La sénescence réplicative (SR) dans les fibroblastes humains primaires est causée par l’érosion des télomères et est contrôlée par p53. La régulation dynamique de l’activation de p53 est essentielle pour l’induction de la sénescence ; cependant, les mécanismes moléculaires sous-jacents ne sont pas clairement établis. Nous montrons, dans les cellules surexprimant les isoformes Δ133/Δ160p53, que ces isoformes s’oligomérisent avec p53, conduisant ainsi à la stabilisation d’une forme inactive de p53. A l’inverse, l’inactivation des isoformes endogènes Δ133/Δ160p53 induit l’accumulation de la protéine p53 et l’activation de son activité transcriptionnelle. La surexpression de Δ133/Δ160p53 inhibe les fonctions de p53, en particulier son activité transcriptionnelle et son rôle dans l’arrêt du cycle après un dommage à l’ADN. Nous avons remarqué que les protéines Δ133/Δ160p53 et p53 sauvage possédaient des conformations différentes. Les protéines Δ133/Δ160p53 sont reconnues pas l’anticorps Pab40 : elles adopteraient une conformation similaire à un mutant de conformation de p53. Enfin, nous observons qu’une faible expression de l’ARNm Δ133/Δ160TP53 coïnciderait avec la durée de l’activation transcriptionnelle de p53 lors de la SR, indiquée par l’accumulation de l’ARNm d’un effecteur majeur de p53, p21. L’augmentation de l’expression de Δ133/Δ160TP53 à un temps tardif au cours de la SR est corrélée à l’accumulation du marqueur de sénescence p16INK4a et à celle de la cytokine pro-inflammatoire IL-6. En conséquence, les isoformes Δ133/Δ160p53 contrôleraient l’activité de p53 dans l’arrêt du cycle et sur le phénotype sécrétoire des cellules sénescentes. / Telomere attrition in primary human fibroblasts induces replicative senescence by activation of the tumour suppressor p53. Fine-tuned activation of p53 is essential for senescence induction; however, the mechanisms underlying the regulation of p53 activity during senescence have not been clearly established yet. We report here that in cells that express the Δ133/Δ160p53 isoforms, these p53 isoforms oligomerize with p53, leading to the stabilization of the transcriptionally inactive form of p53. Conversely, endogenous Δ133/Δ160p53 silencing increases the level of p53 and p53-dependent transcriptional activity to promote cell cycle arrest. Overexpressed Δ133/Δ160p53 repress p53 functions, including gene transcription activation and growth inhibition, upon DNA damage. We also found that Δ133/Δ160p53 and wild-type p53 have different structural conformations. Δ133/Δ160p53 adopt a more unfolded conformation recognized by the Pab240 antibody, indicating that these p53 isoforms have a p53 mutant-like conformation. Finally, we observed that low level of Δ133/Δ160TP53 mRNA coincided with the duration of p53 transcriptional activation in replicatively senescent fibroblasts, as indicated by the upregulation of CDKN1A (p21) mRNA expression, a downstream effector of p53. Δ133/Δ160p53TP53 was upregulated at a later stage when the senescence marker p16INK4a and the pro-inflammatory interleukin-6 (IL-6) were also induced. Therefore, p53 activity on growth suppression and senescence-associated secretory phenotype may be differentially regulated by its Δ133/Δ160p53 isoforms.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23082号 / 医博第4709号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 妹尾 浩, 教授 村川 泰裕 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

O melanoma é um dos tipos de câncer de pele cuja frequência tem crescido nos últimos anos e apresentado elevada taxa de mortalidade, apesar de ter reduzida prevalência. Mesmo havendo um considerável avanço nas propostas terapêuticas nos últimos anos, ainda se vê necessário o desenvolvimento de novas abordagens, sendo a terapia gênica uma promissora possibilidade para tal. Utilizando vetores adenovirais com promotor responsivo à p53 (PGTx beta) para a transferência gênica de p19Arf (proteína supressora de tumor) e interferon-beta (citocina imunomodulatória) em células de melanoma murino com o gene Trp53 selvagem, o nosso grupo demonstrou previamente que a combinação dos dois genes, mas não o tratamento individual, promove efeito citotóxico sinérgico com a liberação de marcadores de morte imunogênica, in vitro; e significativa redução da progressão tumoral acompanhada de uma forte resposta imunológica de linfócitos T CD4+ e CD8+, células NK e neutrófilos contra desafios tumorais, in vivo. Porém, como a translação para modelos de melanomas humanos ainda estava em estágio inicial, ainda não haviam sido confirmamos se esses benefícios também seriam recapitulados. Observações inicias sugeriam que apenas a transferência gênica de interferon-beta seja suficiente para induzir morte celular em linhagens humanas portadoras de TP53 selvagem, sem ainda terem sido identificado o efeito da transferência de p14ARF e nem a necessidade de p53 endógeno para a resposta. Dessa forma, o presente projeto buscou avaliar os efeitos antitumorais provocados pela terapia gênica