TCTP est une protéine de 20 kDa que l’on retrouve souvent sous forme de dimère. Elle est fortement conservée dans la phylogénie et on la trouve dans les levures, les plantes, les invertébrés et les mammifères. Elle est localisée dans tous les compartiments de la cellule : noyau, cytoplasme, et membranes. Il s’agit d’une protéine très abondante dans des cellules souches ainsi que des cellules en croissance exponentielle, y compris les cellules tumorales. Sa fonction principale est celle d’une « protéine de survie ». TCTP a été décrite comme interagissant avec de multiples protéines dont p53, MDM2, Bcl-xL et TSAP6. Le but de mon travail est de permettre de mieux caractériser ces fonctions de TCTP et pour cela, nous avons étudié ses interactions in vitro et surtout, in vivo, dans différents modèles génétiques chez la souris. / TCTP is a 20 kDa protein frequently encountered as a dimer. It is highly conserved through phylogeny and is present inn yeast, plants, invertebrates and mammals. It is localized in all compartments of the cell: nucleus, cytoplasm, membranes. This protein is highly abundant in stem cells and during the exponential growth, including in cancer cells. It mainly functions as a survivor factor. TCTP has been described as interacting with multiple proteins, including p53, MDM2, Bcl-xL and TSAP6. The purpose of my work is to better characterize these functions of TCTP; we therefore studied its interactions in vitro, but mostly in vivo, using different murine genetic models.

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Mitochondrial outer membrane permeabilization (MOMP) is regulated by protein-protein and protein-membrane interactions between Bcl-2 family proteins. These interactions are governed by the concentrations and relative binding affinities of the proteins for each other. These affinities are altered by conformation changes of Bcl-2 family proteins resulting from interactions with each other and with membranes. How Bcl-2 proteins transition into and out of the conformations that controls their functions, and ultimately the fate of the cell, is not well understood. Here, kinetic analysis of the pore-forming Bcl-2 family member, Bax, revealed that Bax undergoes a conformational rearrangement through at least one structurally distinct intermediate that is a necessary precursor to pore formation. We discover that four cancer-associated Bax point mutants are trapped in the intermediate state, suggesting that transitions into and out of this intermediate can be modulated independently with consequences for the execution of apoptosis. Furthermore we report that the conformation changes Bax undergoes can be regulated by phosphorylation of Bax on residue S184 by the pro-survival kinase, Akt. Phosphorylation converts Bax into an anti-apoptotic protein that functions in a dominant-negative fashion. Bioinformatics revealed that in human cancers, higher levels of Bax are positively associated with high levels of PI3K/AKT pathway genes representing an added mechanism for cancer cells to evade apoptosis. Additionally we studied the interactions between Bax, the anti-apoptotic protein Bcl-XL, the sensitizer BH3 protein Bad and the BH3 activator protein Bid. We uncover a new mechanism of apoptosis regulation whereby Bad binds to one monomer of a Bcl-XL dimer eliciting an activating conformation change in a tBid bound to the other monomer of the Bcl-XL dimer. This allows Bad to function as a non-competitive inhibitor of Bcl-XL, and represents a novel mechanism that significantly enhances the potency of Bad to elicit apoptosis. / Thesis / Doctor of Philosophy (PhD) / Every day the human body creates billions of cells replacing damaged or unwanted cells. The death of these cells is tightly controlled and can result in disease when misregulated. Cancers arise when there is too little cell death and neurodegenerative diseases, such as Alzheimer’s, arise from too much cell death. Much research, including this thesis, is focused on understanding how cells die because once understood, cell death can be manipulated to treat disease. Cell death ironically occurs at the mitochondria, a cellular organ normally responsible for creating the energy required for the cell to live. When cell death is initiated, the mitochondria get holes poked into them, releasing pro-death factors that irreversibly commit the cell to dying. The work presented here uncovers new information about the regulation of the hole poking process, how it is blocked in breast cancer and how the process may be modulated to treat cancers.

