Studies have shown that the alternative splicing of caspase 9 and the phospho-status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and de novo ceramide via the action of protein phosphatase-1 (PP1). Two RNA splice variants are derived from the caspase 9 gene, pro-apoptotic caspase 9a and anti-apoptotic caspase 9b, via alternative splicing by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. In this study, the link between SR proteins and the alternative splicing of caspase 9 was established. Sequence analysis of the exon 3, 4, 5, and 6 cassette of the caspase 9 gene identified five possible high affinity sequences for interaction with the SR protein, SRp30a, a well-established regulator of exon inclusion/exclusion. Replacement mutagenesis identified purine-rich sequences between exons 4 and 5 and wthin exon 6 as important for binding SRp30a and required for expression of the caspase 9a splice variant. In vitro binding assays coupled with competitor studies demonstrated specific binding of RNA trans-acting proteins and SRp30a with these sequences. Furthermore, SDS-PAGE analysis of cross-linked RNA trans-acting factors with these possible RNA cis-elements revealed the specific binding of an approximate 66, 56, 45, and 38 kDa protein/protein complex to these sequences. A previous application of RNAi technology to downregulate SRp30a in A549 lung adenocarcinoma cells induced an approximately 75% decrease in SRp30a expression and induced a dramatic change in the ratio of caspase 9a/caspase 9b. Therefore, these studies have identified SRp30a as a major regulator of the alternative splicing of caspase 9 directly linking de novo ceramide generation, PP1, and SRp30a as the signal transduction pathway regulating the expression of caspase 9.

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

Iron is an essential trace element for almost all living organisms. Iron overload in cells and tissues, however, leads to their disruption. Most oftenly damaged are parenchymatic organs such as the liver, pancreas and heart. The aim of this thesis was to create cellular in vitro models for the investigation of effects of excess iron on hepatocytes and pancreatic beta cells and on these models to investigate cellular processes which lead to cellular damage during iron overload. We focused on examining the presence of oxidative and endoplasmic reticulum stress and the activation of apoptotic cell death. For our experiments, we used HEP-G2 cell line which represents human hepatocytes and NES2Y cell line which represents human pancreatic beta cells. To study the mechanisms of cellular damage during iron overload, we used two approaches by which we observed both acute and long-term effects of high levels of iron on damage of the tested cell lines. When studying the acute effect of excess iron on the cells, we applied high doses of iron (using 15 mM ferric citrate in medium) that led to the activation of cell death in hours. Long-term effects of iron overload were tested on cells regularly cultivated in the presence of 50 μM and 100 μM ferric citrate over a period of several months. Iron concentrations...

Apart from CAD, the transient liberation of AIF during day 6 of TF-1 differentiation could pose another threat to the genomic DNA in cells. We have demonstrated the absence of AIF in the nucleus of TF-1 cells despite its release from the mitochondria by using confocal studies. Moreover, the expression of heat shock protein 70 kDa (Hsp70), a well-known antagonist of AIF, was found to be temporarily increased at day 6. Taken together, our results implied a plausible retention of AIF in the cytoplasm by Hsp70. Although Hsp70 is commonly utilized by many cancer cells to counteract AIF and avoid DNA fragmentation, we are the first to demonstrate its role in suppressing AIF during normal erythroid maturation. / As a whole, we have illustrated that the activated caspase-3, mediated most likely by the mitochondrial pathway, is an essential component in the differentiation of TF-1 cells. Its activation was nevertheless not coupled with DNA fragmentation due to some protective mechanisms such as CAD downregulation, Hsp70 upregulation and overexpression of Bcl-XL. Our study therefore provides some insights in the understanding of the relationship between human erythropoiesis and apoptosis and a better understanding in this regard will undoubtedly facilitate the development of new drugs in the treatment of different hematopoietic diseases. / Caspases play a central role in apoptosis. Their activations during the process are accounted for different biochemical and morphological changes in apoptotic cells. Yet in recent years, increasing studies had shown that caspases were also involved in some non-apoptotic cellular events, including T and B-lymphocytes activation, as well as the terminal differentiation of lens cells, megakaryocytes and erythrocytes. / In order to find out other unknown cellular mechanisms in erythropoiesis, mRNA differential display was employed to compare the gene expression pattern of TF-1 cells at different stages of differentiation. Several differentially expressed genes were identified and subsequently confirmed by RT PCR. These genes include formin binding protein 3, destrin and T-complex protein-1 (TCP-1). Their involvement in erythroid differentiation was still not clear at the moment but would