combinada de p14ARF e interferon-beta em modelos de melanoma humano utilizando linhagens com e sem a via da p53 integra. Para isso, foram utilizadas diferentes linhagens celulares com TP53 selvagem ou com distintas mutações e também foram construídos vetores adenovirais com o promotor constitutivo CMV, tornando assim possível a expressão dos transgenes de maneira independente do status do TP53 endógeno. O presente trabalho revelou que a transferência combinada do interferon-beta e p14ARF revelou vantagem quanto ao estímulo citotóxico e regulação negativa na dinâmica da população em ambas as linhagens UACC-62 e SK-Mel-29, independentemente do estado da via da p53. Na avaliação dos mecanismos de morte foi observado que ambas a linhagens apresentaram marcação positiva para marcadores da via da apoptose, porém com possível participação de outras modalidades de morte-celular, como a necrose, para a linhagem com o TP53 mutado (SK-Mel-29). Além disso, mostramos que os tratamentos potencialmente induzem vias de morte com caráter imunogênico pela secreção de ATP e exposição da calreticulina, sendo este último marcador mais significantemente observado mediante o tratamento combinado. Assim, recapitulamos o benefício observado em modelo murino para a transferência gênica do interferon-beta e p14ARF em modelo de melanoma humano, e investigamos marcadores importantes à translação da proposta terapêutica para o melanoma / Melanoma is one of the types of skin cancer whose frequency has grown in the last years and presents a high mortality rate, despite its low prevalence. Although there has been considerable progress in therapeutic proposals in recent years, it is still necessary to develop new approaches, being gene therapy a promising possibility for this. With the use of adenoviral vectors with a p53 responsive promoter (PGTx beta) for the gene transfer of p19Arf (tumor suppressor protein) and interferon-beta (immunomodulatory cytokine) in murine melanoma cells bearing wild-type Trp53 gene, our group previously demonstrated that the combination of the two genes, but not individual treatment, promotes a synergistic cytotoxic effect with the release of immunogenic death markers in vitro; and significant reduction of tumor progression with a strong immune response mediated by CD4+ and CD8+ T lymphocytes, NK cells and neutrophils in tumor challenges in vivo. However, as the translation for human melanoma models was still at an early stage, it still was not possible to confirm whether these benefits would also be recapitulated in a human model. Initial observations suggested that interferon-beta gene transfer is sufficient to induce cell death in wild-type TP53-bearing human melanoma cell lines, with the effect of p14ARF gene transfer and the role for endogenous p53 in this response yet to be investigated. Thus, the present work aimed to evaluate the antitumor effects induced upon the combined gene transfer of p14ARF and interferon-beta in human melanoma cell lines with and without a functional p53 pathway. For this, different cell lines bearing wild-type TP53 or with different mutations were used and adenoviral vectors with the constitutive CMV promoter were also constructed, making possible the expression of the transgenes independently of the endogenous TP53 status. The present work showed that the combined transfer of interferon-beta and p14ARF was advantageous in cytotoxic stimulation and negative regulation in population dynamics for both cell lines UACC-62 and SK-Mel-29, regardless of p53 pathway status. In the evaluation of the triggered cell death mechanisms it was observed that both cell lines presented positive markers of the apoptosis pathway, but with possible participation of other cell death mechanism, such as necrosis, for the mutated TP53 cell line SK-Mel-29. In addition, we showed that the treatments potentially induced cell death pathways with immunogenic features including the secretion of ATP and calreticulin exposure, being the latter marker more significantly presented after the combined treatment. Thus, we recapitulated the benefit observed in murine model for the gene transfer of interferon-beta and p14ARF in the model of human melanoma, and investigated important markers for the translation of the melanoma therapeutic proposal

