La protéine suppresseur de tumeurs PTEN (Phosphatase and tensin deleted on chromosome 10) est une phosphatase lipidique. En déphosphorylant le phosphatidylinositol (3,4,5) trisphosphate (PIP3) en PI(4,5) P2, PTEN contre-régule la voie PI3K/Akt et inhibe la prolifération. D’autres fonctions de PTEN peuvent être indépendantes de son activité phosphatase lipidique, notamment l’inhibition de la migration. Bien que PTEN soit, après p53, le suppresseur de tumeurs le plus muté dans un large panel de cancers (gliomes, prostate, sein, endomètre…), les mécanismes par lesquels ses fonctions sont régulées ne sont pas entièrement élucidés. Par une approche de double-hybride, notre équipe a identifié que les β-arrestines (β-arrs), des protéines d’échafaudage, interagissent avec PTEN. Nos travaux mettent en évidence que l’interaction entre PTEN et les β-arrs permet de moduler ses deux activités dépendantes ou non de son activité phosphatase lipidique. D’une part, les β-arrs augmentent l’activité phosphatase lipidique de PTEN in vitro. La GTPase RhoA et sa kinase d’aval ROCK activent PTEN, et ceci se fait par l’intermédiaire des β-arrs. La stimulation du récepteur à l’acide lysophosphatidique (LPA), qui active la voie RhoA/ROCK, augmente la formation du complexe PTEN/β-arrs et permet le recrutement du complexe à la membrane. Par l’effet positif sur l’activité phosphatase lipidique de PTEN, les β-arrs participent à l’inhibition d’Akt et de la prolifération dans les fibroblastes embryonnaires de souris (MEF). A l’inverse dans les gliomes U373, les β-arrs lèvent l’inhibition de la migration exercée par le domaine C2 de PTEN, indépendamment de son activité phosphatase lipidique. En aval de l’activation de RhoA induite par blessure du tapis cellulaire, les β-arrs interagissent davantage avec PTEN et rétablissent la migration des gliomes. De ce fait, les β-arrs régulent différentiellement les fonctions de PTEN importantes pour le contrôle de la prolifération cellulaire et la migration. Enfin, l’activité et la localisation de PTEN sont modulées par des interactions intramoléculaires entre ses domaines catalytiques, C2 et sa queue C-terminale régulatrice. Ces interactions régulent le passage d’une conformation fermée vers une conformation ouverte et active de PTEN. Grâce au développement d’un biosenseur de PTEN basé sur le transfert d’énergie par résonnance (RET), nous pouvons suivre pour la première fois les changements conformationnels de PTEN dans les cellules vivantes. En utilisant ce biosenseur nous montrons que la mutation des résidus impliqués dans les interactions intramoléculaires entraine des changements de conformation détectés par des variations de RET. De plus, l’activation de voies de signalisation connues pour activer PTEN, entrainent des changements conformationnels qui corrèlent avec l’augmentation de l’activité phosphatase lipidique de PTEN. Nos données montrent que le biosenseur peut être utilisé comme outil pour détecter les changements d’activité de PTEN dans les cellules vivantes. L’axe suppresseur de tumeurs/oncogène PTEN/PI3K/Akt joue un rôle essentiel dans la progression tumorale et constitue une cible thérapeutique pour le cancer. L’ensemble de nos travaux permet d’ajouter un degré de compréhension dans la régulation de PTEN, tant par les β-arrs que par l’interaction intramoléculaire et les changements conformationnels. / The Tumour Suppressor protein PTEN (Phosphatase and tensin deleted on chromosome 10) is a lipid phosphatase. By converting phosphatidylinositol (3,4,5) trisphosphate (PIP3) to PI(4,5)P2, PTEN inhibits the PI3K/Akt signalling pathway and cell proliferation. Other functions attributed to PTEN, including the inhibition of cell migration, can occur independently of its lipid phosphatase activity. Although PTEN function is dysregulated in a broad range of cancers (gliomas, prostate, breast, endometrium…), the mechanisms by which it is regulated are far from being completely elucidated. Using a two-hybrid approach, our team identified that the molecular scaffolds, β-arrestins (β-arrs), interact with PTEN.Our studies demonstrate that β-arrs modulate distinct functional outputs of PTEN that in turn are dependent or independent on its lipid phosphatase activity. β-arrs increase the lipid phosphatase activity of PTEN