be investigated in the near future. Furthermore, aiming at identifying the interacting proteins or inhibitors of caspase-3 in the system, a pull down assay was developed by means of the bacterial expression of a recombinant human caspase-3 mutant protein. With the mutation in the active site, the binding of our recombinant caspase-3 mutant with two known partners ICAD and BIRII (Baculovirus Inhibitor of apoptosis protein Repeat II) domain has been demonstrated. We hope in the near future that it can be employed to fish out some novel caspase-3 substrates from the differentiating TF-1 cell lysate. / In the present study, the participation of caspase in in vitro erythropoiesis was investigated using a human erythroleukemia cell line TF-1. Erythropoietin (EPO) induced erythroid maturation of TF-1 as indicated by the expression of erythroid-lineage markers like glycophorin A (GPA), transferrin receptors (CD71) and synthesis of hemoglobin (Hb). Activation of caspase-3 was observed from day 6 to day 12 during TF-1 differentiation after EPO treatment. With the administration of caspase-3 specific inhibitor, expressions of GPA and CD71 were partially blocked, suggesting that caspase-3 activation is essential in erythropoiesis in our TF-1 model. / Possible involvement of the intrinsic and extrinsic apoptotic pathways was studied by investigating respectively the activation of pro-caspase-9 and -8. It was found that caspase-9, but not -8, was activated at the corresponding time point when caspase-3 was activated. Besides, a transient mitochondrial depolarization coupled with the release of cytochrome c and apoptosis inducing factor (AIF) were detected on day 6, strongly implying a role of mitochondria in triggering the activation of executioner caspase-3. On the other hand, GPA and CD71 expressions were blocked by the application of mitochondrial depolarization inhibitor cyclosporin A (CyA). Also, the recovery of mitochondrial membrane potential was found to be correlated with an overexpression of Bcl-XL at a late stage of TF-1 differentiation, and the role of Bcl-XL was subsequently manifested further by a significant retardation of erythroid differentiation in the siRNA Bcl-XL knocked down TF-1 cells. / The exact role of caspase-3 in erythroid differentiation is far from clear at this moment. Yet, its regulation in the process is equally intriguing. On the course of TF-1 maturation, activated caspase-3 was able to cleave and de-localize the Inhibitor of Caspase-activated DNase (ICAD) from the nucleus, but at the same time DNA fragmentation was not detected by TUNEL assay nor agarose electrophoresis. Furthermore, protection against DNA fragmentation was observed in the EPO-treated TF-1 cells when challenged with a potent apoptotic inducer staurosporine (STS). These observations are in contrast to our understanding that DNA is fragmented by CAD (Caspase-activated DNase) when ICAD in the ICAD-CAD complex is cleaved by caspase-3. For these apparently contradictory observations, we demonstrated that downregulation of CAD occurred at the mRNA and protein levels during the erythroid differentiation in TF-1. This provides a cell rescuing mechanism in non-apoptotic cells with activated caspases. / Lui Chun Kin Julian. / "September 2006." / Adviser: Siu Kai Kong. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1620. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 239-253). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Malignant pleural mesothelioma (MPM) is an aggressive, treatment-resistant tumour. Cisplatin (cis-diamminedichloroplatinum (II)) is the best single-agent chemotherapy for MPM, but platinum-based combination therapies give the best overall response rates. However, cisplatin use is limited by resistance and severe side effects. This thesis has increased the knowledge concerning cisplatin-induced cell death in MPM by describing a novel potential therapeutic target, and three novel phenotypes of cisplatin-resistance in a human MPM cell line (P31) and its cisplatin-resistant sub-line (P31res1.2). The novel potential therapeutic target, and one of the novel phenotypes, was cisplatin-resistant pro-apoptotic BH3-only proteins. In the P31 cells, cisplatin transiently increased pro-apoptotic BH3-only proteins during 6 h of exposure. This response was almost completely abrogated in the P31res1.2 cells. De-regulated caspase activity and activation was the second novel phenotype identified. The P31res1.2 cells had earlier, possibly mitochondria-independent, caspase-3 activation, increased basal caspase-3 activity and increased basal cleavage of caspase-8 and -9. Despite these differences, 6-h equitoxic cisplatin exposures rendered 50-60% of the cells apoptotic in both cell lines. The third novel phenotype was abrogated Na+K+2Cl--cotransporter (NKCC1) activity. Although NKCC1 activity was dispensable for cisplatin-induced apoptosis, balanced potassium transport activity was essential for P31 cell survival. Finally, the survival signalling protein Protein Kinase B (PKB or Akt) isoforms α and γ were constitutively activated in a PI3K-independent manner in P31 cells. In the P31res1.2 cells, PKBα and γ activities were increased, and there was PI3K-dependent activation of PKBβ. However, this increase in PKB isoform activity was not strongly associated to the cisplatin-resistance of the P31res1.2 cells.

