The oncogenic protein Bcl-2 is overexpressed in many cancers and prevents cells from undergoing apoptosis in response to traditional chemotherapeutic agents. Recent research has focussed on the development of novel agents that can disrupt the function of Bcl-2 and trigger apoptosis in cancer cells. The isothiocyanates are a class of naturally-occurring phytochemical with potential for development as both chemopreventive and chemotherapeutic agents. This thesis investigated the ability of the isothiocyanates to induce apoptosis in cells that overexpressed Bcl-2. Initially, phenethyl isothiocyanate was shown to be cytotoxic to the Jurkat Tlymphoma cell line with an LD50 of 7.4 µM. Bcl-2 expression had little protective effect, and even greater than 50-fold overexpression only increased the LD50 to 15.1 µM. Morphological and biochemical assays indicated that death still occurred by apoptosis despite overexpression of Bcl-2. A variety of other isothiocyanates were also screened for cytotoxic activity. While the isothiocyanate moiety was crucial for induction of apoptosis, the chemistry of the side chain attached to the isothiocyanate moiety also profoundly influenced the ability of an isothiocyanate to kill Bcl-2 overexpressing cells. The aromatic isothiocyanates were generally far more cytotoxic than aliphatic isothiocyanates. However, within the aromatic isothiocyanates tested in this study the length of the carbon linker group, between the phenyl ring and the isothiocyanate moiety, also influenced cytotoxic activity. Phenethyl isothiocyanate was identified as the most promising compound when targeting cells that overexpressed Bcl-2. Given that minor structural alterations significantly altered cytotoxic activity it is hypothesised that specific interactions with cellular targets may mediate induction of apoptosis by the isothiocyanates. Finally, using a sensitive proteomic technique to label oxidised thiol proteins a preliminary investigation of the targets of the isothiocyanates was performed. A number of thiol proteins were selectively modified following exposure to phenethyl isothiocyanate. One thiol protein that consistently changed was identified as mitochondrial peroxiredoxin-3. Changes to the oxidation state of peroxiredoxin-3 occurred well before activation of apoptosis and may play a role in mediating induction of apoptosis in cells that overexpress Bcl-2. The results of this thesis have provided a platform to permit further investigation of the chemotherapeutic potential of the isothiocyanates and investigation of the mechanisms that allow the isothiocyanates to induce apoptosis in cells that overexpress the oncogene Bcl-2. In the future, the identification of primary targets of the isothiocyanates may aid the design and testing of novel anticancer drugs, and it will also provide novel insight into the regulation of apoptosis.

The oncogenic protein Bcl-2 is overexpressed in many cancers and prevents cells from undergoing apoptosis in response to traditional chemotherapeutic agents. Recent research has focussed on the development of novel agents that can disrupt the function of Bcl-2 and trigger apoptosis in cancer cells. The isothiocyanates are a class of naturally-occurring phytochemical with potential for development as both chemopreventive and chemotherapeutic agents. This thesis investigated the ability of the isothiocyanates to induce apoptosis in cells that overexpressed Bcl-2. Initially, phenethyl isothiocyanate was shown to be cytotoxic to the Jurkat Tlymphoma cell line with an LD50 of 7.4 µM. Bcl-2 expression had little protective effect, and even greater than 50-fold overexpression only increased the LD50 to 15.1 µM. Morphological and biochemical assays indicated that death still occurred by apoptosis despite overexpression of Bcl-2. A variety of other isothiocyanates were also screened for cytotoxic activity. While the isothiocyanate moiety was crucial for induction of apoptosis, the chemistry of the side chain attached to the isothiocyanate moiety also profoundly influenced the ability of an isothiocyanate to kill Bcl-2 overexpressing cells. The aromatic isothiocyanates were generally far more cytotoxic than aliphatic isothiocyanates. However, within the aromatic isothiocyanates tested in this study the length of the carbon linker group, between the phenyl ring and the isothiocyanate moiety, also influenced cytotoxic activity. Phenethyl isothiocyanate was identified as the most promising compound when targeting cells that overexpressed Bcl-2. Given that minor structural alterations significantly altered cytotoxic activity it is hypothesised that specific interactions with cellular targets may mediate induction of apoptosis by the isothiocyanates. Finally, using a sensitive proteomic technique to label oxidised thiol proteins a preliminary investigation of the targets of the isothiocyanates was performed. A number of thiol proteins were selectively modified following exposure to phenethyl isothiocyanate. One thiol protein that consistently changed was identified as mitochondrial peroxiredoxin-3. Changes to the oxidation state of peroxiredoxin-3 occurred well before activation of apoptosis and may play a role in mediating induction of apoptosis in cells that overexpress Bcl-2. The results of this thesis have provided a platform to permit further investigation of the chemotherapeutic potential of the isothiocyanates and investigation of the mechanisms that allow the isothiocyanates to induce apoptosis in cells that overexpress the oncogene Bcl-2. In the future, the identification of primary targets of the isothiocyanates may aid the design and testing of novel anticancer drugs, and it will also provide novel insight into the regulation of apoptosis.

