received a direct, intraocular injection of MNU into the posterior chamber of one eye whereas the contralateral eye served as sham-injected control. The effects of MNU were determined by standard immunohistochemical methods using known, well-established molecular markers of retinal cells. The MNU induced unilateral, selective, and dose-dependent photoreceptor degeneration: up to ~60% of photoreceptors lost the injected eye of the goldfish within 7 days, followed by nearly complete regeneration by ~50 days post-injection. Repeated MNU treatments did not increase the magnitude of degeneration, but delayed the regeneration. Unlike in mammals, MNU did not destroy all of the photoreceptors in fish. The incomplete photoreceptor degeneration together with the quick regeneration may be responsible for preventing the development of chronic morphological and functional consequences. However, the regeneration observed after MNU treatment is promising. Inducing total photoreceptor degeneration in fish retina, possibly by combining MNU with other factors shown to destroy photoreceptors (i.e. strong light) could provide an all-encompassing natural model for studying the potential of stem cell-based vision rescue approaches after photoreceptor loss

    The dynamical organization of the core pluripotency transcription factors responds to differentiation cues in early S-phase

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    DNA replication in stem cells is a major challenge for pluripotency preservation and cell fate decisions. This process involves massive changes in the chromatin architecture and the reorganization of many transcription-related molecules in different spatial and temporal scales. Pluripotency is controlled by the master transcription factors (TFs) OCT4, SOX2 and NANOG that partition into condensates in the nucleus of embryonic stem cells. These condensates are proposed to play relevant roles in the regulation of gene expression and the maintenance of pluripotency. Here, we asked whether the dynamical distribution of the pluripotency TFs changes during the cell cycle, particularly during DNA replication. Since the S phase is considered to be a window of opportunity for cell fate decisions, we explored if differentiation cues in G1 phase trigger changes in the distribution of these TFs during the subsequent S phase. Our results show a spatial redistribution of TFs condensates during DNA replication which was not directly related to chromatin compaction. Additionally, fluorescence fluctuation spectroscopy revealed TF-specific, subtle changes in the landscape of TF-chromatin interactions, consistent with their particularities as key players of the pluripotency network. Moreover, we found that differentiation stimuli in the preceding G1 phase triggered a relatively fast and massive reorganization of pluripotency TFs in early-S phase. Particularly, OCT4 and SOX2 condensates dissolved whereas the lifetimes of TF-chromatin interactions increased suggesting that the reorganization of condensates is accompanied with a change in the landscape of TF-chromatin interactions. Notably, NANOG showed impaired interactions with chromatin in stimulated early-S cells in line with its role as naĂŻve pluripotency TF. Together, these findings provide new insights into the regulation of the core pluripotency TFs during DNA replication of embryonic stem cells and highlight their different roles at early differentiation stages

    Ameliorative effect of virgin olive oil against nephrotoxicity following sub-chronic administration of ethephon in male rats