in vitro. The small GTPase RhoA and its downstream effector ROCK activate PTEN and this effect requires β-arrs. The stimulation of the lysophosphatidic acid receptor 1 (LPA1-R) receptor, that activates the RhoA/ROCK pathway, was found to increase the association of β-arrs with PTEN and induced plasma membrane translocation of the complex. Through their stimulatory effect on the lipid phosphatase activity of PTEN, β-arrs inhibit the PI3K/Akt pathway and proliferation of mouse embryonic fibroblasts. In contrast, in U373 glioma cells, βarrs release the brake on cell migration, which is mediated by the C2 domain of PTEN independently of its lipid phosphatase activity. Following wounding of a cell monolayer, and RhoA activation, β-arrs show increased association with PTEN, and rescue glioma cell migration. β-arrs therefore differentially regulate functions of PTEN important in the control of cell proliferation and migration.The activity and localization of PTEN are under tight control of intramolecular interactions between its regulatory C-terminal tail, and catalytic and C2 domains. These intramolecular interactions regulate a switch between a closed form of PTEN, and an open and active form that is targeted to the membrane. We have developed a resonance energy transfer (RET)-based biosensor that permits the monitoring of PTEN conformational change in live cells. Using the biosensor we demonstrate that mutation of residues implicated in the intramolecular switch produce conformational rearrangement of PTEN, detected by changes in RET. Furthermore, activation of signalling pathways known to activate PTEN, elicit conformational changes that parallel increased PTEN lipid phosphatase activity in living cells. Combined, these data demonstrate that the biosensor can be used as a tool to detect changes in PTEN tumour suppressor activity in live cells.The tumour suppressor/oncogene PTEN/PI3K/Akt axis plays a key role in tumour progression and represents a major therapeutic target in the treatment of cancer. Our studies help to further our understanding of how tumour suppressor PTEN is controlled by inter- and intramolecular interactions and provide a biosensor that can report changes in PTEN activity.

No rim, a maior parte da carga de glicose filtrada é reabsorvida na porção inicial do túbulo proximal, no qual são co-expressos os transportadores: SGLT2 e GLUT2. No diabetes mellitus ocorre aumento no fluxo transepitelial de glicose, o que decorre de aumento na expressão desses transportadores, e pode ser revertido pelo tratamento com insulina. Os fatores transcricionais HNF1A, HNF4A e FOXA2 são descritos como potenciais reguladores do gene Slc2a2. A proteína AKT medeia efeitos da insulina, e é capaz de ativar fatores transcricionais. O objetivo deste estudo foi investigar em linhagem celular IRPTC, o efeito da alta concentração de glicose e da insulina sobre a expressão de Slc2a2/GLUT2 e Slc5a2/SGLT2, assim como a participação da AKT e dos fatores transcricionais. Observamos que a alta concentração de glicose aumentou a expressão do Slc2a2/GLUT2 e a atividade de ligação dos fatores transcricionais HNF4A e FOXA2 na região promotora do gene Slc2a2, por mecanismo mediado pela AKT. A insulina reverteu o efeito sobre o Slc2a2, porém não alterou o conteúdo de GLUT2. / Glucose filtrated load is reabsorbed in renal proximal tubule by the coordinate action of the glucose transporters SGLT2 and GLUT2. In diabetes, renal glucose reabsorption increases; that involves overexpression of the glucose transporters, and is reversed by insulin therapy. The transcription factors HNF1A, HNF4A and FOXA2 have been proposed as modulators of Slc2a2 gene expression. The AKT protein is an important mediator of insulin action, and has been able to activate transcription factors. The present study investigates in immortalized rat proximal tubule cells the effects of high glucose and insulin concentrations upon the Slc2a2/GLUT2 and Slc5a2/SGLT2 expression, as well as the participation of AKT, HNF1A, HNF4A and FOXA2. On the other hand, 25 mM glucose increased the expression of Slc2a2GLUT2, which was accompanied by increased HNF4A and FOXA2 binding in the Slc2a2 promoter, in an AKT-mediated way. Insulin reversed the Slc2a2 mRNA regulation, but did not alter GLUT2 content.

