Diss. (sammanfattning) Umeå : Umeå universitet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 142-166.

Thesis (Ph.D.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

C-Kit est généralement considéré comme un récepteur à tyrosine kinase et comme un proto-oncogène, dont la surexpression et la mutation conduisent à une progression tumorale médiée par son activité kinase. En clinique, les traitements ciblant l’activité kinase de c-Kit, comme l’Imatinib (Gleevec), ont été largement utilisés pour traiter les patients atteints de maladies liées à c-Kit. Alors que le rôle de c-Kit comme proto-oncogène ne fait aucun doute, certaines études et analyses de bases de données diffèrent avec l’idée d’un rôle pro-tumoral de c-Kit, laissant penser à un rôle différent de c-Kit dans le cancer. Ici, nous montrons que c-Kit appartient à la famille des récepteurs à dépendance, de la même façon que d’autres récepteurs de la famille des tyrosine kinases tel que MET, RET et TrkC. En absence de son ligand Stem Cell Factor (SCF), au lieu de rester inactif, c-Kit déclenche l’apoptose, qui peut être renforcée par l’invalidation de son activité kinase. En parallèle, nous montrons que c-Kit est capable de se lier à la caspase-9 et de l’activer. De plus, à la manière d’autres récepteurs à dépendance, c-Kit est aussi clivé par des protéases de type caspase sur son résidu acide aspartique D816, qui est nécessaire à son activité pro-apoptotique. La mutation du site D816 inhibe l’interaction entre c-kit et la caspase-9 et invalide l’activité pro-apoptotique de c-Kit. De façon intéressante, la mutation D816 est l’une des mutations les plus communes de ce récepteur dans la plupart des cancers liés à c-Kit, et cette mutation favorise la résistance au traitement Gleevec. Nous montrons aussi que la surexpression de c-Kit invalidé pour son activité kinase est capable d’inhiber la croissance tumorale dans des modèles animaux, alors que la mutation du site D816 empêche son effet suppresseur de tumeur. En outre, nous avons développé un outil permettant de bloquer l’interaction entre SCF et c-Kit, déclenchant l’activité pro-apoptotique de c-Kit dans les cancers positifs pour ce récepteur. En utilisant l’activité pro-apoptotique de c-Kit, en combinaison avec des inhibiteurs de kinases comme le Gleevec, nous proposons une nouvelle stratégie thérapeutique. En conclusion, nous démontrons que c-Kit est un membre de la famille des récepteurs à dépendance, présentant une activité pro-apoptotique, et pouvant être utilisé comme un outil alternatif dans le cadre d’un traitement contre le cancer / C-Kit has been generally considered as a receptor tyrosine kinase and a proto-oncogene, whose upregulation and mutation lead to tumor progression through its kinase activity. Clinically, drugs targeting the kinase activity of c-Kit, such as Imatinib (Gleevec), have been wildly used to treat patients with c-Kit related diseases. While the role of c-Kit as a proto-oncogene is of no doubt, some research reports and database analysis do not fit well the tumor promoting role of c-Kit, indicating a possible different role of c-Kit in cancer. Here, we show that c-Kit belongs to the dependence receptor family, similarly to other receptor tyrosine kinases such as MET, RET and TrkC. In the absent of its ligand SCF (stem cell factor), instead of staying inactive, c-Kit triggers apoptosis, which can be enhanced by silencing its kinase activity. Besides, we have shown that c-Kit is able to bind and activate caspase-9. Moreover, similarly to other dependence receptors, c-Kit is also cleaved by caspases-like protease at aspartic acid residue D816, which is crucial for its pro-apoptotic activity. The mutation of D816 site inhibits the c-Kit/caspase-9 binding and silences the pro-apoptotic activity of c-Kit. Of interest, c-Kit D816 mutation is one of the most common mutation of this receptor in many c-Kit related cancers and it promotes resistance against Gleevec treatment. We also show that overexpression of kinase mutated c-Kit is able to inhibit tumor growth in animal models, while the mutation of D816 site impairs the tumor suppressing activity. Furthermore, we develop a tool to block the SCF/c-Kit interaction, which unleashes the pro-apoptotic activity of c-Kit in cancers expressing this receptor. By using the pro-apoptotic activity of c-Kit, in combination with kinase inhibitors like Gleevec, we propose a novel therapeutic strategy. In conclusion, we demonstrate that c-Kit is a member of dependence receptor family, harboring intrinsic pro-apoptotic activity, which can be used as an alternative tool in cancer treatment