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 2002. / Titre de l'écran-titre (visionné le 17 juillet 2006). Publié aussi en version papier.

A Tuberculose é uma das doenças infecciosas mais antiga e bem descrita. Entretanto, ainda permanece como um dos principais problemas de saúde pública a ser enfrentado em âmbito global. A implantação de novas estratégias no controle da Tuberculose requer uma melhor compreensão dos mecanismos que sucedem a fagocitose das micobactérias por macrófagos. Após a fagocitose, as micobactérias dão início a um conjunto de ações para sobreviverem e se replicarem no ambiente intracelular, entre as quais a provável interferência no processo de morte celular. Estudos mostram que M. tuberculosis pode apresentar habilidade de interferir no mecanismo de morte celular. Essa habilidade se tornou um desafio a ser estudado devido às implicações que isso deve ter na patogênese da doença. O nosso estudo teve por objetivo analisar a expressão de genes anti-apoptóticos (bcl-2, bcl-x e mcl-1) e pro-apoptóticos (bak, bax e bid) por PCR em tempo Real em macrófagos humanos derivados de células THP-1 após diferenciação induzida por PMA. Além disso, analisar a porcentagem de fragmentação de DNA nesses macrófagos, utilizando a citometria de fluxo, pois a fragmentação internucleossômica do DNA é uma das características apresentadas por células apoptóticas. Para as infecções foram utilizados isolados clínicos de M. tuberculosis com perfil de suscetibilidade a fármacos diferentes e a cepa padrão H37Rv (ATCC). Os dados de expressão foram analisados pela diferença de entre os isolados clínicos sensíveis, resistentes a três dos fármacos utilizados no tratamento da tuberculose humana e a cepa padrão H37Rv, utilizando-se o método de 2-ΔΔCT. Para comparar os resultados de expressão gênica, bem como a porcentagem de fragmentação de DNA, nos macrófagos infectados com os diferentes isolados clínicos, foram utilizados análise de variância (ANOVA) e o teste de comparação múltipla de Tukey. Os resultados sugerem, que os isolados clínicos resistentes a INH, RIF e EMB utilizados no nosso estudo, bem como a cepa padrão H37Rv (ATCC), não induzem mecanismos anti-apoptóticos para evadir da resposta imune. A ocorrência de fragmentação de DNA nos macrófagos infectados é um indicativo de morte por apoptose ou pyroptose. Além disso, o tempo de infecção éum fator importante e, com certeza, infecções com tempos maiores poderiam induzir ainda mais a morte dos macrófagos infectados. / Tuberculsis, an ancient infection disease, continues to thrive. Although well described, it remains a world health problem to overcome. The development and application of new strategies to control Tuberculosis requires a better understanding of mechanisms involved in mycobacteria-macrophages interaction. Following phagocytosis, mycobacteria initiates a variety of actions to survive and multiply themselves inside macrophages. According to researches, mycobacteria might interfere in the cell death mechanism. This ability became a challenge to be studied due to its implications in the pathogenesis of the disease. The aim of this study was to analyze the gene expression of anti-apoptotic (bcl-2, bcl-x e mcl-1) and pro-apoptotic genes (bak, bax e bid) in PMA-treated THP-1 cells by Real Time qPCR. Moreover, the percentage of macrophage DNA fragmentation was assessed by flow citometry because internucleosomal DNA fragmentation is characteristic of apoptotic and pyroptotic cell death. The infection was carried out using clinical isolates of M. tuberculosis resistent to multiple drugs, drug susceptibility and the M. tuberculosis H37Rv strain. The difference in the expression profile among clinical isolates, susceptible and resistant to three drugs used in the TB treatment, and the M. tuberculosis H37Rv were evaluated with the method 2-ΔΔCT. In order to compare gene expression patterns as well as the percentage of DNA fragmentation in macrophages infected with different clinical isolates, it was used analysis of variance (ANOVA) and Tukey`s multiple comparison test. The results suggest that M. tuberculosis H37Rv and the clinical isolates presenting higher drug resistant profile might induce programmed cell death in macrophages after 24-h infection. This was observed in the gene expression pattern and also in the macrophage DNA fragmentation profile, which indentifies apoptosis or pyroptosis. Therefore, it is suggested these clinical isolates and M. tuberculosis H37Rv do not present anti-apoptotic mechanisms to evade immune response. Moreover, the infection time is an important factor and, definitely, infections for long time could induce increase death of the infectados macrophages.