Cytotoxicity of Multi-Purpose Solutions (MPS) is commonly tested on cells using diluted MPS or extracts from MPS soaked contact lenses. There is evidence that lens type will affect uptake and release of compounds contained in MPS. To assess the cytotoxicity of agents contained in MPS that would be released by contact lens, an in vitro “onlay” model was used, whereby MPS soaked silicone hydrogel lenses were directly set onto a confluent monolayer of corneal cells. Chapter 4 describes the impact of MPS released from contact lenses on immortalized human corneal epithelial cells. MPS-soaked lens interactions with cells were characterized by studying cell viability, cell adhesion and caspase assays. In Chapter 5, mechanisms of cell death induced by exposure to MPS from contact lenses were determined through evaluation of apoptotic markers, such as activation of caspase 3 and 9. In Chapter 6, the impact of the physical properties of silicone hydrogel lenses, specifically surface treatments, on cytotoxicity of MPS were investigated. The development of methods for characterizing the release of MPS from lenses, using absorbance spectra, is also described. The results indicate that exposure to contact lenses soaked in Opti-Free Express (OFX) and ReNu not only induces cell death in vitro, but also has an adverse effect on adhesion phenotype, suggesting that the remaining cells may have a compromised epithelial structure. Borate- buffered MPS were found to be more cytotoxic than phosphate-buffered base solutions. Investigation of the mechanisms of cell death revealed that ReNu and OFX induced corneal epithelial cell death in vitro using different pathways, whereby ReNu induced a necrotic pathway while OFX-induced cell death was mediated by the intrinsic pathway of apoptosis. The in vitro model was also able to identify differences between silicone hydrogels with different surface treatments: the different surface treatments and chemistries of silicone hydrogels lens will affect the release profile of MPS and hence their potential cytotoxicity. By investigating the induction of cell death processes by solution-lens combinations in vitro, we aim to prevent potential adverse effects in the cornea, which may ultimately compromise various visual and barrier functions. The findings indicate the wealth of information in vitro cytotoxicity testing can provide when evaluating the toxicological profile of MPS.

Cytotoxicity of Multi-Purpose Solutions (MPS) is commonly tested on cells using diluted MPS or extracts from MPS soaked contact lenses. There is evidence that lens type will affect uptake and release of compounds contained in MPS. To assess the cytotoxicity of agents contained in MPS that would be released by contact lens, an in vitro “onlay” model was used, whereby MPS soaked silicone hydrogel lenses were directly set onto a confluent monolayer of corneal cells. Chapter 4 describes the impact of MPS released from contact lenses on immortalized human corneal epithelial cells. MPS-soaked lens interactions with cells were characterized by studying cell viability, cell adhesion and caspase assays. In Chapter 5, mechanisms of cell death induced by exposure to MPS from contact lenses were determined through evaluation of apoptotic markers, such as activation of caspase 3 and 9. In Chapter 6, the impact of the physical properties of silicone hydrogel lenses, specifically surface treatments, on cytotoxicity of MPS were investigated. The development of methods for characterizing the release of MPS from lenses, using absorbance spectra, is also described. The results indicate that exposure to contact lenses soaked in Opti-Free Express (OFX) and ReNu not only induces cell death in vitro, but also has an adverse effect on adhesion phenotype, suggesting that the remaining cells may have a compromised epithelial structure. Borate- buffered MPS were found to be more cytotoxic than phosphate-buffered base solutions. Investigation of the mechanisms of cell death revealed that ReNu and OFX induced corneal epithelial cell death in vitro using different pathways, whereby ReNu induced a necrotic pathway while OFX-induced cell death was mediated by the intrinsic pathway of apoptosis. The in vitro model was also able to identify differences between silicone hydrogels with different surface treatments: the different surface treatments and chemistries of silicone hydrogels lens will affect the release profile of MPS and hence their potential cytotoxicity. By investigating the induction of cell death processes by solution-lens combinations in vitro, we aim to prevent potential adverse effects in the cornea, which may ultimately compromise various visual and barrier functions. The findings indicate the wealth of information in vitro cytotoxicity testing can provide when evaluating the toxicological profile of MPS.