L'utilisation d'un agent déméthylant induit la réexpression de la protéine RIP3, une sérine-thréonine kinase, dans un modèle leucémique murin exprimant BCR-ABL humain. La réexpression de RIP3 conduit rapidement les cellules vers la nécroptose. Le mutant délété du domaine kinase est de façon surprenante plus "apoptogène" et induit le clivage de p65/RelA sur le résidu d'acide aspartique D361 par la caspase 6. Pour déterminer l'impact de ce clivage, nous avons construit un mutant non clivable p65/RelA D361E, ainsi que des plasmides exprimant chacun des fragments p65/RelA 1-361 ou p65/RelA 362-549, ou un plasmide exprimant simultanément p65/RelA 1-361 + p65/RelA 362-549. Ces différents plasmides codant pour les différentes formes de la protéine p65/RelA sont incorporés par transfection dans les cellules leucémiques ou de mélanome pour lesquels le gène RIP3 est respectivement méthylé ou exprimé. In vivo, nous mettons en évidence une différence de tumorigénicité entre les deux modèles. Elle est accrue par la présence de p65/RelA D361E par rapport à celle de p65/RelA WT et de p65/RelA 1-361 + p65/RelA 362-549 dans le modèle leucémique. Elle est au contraire faible dans le modèle du mélanome pour lequel la surexpression des fragments p65/RelA 1-361 +362-549 induit la tumorigenèse la plus forte. L'agressivité du mutant non clivable in vivo n'est pas corrélée à l'activité de NF-kB mesurée in vitro. Les fragments comme le mutant p65/RelA D361E induisent des profils d'expression différents dans le modèle murin de leucémie avec la modulation notable d'expression génique de la famille d'inhibiteurs de protéases à cystéine Stefins, ainsi que le transporteur de bicarbonate de sodium SLC4A5 qui joue un rôle majeur dans la régulation du pH intracellulaire. Le mutant p65/RelA D361E induit une expression importante du transporteur de bicarbonate de sodium SLC4A5 dans le modèle leucémique responsable de l'augmentation du pH intracellulaire qui participe au développement tumoral. Par contre, ce sont les deux fragments p65/RelA 1-361 + p65/RelA 362-549 qui induisent simultanément une expression plus forte de la molécule d'immunoéchappement PDL1, vraisemblablement par un mécanisme post-traductionnel. L'étude de la "souchitude" des modèles montre une différence d'activité du mutant p65/RelA D361E selon le modèle. On observe une augmentation de l'activité ALDH dans le modèle leucémique et une diminution de la formation de sphères dans le modèle de mélanome. En conclusion, ces résultats indiquent que les fragments issus du clivage de p65/RelA par l'activité de RIP3 indépendante de la kinase possèdent un rôle différent de celui de la forme sauvage sur la souchitude des cellules cancéreuses, et qu'elle dépend du modèle étudié. Ils confirment que le mutant non clivable possède la plus forte activité tumorigénique. Ils laissent également supposer que les fragments Nter et Cter puissent avoir une activité dans des cellules tumorales possédant une protéine RIP3 fonctionnelle et active, probablement par des mécanismes inflammatoires ou autres qui doivent être caractérisés. / The receptor-interacting protein kinase 3 (RIPK3) can induce necroptosis, apoptosis, or cell proliferation, and is silenced in several hematological malignancies. We previously reported that RIPK3 activity independent of its kinase domain induces p65/RelA caspase-mediated cleavage resulting in N-terminal 1-362 and C-terminal 362-549 fragments. We show here that a non-cleavable p65/RelA D361E mutant expressed in DA1-3b leukemia cells decrease mouse survival and that coexpressed p65/RelA fragments increase tumoriginicty of B16/F1 melanoma cells that did not correlated with in vitro measured Nf-kB activity. Fragments and p65/RelA fragments display different expression profiles in DA1-3b leukemic cells, with the notable modulation of gene expression of the Stefin cysteine protease inhibitor family and of SLC4A5, a Na+-coupled HCO−3 transporter. DA1-3b cells expressing p65/RelA D361E mutant showed more basic intracellular pH. p65/RelA fragments induced ovexpression of PD-L1 immunoescape molecule in DA1-3b cells. Markers of stemness were also affected: p65/RelA D361E induced increased ALDH activity in DA1-3b cells and fragments expression resulted in increased melanoma sphere formation in B16/F1 cells. Thus, far from being neutral, p65/RelA cleavage initiated by kinase independent activity of RIPK3 induced a pleiotropic range of effects in vitro and in vivo in cancer cells, that may vary across tumor types.