A Tuberculose é uma das doenças infecciosas mais antiga e bem descrita. Entretanto, ainda permanece como um dos principais problemas de saúde pública a ser enfrentado em âmbito global. A implantação de novas estratégias no controle da Tuberculose requer uma melhor compreensão dos mecanismos que sucedem a fagocitose das micobactérias por macrófagos. Após a fagocitose, as micobactérias dão início a um conjunto de ações para sobreviverem e se replicarem no ambiente intracelular, entre as quais a provável interferência no processo de morte celular. Estudos mostram que M. tuberculosis pode apresentar habilidade de interferir no mecanismo de morte celular. Essa habilidade se tornou um desafio a ser estudado devido às implicações que isso deve ter na patogênese da doença. O nosso estudo teve por objetivo analisar a expressão de genes anti-apoptóticos (bcl-2, bcl-x e mcl-1) e pro-apoptóticos (bak, bax e bid) por PCR em tempo Real em macrófagos humanos derivados de células THP-1 após diferenciação induzida por PMA. Além disso, analisar a porcentagem de fragmentação de DNA nesses macrófagos, utilizando a citometria de fluxo, pois a fragmentação internucleossômica do DNA é uma das características apresentadas por células apoptóticas. Para as infecções foram utilizados isolados clínicos de M. tuberculosis com perfil de suscetibilidade a fármacos diferentes e a cepa padrão H37Rv (ATCC). Os dados de expressão foram analisados pela diferença de entre os isolados clínicos sensíveis, resistentes a três dos fármacos utilizados no tratamento da tuberculose humana e a cepa padrão H37Rv, utilizando-se o método de 2-ΔΔCT. Para comparar os resultados de expressão gênica, bem como a porcentagem de fragmentação de DNA, nos macrófagos infectados com os diferentes isolados clínicos, foram utilizados análise de variância (ANOVA) e o teste de comparação múltipla de Tukey. Os resultados sugerem, que os isolados clínicos resistentes a INH, RIF e EMB utilizados no nosso estudo, bem como a cepa padrão H37Rv (ATCC), não induzem mecanismos anti-apoptóticos para evadir da resposta imune. A ocorrência de fragmentação de DNA nos macrófagos infectados é um indicativo de morte por apoptose ou pyroptose. Além disso, o tempo de infecção éum fator importante e, com certeza, infecções com tempos maiores poderiam induzir ainda mais a morte dos macrófagos infectados. / Tuberculsis, an ancient infection disease, continues to thrive. Although well described, it remains a world health problem to overcome. The development and application of new strategies to control Tuberculosis requires a better understanding of mechanisms involved in mycobacteria-macrophages interaction. Following phagocytosis, mycobacteria initiates a variety of actions to survive and multiply themselves inside macrophages. According to researches, mycobacteria might interfere in the cell death mechanism. This ability became a challenge to be studied due to its implications in the pathogenesis of the disease. The aim of this study was to analyze the gene expression of anti-apoptotic (bcl-2, bcl-x e mcl-1) and pro-apoptotic genes (bak, bax e bid) in PMA-treated THP-1 cells by Real Time qPCR. Moreover, the percentage of macrophage DNA fragmentation was assessed by flow citometry because internucleosomal DNA fragmentation is characteristic of apoptotic and pyroptotic cell death. The infection was carried out using clinical isolates of M. tuberculosis resistent to multiple drugs, drug susceptibility and the M. tuberculosis H37Rv strain. The difference in the expression profile among clinical isolates, susceptible and resistant to three drugs used in the TB treatment, and the M. tuberculosis H37Rv were evaluated with the method 2-ΔΔCT. In order to compare gene expression patterns as well as the percentage of DNA fragmentation in macrophages infected with different clinical isolates, it was used analysis of variance (ANOVA) and Tukey`s multiple comparison test. The results suggest that M. tuberculosis H37Rv and the clinical isolates presenting higher drug resistant profile might induce programmed cell death in macrophages after 24-h infection. This was observed in the gene expression pattern and also in the macrophage DNA fragmentation profile, which indentifies apoptosis or pyroptosis. Therefore, it is suggested these clinical isolates and M. tuberculosis H37Rv do not present anti-apoptotic mechanisms to evade immune response. Moreover, the infection time is an important factor and, definitely, infections for long time could induce increase death of the infectados macrophages.