L'athérosclérose est une pathologie artérielle complexe à évolution lente définie par un remodelage de la paroi vasculaire, associée à des réactions inflammatoires, à des processus de prolifération, de mort cellulaire et à une accumulation de lipides oxydés tels que les oxystérols (dérivés oxydés du cholestérol) dont le rôle est fortement suspecté dans le développement de l'athérome.<br /><br />Le travail réalisé a été effectué sur des cellules monocytaires humaines U937 et THP-1, aortiques de rat A7R5 et carcinomateuses humaines MCF-7 (déficientes en caspase-3). Différents oxystérols, présents en quantité importante dans les lésions athéromateuses, ont été utilisés (7-cétocholestérol (7KC), 7Β-hydroxycholestérol, 25-hydroxycholestérol, cholestérol-5Α, 6Α-epoxide, cholestérol-5Β, 6Β-epoxide) ainsi que du cholestérol.<br /><br />La première partie du travail a montré que la mort cellulaire induite par le 7KC est un phénomène complexe présentant des caractéristiques apoptotiques accompagnées d'une synthèse de structures multilamellaires cytoplasmiques appelées corps myéliniques visualisés par microscopie électronique à transmission. La synthèse rapide de ces structures précède les effets cytotoxiques : chute du potentiel membranaire mitochondrial, augmentation de la perméabilité à l'iodure de propidium et modifications de la morphologie nucléaire (condensation, fragmentation, gonflement des noyaux). L'isolement de ces structures par ultracentrifugation après coloration à la monodansylcadavérine (fluorochrome lysosomotropique acide) ou au Nile Red (fluorescence jaune en présence de lipides neutres et rouge en présence de lipides polaires) a permis de montrer qu'elles sont riches en lipides polaires (sphingomyéline, phosphatidylcholine) et en cholestérol et qu'elles accumulent le 7KC. Au sein de ces corps myéliniques, une co-localisation du 7KC avec les lipides polaires a été démontrée par la technique de FRET réalisée par microscopie confocale mono- et bi-photonique. Les caractéristiques morphologiques et biochimiques des figures myéliniques ont permis d'établir que le 7-cétocholestérol est un puissant inducteur de phospholipidose.<br /><br />Compte tenu des modifications lipidiques spatiotemporelles, quantitatives et qualitatives, induites par le 7KC et révélé par le Nile Red, la seconde partie du travail a précisé les relations entre la mort cellulaire, la synthèse de corps myéliniques, l'accumulation de lipides polaires et l'activité caspase. Avec les différents oxystérols étudiés, les corps myéliniques ne sont observés qu'avec des composés cytotoxiques (7KC, 7Β-hydroxycholestérol, cholestérol-5Β, 6Β-epoxide) et leur synthèse est indépendante d'activité caspase. En revanche, l'accumulation de lipides polaires induite par le 7KC est inhibée en présence de z-VAD-fmk (inhibiteurs de caspases large spectre) et de z-VDVAD-fmk (inhibiteur de caspase-2). Certaines caspases et en particulier la caspase-2 pourraient contribuer à l'accumulation de lipides polaires.<br /><br />La troisième partie du travail a conduit à étudier les effets de la Vitamine-E (VitE) sur la mort cellulaire induite par le 7KC en raison de ses propriétés anti-apoptotiques et anti-oxydantes. La VitE protège de la mort cellulaire induite par le 7KC. Les effets protecteurs pourraient en partie être dus à la capacité de la VitE à maintenir fonctionnelle la voie PI3-K/c-Akt en s'opposant aux déphosphorylations de PDK-1 et de c-Akt, et en préservant l'activité PI3-K. Par ailleurs, la VitE s'oppose aux modifications lipidiques au niveau de la membrane cytoplasmique et à l'accumulation de lipides polaires. La VitE réduit aussi la dégradation de la pro-forme de la caspase-2L et augmente les taux d'ARNm correspondants.<br /><br />Ces travaux montrent des relations entre la mort cellulaire induite par des oxystérols et le métabolisme des lipides. Ils révèlent aussi que la VitE protège de la mort induite par le 7KC en agissant au niveau de la voie PI3-K/c-Akt. Par ailleurs, la VitE s'oppose aux modifications lipidiques membranaires et cytoplasmiques associées à la mort cellulaire induite par le 7KC.

