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

No description available.

No description available.

No description available.

No description available.

Mehrere Studien haben gezeigt, daß die Cyclooxygenase-2 (COX-2) eine bedeutende Rolle sowohl bei Entstehung als auch Progression maligner Tumoren spielt. COX-2-Inhibitoren werden bereits in klinischen Studien zur Krebstherapie getestet. COX-2 ist die induzierbare Isoform der Cyclooxygenase - dem Schlüsselenzym der Synthese von Prostaglandinen und anderen Eicosanoiden. Im Tier- und Zellkulturmodell konnten COX-Hemmer anti-Tumor-Effekte hervorrufen. Es ist jedoch unklar, ob diese Effekte durch Hemmung des COX-Enzyms oder durch COX-unabhängige Mechanismen vermittelt werden. Wir untersuchten daher die Auswirkung der COX-Inhibition zum einen durch den selektiven COX-2-Hemmer NS-398 sowie zum anderen durch COX-Isoform-spezifische RNA-Interferenz (RNAi) in zwei humanen Ovarialkarzinomzellinien (OVCAR-3 und SKOV-3). OVCAR-3 zeigte eine konstitutive COX-1-Expression und eine durch IL-1beta induzierbare COX-2-Expression. SKOV-3 war COX-1- und COX-2-negativ. IL-1beta führte bei OVCAR-3 zu einer vermehrten Produktion von Prostaglandin E2 (PGE2), die durch eine gegen die COX-2 gerichtete siRNA gehemmt werden konnte, wohingegen COX-1-siRNA keinen Effekt hatte. Das deutet darauf hin, daß die COX-2 die Hauptquelle von PGE2 in OVCAR-3 ist. 1mikroM NS-398 waren ausreichend, um die PGE2-Produktion und somit auch die COX-2 in OVCAR-3 zu inhibieren. Höhere Konzentrationen NS-398 (>10mikroM) hatten einen antiproliferativen Effekt. Auch in der COX-2-negativen Zellinie SKOV-3 trat diese Wachstumshemmung auf; sie war nicht durch exogene Zufuhr von PGE2 (10mikroM) reversibel. Durchflußzytometrische Zellzyklusanalyse ergab, daß der Wachstumshemmung in beiden Zellinien ein G0/G1-Zellzyklusarrest zugrunde liegt. Dagegen führten weder COX-1- noch COX-2-Ausschaltung durch RNAi zu ähnlichen Auswirkungen auf Proliferation bzw. Zellzyklus. Diese Ergebnisse zeigen, dass ein COX-2-unabhängiger Mechanismus für den durch NS-398 induzierten G0/G1-Arrest verantwortlich ist. / Several studies have provided evidence that the enzyme Cyclooxygenase-2 (COX-2) plays an important role in tumor development and progression. COX-2-inhibitors are already evaluated in clinical trials as cancer therapeutics. COX-2 is the inducible isoform of cyclooxygenase - the rate-limiting enzyme in the synthesis of prostaglandins and other eicosanoids. COX-inhibitors cause antitumor effects in animal models and in cell culture experiments. However, it is not clear, whether these effects are due to inhibition of the COX-enzyme or mediated via a COX-independent mechanism. We therefore investigated the effects of COX inhibition by the selective COX-2-inhibitor NS-398, as well as by COX-isoform specific RNA interference (RNAi) in the human ovarian carcinoma cell lines OVCAR-3 and SKOV-3. OVCAR-3 cells showed a constitutive expression of COX-1, and an inducible COX-2 expression. COX-2 was induced through stimulation with Interleukin-1beta, leading to production of high levels of Prostaglandin E2 (PGE2). SKOV-3 cells were negative for both COX isoforms. Selective COX-2-suppression by RNAi reduced PGE2 production in OVCAR-3, whereas COX-1-siRNA had no effect on PGE2 synthesis. Thus, COX-2 is the main source of PGE2 in OVCAR-3 cells. In these cells, 1microM NS-398 was sufficient to completely inhibit PGE2-synthesis - and thus the activity of the COX-2 enzyme. Increasing amounts of NS-398 (>10microM) had an antiproliferative effect. This growth inhibition was also observed in the COX-negative cell line SKOV-3, it could not be reverted by exogenous addition of PGE2 (10microM). Flowcytometric analysis of the cell cycle revealed that this growth inhibition was based on a G0/G1-cell-cycle-arrest. In contrast, suppression of COX-1 or COX-2 by RNAi had no effect on proliferation or cell cycle progression. These results suggest that a COX-independent mechanism is responsible for the G0/G1-arrest induced by NS-398.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