A proteína P53 pode estar virtualmente alterada em todos os cânceres humanos e portanto, na ausência de mutação, uma possibilidade para a inativação da p53 é a formação de complexo com outras proteínas, tal como a proteína Mdm2. Estudos prévios realizados em nosso laboratório demonstraram a superexpressão de Mdm2 na ausência de expressão da proteína P53 em adenomas pleomórficos. O objetivo deste estudo foi analisar a expressão das proteínas Mdm2, P53, P21 e Akt em adenoma pleomórfico e o mioepitelioma através das técnicas de imunoistoquímica, western blotting e imunofluorescência. A superexpressão de Mdm2 e Akt foi encontrada na maioria das linhagens e lesões utilizadas neste estudo enquanto as proteínas P53 e P21 não demonstraram expressão nas neoplasias estudadas. As superexpressões das proteínas Mdm2 e Akt estão relacionadas à tumorigênese em neoplasias benignas de glândula salivar. / The P53 protein can be altered in virtually all human cancers and in the absence of mutations, P53 inactivation is possible via complex formation with others proteins, such as the Mdm2. Previous studies from our laboratory showed overexpression of mdm2 and lack of p53 expression in pleomorphic adenomas. The aim of this study was to analyze the expression of Mdm2, P53, P21 and Akt proteins in pleomorphic adenomas and myoepiteliomas by Western blotting, immunohistochemistry and immunofluorescence techniques. Overexpression of Mdm2 and Akt was present in the majority of cell lineages and tumors studied, while the expression of P53 and P21 proteins was considered absent. Overexpression of Mdm2 and Akt are related to the tumorigenesis of benign salivary gland neoplasms.

A proteína Akt modula a função de numerosos substratos envolvidos na regulação da sobrevivência celular, progressão do ciclo celular e crescimento celular. Estudos prévios realizados em nosso laboratório demonstraram a superexpressão de Akt em adenoma pleomórfico, mioepitelioma e carcinoma adenóide cístico. O objetivo deste estudo foi analisar a via da proteína Akt através da avaliação da expressão das proteínas NFkB e PTEN em neoplasias benignas e malignas de glândulas salivares através das técnicas de imunohistoquímica, western blotting e imunofluorescência, e a possível interação protéica direta entre p-Akt/Mdm2 e p-Akt/PTEN em linhagem de carcinoma adenóide cístico. A superexpressão nuclear na proteína PTEN foi encontrada nas duas neoplasias malignas estudadas. Além disso, não foi observada interação direta entre as proteínas p-Akt/Mdm2 e p-Akt/PTEN, as quais apresentam localização nuclear em neoplasias de glândulas salivares. / The Akt protein modulates the function of numerous substrates involved in the regulation of cell survival, cell cycle progression and cell growth. Previous studies from our laboratories showed overexpression of Akt and Mdm2 followed by the lack of p53 expression in pleomorphic adenoma, myoepithelioma and adenoid cystic carcinoma. The aim of this study was to analyze the Akt pathway through evaluation of expression of NFkB and PTEN proteins in pleomorphic adenoma, carcinoma ex pleomophic adenoma and adenoid cystic carcinoma by western blotting, immunofluorescence and immunohistochemical techniques, and we have intended to analyse a possible direct interaction between p-Akt/Mdm2 and p-Akt/ PTEN protein in salivary gland tumours. Overexpression of nuclear PTEN was present in both carcinomas studied. In addition, there was no direct interaction between p-Akt/Mdm2 and p-Akt/ PTEN protein, which presents a nuclear localization in salivary gland tumours.

