Les cellules tumorales circulantes (CTC) représentent une source de matériel tumoral accessible de manière non invasive, susceptible de fournir des informations cliniques et fondamentales. Ces cellules issues de tumeurs primitives ou métastatiques représentent une population hétérogène d’éléments très rares circulant dans le sang. La personnalisation des traitements en oncologie repose sur la caractérisation moléculaire de biopsies tumorales mais celles-ci peuvent être difficiles à réaliser ou peu informatives. De ce fait, la caractérisation moléculaire et fonctionnelle des CTC présente un double intérêt, clinique pour identifier des biomarqueurs de sensibilité à des traitements, et fondamental pour étudier les mécanismes qui sous-tendent leur potentiel à initier des tumeurs.Les objectifs de ma thèse ont été d’une part de caractériser par séquençage de l’exome (WES) les CTC à l’échelle de cellule unique de patients atteints de cancers de la prostate (PCa) métastatiques et d’autre part d’établir puis caractériser des modèles de xénogreffes dérivés de CTC (CDX) chez des patients atteints de cancers bronchiques non à petites cellules (CBNPC) ou de PCa.Pour répondre au premier objectif, nous avons développé une méthode expérimentale globale incluant trois approches technologiques permettant d’enrichir et d’isoler des CTC individuelles de différents phénotypes (épithélial, épithélio-mésenchymateux et mésenchymateux), d’amplifier la totalité du génome (WGA) et de le séquencer. Le WES a été réalisé pour 34 échantillons de CTC sélectionnés sur des critères de qualité du WGA, ainsi que pour les biopsies de métastases correspondantes chez sept patients. Deux patients présentant une hétérogénéité phénotypique de leurs CTC, ont été analysés en profondeur. Nous avons mis en évidence des mutations partagées entre les CTC et les biopsies tumorales correspondantes ainsi que des mutations uniquement retrouvées dans les CTC. Ces mutations spécifiques aux CTC sont présentes dans tous les phénotypes et affectent particulièrement les gènes impliqués dans le remodelage du cytosquelette, la réparation de l’ADN ou l’invasion. L’existence de mutations communes entre les CTC de différents phénotypes suggère une relation phylogénique entre ces cellules mais une évolution divergente pendant le processus métastatique. Ce travail est soumis pour publication.Dans la seconde partie de ma thèse, nous avons implantés les CTC de 67 patients atteints de CBNPC et 24 patients atteints de PCa chez des souris immunodéprimées. Nous avons établis quatre CDX de CBNPC et un CDX de PCa. La caractérisation de ces modèles, des biopsies tumorales, des CTC collectées au moment de la xénogreffe, des CDX et des lignées cellulaires établies à partir du CDX, ont révélé que les CTC, le CDX et les lignées cellulaires « miment » le phénotype et le profil mutationnel des biopsies tumorales. La caractérisation plus approfondie de l’une des lignées cellulaires montre la présence d’un stress réplicatif et d’une instabilité génomique élevée. Ce résultat nous oriente sur l’hypothèse d’un rôle éventuel de l’instabilité génomique dans la tumorigénicité des CTC.Dans ce travail, nous avons montré que le profil mutationnel des CTC présente de fortes similitudes avec les biopsies tumorales des patients dans les patients atteints de PCa étudiés. De plus, nous avons observé l’existence de mutations spécifiques aux CTC, non détectées dans les biopsies tumorales. Également, nous montrons que des CTC issues de CBNPC et de PCa sont tumorigéniques in vivo et qu’elles reflètent le profil mutationnel des biopsies tumorales des patients. Ces modèles constituent des outils originaux et intéressants pour identifier de nouvelles cibles thérapeutiques et stratégies anti-cancéreuses, et comprendre les mécanismes qui supportent le potentiel des CTC à initier des tumeurs. / Circulating tumor cells (CTCs) represents an non invasive source of tumor material which may provide clinical and basic information. These cells derived from primary or metastatic tumors represents an heterogeneous population of very rare events which circulates in the blood. Oncology personnalized medicine is based on biopsies molecular characterization but these are sometimes which difficult to realize and poorly informative. Thereby molecular and functional characterization of CTCs presents a double interest, clinical to identify treatments biomarkers sensitivity and basic to study mechanisms underlying their tumor inititiating cell (TIC) potential. The two goals of my thesis were on the one hand to characterize by whole-exome sequencing (WES) at the single level the CTCs from patients with metastatic prostate cancers (mPCa) and on the other hand to establish and characterize CTC-derived xenografts (CDX) from patients with non-small-cell lung cancer (NSCLC) or mPCa. For the first goal we developped a global workflow which include three technological approaches to enrich and isolate individual CTCs from different phenotype (epithelial, epithelial and mesenchymal, mesenchymal), to perform whole genome amplification (WGA) and to sequence them. WES was performed on 34 CTC samples selected according to WGA quality and on corresponding metastasis biopsies from seven patients. Two patients with phenotypic heterogeneity of CTCs were deeply analyzed. We highlighted shared mutations between CTCs and matched biopsies as well as mutations only detected in CTCs. These private CTC mutations are detected in all phenotype and particularly affect genes invlved in cytoskeleton remodeling, DNA repair or invasion. The existence of common mutations between CTCs from various phenotype suggests a phylogenic link between these cells but a divergent evolution during metastatic process. This work is submitted for publication. For the second goal, we implanted CTCs from 67 NSCLC patients and 28 mPCa patients in immunocompromised mice. We established four NSCLC CDX and one mPCa CDX. The characterization of tumor biopsies, CTCs collected at the time of xenograft, CDX and CDX-derived cell lines revealed that CTCs, CDX and cell lines miror the phenotype and mutational landscape of tumor biopsies. The more deeply characterization of one cell line show the presence of a high replicative stress and genomic instability. This result directs us to the hypothesis of a possible role of the genomic instability in CTC tumorigenicity.We demonstrated in this work that CTCs mutational landscape harbors high similairities with patients tumor biopsies in mPCa. Furthermore we observed CTC private mutations not detected in tumor biopsies. Also we showed that some CTCs from NSCLC and mPCa are tumorigenic in vivo and that these CTCs mirror mutational profile of patients tumor biopsies. These models are original and interesting tools to identify new therapeutic targets and anti-tumoral strategies and understand mechanisms underlying the TIC potential of CTCs.