L’évolution d’une cellule tumorale initiée à une tumeur solide nécessite, à chaque étape, un microenvironnement favorable à sa survie et à sa croissance. Le microenvironnement tumoral est comparé à un foyer d’inflammation chronique dont la composition cellulaire et moléculaire est complexe. Les cellules souches mésenchymateuses (CSM) représentent l’un des principaux acteurs cellulaires présents. Elles migrent vers les sites tumoraux où elles soutiennent l’inflammation, l’angiogenèse et le développement tumoral en activant plusieurs voies de signalisation. Une des voies majeures qui contribuent à l’inflammation est la voie de signalisation NF-B. L’initiation de cette voie provient de la membrane cellulaire entre autres des cavéoles. Nous soumettons l’hypothèse que l’une des cavines, protéines associées aux cavéoles, modulerait le phénotype inflammatoire etou migratoire dans les CSM traitées à la cytokine TNF- (facteur de nécrose tumorale ) en modulant la voie de signalisation NF-B. En effet, nous avons observé une régulation à la hausse de l’expression de la COX-2 (cyclooxygénase-2) et une diminution de l’expression d’IκB qui sont synonymes de l’activation de la voie NF-B dans les CSM que nous avons traitées au TNF-. Nous avons trouvé que le TNF- induit la migration des CSM, et que la répression génique de la Cavine-2 augmente significativement la migration des CSM traitées par le TNF-. La répression génique de la Cavine-2 vient aussi amplifier la tubulogenèse dans les CSM en réponse au TNF-. D’un point de vue moléculaire, la répression génique de la Cavine-2 a montré une très forte amplification de l'expression protéique de la COX-2 dans les CSM en réponse au TNF-. Dans ces mêmes cellules où la Cavine-2 a été réprimée, et suite à un traitement au TNF-, le pic de phosphorylation est plus intense et la courbe de phosphorylation est plus prolongée dans le temps. Ces observations nous permettent d’affirmer que la Cavine-2 a un rôle répresseur sur l’expression de COX-2. Collectivement, nos résultats montrent que la Cavine-2 peut être proposée comme un gène suppresseur de tumeur et est de ce fait, une bonne cible thérapeutique dans les CSM qui permettraient d’agir à des stades précoces du développement tumoral. / The evolution of an initiated tumor cell into a solid tumor requires at each stage a favorable microenvironment for its survival and growth. The tumor microenvironment is compared to a chronic inflammation site with a cellular and molecular complex composition. Mesenchymal stem cells (MSC) have important roles in tumor microenvironment. They migrate to tumor sites where they maintain the inflammation, angiogenesis and tumor development by activating multiple signaling pathways. One of the major pathways that contribute to inflammation is the NF-B signaling pathway. The initiation of this pathway comes from the cell membrane and caveolae. Our hypothesis is that one of cavins, proteins associated to caveolae, modulates the inflammatory phenotype and migration in MSC treated with TNF-. We suggest that this process is modulated by a NF-B signaling pathway. Indeed, we observed an up-regulation of the expression of the cyclooxygenase-2 (COX-2) and a decrease in the expression of IκB which suggest that activation of the NF-B pathway is involved in the MSC treated with TNF. We found that the TNF- induced migration in the MSC, and the knockout of Cavin-2 significantly increased migration of MSC treated with TNF-. The silencing of Cavin-2 considerably increased tubulogenesis of MSC treated with TNF-. At the molecular level, knockout of Cavin-2 showed a very strong amplification of protein expression of COX-2 in the MSC in response to TNF-. In these same cells where Cavin-2 was repressed and treated with TNF-, the peak of phosphorylation of pIB is more intense and the phosphorylation curve is sustained in time. These observations allow us to assert that Cavin-2 has a repressing role on the expression of COX-2. Collectively, our results show that the gene encoding Cavin-2 can be proposed as tumor suppressor gene. This study allowed us to identify new therapeutic targets: Cavins proteins.