[ES] La familia de proteínas Bcl-2 regula la apoptosis a través de una compleja red de interacciones. Las células tumorales suelen presentar mutaciones que afectan a su expresión o sus interacciones para mejorar la progresión tumoral. Además, alteraciones en su regulación también promueven la migración de células cancerígenas, la invasión y la metástasis. Para llevar a cabo sus funciones, las proteínas Bcl 2 interaccionan entre sí tanto en el citoplasma como en las membranas intracelulares. Los equilibrios de interacción de los dominios Bcl citosólicos se han investigado ampliamente y recientemente, se han propuesto como dianas terapéuticas. Sin embargo, el interactoma de los dominios transmembrana (TMD, del inglés transmembrane domains) sigue siendo poco conocido. Por ello, un conocimiento profundo de la biología de las proteínas Bcl-2 es necesario para explotar eficientemente sus superficies de unión en el tratamiento del cáncer. Para llevar a cabo este objetivo, nos hemos centrado en tres áreas: 1. La comprensión detallada de la contribución del TMD de Mcl-1 a su interactoma en membrana y su función. 2. El descubrimiento de nuevos inhibidores de Mcl-1 que actúen sobre su TMD y que permitan desarrollar una clase de drogas anticancerígenas aún por explorar. 3. La caracterización molecular de mutaciones relacionadas con el cáncer descritas en los TMD de Bcl-2 y Bcl-xL y sus implicaciones en la supervivencia de las células tumorales. La proteína antiapoptótica Mcl-1 inhibe a los miembros proapoptóticos Bak, Bax, Bok, Noxa, etc. Aunque se ha estudiado en detalle su actividad promoviendo la supervivencia celular, el mecanismo molecular por el cuál previene la apoptosis mediada por Bok aún no está claro. Además, el conocimiento de las actividades de Mcl-1, descritas hasta ahora, se basa exclusivamente en las estructuras resueltas de las regiones solubles en agua y en estudios centrados en los dominios citosólicos. Por primera vez, hemos demostrado la relevancia del TMD de Mcl-1 en su equilibrio de interacción. En este trabajo describimos su capacidad específica para homo- y hetero-oligomerizar con el TMD de Bok. También ponemos de manifiesto la influencia de estas interacciones en la modulación de apoptosis y resaltamos la relevancia clínica de los mutantes del TMD de Mcl-1 identificados en pacientes con cáncer. Muchos tumores hematológicos y sólidos sobre-expresan Mcl-1 como mecanismo para adquirir quimiorresistencia. Se han desarrollado miméticos de BH3 específicos para modular su actividad antiapoptótica en células cancerosas. Sin embargo, aún no disponemos de datos científicos que informen sobre su toxicidad y eficacia en humanos. En este trabajo, proponemos la novedosa interacción de los TMDs de Mcl-1 y Bok como un nuevo sitio de acción de fármacos quimioterapéuticos. Hemos identificado tres inhibidores de esta interacción con características que los hacen prometedores candidatos para el desarrollo farmacéutico, así como buenas herramientas moleculares para estudiar la interacción de los TMDs de Mcl-1 y Bok. Para modular la apoptosis, las células tumorales también presentan versiones mutadas de las proteínas antiapoptóticas Bcl-2 y Bcl-xL. En nuestro conocimiento, este es el primer estudio que analiza mutaciones somáticas de sus TMDs. Nuestro trabajo demuestra cómo estas mutaciones alteran el equilibrio en membrana de las proteínas. Además, nuestros resultados explican la influencia que algunos mutantes somáticos ejercen en la regulación de la apoptosis. En general, los resultados científicos que aparecen en esta tesis resaltan el papel de los Bcl TMDs en el interactoma de las proteínas Bcl-2. Estos hallazgos corroboran que las interacciones laterales entre los TMDs son específicas y contribuyen activamente a la funcionalidad de la proteína. Por lo tanto, comprender los Bcl TMDs puede proporcionar nuevos conocimientos sobre la biología de las proteínas Bcl. / [CA] La família de proteïnes Bcl-2 regula l'apoptosi a través d'una complexa xarxa d'interaccions. Les cèl·lules tumorals solen presentar mutacions que afecten la seua expressió o les seues interaccions per a millorar la progressió tumoral. A més, alteracions en la seua regulació també promouen la migració de cèl·lules cancerígenes, la invasió i la metàstasi. Per a dur a terme