O melanoma é um dos tipos de câncer de pele cuja frequência tem crescido nos últimos anos e apresentado elevada taxa de mortalidade, apesar de ter reduzida prevalência. Mesmo havendo um considerável avanço nas propostas terapêuticas nos últimos anos, ainda se vê necessário o desenvolvimento de novas abordagens, sendo a terapia gênica uma promissora possibilidade para tal. Utilizando vetores adenovirais com promotor responsivo à p53 (PGTx beta) para a transferência gênica de p19Arf (proteína supressora de tumor) e interferon-beta (citocina imunomodulatória) em células de melanoma murino com o gene Trp53 selvagem, o nosso grupo demonstrou previamente que a combinação dos dois genes, mas não o tratamento individual, promove efeito citotóxico sinérgico com a liberação de marcadores de morte imunogênica, in vitro; e significativa redução da progressão tumoral acompanhada de uma forte resposta imunológica de linfócitos T CD4+ e CD8+, células NK e neutrófilos contra desafios tumorais, in vivo. Porém, como a translação para modelos de melanomas humanos ainda estava em estágio inicial, ainda não haviam sido confirmamos se esses benefícios também seriam recapitulados. Observações inicias sugeriam que apenas a transferência gênica de interferon-beta seja suficiente para induzir morte celular em linhagens humanas portadoras de TP53 selvagem, sem ainda terem sido identificado o efeito da transferência de p14ARF e nem a necessidade de p53 endógeno para a resposta. Dessa forma, o presente projeto buscou avaliar os efeitos antitumorais provocados pela terapia gênica combinada de p14ARF e interferon-beta em modelos de melanoma humano utilizando linhagens com e sem a via da p53 integra. Para isso, foram utilizadas diferentes linhagens celulares com TP53 selvagem ou com distintas mutações e também foram construídos vetores adenovirais com o promotor constitutivo CMV, tornando assim possível a expressão dos transgenes de maneira independente do status do TP53 endógeno. O presente trabalho revelou que a transferência combinada do interferon-beta e p14ARF revelou vantagem quanto ao estímulo citotóxico e regulação negativa na dinâmica da população em ambas as linhagens UACC-62 e SK-Mel-29, independentemente do estado da via da p53. Na avaliação dos mecanismos de morte foi observado que ambas a linhagens apresentaram marcação positiva para marcadores da via da apoptose, porém com possível participação de outras modalidades de morte-celular, como a necrose, para a linhagem com o TP53 mutado (SK-Mel-29). Além disso, mostramos que os tratamentos potencialmente induzem vias de morte com caráter imunogênico pela secreção de ATP e exposição da calreticulina, sendo este último marcador mais significantemente observado mediante o tratamento combinado. Assim, recapitulamos o benefício observado em modelo murino para a transferência gênica do interferon-beta e p14ARF em modelo de melanoma humano, e investigamos marcadores importantes à translação da proposta terapêutica para o melanoma / Melanoma is one of the types of skin cancer whose frequency has grown in the last years and presents a high mortality rate, despite its low prevalence. Although there has been considerable progress in therapeutic proposals in recent years, it is still necessary to develop new approaches, being gene therapy a promising possibility for this. With the use of adenoviral vectors with a p53 responsive promoter (PGTx beta) for the gene transfer of p19Arf (tumor suppressor protein) and interferon-beta (immunomodulatory cytokine) in murine melanoma cells bearing wild-type Trp53 gene, our group previously demonstrated that the combination of the two genes, but not individual treatment, promotes a synergistic cytotoxic effect with the release of immunogenic death markers in vitro; and significant reduction of tumor progression with a strong immune response mediated by CD4+ and CD8+ T lymphocytes, NK cells and neutrophils in tumor challenges in vivo. However, as the translation for human melanoma models was still at an early stage, it still was not possible to confirm whether these benefits would also be recapitulated in a human model. Initial observations suggested that interferon-beta gene transfer is sufficient to induce cell death in wild-type TP53-bearing human melanoma cell lines, with the effect of p14ARF gene transfer and the role for endogenous p53 in this response yet to be investigated. Thus, the present work aimed to evaluate the antitumor effects induced upon the combined gene transfer of p14ARF and interferon-beta in human melanoma cell lines with and without a functional p53 pathway. For this, different cell lines bearing wild-type TP53 or with different mutations were used and adenoviral vectors with the constitutive CMV promoter were also constructed, making possible the expression of the transgenes independently of the endogenous TP53 status. The present work showed that the combined transfer of interferon-beta and p14ARF was advantageous in cytotoxic stimulation and negative regulation in population dynamics for both cell lines UACC-62 and SK-Mel-29, regardless of p53 pathway status. In the evaluation of the triggered cell death mechanisms it was observed that both cell lines presented positive markers of the apoptosis pathway, but with possible participation of other cell death mechanism, such as necrosis, for the mutated TP53 cell line SK-Mel-29. In addition, we showed that the treatments potentially induced cell death pathways with immunogenic features including the secretion of ATP and calreticulin exposure, being the latter marker more significantly presented after the combined treatment. Thus, we recapitulated the benefit observed in murine model for the gene transfer of interferon-beta and p14ARF in the model of human melanoma, and investigated important markers for the translation of the melanoma therapeutic proposal