Made available in DSpace on 2015-07-22T20:49:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-31 / Objetivo: Investigar os efeitos da oxigenação hiperbárica (OHB) e da Nacetilcisteína (NAC) isoladamente ou em associação sobre a viabilidade de retalhos cutâneos em ratos. Métodos: 32 ratos Wistar machos foram distribuídos randomicamente em GS (sham/n=8), GNAC (N-acetilcisteína/n=8), GOHB (oxigenação hiperbárica/n=8) e GNH (NAC+OHB/n=8). Sob anestesia geral foi dissecado um retalho dorsal de base cranial medindo (2x8cm) conforme modelo preconizado por McFarlane; foi interposta uma lâmina de polietileno entre o retalho e seu leito impedindo a vascularização a partir do mesmo; o retalho foi suturado de novo sobre seu leito. Nos sete dias consecutivos cada animal recebeu: GS solução salina intraperitoneal, o grupo GNAC 300mg de NAC intraperitoneal, o grupo GOHB 2 horas de oxigenação hiperbárica a 2,4 ATA e o grupo GNH recebeu a associação dos tratamentos. No oitavo dia do experimento os animais foram novamente anestesiados e foram coletadas biópsias dos terços proximal, médio, distal e controle para análise imuno-histoquímica bem como fotografias digitais para posterior análise por meio do programa Image-Lab. Os dados obtidos foram estatisticamente analisados pelos testes ANOVA e Bonferroni (p<0,05). Resultados: A média da área de necrose no grupo GS foi de 18,3%, no grupo GNAC 24,3%, no grupo GOHB 12,6% e no grupo GNH 14,9%. A expressão do VEGF na epiderme, derme, tecido submuscular e vasos não demonstrou diferenças significativas nos diferentes grupos de tratamento. A acumulação de células em apoptose foi significativamente menor no terço médio em todos os grupos tratados, mas significativamente menor nos grupos tratados com OHB. Os resultados menos favoráveis foram observados no grupo GS e GNAC. Conclusão: A OHB está associada à menor expressão de apoptose celular e menor área de necrose de pele. A NAC não apresentou efetividade na proteção do retalho randômico quer pela avaliação da apoptose ou da necrose celular. A associação dos dois procedimentos não produziu potencialização dos resultados favoráveis do uso de ambos separadamente. Os achados sugerem que a difusão do oxigênio pelo interstício celular foi o fator determinante dos resultados mais favoráveis da OHB. / Objective: To investigate the effects of hyperbaric oxygen (HBO) and N-acetylcysteine (NAC) alone or in association on the viability of skin flaps in rats. Methods: 32 male Wistar rats were randomized in: GS (sham/n=8), GNAC (Nacetylcysteine/ n=8), GHBO (hyperbaric oxygen/n=8) and GNH (NAC+OHB/n= 8). A skin flap measuring cranial base (2x8cm) was performed (McFarlane) and brought a polyethylene film between the skin flap and its surgical bed prior to closure with interrupted polyamide sutures. For seven days the animals received injections of saline intraperitoneally (GS) and NAC at a dose of 300mg/kg (GNAC). HBO was performed for periods of two hours with 2.4 ATA (GOHB) for seven days and the association of NAC and HBO was carried out in group GNH. On the eighth day the dorsum of the animal was photographed and collected biopsy specimen of skin from the flap at thirds proximal, middle and distal flap. The areas of necrosis were evaluated (photos processed by the Image-Lab), assessment of apoptosis (cleaved caspase 3) and angiogenesis (VEGF). The data obtained were statistically analyzed by ANOVA and Bonferroni (p <0.05). Results: The mean areas of necrosis (mm2) were: GS 18.3%, GNAC 24.3%, GOHB 12.6% and GNH 14.9%. The expression of VEGF in the epidermis, dermis, submuscular tissue and vessels was not significant in the different treatment groups. The presence of cells undergoing apoptosis was significantly lower in the middle third of all treated groups compared to sham group, but significantly lower in the group treated with HBO alone or in combination with NAC. Conclusion: HBO is associated with reduced expression of apoptosis and reduced area of necrosis of skin flap. The NAC is not associated with lower expression of apoptosis and alone had the worst results in this experiment. The association of the two procedures did not produce potentiating of the favorable results of the use of both separately. The findings suggest that the diffusion of oxygen through the interstitial space was the determining factor of more favorable results of HBO. / TEDE