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    Background: Ethephon (EP) is the most famous plant growth regulator with different adverse effects on kidney function. Virgin Olive Oil (VOO) is considered as a natural source of antioxidant with beneficial effects. Thus, this study was conducted to investigate the effects of VOO on nephrotoxicity induced by EP in rats. Methods and materials: In this study, 80 male rats (weighing 200Ăąïżœïżœ250 g) were divided into four groups including I: control group received normal saline as vehicle, II: received VOO, III: received EP (150 mg/kg/day) for 2 months, IV: received EP (150 mg/kg/day for 2 months, after 2-month pretreatment with VOO. VOO (2 mL/kg/day) and vehicle were administered by gastric gavage for 2 months. At the end, the animals were sacrificed, and their blood and kidneys were used for examinations. Isolated kidneys were used for histopathological and oxidative stress studies. Results: Significant increases were recorded in blood (neutrophils, monocytes) and urinary parameters as well as malondialdehyde (MDA) content in the group III compared to groups II and I (PĂ‹ïżœ0.05). Antioxidant enzymes significantly declined and histopathological alterations increased in the group III. In the group IV, significant decreases were recorded in blood and urinary parameters, MDA, and histopathological alterations and a significant increase were found in antioxidant enzymes compared to group III (PĂ‹ïżœ0.05). Conclusions: Findings of the present study demonstrated protective effects of VOO in prevention of kidneys against EP -induced toxicity in albino rats. © 2019 Center for Food and Biomolecules, National Taiwan Universit

    Glucocorticoid effects in the regenerating fin reflect tissue homeostasis disturbances in zebrafish by affecting Wnt signaling

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    As a treatment for various immune-mediated diseases, the use of glucocorticoids as anti-inflammatory and immunosuppressive agents is common practice. However, their use is severely hampered by the risk of the development of adverse effects such as secondary osteoporosis, skin atrophy, and peptic ulcer formation. The exact molecular and cellular mechanisms underlying those adverse effects, which involve most major organ systems, are not yet fully understood. Therefore, their investigation is of great importance to improve treatment regimens for patients. Here, we investigated the effects of the glucocorticoid prednisolone on cell proliferation and Wnt signaling in homeostatic skin and intestinal tissue and compared them to the anti-regenerative effects in zebrafish fin regeneration. We also investigated a potential recovery from the glucocorticoid treatment and the impact of short-term treatment with prednisolone. We identified a dampening effect of prednisolone on Wnt signaling and proliferation in highly proliferative tissues, namely the skin and intestine, as well as reduced fin regenerate length and Wnt reporter activity in the fin. The presence of the Wnt inhibitor Dickkopf1 was enhanced in prednisolone treated skin tissue. A decreased number of mucous producing goblet cells was observed in the intestine of prednisolone treated zebrafish. Unexpectedly, proliferation in bone forming osteoblasts of the skull, homeostatic scales, as well as the brain was not decreased, opposite to the observed effects in the skin, fin, and intestine. Short-term treatment with prednisolone for a few days did not significantly alter fin regenerate length, skin cell proliferation, intestinal leukocyte number and proliferation of intestinal crypt cells. However, it affected the number of mucous-producing goblet cells in the gut. Likewise, discontinuation of prednisolone treatment for a few days saved the skin and intestine from a significant reduction of skin and intestinal cell proliferation, intestinal leukocyte number and regenerate length, but did not rescue goblet cell number. The suppressive effects of glucocorticoids in highly proliferative tissues may be relevant in the context of their therapeutic applications in patients with inflammatory diseases

    The role of different subtypes of olfactory bulb interneurons in olfactory behavior