Dados recentes do nosso laboratório demonstram que somente a co-transdução, não a tradução individual, com vetores adenovirais portadores de Interferon-beta (IFN?) (citocina imuno modulatória) e p19Arf (parceira funcional da proteína supressora de tumor p53) resulta na morte celular massiva do melanoma murino B16F10. A capacidade desse tratamento combinado de induzir uma resposta imune antitumoral ainda não foi avaliada. Dessa maneira, o objetivo do presente trabalho foi investigar se células B16F0 tratadas por essa combinação são capazes de induzir uma resposta imune antitumoral em um modelo de vacinação profilático de melanoma. Para isso, essas células foram co-transduzidas com os vetores AdPGp19 e AdPGIFN? e 48 horas depois, inoculadas como agente vacinal no flanco esquerdo (sítio da vacina) de camundongos C57BL/6 imunocompetentes. Sete dias após a última vacinação, esses animais foram desafiados com células B16F10 naïve no flanco direito (sítio do desafio). A progressão tumoral do desafio foi significativamente reduzida, mesmo quando o desafio tumoral foi feito 73 dias após da vacinação. Porém, como os animais imunizados desenvolveram tumores no sítio da vacina, condições para o uso dessas células tratadas foram avaliadas, revelando que: o número de células e de aplicações usadas durante a vacinação tem influência no aparecimento desse tumores, e que apenas com o tratamento combinado os camundongos permanecem livres de tumor. A influência do sistema imune para este resultado foi revelada após protocolo de imunussupressão. Em seguida, o papel da p19Arf e do IFN? na proteção antitumoral da combinação foi estudado. In vitro, os efeitos antitumorais da combinação parecem ser mais influentes da reposição de p19Arf do que da expressão de IFN?, mas já in vivo, na presença do sistema imune, foram mais dependentes do IFN?. Com a combinação estes efeitos mostraram-se mais pronunciados, induzindo uma proteção antitumoral e maior sobrevida aos animais vacinados. Estes resultados indicam que a combinação p19Arf e IFN? pode ser aplicada como um agente imunoterápico e sugerem que a associação entre morte celular e imuno estimulação pode beneficiar o tratamento contra o câncer / Previously, we have shown in a mouse melanoma model of in situ gene therapy that co-transduction, but not individual application, with adenovirus vectors expressing the Interferon-beta (IFN?) (immune modulatory cytokine) and p19Arf (functional partner of the p53 tumor suppressor) transgenes results in massive cell death and reduced tumor progression. However, the capability of this combined treatment to stimulate an antitumor immune response has not been evaluated. Therefore, the aim of this work was to investigate, trough a prophylactic vaccine model, if B16F10 cells treated by the p19Arf and IFN? combination could induce such immune response. To do so, these cells were co-transduced by the AdPGp19 e AdPGIFN? adenoviral vectors and 48 hours after, inoculated as a vaccine agent in the left flank (vaccine site) of immune competent C57BL/6 mice. Seven days after the last vaccine, a tumor challenge was done with naïve B16F10 cells in the right flank (challenge site). Tumor progression was markedly reduced, even when challenge was done 73 days after the vaccination. However, since these animals developed tumors where the vaccine was applied, more appropriate conditions for the use of these treated cells were pursued, thus revealing that: the number of cells and inoculations can dictate tumor development, and also, that only with the combined treatment was tumor formation abolished. The influence of the immune system for this result was revelead by performing an immune supression protocol. Next, the roles of p19Arf and of IFN? were studied. In vitro, the antitumor effects were stronger upon the introduction of p19Arf than IFN?, but in vivo, in the presence of the immune system, the effects were more IFN? dependent. In fact, these effects were more pronouced with the combined treatment, inducing protection against tumor formation and progression and increasing survival in the vaccinated animals. Taken together, these results demonstrate the application of cells treated by the p19Arf e IFN? combination as an effective vaccine agent and also indicates that the association between cell death and immune stimulation may benefit the treatment of cancer