Quelque soit le type de dégénérescences rétiniennes, les cellules photoréceptrices à l'origine de la genèse du signal lumineux, meurent par un mécanisme commun : l'apoptose. Au laboratoire, nous avons mis en évidence que l'inhibition de la caspase-3, une caspase effectrice de l'apoptose, permet de réduire l'apoptose des cellules photoréceptrices (Perche et al. 2007). Dans la continuité de ces résultats, le but de nos travaux de thèse est d'identifier les molécules impliquées en amont de la caspase-3. Pour mener à bien notre projet, nous avons utilisées un modèle expérimentale de dégénérescence rétinienne induite par une exposition à la lumière (modèle de photodégénérescence rétinienne). Les atteintes rétiniennes sont quantifiées par : l'électrorétinographie in-vivo permettant d'évaluer la fonction rétinienne, l'histologie pour l'analyse morphométrique de la rétine aux quelles sont associés des dosages enzymatiques. Ainsi, nous avons montré que l'injection d'un inhibiteur de la caspase-12 à 0,4 ou à 0,8 mM, de la caspase-9 à 0,2 ou à 0,4 mM, ou de la caspase-8 à 0,2 mM, injecté dans le vitré n'a aucun effet toxique sur la rétine et n'a aucun effet protecteur contre l'apoptose des cellules photoréceptrices induites par la lumière. Ces résultats suggèrent que les caspases-8, 9 et 12 ne sont pas impliquées dans l'activation de la caspase-3 et donc dans l'initiation de l'apoptose des photorécepteurs induite par la lumière. Toutefois, après injection dans le vitré, les inhibiteurs inhibent leur cible respective uniquement transitoirement. Par conséquent, pour pouvoir conclure sur le rôle de ces caspases dans le processus dégénératif, il faudrait pouvoir inhiber les caspases de façon plus persistante. Il serait donc intéressant de reproduire des expérimentations similaires en augmentant la concentration de l'inhibiteur injecté ou en réduisant le délai entre l'injection de l'inhibiteur et l'induction du stress. De plus, la caspase-3 peut être activée indépendamment des caspases initiatrices, comme par exemple : les céramides, les cathepsines et les calpaïnes. / No abstract available

In the last decade, a tremendous gain in knowledge concerning the molecular events of apoptosis signaling and execution has been achieved. The aim of this thesis was to clarify the role of lysosomal membrane permeabilization and lysosomal proteases, cathepsins, in signaling for apoptosis. We identified cathepsin D as an important factor in staurosporine-induced human fibroblast cell death. After release to the cytosol, cathepsin D promoted mitochondrial release of cytochrome c by proteolytic activation of Bid. Cathepsin D-mediated cleavage of Bid generated two fragments with the apparent molecular mass of 15 and 19 kDa. By sequence analysis, three cathepsin D-specific cleavage sites, Phe24, Trp48, and Phe183, were identified. Moreover, we investigated the mechanism by which cathepsins escape the lysosomal compartment, and found that Bax is translocated from the cytosol to lysosomes upon staurosporine treatment. In agreement with these data, recombinant Bax triggered release of cathepsins from isolated rat liver lysosomes. Conceivably, the Bcl-2 family of proteins may govern release of pro-apoptotic factors from both lysosomes and mitochondria. The importance of lysosomal cathepsins in apoptosis signaling was studied also in oral squamous cell carcinoma cells following exposure to the redox-cycling drug naphthazarin or agonistic anti-Fas antibodies. In this experimental system, cathepsins were released to the cytosol, however, inhibition of neither cathepsin D, nor cysteine cathepsin activity suppressed cell death. Interestingly, cysteine cathepsins still