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Les homologues Bcl-2 constituent une famille de protéines structurellement apparentées qui jouent un rôle central dans la régulation de l'apoptose. Les mécanismes de régulation de l'apoptose entérocytaire sont peu connus. Afin de mieux comprendre cette régulation, le but de ce travail était d'analyser l'établissement des mécanismes de contrôles de l'apoptose entérocytaire au cours du développement de l'intestin humain. Pour ce faire nous avons étudié l'expression épithéliale des homologues Bcl-2 proapototiques (Bax, Bak et Bad) et anti-apoptotiques (Bcl-2, Mcl-1 et BCI-X L ), ainsi que la molécule associée anti-apoptotique Bag-1, le long de l'axe cryptevillosité et pour les différents segments (jéjunum, iléon et côlon) de spécimens foetaux humains âgés de 9 à 20 semaines de gestation. Dans un premier temps, afin d'analyser l'expression épithéliale des homologues Bcl-2 le long de l'axe crypte-villosité, nous avons effectué des immunofluorescences indirectes en utilisant des anticorps spécifiques pour les homologues étudiés. Dans un deuxième temps, afin d'analyser l'expression épithéliale des homologues Bcl-2, nous avons effectué des Western blots."--Résumé abrégé par UMI.

Plusieurs évidences suggèrent que des membres impliqués dans le contrôle de l'activation de la cascade apoptotique et particulièrement les membres de la famille de protéines Bcl-2 pourraient jouer un rôle dans le phénomène de résistance observé dans les tumeurs ovariennes. Le but de cette étude est de déterminer l'importance relative de la protéine Bcl-2 dans le phénomène de résistance clinique à la chimiothérapie dans le cancer ovarien, plus précisément au cisplatin. L'analyse de l'expression de la protéine Bcl-2 dans des cellules d'ovaire normales et cancéreuses a démontré une surexpression de la protéine dans les cellules cancéreuses par rapport aux cellules d'ovaire normales, mais aucune corrélation entre l'expression de la protéine Bcl-2 et la sensibilité au cisplatin des cellules d'ovaire cancéreuses n'a pu être établie. Pour pouvoir mieux évaluer le rôle de la protéine Bcl-2 dans le phénomène de résistance, nous avons utilisé un anticorps monovalent modifié (scFv) dirigé contre cette dernière. Ce scFv agit essentiellement comme un inhibiteur spécifique de la protéine Bcl-2 ans les cellules.

Background Cisplatin is used for treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC) but treatment with cisplatin often leads to acquired resistance to cisplatin, resulting in poor patient survival. Globotriaosylceramide (Gb3) and multidrug resistance protein 1 (MDR1) have been associated with cisplatin resistance. Gb3 serves as a receptor for verotoxin-1 (VT-1), which induces apoptosis, and has been shown to have a functional dependency to MDR1 and heat shock protein 70 (HSP7o). The Bcl-2 family of proteins and inhibitors of apoptosis (IAPs) are key regulators of apoptosis. BH3-mimetics mimic pro-apoptotic BH3-only proteins, while Smac mimetics mimic the IAP-binding protein Smac/Diablo. These drugs have shown great promise in reversing cisplatin resistance. Exosomes are small bio-nanoparticles secreted and taken up by both cancer cells and normal cells. They have the ability to transfer properties between cells and have been shown to confer resistance to cisplatin. Methods In this thesis, NSCLC cell line H1299 and MPM cell line P31 were studied using western blot, flow cytometry, proteome profilers, confocal microscopy and gene expression arrays to investigate changes in protein and gene expression after acquisition of cisplatin resistance (P31res and H1299res) or after incubation with exosomes or drugs that target these. The cytotoxic and apoptotic effects were studied using fluorometric cytotoxicity assay (FMCA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results This thesis confirms that Gb3 is a potential target for cisplatin resistance reversal. Incubation with glycosphingolipid production inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) and VT-1 led to reduced Gb3 cell surface expression and increased cytotoxic effect of cisplatin in all cell lines. Gb3 and MDR1 was not co-localized in any studied cell line, but Gb3 and HSP70 were co-localized on the cell surface and PPMP and VT-1 led to a decrease of both Gb3 and HSP70. Both BH3-mimetic obatoclax and Smac mimetic AT-406 had an additive effect on cisplatin-induced cytotoxicity and apoptosis in P31 and a synergistic effect in P31res. Results indicate that exosomes from cisplatin-resistant cell lines can transfer HSP70 to the surface of cells. Conclusion Cell surface Gb3 and HSP70, the Bcl-2/IAP-family proteins and exosomal transfer of cisplatin resistance characteristics are potential targets in combatting cisplatin resistance that show therapeutic promise and warrant further research.