les seues funcions, les proteïnes Bcl-2 interaccionen entre si tant en el citoplasma com en les membranes intracel·lulars. Els equilibris d'interacció dels dominis Bcl citosòlics s'han investigat àmpliament i recentment, s'han proposat com a dianes terapèutiques. No obstant això, l'interactoma dels dominis transmembrana (TMD, de l'anglés transmembrane domains) continua sent poc conegut. Per això, un coneixement profund de la biologia de les proteïnes Bcl-2 és necessari per a explotar eficientment les seues superfícies d'unió en el tractament del càncer. Per a dur a terme aquest objectiu, ens hem centrat en tres àrees: 1. La comprensió detallada de la contribució del TMD de Mcl-1 al seu interactoma en membrana i la seua funció. 2. El descobriment de nous inhibidors de Mcl-1 que actuen sobre el seu TMD i que permeten desenvolupar una classe de drogues anticanceroses encara per explorar. 3. La caracterització molecular de mutacions relacionades amb el càncer descrites en els TMD de Bcl-2 i Bcl-xL i les seues implicacions en la supervivència de les cèl·lules tumorals. La proteïna anti apoptòtica Mcl-1 inhibeix als membres pro apoptòtics Bak, Bax, Bok, Noxa, etc. Encara que s'ha estudiat detalladament la seua activitat promovent la supervivència cel·lular, el mecanisme molecular pel qual prevé l'apoptosi mediada per Bok encara no és clar. A més, el coneixement de les activitats de Mcl-1, descrites fins ara, es basa exclusivament en les estructures resoltes solubles en aigua i en estudis centrats en els dominis externs a la membrana. Per primera vegada, hem demostrat la rellevància del TMD de Mcl-1 el seu equilibri d'interacció. En aquest treball descrivim la seua capacitat específica per a unir-se amb si mateix i per a hetero-oligomeritzar amb el TMD de Bok. També expliquem la influència d'aquestes interaccions en l'apoptosi i ressaltem la rellevància clínica dels mutants del TMD de Mcl-1 identificats en pacients amb càncer. Molts tumors hematològics i sòlids sobre-expressen Mcl-1 com un mecanisme per a adquirir quimioresistència. S'han desenvolupat mimètics de BH3 específics per a modular la seua activitat anti apoptòtica en cèl·lules canceroses. No obstant això, encara no disposem de dades científiques que informen sobre la seua toxicitat i eficàcia en humans. Per això, proposem la nova interacció dels TMDs de Mcl-1 i Bok com un lloc d'actuació de fàrmacs quimioterapèutiques. Hem identificat tres inhibidors d'aquesta interacció amb característiques que els fan prometedors candidats per al desenvolupament farmacèutic, així com bones eines moleculars per a estudiar la interacció dels TMDs de Mcl-1 i Bok. Per a modular l'apoptosi, les cèl·lules tumorals també presenten versions mutades de les proteïnes anti apoptòtiques Bcl-2 i Bcl-xL. En el nostre coneixement, aquest és el primer estudi que analitza mutacions somàtiques de les seues TMDs. El nostre treball demostra com aquestes mutacions alteren l'equilibri en membrana de les proteïnes. A més, els nostres resultats expliquen la influència que alguns mutants somàtics exerceixen en la regulació de l'apoptosi. En general, els resultats científics que apareixen en aquesta tesi ressalten el paper dels Bcl TMDs en l'interactoma de les proteïnes Bcl-2. Aquestes troballes corroboren que les interaccions laterals entre els TMDs són específiques de la seqüència i contribueixen activament a la funcionalitat de la proteïna. Per tant, comprendre els Bcl TMDs pot proporcionar nous coneixements sobre la biologia de les proteïnes Bcl / [EN] The family of the Bcl-2 proteins modulates the apoptotic pathway by a complex network of interactions. Tumor cells frequently present mutations that affect Bcl-2 proteins expression or interactions to enhance cancer progression. Dysregulation of these proteins also promotes cancer cell migration, invasion, and metastasis. To execute their functions, Bcl-2 proteins interact in both the cytosol and intracellular membranes. Binding equilibria of Bcl extramembrane domains has been largely investigated and recently proposed as chemotherapeutic targets. However, the interactome of transmembrane domains (TMDs) remains poorly understood. In this scenario, a deep knowledge of the biology of Bcl-2 proteins is needed to exploit efficiently their binding surfaces for cancer treatment. To address this aim, our research focuses on three areas: 1. The detailed comprehension of the TMD contribution to both the Mcl-1 membrane interactome and protein functionality. 