La toxine stable à la chaleur de type b (STb) est une des toxines produites par les souches Enterotoxigenic Escherichia coli (ETEC) impliquée dans le développement de la diarrhée. Une étude antérieure par Goncalves et al. (2009) a démontré que les cellules ayant internalisé la toxine STb démontraient une morphologie qui rappelle l’apoptose. Le changement du potentiel membranaire observé par Goncalves et al. (2009) nous a incité à vérifier la capacité de la toxine STb à induire l’apoptose des cellules HRT-18 et IEC-18 par la voie intrinsèque. Les cellules HRT-18 et IEC-18 ont été traitées avec de la toxine purifiée pour une durée de 24 heures puis ells ont été récoltées et examinées pour des caratéristiques de l’apoptose. L’activation des caspases-9 et -3, mais pas de la caspase-8, a été observée dans les deux lignées cellulaires à l’aide des substrats fluorescents spécifiques pour chaque caspase. L’ADN extrait des cellules HRT-18 et IEC-18 a révélé une fragmentation lorsque migré sur gel d’agarose. La condensation et la fragmentation des noyaux ont été observées en microscopie à fluorescence suite à une coloration de l’ADN au Hoechst 33342. Les indices apoptotiques des cellules HRT-18 et IEC-18 traitées avec des quantités croissantes de STb montrent une dose-réponse pour les deux lignées. L’activation de la caspase-9 est une indication que la voie intrinsèque de l’apoptose est activée dans les cellules HRT-18 et IEC-18. L’absence de l’activation de la caspase-8 démontre que la voie extrinsèque n’est pas impliquée dans la mort cellulaire médiée par STb. / Heat-stable toxin b (STb) is one of the toxins produced by Enterotoxigenic Escherichia coli (ETEC) strains implicated in the development of diarrhea. A previous study conducted by Goncalves et al. (2009) showed that cells having internalized STb toxin demonstrated apoptotic-like morphology. The change in the mitochondrial membrane potential observed by Goncalves et al. (2009) prompted us to verify the ability of STb toxin to induce apoptosis via the intrinsic pathway in HRT-18 and IEC-18 cells. Both cell lines were treated with purified STb toxin for a period of 24 hours, harvested, and examined for apoptotic features. Activation of caspases-9 and -3, but not -8, was observed in HRT-18 and IEC-18 cells as determined with the use of fluorescent substrates specific to each caspase. Extracted DNA revealed DNA laddering when migrated on agarose gels. Nuclear condensation and fragmentation of Hoechst 33342 stained DNA of HRT-18 and IEC-18 cells were visualized by fluorescence microscopy. Apoptotic indexes of HRT-18 and IEC-18 cells treated with increasing amounts of STb toxin revealed dose-dependent responses in both cell lines. The activation of caspase-9 is an indication of the intrinsic pathway being activated in HRT-18 and IEC-18 cells by STb toxin. The lack of caspase-8 activation demonstrates that the extrinsic pathway of apoptosis is not involved in the programmed cell death mediated by STb.

Exposure cigarette smoke (CS) during prenatal life is the leading cause of preventable premature death. In this study, we explored the hypothesis that in vitro exposure of fetal lung cells to cigarette smoke extract (CSE) may result in the alteration of apoptosis through activation of caspase-3. Alongside we compared the responses of fetal lung cells with A549 cells and rat periodontal ligament (PDL) fibroblasts exposed to CSE in a dose dependent manner. Caspase-3 activity and inhibition was measured using a fluorometric assay. Cell viability in smoke exposed cells was measured using MTT formazan assay. Caspase-3 expression and cellular localization was detected by western blot analysis and immunofluorescence. Our results indicate that caspase-3 activity was significantly (p < 0.05) elevated and cell viability was significantly inhibited in fetal rat lung cells exposed to 10% or 15 % (v/v) CSE. No significant differences were observed in the caspase-3 activity or cellular viability in A549 cells and rat PDL fibroblasts exposed to 5%, 10% or 15% (v/v) CSE. Activation of caspase-3 in fetal lung connective tissue and alveolar epithelial cells may be one of the reasons for the developmental pulmonary toxicity induced by CSE.