Exposure of cells to hydrogen peroxide can cause oxidative damage to cellular constituents including lipids, protein, and DNA. At elevated concentrations, hydrogen peroxide can trigger cell death by apoptosis or necrosis. Apoptotic cell death can be prevented by overexpression of the oncoprotein Bcl-2. The exact mechanism by which Bcl-2 blocks cell death is controversial. Some researchers believe that Bcl-2 possesses antioxidant properties that protect cells from apoptosis. The purpose of this thesis was to assess oxidative stress and apoptosis following hydrogen peroxide exposure in Jurkat T cells overexpressing Bcl-2. One of the major objectives was to ascertain whether or not Bcl-2 overexpression elevated the antioxidant capacity of Jurkat T cells to provide protection from oxidant-induced cell death. Hydrogen peroxide treated Jurkat cells became apoptotic at moderate levels of oxidant (25-100 uM H2O2), and necrotic at higher doses (greater than 200 uM H2O2). Bcl-2 overexpression prevented caspase activation and cell death at the apoptotic doses of H2O2, but not the necrotic doses. Caspase inhibition studies demonstrated that Bcl-2 overexpression provided a greater level of resistance from H2O2-induced cell death than the broad-spectrum caspase inhibitor z-VAD.fmk. A systematic study was carried out examining the antioxidant status of Jurkat cells overexpressing Bcl-2. Several Bcl-2 transfectants were utilised for the study, so that any differences seen could be correlated to the level of Bcl-2 expression. Surprisingly, there were no statistically significant differences among the Bcl-2 transfectants for any of the antioxidant enzymes. Jurkat cells overexpressing Bcl-2 exhibited the same level of oxidative damage to lipids and protein in response to H2O2 exposure as the parental Jurkat cells. Interestingly, Jurkat cells overexpressing Bcl-2 continued to grow in culture after H2O2 exposure, despite harboring damage to cellular constituents. Consistent with these results, H2O2 treated Jurkat cells overexpressing Bcl-2, which failed to undergo apoptosis, were more prone to genomic instability. Together, these findings suggest that Bcl-2 overexpression protects Jurkat cells from H2O2-induced cell death by blocking apoptosis. Jurkat cells overexpressing Bcl-2 were no better at detoxifying oxidants and showed the same level of oxidative damage following H2O2 exposure. As a result, the overexpression of Bcl-2 considerably enhanced the mutagenicity of H2O2.

In this dissertation, we explore the transcriptional regulatory roles of Gquadruplex- forming motifs and the involvement of specific transcriptional factors, which interact with the same elements, in the control of human c-myc and bcl-2 gene expression. The G-quadruplex structures within the NHE III1 region of the c-myc promoter and their ability to repress transcription has been well established. However, a longstanding unanswered question is how these stable DNA secondary structures are transformed to activate c-myc transcription. NDPK-B has been recognized as an activator of c-myc transcription via interactions with NHE III1 region of the c-myc gene promoter. Through the use of RNAi, we confirmed the transcriptional regulatory role of NDPK-B. We demonstrate that NDPK-B has DNA binding activity and the nuclease activity results from a contaminating protein. NDPK-B preferentially binds to the singlestranded guanine-rich strand of the c-myc NHE III₁. Potassium ions and G-quadruplexinteractive agents, which stabilize G-quadruplex structures, had an inhibitory effect on NDPK-B DNA binding activity. Based on our studies, we have proposed a stepwise trapping-out of the NHE III1 region in a single-stranded form, thus allowing singlestranded transcription factors to bind and activate c-myc transcription. This model provides a rationale for how the stabilization of G-quadruplexes within the c-myc gene promoter region can inhibit NDPK-B from activating c-myc transcription. Similarly, the human bcl-2 gene contains a GC-rich region within its promoter region, which is critical in the regulation of bcl-2 expression. We demonstrate that the guanine rich strand within this region can form three intramolecular G-quadruplex structures. Based on NMR studies, the central G-quadruplex forms a mixed parallel/antiparallel structure with three tetrads connected by loops of one, seven, and three bases. The Gquadruplex structures in the bcl-2 promoter extends beyond the ability to form any one of three separate G-quadruplexes to each having the capacity to form either three or six different loop isomers. This suggests that targeting these individual structures could lead to different biological outcomes. We also found that Telomestatin upregulates bcl-2 gene expression, which we propose is a result of inhibiting the binding of the WT1 repressor protein by the formation of a drug-stabilized G-quadruplex structure.