2. The discovery of new Mcl-1 inhibitors that target the transmembrane surface to develop a class of anticancer drugs currently unexplored. 3. The molecular characterization of cancer-related mutations within the Bcl-2 and Bcl-xL TMDs and their implications for the survival of cancer cells. Antiapoptotic Mcl-1 protein inhibits the proapoptotic members Bak, Bax, Bok, and Noxa, among others. Although its prosurvival activity has been well studied, the molecular mechanism to prevent Bok-mediated apoptosis remains unclear. Furthermore, understanding of Mcl-1 activities described to date is only based on water-soluble structures and studies focused on extramembrane domains. For the first time, we uncover the relevance of the Mcl-1 TMD in the interaction equilibria of the protein. In the present work, we describe its specific capacity to self-associate and hetero-oligomerize with the Bok TMD. We also explain the influence of these interactions in the apoptotic pathway and highlight the clinical relevance of Mcl-1 TMD mutants identified in tumor patients. Many hematological and solid malignancies overexpress Mcl-1 as an acquired chemoresistance mechanism. To modulate its antiapoptotic activity in cancer cells, specific BH3 mimetics have been developed; however, there is no scientific data yet regarding human toxicity and efficacy. In this work, we propose the novel Mcl-1 and Bok TMDs interaction interface as a drugging site in the development of chemotherapeutics. We identify three potential inhibitors of such molecular interface with promising features to become both drug candidates for pharmaceutical development and research toosl for the molecular study of the Mcl-1 and Bok TMDs interaction. To take advantage of apoptosis modulation, tumor cells also present mutated versions of the antiapoptotic members Bcl-2 and Bcl-xL. To our knowledge, this is the first study that analyzes patient-derived mutations within Bcl-2 and Bcl-xL TMDs and demonstrates how said mutations alter the membrane equilibria of these proteins. The results presented here also explain the functional influence of some somatic mutants in apoptosis regulation. Overall, the scientific results exhibited in this Thesis highlight the role of Bcl TMDs in the interactome of Bcl-2 proteins. These findings corroborate that lateral interactions between TMDs are sequence-specific and actively contribute to protein functionality. Therefore, understanding of Bcl transmembrane segments may provide new insights into the biology of Bcl 2 proteins for their pharmaceutical modulation in antitumoral therapy. / The student has been granted with a PhD fellowship and a short-term fellowship from the Generalitat Valenciana (Subvenciones para la contratación de personal investigador de carácter predoctoral, 2016-2019, and Grant for predoctoral stays out of the Comunitat Valenciana, 2019). This work has been supported by the Spanish Ministry of Economy and Competitiveness (projects SAF2014-52614-R and SAF2017-84689-R / Lucendo Gutiérrez, E. (2020). Understanding and Drugging the Bcl-2 Transmembrane Interactome for Tumor Treatment [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/155914 / TESIS

Abstract In this work the extent of apoptosis was studied in a set of 504 benign and malignant breast lesions to elucidate its role in breast tumor development and progression. Also the correlation of apoptosis with estrogen and progesterone receptor positivity, cell proliferation and patients' prognosis was studied. The breast lesions were also analyzed immunohistochemically with antibodies to apoptosis regulating proteins bcl-2 and bax, and caspases 3, 6 and 8. In addition, the immunohistochemical expression of NO• synthesizing enzyme iNOS in relation to apoptosis and angiogenesis was studied. Furthermore, the expression of the antioxidative enzyme MnSOD was studied in relation to apoptosis and cell proliferation. According to the results, the apoptotic index was lowest in benign breast lesions. It was higher in in situ carcinomas, where a gradual increase in the extent of apoptosis from grade I to III in situ carcinoma