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    Le bulbe olfactif (BO) reprĂ©sente dans le cerveau le premier relai dans le traitement des informations olfactives. Au niveau de cette structure, plusieurs types de neurones sont impliquĂ©s dans la modulation de l’information odorante, avant mĂȘme que celle-ci ne soit envoyĂ©e vers des structures corticales supĂ©rieures. Parmi eux se trouvent les cellules granulaires (CGs), une population d’interneurones rĂ©gulant de maniĂšre importante l’activitĂ© des cellules principales du BO. De maniĂšre intĂ©ressante, le BO est capable Ă  l’ñge adulte de produire et rĂ©gĂ©nĂ©rer une partie de sa population interneuronale via le processus de neurogĂ©nĂšse adulte. Il est ainsi possible de faire la distinction entre les CGs gĂ©nĂ©rĂ©es au cours de la pĂ©riode postnatale (CGs postnatales) des CGs gĂ©nĂ©rĂ©es Ă  l’ñge adulte (CGs nouvellement gĂ©nĂ©rĂ©es). Le rĂŽle que jouent ces CGs dans le traitement olfactif mais aussi dans les diffĂ©rents comportements olfactifs a pendant trĂšs longtemps donnĂ© lieu Ă  des interprĂ©tations contradictoires. Le manque de cohĂ©rence au niveau des donnĂ©es peut s’expliquer par le fait que pendant longtemps, les CGs ont Ă©tĂ© considĂ©rĂ©es comme Ă©tant une population homogĂšne de cellules. NĂ©anmoins, des Ă©tudes ont montrĂ© que les CGs peuvent exprimer diffĂ©rents marqueurs neurochimiques. Notamment, nous nous sommes intĂ©ressĂ©s dans le cadre de notre Ă©tude Ă  deux de ces marqueurs : la protĂ©ine kinase calcium calmoduline dĂ©pendante IIα (CaMKIIα) et la CalrĂ©tinine (CR). Une telle hĂ©tĂ©rogĂ©nĂ©itĂ© au sein des cellules interneuronales du BO pourrait Ă©galement reflĂ©ter une hĂ©tĂ©rogĂ©nĂ©itĂ© fonctionnelle, chaque sous-population de CGs pouvant contribuer de façon propre et unique au traitement des informations olfactives et donc au comportement olfactif. Dans la premiĂšre partie de ces travaux, nous avons Ă©tudiĂ© le rĂŽle fonctionnel des cellules exprimant la CaMKIIα et l’avons comparĂ© Ă  la population gĂ©nĂ©rale de CGs. De maniĂšre intĂ©ressante, nous montrons que, bien que ces deux populations de cellules soient en tous points semblables au niveau morphologique, les cellules CaMKIIα reçoivent un niveau d’inhibition moindre par rapport Ă  leurs homologies nĂ©gatives, les rendant plus susceptibles d’ĂȘtre activĂ©es suite Ă  des tĂąches comportementales spĂ©cifiques. De plus, l’inhibition spĂ©cifique des cellules CaMKIIα- positive entraine une perturbation des performances de discrimination fine. Dans la seconde partie de ces travaux, nous nous sommes intĂ©ressĂ©s cette foisci Ă  la sous-population de CGs exprimant la CR, en tenant compte Ă©galement de la pĂ©riode dĂ©veloppementale de ces cellules (i.e CGs post-natales ou nouvellement gĂ©nĂ©rĂ©es). Nous montrons que les cellules nouvellement gĂ©nĂ©rĂ©es exprimant ou non la CR, ainsi que les cellules CR-positives postnatales diffĂšrent quant Ă  leurs propriĂ©tĂ©s Ă©lectrophysiologiques. De plus, tout comme les cellules exprimant la CaMKIIα, les cellules exprimant la CR prĂ©sentent un niveau d’activation plus important Ă  la suite de certaines tĂąches comportementales et sont Ă©galement nĂ©cessaires Ă  la bonne rĂ©alisation de tĂąches de discrimination olfactive.The olfactory bulb (OB) is considered as the first relay in the brain during olfactory processing. Several types of neurons are involved at the level of this structure in the refinement of the olfactory information before it is sent to higher cortical structures. Among the cell types involved is the population of granule cells (GC), a population of interneurons largely regulating the activity of OB principal cells. Interestingly, the OB retain during adulthood the ability to produce and renew part of its interneuronal pool through a process called adult neurogenesis. Therefore, it is possible to distinguish in the adult OB between GCs born during the early postnatal period (early-born GCs) to the one that were generated during adulthood (adult-born GCs). Several studies aimed at determining the precise role played by GC in olfactory processing and olfactory behavior, giving rise quite often to conflicting results. This absence of coherence in the data could come from the fact that for long, the population of GCs was considered as a homogeneous cell population. However, GCs were shown to express diverse neurochemical markers. In this study we investigated more particularly into two of those markers, showed to be expressed by GCs: the Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) and Calretinin (CR). Hence, such a heterogeneity in the phenotype of OB interneurons could also underlie a functional heterogeneity of the different GC subpopulation, each one contributing in a unique way to olfactory processing and thus olfactory behavior. In the first part of this work, we investigated the functional role of CaMKIIα- expressing cells and compared it to the general population of GCs. Interestingly we revealed that CaMKIIα-positive GCs are more prone to activation following specific behavioral tasks, likely due to a decreased level of inhibition as compared to their negative counterparts. Moreover, the specific inhibition of this GC subpopulation let to alteration of animals’ fine discrimination abilities. In the second part of our work, when focusing this time on the subpopulation of CR-expressing GCs, taking this time also into account the developmental period at which they were generated (i.e early- versus adult-born cells), we showed that adultborn CR-expressing and non-expressing GCs, but also early-born CR-expressing GCs display different electrophysiological characteristics. Moreover, as for CaMKIIα- positive GCs, CR-positive GCs present a higher level of activation following specific olfactory tasks and are also important for a proper ability to perform olfactory discrimination tasks

    Charting PARP-1 dependent mechanisms for DNA double-strand break resection

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    L'intĂ©gritĂ© de l'ADN gĂ©nomique humain est maintenue par des systĂšmes de rĂ©paration de l'ADN qui protĂšgent les cellules des dommages causĂ©s par des agents environnementaux ou des lĂ©sions spontanĂ©es de l'ADN. Chaque cellule peut subir jusqu'Ă  10⁔ lĂ©sions par jour, y compris les cassures double-brin de l'ADN (CDB). La poly(ADPribosyl)ation (PARylation) est l'un des premiers Ă©vĂ©nements de signalisation molĂ©culaire survenant aux CDBs. Il est catalysĂ© par les poly(ADP-ribose)polymĂ©rases (PARP) qui sont directement activĂ©es par ces lĂ©sions d'ADN. Le fait de ne pas gĂ©nĂ©rer de poly(ADP)ribosyl (pADPr) en rĂ©ponse Ă  des dommages Ă  l'ADN par une inhibition chimique ou par l'absence de PARP-1 augmente la sensibilitĂ© cellulaire au stress gĂ©notoxique, indiquant que la pADPr elle-mĂȘme est une molĂ©cule clĂ© de signalisation des dommages Ă  l'ADN. L'inhibition de l'enzyme de signalisation des dommages Ă  l'ADN, la poly(ADP-ribose) polymĂ©rase-1 (PARP-1) est l'une des nouvelles thĂ©rapies les plus prometteuses contre le cancer. Les inhibiteurs de PARP sensibilisent les cellules cancĂ©reuses aux agents endommageant l'ADN et tuent efficacement les cellules cancĂ©reuses du sein, des ovaires et du pancrĂ©as dĂ©ficientes en BRCA1 (Breast Cancer gene 1) et BRCA2 (Breast Cancer gene 2), ce qui suggĂšre que les cellules dĂ©ficientes en rĂ©paration des CDBs sont extrĂȘmement sensibles Ă  l'inhibition de PARP. Pourtant, les mĂ©canismes sous-jacents Ă  cette lĂ©talitĂ© synthĂ©tique entre le dĂ©ficit de rĂ©paration du CDB et l'inhibition de PARP restent mal dĂ©finis. Il y a un dĂ©bat considĂ©rable sur le mĂ©canisme par lequel l'inhibition de PARP tue les cellules dĂ©ficientes en rĂ©paration de l'ADN, et le plein potentiel des inhibiteurs de PARP dans le traitement du cancer ne peut ĂȘtre obtenu que par une comprĂ©hension claire des voies de rĂ©ponse aux dommages de l'ADN (DDR) aux CDB et comment