Introdução: A epilepsia do lobo temporal (ELT) é a forma mais frequente de epilepsia em adultos. Um modelo experimental de ELT consiste na indução de status epilepticus (SE) em animais por administração de Pilocarpina. Este modelo induz mudanças patofisiológicas e comportamentais em ratos muito semelhantes às observadas em seres humanos com ELT. Apesar da literatura apresentar dados relacionados às respostas celulares, pouco se conhece a respeito do envolvimento de cascatas de sinalização com insultos epileptogênicos no sistema nervoso. A enzima fosfatidilinositol-3-cinase (PI3K) está envolvida na ativação da cascata de sinalização intracelular da Akt. A Akt é uma proteína cinase especifíca de serina/treonina cuja forma ativa proporciona um controle no crescimento e proliferação celular, bem como induz um sinal de sobrevivência para a proteção de células contra a apoptose. Alguns estudos mostram que a ativação da PI3K é inibida por potentes drogas, tais como a LY294002 e a Wortmanina. A PI3K ativa a Akt e a cascata de sinais extra e intracelulares neuroprotetores pós-insultos. O estudo da ação de inibidores da PI3K em modelos de epilepsia pode fornecer dados sobre o envolvimento da Akt em sinais neuroprotetores. Objetivos: Avaliar o efeito do SE por injeção intra-hipocampal de Pilocarpina na ativação da Akt, bem como os efeitos do bloqueio desta cascata sobre as alterações patológicas observadas no hipocampo de ratos na fase aguda pós-SE. Metodologia: Ratos da cepa Wistar machos (250-300 g) foram divididos em grupos de tratamento, sendo tratados com injeções ipsilaterais na região posterior do hipocampo, com uma hora de intervalo entre drogas, das drogas: Salina e Pilocarpina (grupo Sal+Pilo), LY294002 e Pilocarpina (LY+Pilo) e Wortmanina e Pilocarpina (Wort+Pilo). No grupo considerado como controle foram injetadas as seguintes drogas: Salina e Salina (grupo Sal+Sal) ou Dimetilsulfóxido e Salina (grupo DMSO+Sal). O tempo de SE induzido por Pilocarpina foi fixado em 2 horas. Grupos de animais foram sacrificados nos instantes de 1 dia e 7 dias após o SE e seus encéfalos foram processados objetivando a imuno-histoquímica para NeuN, GFAP e Akt (pan). Resultados: A densidade neuronial na região do hilo do hipocampo foi menor no grupo Sal+Pilo, seguido dos grupos LY+Pilo e Wort+Pilo, avaliando-se os vários níveis de formação hipocampal e região posterior. Para a análise da astrogliose, na camada granular, o grupo LY+Pilo apresentou maior número de astrócitos positivos; na região do hilo hipocampal, os grupos LY+Pilo e Wort+Pilo apresentaram baixa expressão para a proteína Akt, comparados aos grupos Controles; assim como o grupo Wort+Pilo, em CA2, apresentou baixa expressão da proteína Akt somente na região posterior. Os animais Sal+Pilo sobrevida-7dias pós-SE revelaram maior expressão da Akt quando comparados com o grupo Sal+Pilo sobrevida-1dia pós-SE. Quanto à análise comportamental, o grupo Wort+Pilo apresentou maior latência para o início do SE que o grupo Sal+Pilo, não sendo observado diferença de severidade, uma vez atingido o SE. Conclusões: Na hipótese de trabalho, o uso de inibidores da fosforilação de Akt resultaria em maior morte neuronial, astrogliose e expressão inalterada da Akt. Ao contrário do esperado, os grupos que receberam injeções intra-hipocampais de inibidores de Akt, antes da indução de SE, exibiram menor perda neuronial, menor astrogliose e menor expressão da Akt para os vários níveis de formação hipocampal e no hipocampo posterior. Porém, o grupo Sal+Pilo sobrevida-7 dias pós-SE exibiu maior expressão para Akt quando comparado com o grupo Sal+Pilo de sobrevida-1 dia pós-SE. Além disso, o pré-tratamento com Wortmanina demonstrou um maior tempo de latência para o início do SE, o que nos sugere uma maior neuroproteção do que LY294002. / Introduction: Temporal lobe epilepsy (TLE) is the most frequent type of adult human epilepsy. An experimental model of TLE, the Pilocarpine induced epilepsy, followed by pathofisiologic and behavioural alterations in rats resembling human diagnosis with TLE. Although there are several data on cell response in literature, few information exist on the cascade of signals involved in epileptogenesis process of central nervous system. The phosphatidylinositol-3-kinase (PI3K) is involved in activation of Akt intracellular signaling cascade. The serine/threonine kinase Akt, that in active form promote a control of growth and cell proliferation, such as survival sign protecting cells from apoptosis. Some studies have shown that activation of PI3K is blocked by potents pharmacological inhibitors, for example the LY294002 and Wortmannin. PI3K activate Akt and both extra and intracellular cascade signals involved in neuroprotection after seizures. The study of inhibitors mechanism in model of epilepsy can provide information on the involvement of Akt in signals of neuroprotection. Objectives: To evaluate the effects of SE by the intrahippocampal injection of Pilocarpine in Akt activation, and the effects of the blockade of this cascade on pathologic alterations observed in hippocampus of rats in acute phase after SE as well. Methods: Male Wistar rats (weighing 250 to 300 g) were divided in groups treated ipsilaterally with injections in the posterior region of hippocampus with an elapsed time of one hour subsequently after each injection of following substances: Physiological Saline and Pilocarpine (group Sal+Pilo), LY294002 and Pilocarpine (LY+Pilo), and Wortmannin and Pilocarpine (Wort+Pilo). Control groups were treated with the following drugs Saline + Saline (group Sal+Sal) or Dimethylsulphoxide + Saline (DMSO+Sal). A fixed time of 2 hours was considered for evaluating the SE induced by Pilocarpine. Animals were sacrificed 1 day and 7 days after SE, and their brains were processed imunohistochemically for NeuN, GFAP and Akt (pan) detecting. Results: The neuronal density in the hippocampal hilus was lower in the Sal+Pilo group, followed by LY+Pilo and Wort+Pilo groups, this evaluate various levels of hippocampal formation and posterior region. For the analysis of reactive astrogliosis of the granular cell layer, the LY+Pilo group presented a great number of GFAP-positive astrocytes. In the hilar region, the LY+Pilo and Wort+Pilo groups presented a reduced Akt expression compared to the Control group, such as the group Wort+Pilo in CA2, presented a reduced Akt expression only in posterior region. The Sal+Pilo animals with a survival time of 7 days after SE revealed higher Akt expression when compared to the Sal+Pilo animals with a survival time of 1 day. In behavioural analysis, the Wort+Pilo group presented major time of latency to SE than Sal+Pilo group, without differences in the disease severity, once reached the SE. Conclusions: On the contrary to the original hypothesis of this work, the use of inhibitors of Akt phosphorylating resulted in an unaltered neuronal death, astrogliosis and Akt expression. The groups that received intrahippocampal injection of Akt inhibitors before inducing SE exhibited a reduced neuronal loss, astrogliosis and Akt expression to the various levels of hippocampal formation and posterior region. But, to the Sal+Pilo group with a survival time of 7 days after SE induction exhibited greater Akt expression than Sal+Pilo group with a survival time of 1 day after SE induction. However, the pretreatment with Wortmannin displayed major time of latency to SE induction, suggesting this substance as a better neuroprotector than LY294002, according to the methodology applied in this study.

Diversas alterações genéticas estão associadas à patogênese do carcinoma epidermoide (CE), neoplasia maligna mais comum de cabeça e pescoço. Algumas dessas alterações comprometem proteínas pertencentes à via de sinalização do Akt, envolvida em diferentes fenômenos celulares. Este trabalho teve como objetivo estudar a expressão da proteína pAkt em linhagens celulares de carcinomas epidermoides de cabeça e pescoço, de forma a verificar possíveis alterações na transcrição dessa molécula em células de CE tratadas com Curcumina. Foram utilizadas duas linhagens celulares de CE de cabeça e pescoço (FaDu e SCC9) e uma linhagem de queratinócitos normais (HaCat) divididas em dois grupos: a. Grupo controle não tratado; b. Células tratadas com Curcumina. A proliferação celular foi monitorada através do teste de viabilidade celular e a análise da expressão de proteína foi realizada através da técnica do Western Blotting que revelou supressão do pAkt na linhagem celular SCC9 nos tempos de 24 e 48 horas. Desta forma, conclui-se que a Curcumina na via do Akt em carcinomas epidermoides de cabeça e pescoço tem importante ação supressora do gene pAkt. / Several genetic alterations are associated with the pathogenesis of squamous cell carcinoma (SCC), the most common malignant neoplasm of the head and neck. Some of these changes compromise the proteins belonging to the Akt signaling pathway, involved in various cellular phenomena. The objective of this study to explores the expression of the pAkt protein in cell lines of the squamous cell carcinomas of the head and neck to verify possible changes in the transcription of this molecule in EC cells treated with curcumin. The study used two cell lines of EC head and neck (FaDu and SCC9) and a normal line of keratinocytes (HaCat), split into two groups: A. the controlled group, untreated; B. Cells treated with curcumin. The cell proliferation it was observed by cell viability test and analysis of protein expression performed through Western blotting technique revealed suppression of pAkt in SCC9 cell line at 24 and 48 hours. Thus, it is concluded that the Curcumin on the path of Akt in squamous cell carcinoma of the head and neck has a significant suppressive effect of gene Akt.