was seen. The apoptotic index in invasive carcinomas was higher than in in situ carcinomas, and also in invasive carcinomas there was a gradual increase in apoptosis from grade I to III carcinomas. The apoptotic index was highest in recurrent carcinomas. Strong bcl-2 expression was usually found in benign breast lesions but the immunoreactivity decreased in in situ and invasive carcinomas. There was a significant inverse association between bcl-2 immunoreactivity and the extent of apoptosis. Low bcl-2 immunoreactivity also associated with estrogen- or progesterone receptor negativity. In contrast, bax expression did not show any significant association with apoptosis, hormone receptors or the histologic types of tumors. Strong cytoplasmic caspase 3, 6 and 8 immunoreactivity was found in most carcinomas. It was weaker in in situ carcinomas and only weak immunoreactivity could be seen in benign breast lesions. There was a significant association between the extent of apoptosis and caspase immunoreactivity. iNOS expression was found in both tumor and stromal cells. iNOS expression in tumor cells was more frequently found in invasive than in in situ carcinomas. Its expression correlated significantly with a high apoptotic index and high vascularization of the lesion. There was significantly less MnSOD immunoreactivity in invasive breast carcinomas compared to in situ carcinomas or benign hyperplasias. MnSOD immunoreactivity did not associate with the extent of apoptosis, but there was a marginal inverse association between cell proliferation and MnSOD expression. Increased apoptosis was significantly associated with a high cell proliferation, and inversely associated with a positive estrogen status. A high apoptotic index (< 0.50%) was associated with a decreased survival of the patients. The results of this study show that apoptosis plays a decisive role in the development and progression of breast carcinoma. It is influenced not only by apoptosis regulating proteins, such as bcl-2 and caspases, but also by the estrogen receptor status. Apoptosis was also associated with iNOS positivity, the effect of which is mediated through increased NO• production. In line with the suggested role of MnSOD as a tumor suppressor gene, its expression was downregulated in invasive breast carcinoma. In conclusion, the association of apoptosis with patient survival in breast carcinoma may be secondary to its association with tumor cell proliferation and high tumor grade, not necessarily suggesting any causal association between apoptosis and survival.

In Tumoren führt die Deregulation von Proteinen der Bcl-2 Familie zur Apoptoseresistenz. Diese kann aus einem Verlust der pro-apoptotischen Proteine Bax bzw. Bak, einer Hochregulation von anti-apoptotischen Bcl-2 Proteinen oder aus einer Inaktivierung von BH3-only Proteinen heraus erfolgen. BH3-only Proteine sequestrieren anti-apoptotische Bcl-2 Proteine und induzieren dadurch die Aktivierung von Bax bzw. Bak. Ziel der vorliegenden Arbeit war daher, die Regulation von Apoptose durch das pro-apoptotische BH3-only-Protein Noxa zu untersuchen. Zusätzlich sollte der Mechanismus der Apoptoseinduktion durch niedermolekulare Bcl-2 Inhibitoren in Tumorzellen untersucht werden. Hier wird gezeigt, dass die putativen Bcl-2 Inhibitoren Gossypol, HA14-1 und zum Teil BH3I-2 unabhängig von Bax und Bak Zelltod induzierten. Dies weist darauf hin, dass die Apoptoseinduktion durch diese niedermolekularen Substanzen zumindest teilweise "off-target" ist, d.h. unabhängig von den Zielstrukturen der Bcl-2 Familie erfolgt. Eine andere Strategie zur Aktivierung des mitochondrialen Apoptosewegs ist die Expression von BH3-only Proteinen. Zunächst wurde die Expression putativer Spleißvarianten des Noxa-Gens auf mRNA- und Proteinebene durchgeführt. Hierbei wurde das bekannte BH3-only Protein Noxa als kurze Spleißvariante (NoxaS) und eine bisher unbekannte, lange Spleißvariante (NoxaL) gefunden. Zur funktionellen Analyse wurden konditionale Expressionsvektoren für beide Noxa-Varianten hergestellt. Die Expression von NoxaS führte zu einer differenziellen Aktivierung von Bak und/oder Bax. NoxaL hingegen verfügt über keine BH3-Domäne, induzierte aber dennoch Apoptose in einem Prostatakarzinom-Zelllinienmodell. Weitergehende Untersuchungen