Diversos estudos têm demonstrado que a atividade coordenada das células epiteliais com o estroma é fundamental no crescimento e diferenciação em situações fisiológicas e patológicas, inclusive no câncer. Vários relatos acentuam a importância do compartimento estromal nos tumores malignos e indicam fortemente que interações contínuas entre o carcinoma e as células estromais (resultando em regulamento e modulação recíproca) são condições prévias para desenvolvimento e progressão de carcinomas. Comparativamente, pouca informação está disponível sobre as características e o papel do estroma durante o processo carcinogênico e a maioria dos dados são baseados em estudos isolados. Nos animais tratados com o carcinógeno Dimetilhidrazina foi identificado na mucosa colônica o aparececimento de “Focos de Estroma Ativado" (FEA) que diferem do foco inflamatório esporádico encontrado na mucosa normal dos animais controles devido à imuno-expressão aumentada de células CD34, CD68, α-actina de músculo liso (ASMA), COX-2 positivas e densidade microvascular. Além disso, o FEA cercou um número aumentado de criptas colônicas em fissão que freqüentemente apresentavam células epiteliais com núcleos hipercromáticos. Este último achado pode sugerir correlação entre as alterações estromais e epiteliais dentro dos FEA. Embora esses achados sejam novos, são consistentes com observações prévias que o estroma tem um papel significante na carcinogênese. Juntamente com dados da literatura, este trabalho sugere que, no cólon, a “field cancerization" epitelial pode ser acompanhada através de alterações estromais e isto pode apontar novos marcadores de transformação neoplásica. / There has been considerable that the activity of epithelial cells with their stroma is fundamental in controlling growth and differentiation in normal and pathological situations, including cancer. A number of reports stress the importance of the stromal compartment in malignant tumors and strongly indicate that continuous interactions between the carcinoma and stromal cells (resulting in their reciprocal regulation and modulation) are prerequisites for carcinoma development and progression. Comparatively, less information is available about the features and role of the stroma for the carcinogenic process. In animals treated with the carcinogen Dimethyl-hydrazine we identified the appearing of mucosal “Activated Stromal Foci" (ASF) that differ from the sporadic inflammatory foci found in the normal mucosa of the control animals because of the presence of increased immune-expression of CD34, CD68, α-smooth muscle actin (ASMA), COX-2 positive cells and microvessel density. Furthermore, the ASF surrounded a increased number of colonic crypts in fission when compared to areas of normal stroma. This last finding suggests that stromal activation and epithelial changes may be correlated. These findings are novel but expected and consistent with previous observations that the stroma has a significant role in carcinogenesis. Taken together with literature data, our findings suggest that in the colon, the epithelial field cancerization may be accompanied by stromal changes and this may point to the finding of new markers of neoplastic transformation.

Diversos estudos têm demonstrado que a atividade coordenada das células epiteliais com o estroma é fundamental no crescimento e diferenciação em situações fisiológicas e patológicas, inclusive no câncer. Vários relatos acentuam a importância do compartimento estromal nos tumores malignos e indicam fortemente que interações contínuas entre o carcinoma e as células estromais (resultando em regulamento e modulação recíproca) são condições prévias para desenvolvimento e progressão de carcinomas. Comparativamente, pouca informação está disponível sobre as características e o papel do estroma durante o processo carcinogênico e a maioria dos dados são baseados em estudos isolados. Nos animais tratados com o carcinógeno Dimetilhidrazina foi identificado na mucosa colônica o aparececimento de “Focos de Estroma Ativado” (FEA) que diferem do foco inflamatório esporádico encontrado na mucosa normal dos animais controles devido à imuno-expressão aumentada de células CD34, CD68, α-actina de músculo liso (ASMA), COX-2 positivas e densidade microvascular. Além disso, o FEA cercou um número aumentado de criptas colônicas em fissão que freqüentemente apresentavam células epiteliais com núcleos hipercromáticos. Este último achado pode sugerir correlação entre as alterações estromais e epiteliais dentro dos FEA. Embora esses achados sejam novos, são consistentes com observações prévias que o estroma tem um papel significante na carcinogênese. Juntamente com dados da literatura, este trabalho sugere que, no cólon, a “field cancerization” epitelial pode ser acompanhada através de alterações estromais e isto pode apontar novos marcadores de transformação neoplásica. / There has been considerable that the activity of epithelial cells with their stroma is fundamental in controlling growth and differentiation in normal and pathological situations, including cancer. A number of reports stress the importance of the stromal compartment in malignant tumors and strongly indicate that continuous interactions between the carcinoma and stromal cells (resulting in their reciprocal regulation and modulation) are prerequisites for carcinoma development and progression. Comparatively, less information is available about the features and role of the stroma for the carcinogenic process. In animals treated with the carcinogen Dimethyl-hydrazine we identified the appearing of mucosal “Activated Stromal Foci” (ASF) that differ from the sporadic inflammatory foci found in the normal mucosa of the control animals because of the presence of increased immune-expression of CD34, CD68, α-smooth muscle actin (ASMA), COX-2 positive cells and microvessel density. Furthermore, the ASF surrounded a increased number of colonic crypts in fission when compared to areas of normal stroma. This last finding suggests that stromal activation and epithelial changes may be correlated. These findings are novel but expected and consistent with previous observations that the stroma has a significant role in carcinogenesis. Taken together with literature data, our findings suggest that in the colon, the epithelial field cancerization may be accompanied by stromal changes and this may point to the finding of new markers of neoplastic transformation.