Introdução: De origem controversa e repercussões imprevisíveis, o acometimento ovariano pela endometriose é considerado importante marcador de extensão da doença, pois pode se associar a endometriose profunda. Inúmeras teorias etiopatogênicas tentam explicar a gênese da endometriose ovariana e, duas delas recentemente tem sido reativadas, como a da metaplasia celômica que justificaria o conceito atual de endometriose intra-ovariana profunda e a da menstruação retrógrada, que explica a origem tubárea dos endometriomas. Estima-se em 5% a 10% de câncer ovariano em lesões de endometriose de ovário; enquanto, a frequência total de transformação maligna foi estimada entre 0,3 a 2,5%. Objetivo: Avaliar as formas de apresentação da endometriose ovariana e possíveis associações com o quadro clínico, com outros locais de doença, com os marcadores de atividade proliferativa (Ki-67), com a expressão de alterações moleculares dos mecanismos apoptóticos consideradas importantes no processo de carcinogênese das lesões de endometriose (p53 e Bcl-2) e com os receptores de estrogênio (dependência hormonal). Métodos: Estudo de coorte retrospectivo exploratório, com 63 pacientes operadas entre 2002 a 2012, com diagnóstico de endometriose ovariana preenchendo os critérios de inclusão e exclusão. Os preparados histológicos foram reavaliados e reclassificados de acordo com o tipo histológico, com a forma de apresentação e com a presença de infiltração do parênquima ovariano, sendo divididas em endometriose ovariana peritoneal, cistica e intraparenquimatosa. Foram avaliados a expressão do Ki-67, do p53, do Bcl- 2 e dos receptores de estrogênio no epitélio e no estroma tecidual. As pacientes ainda foram avaliadas de acordo com os sintomas clínicos e locais concomitantes de doença. Resultados: A forma de apresentação da endometriose ovariana mais frequente foi a cística (72,2%), seguida pela intraparenquimatosa (22,2%) e pela forma peritoneal (5,6%). Todas podem apresentar componente infiltrativo. A endometriose ovariana infiltrativa esteve presente em 30,5% dos casos. Não se evidenciou associação entre sintomas, distribuição anatômica do doença e expressão dos marcadores com as diferentes formas de apresentação ou com a infiltração do parênquima adjacente. Conclusão: A endometriose ovariana apresenta três formas distintas de apresentação, cística, intraparenquimatosa e peritoneal. Todas podem apresentar componente infiltrativo. Apesar da clara diferenciação histológica, ainda se deve identificar o significado clínico destes achados / Introduction: Of controversial origin and unpredictable repercussions, ovarian endometriosis is an important marker of disease extensiveness, as it may be related to deep infiltrating endometriosis. Numerous theories try to explain its origin, but two of them have been recently reactivated, such as celomic metaplasia, which would justify the concept of deep ovarian endometriosis, and retrograde menstruation, which can explain the tubal origin of ovarian endometriosis. It is estimated 5% to 10% of ovarian cancer in ovarian endometriosis, but malignant transformation may occur in 0.3 to 2.5% of the cases. Objective: Identify the presenting forms of ovarian endometrisosis and its possible relations to clinical symptoms, to other sites of disease, to proliferative activity markers (Ki-67), to the molecular expression of apoptotic mechanisms, considered important to the process of malignant transformation (p53 and Bcl-2) and to estrogen receptors (hormonal dependency). Methods: This is a retrospective exploratory cohort study, done between 2002 and 2012, including 63 women with laparoscopic diagnosis of ovarian endometriosis which fullfilled inclusion and exclusion criteria. The histologic specimens were reanalysed and reclassified according to the histologic pattern, to its presenting form and to the presence of parenchyma infiltration. The expression of Ki-67, p53, Bcl-2 and estrogen receptors were evaluated in the tissue epithelium and stroma. Clinical symptoms and concomitant sites of disease were also evaluated. Results: The most frequent form of ovarian endometriosis was cystic (72.2%), followed by intra-parenchymatous (22.2%) and peritoneal (5.6%). All of them can be infiltrative. The prevalence of infiltrative ovarian endometriosis was 30.5%. No association were found between symptoms, anatomical distribution of disease, markers expression and the presenting forms of ovarian endometriosis as well as adjacent parenchymal infiltration. Conclusion: Ovarian endometriosis has three distinct presenting forms, cystic, intra-parenchymatous and peritoneal. All of them can be infiltrative. Even though there is a clear histologic differentiation, its clinical significance is still to be determined