L’hématopoïèse est le processus physiologique qui permet le développement de l’ensemble des cellules sanguines matures, leur renouvellement et leur homéostasie tout au long de la vie. L’hématopoïèse est généralement décrite de façon hiérarchique avec, au sommet, les cellules souches hématopoïétiques qui s’autorenouvellent et se différencient en progéniteurs puis en cellules matures. La voie de signalisation NOTCH canonique, contrôle l’activité du facteur de transcription RBPJ. Elle joue un rôle dans le développement des lymphocytes T et la spécification de la différenciation des cellules souches hématopoïétiques normales vers la lignée mégacaryocytaire. Les protéines de la famille OTT1 (OTT1, OTT3 et SHARP) s’expriment de façon ubiquitaire et sont impliquées dans le contrôle de l’activité de RBPJ. Les modalités de régulation de ces activités et l’intégration de signaux provenant d’autres voies de signalisation sont mal caractérisées. L’utilisation d’un modèle de différenciation in vitro de cellules souches hématopoïétiques sur des cellules stromales (OP9) exprimant le ligand NOTCH Delta-like 1 (DL1) ainsi que l’utilisation de modèles murins, nous a permis de montrer un lien entre la voie NOTCH et la voie PI3K/AKT dans le développement mégacaryocytaire. Nos résultats indiquent que la différenciation mégacaryocytaire peut être engagée à partir de progéniteurs myéloïdes engagés dépendant principalement de la voie PI3K/AKT, mais également directement à partir de cellules souches hématopoïétiques pour lesquelles une activation de la voie PI3K/AKT conduit à une synergie avec la voie NOTCH, mais n’est pas essentielle à la spécification mégacaryocytaire. D’autre part, pour comprendre le mécanisme de régulation de la protéine OTT1, j’ai recherché ses partenaires protéiques par crible double hybride chez la levure, et identifié des interactions avec, entre autres, des protéines à activité tyrosine kinase de la famille SRC (dont LYN) et SHARP. La spécificité d’interaction entre OTT1 et LYN a été validée dans un modèle de surexpression ainsi que dans une lignée modélisant la leucémie aigüe mégacaryocytaire. Dans nos modèles, l’interaction avec LYN conduit à la phosphorylation d’OTT1. Les analyses fonctionnelles préliminaires n’ont pas permis à ce jour de mettre en évidence un rôle essentiel de cette interaction dans le développement mégacaryocytaire. / Hematopoiesis is generally described as a hierarchical system, with at the top hematopoietic stem cells which self-renew and differentiate in progenitors, then in mature cells. Canonical Notch signaling controls RBPJ transcriptional activity. It plays a role in T lymphocyte development and stem cell fate. OTT1 family proteins (OTT1, OTT3 and SHARP) are expressed ubiquitously and are implied in control of RBPJ activity. The regulation of these activities and signal integration are all not well characterised. The use of an in vitro model of differentiation for hematopoietic stem cells on OP9 stroma cells expressing the NOTCH Delta-like-1 (DL1) ligand and the use of murine models, allowed us to show a link between NOTCH and PI3K/AKT in megakaryocytic development. Our results indicate that megakaryocytic differentiation can be engaged from myeloid progenitors depending mostly on the PI3K pathway but also from hematopoietic stem cells for which, an activation of PI3K/AKT lead to a synergy with NOTCH, but is not essential for megakaryocytic specification. On the other hand, to understand OTT1’s mechanisms of regulation, I looked for proteic binding partners by the double hybrid screen technique. Among the candidates I identified SHARP and SRC family kinases as LYN. The specific interaction between OTT1 and LYN was validated in a overexpression model and in a cell line modeling acute megakaryoblastic leukemia. In our models, the interaction with LYN lead to the phosphorylation of OTT1. However, the first analysis did not point out an essential role of this interaction in megakaryocytic development.