zeigten, dass es hier zu einer BH3-unabhängigen Aktivierung eines Bak-vermittelten Zelltod-Signalwegs kommt. Die vorliegende Arbeit liefert somit neue Erkenntnisse zur Zelltodregulation und experimentellen Apoptoseinduktion über BH3-unabhängige Signalmechanismen. / In many tumors deregulation of Bcl-2 family proteins lead to apoptosis resistance. This deregulation can occur through the loss of pro-apoptotic proteins Bax respectively Bak, through upregulation of anti-apoptotic Bcl-2 proteins or from the inactivation of BH3-only proteins. BH3-only proteins sequester anti-apoptotic Bcl-2 proteins and hereby activate Bax and Bak. Therefore aim of the present work was to study the regulation of apoptosis by the pro-apoptotic BH3-only protein Noxa. In addition the mechanism of apoptosis induction in different tumor cells by small molecular substances mimicking the function of BH3-only proteins ought to be investigated. Here we show that the putative Bcl-2 inhibitors Gossypol, HA14-1 and partly BH3I-2 induce cell death independent from Bax and Bak. This shows that the induction of apoptosis by these substances is at least partly off target, i.e. occurs independent of the target structures of the Bcl-2 family. Another strategy to activate the mitochondrial signaling pathway is the expression of BH3-only proteins. Initially the expression of putative splicing variants of the noxa gene was carried out on mRNA and protein level. Here the known NoxaS as short splicing variant (NoxaS) and an up to that point unknown long splicing variant (NoxaL) have been found. For functional analysis of the two splicing variants conditional expression vectors have been generated. The overexpression of NoxaS led to a differential activation of Bax and/or Bak. However NoxaL does not contain a BH3 domain but still induced apoptosis in a prostate carcinoma cell system. Advanced studies showed a BH3 independent activation of a Bak mediated cell death signaling pathway. The present work therefore provides new insights into cell death regulation and experimental apoptosis induction via BH3 independent signaling mechanisms.

The Bcl-2 family of proteins strictly regulates the intrinsic pathway of apoptosis. Direct physical interactions between Bcl-2 proteins regulate mitochondrial outerpermeabilisation (MOMP), which occurs in response to various cell stresses andapoptotic stimuli. How changes in Bcl-2 protein activity regulate apoptosiscommitment is still unclear, especially with regard to how they interact with eachother within the context of the mitochondrial membrane. Recent studies haveshown that Bcl-2 proteins exist in a dynamic equilibrium between the mitochondriaand the cytosol. In this thesis, by using FRAP, I have measured changes in Bcl-XLand Mcl-1 dynamics in single cells. Surprisingly, individual cells within a populationshow widely differing Bcl-XL and Mcl-1 dynamics. There is a corelation betweenBcl-XL and Mcl-1 dynamics with BH3-only protein expression. Anti-apoptotic andpro-apoptotic Bcl-2 proteins stabilise each other on the OMM. Together, theseresults indicate that cells constantly fine tune mitochondrial priming and thatanalysing anti-apoptotic Bcl-2 proteins by FRAP allows this to be measured at asingle cell level in real time before MOMP.

Head and neck cancer accounts for approximately 3 percent of all cancers in the United States, and over 90% of them are head and neck squamous cell carcinoma (HNSCC). Chemotherapeutic drugs that treat HNSCC can activate BCL-2 family dependent apoptosis. Pro-apoptotic NOXA induced by adenovirus (Ad-NOXA) or fenretinide inactivates anti-apoptotic MCL-1, while ABT-263 can inactivate other anti-apoptotic BCL-2 family members such as BCL-2 and BCL-XL. We used p53 inactive HN8 and HN12, p53 wild-type UMSCC1, and HPV-positive UMSCC47 human HNSCC cell lines and five mouse HNSCC cell lines. Cells were treated with Ad-NOXA, ABT-263, and fenretinide alone or in combinations. Combinational treatment of ABT-263 with Ad-NOXA or fenretinide enhanced cell death among all cell lines we tested regardless of p53 status. These findings support the hypothesis that combinational treatment of Ad-NOXA or fenretinide with ABT-263 increases cell death by simultaneously inhibiting all anti-apoptotic BCL-2 family proteins in HNSCC cells.