appeared to be involved in activation of the caspase cascade. Cathepsins are often overexpressed and secreted by cancer cells, and it has been reported that extracellular cathepsins promote tumor growth and metastasis. Here, we propose that cathepsin B secreted from cancer cells may suppress cancer cell death by shedding of the Fas death receptor. Defects in the regulation of apoptosis contribute to a wide variety of diseases, such as cancer, neurodegeneration and autoimmunity. Increased knowledge of the molecular details of apoptosis could lead to novel, more effective, treatments for these illnesses. This thesis emphasizes the importance of the lysosomal death pathway, which is a promising target for future therapeutic intervention. / In the last decade, a tremendous gain in knowledge concerning the molecular events of apoptosis signaling and execution has been achieved. The aim of this thesis was to clarify the role of lysosomal membrane permeabilization and lysosomal proteases, cathepsins, in signaling for apoptosis. We identified cathepsin D as an important factor in staurosporine-induced human fibroblast cell death. After release to the cytosol, cathepsin D promoted mitochondrial release of cytochrome c by proteolytic activation of Bid. Cathepsin D-mediated cleavage of Bid generated two fragments with the apparent molecular mass of 15 and 19 kDa. By sequence analysis, three cathepsin D-specific cleavage sites, Phe24, Trp48, and Phe183, were identified. Moreover, we investigated the mechanism by which cathepsins escape the lysosomal compartment, and found that Bax is translocated from the cytosol to lysosomes upon staurosporine treatment. In agreement with these data, recombinant Bax triggered release of cathepsins from isolated rat liver lysosomes. Conceivably, the Bcl-2 family of proteins may govern release of pro-apoptotic factors from both lysosomes and mitochondria. The importance of lysosomal cathepsins in apoptosis signaling was studied also in oral squamous cell carcinoma cells following exposure to the redox-cycling drug naphthazarin or agonistic anti-Fas antibodies. In this experimental system, cathepsins were released to the cytosol, however, inhibition of neither cathepsin D, nor cysteine cathepsin activity suppressed cell death. Interestingly, cysteine cathepsins still appeared to be involved in activation of the caspase cascade. Cathepsins are often overexpressed and secreted by cancer cells, and it has been reported that extracellular cathepsins promote tumor growth and metastasis. Here, we propose that cathepsin B secreted from cancer cells may suppress cancer cell death by shedding of the Fas death receptor. Defects in the regulation of apoptosis contribute to a wide variety of diseases, such as cancer, neurodegeneration and autoimmunity. Increased knowledge of the molecular details of apoptosis could lead to novel, more effective, treatments for these illnesses. This thesis emphasizes the importance of the lysosomal death pathway, which is a promising target for future therapeutic intervention.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Apoptosis is a process of the programmed cell death in response to severe mutations in DNA or cell stress. Apoptosis plays a key role in tissue maintenance by eliminating senescent and damaged cells. Various molecules take part in apoptosis, main participants are Bcl-2 protein family and caspases. The latter one are responsible for apoptosis execution, while Bcl-2 protein family regulates apoptotic pathway. Failure of this regulation may cause several pathologies, including development of neoplastic tissue. Human endometrium is a speci c tissue, in which apoptosis is present in cycling pattern. Present study shows expression level of Bcl-2, Bax, Bad, Bid, pro-caspase-3, caspase-3 and PARP in normal, atrophic, hyperplastic and cancerous (Grade I and II) human endometrium. Bad and Bid proteins can be possible breakpoints in neoplastic transfer due to opposit expression in cancerous and hyperplastic endometrium.