ils sont affectĂ©s par les inhibiteurs de PARP. L'objectif gĂ©nĂ©ral de ma thĂšse est d'Ă©tudier le rĂŽle de PARP-1 dans la rĂ©paration DSB et d'identifier les interacteurs de PARP-1 qui jouent Ă©galement un rĂŽle dans ce processus. Les cellules eucaryotes rĂ©parent les CDBs par deux voies principales, la jonction d'extrĂ©mitĂ© non homologue (NHEJ) et la recombinaison homologue (HR). La HR est initiĂ©e par la liaison des CDBs par BRCA1 et le complexe MRE11-RAD50-NBS1 et des nuclĂ©ases EXO1/DNA2 pour gĂ©nĂ©rer de l'ADN simple-brin, qui est ensuite utilisĂ© par la recombinase RAD51 et le complexe BRCA1-PALB2-BRCA2. Une question clĂ© dans notre domaine concerne les facteurs critiques pour rĂ©guler le choix de la voie CDB. HR est initiĂ©e Ă  partir d'extrĂ©mitĂ©s DSB hautement rĂ©sectĂ©es, tandis que dans le NHEJ, la rĂ©section est empĂȘchĂ©e par des facteurs de rĂ©paration clĂ©s incluant RIF1 et 53BP1. En utilisant des cellules dĂ©ficientes en PARP-1, nous avons observĂ© que deux inhibiteurs de la rĂ©section de l'ADN et des rĂ©gulateurs de choix de voie, RIF1 et 53BP1, la formation de foyers induits par des dommages Ă  l'ADN sont fortement altĂ©rĂ©s. Cela confirme notre hypothĂšse selon laquelle PARP-1 participe Ă  la rĂ©paration du DSB en influençant la rĂ©section de l'ADN. Afin de mieux comprendre le mĂ©canisme de rĂ©section et le rĂŽle que PARP-1 y joue, nous avons identifiĂ© d'autres protĂ©ines qui interagissent avec PARP-1 et modulent ce processus. Pour ce faire, nous avons utilisĂ© des donnĂ©es sur les protĂ©ines de liaison au pADPr gĂ©nĂ©rĂ©es Ă  la fois dans notre laboratoire et celui de notre collaborateur Ted Dawson de Johns Hopkins. Les candidats sĂ©lectionnĂ©s Ă  partir de ces listes ont Ă©tĂ© criblĂ©s pour identifier une seule cible qui dĂ©montrerait un phĂ©notype similaire Ă  la perte de PARP-1. Deux cibles initiales ont Ă©tĂ© explorĂ©es et finalement une seule protĂ©ine Ă  doigt de zinc a Ă©tĂ© choisie comme cible principale. Nous devons relever la fonction de ce doigt de zinc en HR, dans l'espoir qu'il permettra de dĂ©couvrir davantage les mĂ©canismes de PARP-1 en rĂ©section. En rĂ©sumĂ©, cette thĂšse Ă©lucide le rĂŽle de PARP-1 dans la rĂ©section de l'ADN et identifie une protĂ©ine Ă  doigt de zinc non Ă©tudiĂ©e auparavant qui interagit avec PARP-1 et partage une fonction similaire Ă  PARP-1 dans la rĂ©section de l'ADN.The integrity of human genomic DNA is maintained by DNA repair systems that will protect cells from damage by environmental agents or spontaneous DNA lesions. Each cell can experience up to 10⁔ lesions daily, including DNA double-strand breaks (DSB)s. Poly(ADP-ribosyl)ation (PARylation) is one of the earliest molecular signalling events occurring at DNA DSBs. It is catalysed by poly(ADP-ribose) polymerases (PARPs) that are directly activated by those DNA lesions. Failure to generate pADPr in response to DNA damage by either chemical inhibition or absence of PARP-1 increases the cellular sensitivity to genotoxic stress, indicating that pADPr itself is a key DNA damage signalling molecule. Inhibition of the DNA damage signalling enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is among the most promising new therapies in cancer. PARP inhibitors sensitize cancer cells to DNA damaging agents and efficiently kill BRCA1- and BRCA2-deficient breast, ovarian and pancreatic cancer cells, suggesting that cells deficient in DSB repair are exquisitely sensitive to PARP inhibition. Yet, the mechanisms underlying this synthetic lethality between DSB repair deficiency and PARP inhibition remain poorly defined. There is considerable debate about the mechanism through which PARP inhibition kills DNA repair-deficient cells, and the full benefit of PARP inhibitors in cancer therapy can only be achieved by a clear understanding of the DNA damage response (DDR) pathways to DSBs and how these are affected by PARP inhibitors. The overall aim of my PhD is to investigate