Introdução: De origem controversa e repercussões imprevisíveis, o acometimento ovariano pela endometriose é considerado importante marcador de extensão da doença, pois pode se associar a endometriose profunda. Inúmeras teorias etiopatogênicas tentam explicar a gênese da endometriose ovariana e, duas delas recentemente tem sido reativadas, como a da metaplasia celômica que justificaria o conceito atual de endometriose intra-ovariana profunda e a da menstruação retrógrada, que explica a origem tubárea dos endometriomas. Estima-se em 5% a 10% de câncer ovariano em lesões de endometriose de ovário; enquanto, a frequência total de transformação maligna foi estimada entre 0,3 a 2,5%. Objetivo: Avaliar as formas de apresentação da endometriose ovariana e possíveis associações com o quadro clínico, com outros locais de doença, com os marcadores de atividade proliferativa (Ki-67), com a expressão de alterações moleculares dos mecanismos apoptóticos consideradas importantes no processo de carcinogênese das lesões de endometriose (p53 e Bcl-2) e com os receptores de estrogênio (dependência hormonal). Métodos: Estudo de coorte retrospectivo exploratório, com 63 pacientes operadas entre 2002 a 2012, com diagnóstico de endometriose ovariana preenchendo os critérios de inclusão e exclusão. Os preparados histológicos foram reavaliados e reclassificados de acordo com o tipo histológico, com a forma de apresentação e com a presença de infiltração do parênquima ovariano, sendo divididas em endometriose ovariana peritoneal, cistica e intraparenquimatosa. Foram avaliados a expressão do Ki-67, do p53, do Bcl- 2 e dos receptores de estrogênio no epitélio e no estroma tecidual. As pacientes ainda foram avaliadas de acordo com os sintomas clínicos e locais concomitantes de doença. Resultados: A forma de apresentação da endometriose ovariana mais frequente foi a cística (72,2%), seguida pela intraparenquimatosa (22,2%) e pela forma peritoneal (5,6%). Todas podem apresentar componente infiltrativo. A endometriose ovariana infiltrativa esteve presente em 30,5% dos casos. Não se evidenciou associação entre sintomas, distribuição anatômica do doença e expressão dos marcadores com as diferentes formas de apresentação ou com a infiltração do parênquima adjacente. Conclusão: A endometriose ovariana apresenta três formas distintas de apresentação, cística, intraparenquimatosa e peritoneal. Todas podem apresentar componente infiltrativo. Apesar da clara diferenciação histológica, ainda se deve identificar o significado clínico destes achados / Introduction: Of controversial origin and unpredictable repercussions, ovarian endometriosis is an important marker of disease extensiveness, as it may be related to deep infiltrating endometriosis. Numerous theories try to explain its origin, but two of them have been recently reactivated, such as celomic metaplasia, which would justify the concept of deep ovarian endometriosis, and retrograde menstruation, which can explain the tubal origin of ovarian endometriosis. It is estimated 5% to 10% of ovarian cancer in ovarian endometriosis, but malignant transformation may occur in 0.3 to 2.5% of the cases. Objective: Identify the presenting forms of ovarian endometrisosis and its possible relations to clinical symptoms, to other sites of disease, to proliferative activity markers (Ki-67), to the molecular expression of apoptotic mechanisms, considered important to the process of malignant transformation (p53 and Bcl-2) and to estrogen receptors (hormonal dependency). Methods: This is a retrospective exploratory cohort study, done between 2002 and 2012, including 63 women with laparoscopic diagnosis of ovarian endometriosis which fullfilled inclusion and exclusion criteria. The histologic specimens were reanalysed and reclassified according to the histologic pattern, to its presenting form and to the presence of parenchyma infiltration. The expression of Ki-67, p53, Bcl-2 and estrogen receptors were evaluated in the tissue epithelium and stroma. Clinical symptoms and concomitant sites of disease were also evaluated. Results: The most frequent form of ovarian endometriosis was cystic (72.2%), followed by intra-parenchymatous (22.2%) and peritoneal (5.6%). All of them can be infiltrative. The prevalence of infiltrative ovarian endometriosis was 30.5%. No association were found between symptoms, anatomical distribution of disease, markers expression and the presenting forms of ovarian endometriosis as well as adjacent parenchymal infiltration. Conclusion: Ovarian endometriosis has three distinct presenting forms, cystic, intra-parenchymatous and peritoneal. All of them can be infiltrative. Even though there is a clear histologic differentiation, its clinical significance is still to be determined

Dados recentes do nosso laboratório demonstram que somente a co-transdução, não a tradução individual, com vetores adenovirais portadores de Interferon-beta (IFN?) (citocina imuno modulatória) e p19Arf (parceira funcional da proteína supressora de tumor p53) resulta na morte celular massiva do melanoma murino B16F10. A capacidade desse tratamento combinado de induzir uma resposta imune antitumoral ainda não foi avaliada. Dessa maneira, o objetivo do presente trabalho foi investigar se células B16F0 tratadas por essa combinação são capazes de induzir uma resposta imune antitumoral em um modelo de vacinação profilático de melanoma. Para isso, essas células foram co-transduzidas com os vetores AdPGp19 e AdPGIFN? e 48 horas depois, inoculadas como agente vacinal no flanco esquerdo (sítio da vacina) de camundongos C57BL/6 imunocompetentes. Sete dias após a última vacinação, esses animais foram desafiados com células B16F10 naïve no flanco direito (sítio do desafio). A progressão tumoral do desafio foi significativamente reduzida, mesmo quando o desafio tumoral foi feito 73 dias após da vacinação. Porém, como os animais imunizados desenvolveram tumores no sítio da vacina, condições para o uso dessas células tratadas foram avaliadas, revelando que: o número de células e de aplicações usadas durante a vacinação tem influência no aparecimento desse tumores, e que apenas com o tratamento combinado os camundongos permanecem livres de tumor. A influência do sistema imune para este resultado foi revelada após protocolo de imunussupressão. Em seguida, o papel da p19Arf e do IFN? na proteção antitumoral da combinação foi estudado. In vitro, os efeitos antitumorais da combinação parecem ser mais influentes da reposição de p19Arf do que da expressão de IFN?, mas já in vivo, na presença do sistema imune, foram mais dependentes do IFN?. Com a combinação estes efeitos mostraram-se mais pronunciados, induzindo uma proteção antitumoral e maior sobrevida aos animais vacinados. Estes resultados indicam que a combinação p19Arf e IFN? pode ser aplicada como um agente imunoterápico e sugerem que a associação entre morte celular e imuno estimulação pode beneficiar o tratamento contra o câncer / Previously, we have shown in a mouse melanoma model of in situ gene therapy that co-transduction, but not individual application, with adenovirus vectors expressing the Interferon-beta (IFN?) (immune modulatory cytokine) and p19Arf (functional partner of the p53 tumor suppressor) transgenes results in massive cell death and reduced tumor progression. However, the capability of this combined treatment to stimulate an antitumor immune response has not been evaluated. Therefore, the aim of this work was to investigate, trough a prophylactic vaccine model, if B16F10 cells treated by the p19Arf and IFN? combination could induce such immune response. To do so, these cells were co-transduced by the AdPGp19 e AdPGIFN? adenoviral vectors and 48 hours after, inoculated as a vaccine agent in the left flank (vaccine site) of immune competent C57BL/6 mice. Seven days after the last vaccine, a tumor challenge was done with naïve B16F10 cells in the right flank (challenge site). Tumor progression was markedly reduced, even when challenge was done 73 days after the vaccination. However, since these animals developed tumors where the vaccine was applied, more appropriate conditions for the use of these treated cells were pursued, thus revealing that: the number of cells and inoculations can dictate tumor development, and also, that only with the combined treatment was tumor formation abolished. The influence of the immune system for this result was revelead by performing an immune supression protocol. Next, the roles of p19Arf and of IFN? were studied. In vitro, the antitumor effects were stronger upon the introduction of p19Arf than IFN?, but in vivo, in the presence of the immune system, the effects were more IFN? dependent. In fact, these effects were more pronouced with the combined treatment, inducing protection against tumor formation and progression and increasing survival in the vaccinated animals. Taken together, these results demonstrate the application of cells treated by the p19Arf e IFN? combination as an effective vaccine agent and also indicates that the association between cell death and immune stimulation may benefit the treatment of cancer

p53 is a nodal tumor suppressor protein that acts as a major defense against cancers. Approximately 50% of human tumours have mutations in p53 gene. Among its myriad features, the most distinctive is the ability to elicit both apoptotic death and cell cycle arrest. p53 has several isoforms. Most of them are produced by either internal promoter activity of the gene or alternate splicing of the pre-mRNA. Apart from these mechanisms, p53 mRNA has also been shown to be translated into two isoforms, the full-length p53 (FL-p53) and a truncated isoform ΔN-p53, which acts as a dominant-negative inhibitor of FL-p53. Under conditions of cellular stress, the canonical mode of translation initiation is compromised. To maintain the synthesis of proteins important for cell survival and cell-fate decisions, a subset of cellular mRNAs utilizes a non-canonical mode of translation initiation. The 5’ untranslated region of these mRNAs are highly structured and function as Internal Ribosome Entry Site (IRES). Previously, from our laboratory it has been shown that translation of p53 and its N-terminally truncated isoform ΔN-p53 can be initiated by IRES mediated mechanism. IRES mediated translation of ΔNp53 was maximum at G1-S phase but that of FL-p53 was maximum at the G2-M phase. Interestingly in case of a human genetic disorder X-linked dyskeratosis congenita (X-DC), aberrant IRES mediated p53 translation has been reported. It has also been reported that during oncogenic induced senescence (OIS) a switch between cap-dependent to IRES meditated translation occurs in p53 mRNA. From our