Pas de résumé en français disponible. / Pas de résumé disponible.

A proteína P53 pode estar virtualmente alterada em todos os cânceres humanos e portanto, na ausência de mutação, uma possibilidade para a inativação da p53 é a formação de complexo com outras proteínas, tal como a proteína Mdm2. Estudos prévios realizados em nosso laboratório demonstraram a superexpressão de Mdm2 na ausência de expressão da proteína P53 em adenomas pleomórficos. O objetivo deste estudo foi analisar a expressão das proteínas Mdm2, P53, P21 e Akt em adenoma pleomórfico e o mioepitelioma através das técnicas de imunoistoquímica, western blotting e imunofluorescência. A superexpressão de Mdm2 e Akt foi encontrada na maioria das linhagens e lesões utilizadas neste estudo enquanto as proteínas P53 e P21 não demonstraram expressão nas neoplasias estudadas. As superexpressões das proteínas Mdm2 e Akt estão relacionadas à tumorigênese em neoplasias benignas de glândula salivar. / The P53 protein can be altered in virtually all human cancers and in the absence of mutations, P53 inactivation is possible via complex formation with others proteins, such as the Mdm2. Previous studies from our laboratory showed overexpression of mdm2 and lack of p53 expression in pleomorphic adenomas. The aim of this study was to analyze the expression of Mdm2, P53, P21 and Akt proteins in pleomorphic adenomas and myoepiteliomas by Western blotting, immunohistochemistry and immunofluorescence techniques. Overexpression of Mdm2 and Akt was present in the majority of cell lineages and tumors studied, while the expression of P53 and P21 proteins was considered absent. Overexpression of Mdm2 and Akt are related to the tumorigenesis of benign salivary gland neoplasms.

A proteína Akt modula a função de numerosos substratos envolvidos na regulação da sobrevivência celular, progressão do ciclo celular e crescimento celular. Estudos prévios realizados em nosso laboratório demonstraram a superexpressão de Akt em adenoma pleomórfico, mioepitelioma e carcinoma adenóide cístico. O objetivo deste estudo foi analisar a via da proteína Akt através da avaliação da expressão das proteínas NFkB e PTEN em neoplasias benignas e malignas de glândulas salivares através das técnicas de imunohistoquímica, western blotting e imunofluorescência, e a possível interação protéica direta entre p-Akt/Mdm2 e p-Akt/PTEN em linhagem de carcinoma adenóide cístico. A superexpressão nuclear na proteína PTEN foi encontrada nas duas neoplasias malignas estudadas. Além disso, não foi observada interação direta entre as proteínas p-Akt/Mdm2 e p-Akt/PTEN, as quais apresentam localização nuclear em neoplasias de glândulas salivares. / The Akt protein modulates the function of numerous substrates involved in the regulation of cell survival, cell cycle progression and cell growth. Previous studies from our laboratories showed overexpression of Akt and Mdm2 followed by the lack of p53 expression in pleomorphic adenoma, myoepithelioma and adenoid cystic carcinoma. The aim of this study was to analyze the Akt pathway through evaluation of expression of NFkB and PTEN proteins in pleomorphic adenoma, carcinoma ex pleomophic adenoma and adenoid cystic carcinoma by western blotting, immunofluorescence and immunohistochemical techniques, and we have intended to analyse a possible direct interaction between p-Akt/Mdm2 and p-Akt/ PTEN protein in salivary gland tumours. Overexpression of nuclear PTEN was present in both carcinomas studied. In addition, there was no direct interaction between p-Akt/Mdm2 and p-Akt/ PTEN protein, which presents a nuclear localization in salivary gland tumours.