the role of PARP-1 in DSB repair and identify interactors of PARP-1 which also play a role in this process. Eukaryotic cells repair DSBs by two major pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). HR is initiated by the binding of DSB by BRCA1 and the end resection of the DSB by MRE11 (and the associated NBS1, RAD50, CtIP, and EXO1) to generate single-stranded DNA, which is further processed by RAD51 and BRCA1-PALB2-BRCA2. A key question in our field regards which factors are critical for regulating the DSB pathway choice. HR is initiated from highly resected DSB ends, whereas in NHEJ, resection is prevented by key repair factors that include RIF1 and 53BP1. Using PARP-1-deficient cells, we have observed that two inhibitors of DNA resection and regulators of pathway choice, RIF1 and 53BP1, are strongly impaired in forming DNA damage-induced foci. This supports our hypothesis that PARP-1 participates in DSB repair by influencing DNA resection. In order to further understand the mechanism of resection and the role that PARP-1 plays in it we also aim to identify other proteins which interact with PARP-1 and modulate this process. To accomplish this, we made use of data on PAR binding proteins generated both in our lab and that of our collaborator Ted Dawson. The candidates selected from these lists were screened to identify a single target that would demonstrate a similar phenotype to PARP-1 loss. Two initial targets were further explored and finally a single zinc finger protein was selected as our primary target. We aim to characterize the function of this zinc finger in HR, in the hopes that it will further uncover the mechanisms of PARP-1 in resection. In summary this thesis elucidates the role of PARP-1 in DNA resection and identifies a previously unstudied zinc finger protein which interacts with PARP-1 and shares a similar function to PARP-1 in DNA resection

    RÎle du gÚne Polycomb BMI1 dans le maintien et la radiorésistance des cellules souches cancéreuses

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    Le glioblastome multiforme (GBM) est la tumeur cĂ©rĂ©brale la plus commune et lĂ©tale chez l’adulte. MalgrĂ© les avancĂ©s fulgurantes dans la derniĂšre dĂ©cennie au niveau des thĂ©rapies contre le cancer, le pronostique reste inchangĂ©. Le manque de spĂ©cificitĂ© des traitements est la cause premiĂšre de la rĂ©currence de cette tumeur. Une meilleure comprĂ©hension au niveau des mĂ©canismes molĂ©culaires et biologiques de cette tumeur est impĂ©rative. La dĂ©couverte des cellules souches cancĂ©reuses (CD133+) au niveau du GBM offre une nouvelle opportunitĂ© thĂ©rapeutique contre cette tumeur. Effectivement, les cellules CD133+ seraient responsables de l’établissement, le maintien et la progression du GBM. De plus, elles sont Ă©galement la cause de la rĂ©sistance du GBM faces aux traitements de radiothĂ©rapies. Ces cellules reprĂ©sentent une cible de choix dans le but d’éradiquer le GBM. L’oncogĂšne BMI1 a Ă©tĂ© associĂ© Ă  plusieurs types de tumeurs et est Ă©galement essentielle au maintien de diffĂ©rentes populations de cellules souches normales et cancĂ©reuses. Une forte expression de BMI1 est observĂ©e au niveau du GBM et plus prĂ©cisĂ©ment, un enrichissement prĂ©fĂ©rentiel de cette protĂ©ine est notĂ© au niveau des cellules CD133+. L’objectif principal de cette thĂšse est d’évaluer le rĂŽle potentiel de BMI1 dans le maintien et la radiorĂ©sistance des cellules souches cancĂ©reuses (CSC), CD133+ du GBM. La fonction principale de BMI1 est la rĂ©gulation nĂ©gative du locus INK4A/ARF. Ce locus est impliquĂ© dans l’activation de deux voies majeurs anti-tumorales : P53 et RB. Or, la perte de BMI1 induit in vitro une diminution des capacitĂ©s prolifĂ©ratives, une augmentation de la diffĂ©rentiation et de l’apoptose, ainsi qu’une augmentation de la radiosensibilitĂ© des CSC du GBM indĂ©pendamment de la prĂ©sence du locus INK4A/ARF. Effectivement, deux tumeurs