laboratory, we have also demonstrated that polypyrimidine tract binding protein (PTB) positively regulates the IRES activities of both the p53 isoforms by shuttling from nucleus to the cytoplasm during genotoxic stress conditions. It is very important to understand how these two isoforms are regulated and in turn control the cellular functions. In the first part of the thesis, to investigate the importance of the structural integrity of the cis acting elements within p53 RNA, we have compared the secondary structure of the wild-type RNA with cancer-derived silent mutant p53 RNAs having mutations in the IRES elements such as L22L (CTA to CTG) a natural cancer mutation and Triple Silent Mutation (mutations were present at the wobble position of codon 17, 18, 19). These mutations result in the conformational alterations of p53 IRES RNA that abrogates the IRES function ex vivo significantly. It appears that these mutant RNAs failed to bind some trans-acting factors (p37, p41/44 etc) which might be critical for the IRES function. By super-shift assay using anti hnRNPC1/C2 antibody, we have demonstrated that the TSM mutant showed reduced binding to this protein factor. Partial knockdown of hnRNP C1/C2 showed significant decrease in p53 IRES activity and reduced synthesis of ΔN-p53. Also we have showed that introducing compensatory mutations in TSM mutant RNA rescued the secondary structure as well as function of p53 IRES. Further, the role of another silent point mutation in the coding sequence of p53 was investigated. Silent mutation (CCG to CCA) at codon 36 (P36P) showed decreased IRES activity. The mutation also resulted in differential binding of cellular proteins. Taken together, our observations suggest pivotal role of some specific trans acting factors in regulating the p53-IRES function, which in turn influences the synthesis of different p53 isoforms. In the second part of the thesis, p53 IRES RNA interacting proteins were identified using RNA affinity approach. Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) were identified and their interaction with p53 IRES RNA in vitro and ex vivo was studied. Interestingly, in the presence of Ca2+ ions Annexin A2 showed increased binding with p53 IRES. By competition UV crosslinking we have showed Annexin A2 and PSF interact specifically with p53 IRES. Toe printing assay results showed the putative contact points of Annexin A2 and PSF proteins on p53 IRES RNA. Interestingly, both proteins showed extensive toe-prints in the neighbourhood of the initiator AUG region of p53. Further, competition UV-crosslinking reveals the interplay of these two proteins. Annexin A2 and PSF appear to compete each other for binding with p53 IRES. PSF is known to interact with PTB protein. Since PTB also interacts with p53 IRES and positively regulates the translation, we wanted to study the interplay between PTB and PSF proteins binding with p53 IRES. To address this, we have performed competition UV crosslinking experiment and showed that increasing concentrations of PTB decreases PSF and p53 IRES interaction. However, increasing concentrations of PSF does not decrease or increase in PTB p53 IRES interaction. Results suggest that both Annexin A2 and PSF proteins play important role in regulation of p53 IRES activity. To address the physiological role of Annexin A2 and PSF proteins on p53 IRES activity, these proteins were partially knocked down in cellulo. This in turn showed decrease in p53 IRES activity in dual luciferase assays as well as in the steady state levels of both the p53 isoforms in transient transfection experiments. Heightened or continued expression of p53 protein is very important under stress where IRES-dependent translation supersedes normal cap-dependent translation. Results showed that expression of Annexin A2 under doxorubicin and thapsigargin induced stress are important for maintenance of both p53 IRES activity and steady state levels of p53 isoforms. Earlier from our laboratory we have showed that the IRES responsible for ∆N-p53 translation is active at G1/S phase while the IRES responsible for full length p53 translation is active at G2/M phase. Subcellular localization of the trans-acting factors plays a pivotal role in regulation of IRES activity of cellular mRNA. In this context we wanted to study the nuclear and cytoplasm localization of Annexin A2 under different cell cycle stages. We have seen Annexin A2 protein is dispersed in nucleus and cytoplasm at G1/S boundary, but post-G2 phase it moved from nucleus to cytoplasm. Further we wanted to investigate the effect of Annexin A2 and PSF on expression of p53 transactivated genes. Partial knock down of Annexin A2 and PSF proteins showed decrease in p21 luciferase activity. By real-time PCR analysis, we have also showed decrease in expression of different p53 targets upon silencing of Annexin A2 protein. Taken together, our observations suggest pivotal role of cis acting and trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.