sur trois possĂšdent une dĂ©lĂ©tion de ce locus, ce qui suggĂšre que BMI1 possĂšde d’autre(s) cible(s) transcriptionnelle(s). Parmi ces nouvelles cibles ont retrouve la protĂ©ine P21, un rĂ©gulateur nĂ©gatif du cycle cellulaire. De plus, la perte de BMI1 inhibe l’établissement d’une tumeur cĂ©rĂ©brale lors d’études de xĂ©nogreffe chez la souris NOD/SCID. Également, une nouvelle fonction de BMI1 indĂ©pendante de son activitĂ© transcriptionnel a Ă©tĂ© dĂ©montrĂ©e. Effectivement, suite Ă  l’induction d’un bris double brin (BDB) de l’ADN, BMI1 est rapidement recrutĂ© au niveau de la lĂ©sion et influence le recrutement des protĂ©ines de reconnaissance du dommage Ă  l’ADN. La perte de BMI1 mĂšne Ă  un dĂ©faut au niveau de la reconnaissance et la rĂ©paration de l’ADN, alors que sa surexpression induit plutĂŽt une augmentation de ces mĂ©canismes et procure une radiorĂ©sistance. Ces rĂ©sultats dĂ©crivent pour la premiĂšre fois l’importance de BMI1 au niveau du maintien, de l’auto-renouvellement et la radiorĂ©sistance des CSC du GBM. Ainsi, ces travaux dĂ©montrent que la protĂ©ine BMI1 reprĂ©sente une cible thĂ©rapeutique de choix dans le but d’éradiquer le GBM, une tumeur cĂ©rĂ©brale lĂ©tale.Glioblastoma multiform (GBM) is the most common and lethal primary brain tumor found in adults. Despite the advances made in the field of cancer therapy in the last decade, the median survival rate remains less than a year. Therefore, a better understanding of the molecular biology of GBM will reveal the mechanisms responsible for the initiation and progression of the tumor, and allow the development of new therapeutic strategies. GBM contains a minority cell population, characterized by tumor initiating cells expressing the stem cell marker, CD133. The CD133+ GBM cells are responsible for tumor initiation, maintenance, progression and resistance to chemo/radiotherapy. The CD133+ cells represent a valuable and specific therapeutic target against GBM. The Polycomb (PcG) group family of transcriptional repressors have been involved in a vast range of cancers. The PcG protein and oncogene BMI1 is the best-characterized PcG protein. The implication of BMI1 in normal and cancer stem cell survival, self-renewal and maintenance has been thoroughly investigated. BMI1 is highly expressed in GBM and more precisely; it is enriched specifically in CD133+ cell populations. The main goal of this thesis was to elucidate the potential role of BMI1 in GBM CD133 + cancer stem cell (CSC) maintenance and radioresistance. The main function of BMI1 is to repress the expression of the genes encoded by the INK4A/ARF locus, which is implicated in the activation of two major tumor suppressor pathways, P53 and RB. However, BMI1 depletion in vitro induces a reduction in proliferation potential, as well as an increase in differentiation, apoptosis, and radiosensitivity regardless of INK4A/ARF status. Indeed, two-thirds of all tumors posses a deletion of this locus, suggesting that BMI1 regulates other targets. P21, a cell cycle regulator, was identified as a new BMI1 target. Moreover, we have observed that the loss of BMI1 inhibits the establishment of a cerebral tumor in a xenograft mouse model. In addition to transcription related activity, we identified a new transcription independent function of BMI1. After the induction of a DNA double-strand-break, BMI1 is rapidly recruited to the damage site and influences the recruitment of DNA damage response proteins. Furthermore, defects in DNA damage recognition and repair are observed after BMI1 knockdown. Consistent with these results, BMI1 overexpression induces DNA damage response and increases radioresistance potential. These results emphasize for the first time the requirement of BMI1 for the maintenance, self-renewal, and radioresistance in GBM CSC, thus providing a potential target for future therapeutic strategies against GBM
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