In general, ionizing radiation promotes cytoprotective autophagy in a majority of breast tumor cells. Previous studies from our laboratory indicated that radiation (5x2 Gy) induces cytoprotective autophagy in MCF-7 cells. In the current work, inhibition of autophagy by silencing of Beclin-1 in MCF-7 cells resulted in an increase in sensitivity to radiation based both on cell number and clonogenic survival; however, there was no increase in apoptosis and the basis for this sensitization is currently under investigation. Unexpectedly, enhancement of autophagy by silencing of Bcl-2 also led to an increase in sensitivity to radiation, possibly through the conversion of cytoprotective to cytostatic autophagy. In contrast to the MCF-7 cells, radiation (5x2 Gy) induces non-protective autophagy in Hs578t cells. Interference with autophagy through silencing of Beclin-1 or induction of Bcl-2 did not alter radiation sensitivity in the Hs578t cells. Since the induction of cytoprotective autophagy can represent an impediment to radiation therapy, it is important to understand the types of autophagy that occur in response to radiation in specific cellular settings and whether interference with autophagy can increase sensitivity to different forms of cancer treatment.

Les cellules tumorales circulantes (CTC) sont des cellules présentes en très faible proportion (une cellule pour un million de cellules normales) dans la circulation sanguine, et qui jouent un rôle important dans le processus de métastase responsable de la majorité des décès de patients atteints de cancer. La détection du cancer à un stade précoce augmente les chances de survie des patients. Le but de ce travail a été de développer un ensemble de technologies permettant de mieux caractériser et détecter les CTC.Nous avons concentré notre étude sur les cellules ayant un phénotype hybride, entre épithélial et mésenchymal, qui pourraient correspondre à des CTC de plus fort potentiel métastatique compte tenu du rôle joué par la transition épithelio-mésenchymateuse dans ce processus. Nous avons tout d’abord isolé, par immunofluorescence et cytométrie en flux, une lignée cellulaire de cancer (A549, le carcinome de poumon humain) co-exprimant la E- et la N-cadhérine, de sorte qu’elle puisse être utilisée comme modèle de CTC dans le développement de nouvelles techniques de détection. Nous avons en particulier adapté le système de microscopie de fluorescence et d’analyse d'images PathfinderTM à haut-débit de la société Imstar S.A. pour identifier efficacement quelques milliers de cellules A549 mélangées à du sang de patient, après une étape de filtration par la taille. Afin d’améliorer l’identification des cellules hybrides, nous avons évalué la technique de transfert de Förster résolue en temps qui pourrait révéler avec un excellent rapport signal/bruit la présence à la membrane cellulaire d’agrégats compacts de N- et E-cadhérines. Enfin, afin d’augmenter le nombre de biomarqueurs simultanément détectés par immunofluorescence nous avons contribué à la mise au point de nanocristaux semi-conducteurs fluorescents conjugués avec un anticorps dirigé contre une protéine d'intérêt. Au final, nos résultats fournissent un ensemble de technologies qui pourront être utilisées pour améliorer la détection et la caractérisation des CTC. / Circulating tumor cells (CTCs) are rare cells (one in millions of normal cells) in blood circulatory system playing a key role in the process of metastasis, which is responsible for the majority of death of patients with cancer. Detecting cancer at early stage can give patients higher chances of survival. The aim of this work is to develop a set of technologies capable of characterizing and detecting the CTCs. We restricted our study to CTCs with hybrid phenotype, between epithelial and mesenchymal, that could correspond to circulating cells with the highest metastatic potential, considering the relation of the Epithelial to Mesenchymal Transition to cancer. Using immunofluorescence and flow cytometry, we first isolated a cancer cell line (A549, human lung carcinoma) co-expressing E- and N-cadherin, which is further used as a CTC model in the development of new detection techniques. In particular, we showed that the high throughput automated fluorescence microscope and image processing Imstar S.A. PathfinderTM system can recover efficiently a few thousands of A549 cells spiked in a blood sample, after an initial size-filtering step. We also used time-gated Fluorescence Resonant Energy Transfer to investigate the presence of E- and N-cadherin clusters at the cell membrane that could enhance the detection sensitivity of hybrid phenotype. Finally, in view of increasing the number of simultaneous biomarkers detection by immunofluorescence we contributed to the development of fluorescent semiconductor nanocrystals conjugated with antibody directed against the protein of interest. Altogether, our results provide a set of technologies that can be used to improve the detection and characterization of CTCs.

No presente trabalho, desenvolvemos uma estratégia vacinal baseada em vacinas de DNA que codificam proteínas do HPV-16, o tipo viral de maior relevância epidemiológica, fusionadas à glicoproteína D (gD) do vírus herpes simplex tipo 1 (HSV-1). A vacina que contêm o gene da E7 de HPV-16 fusionada à gD de HSV-1 (pgDE7), administrada em regime vacinal de quatro doses, foi capaz de gerar significativa ativação de células T CD8+ E7-específicas e apresentar 40% de efeito protetor anti-tumoral terapêutico em camundongos desafiados com células transformadas que expressam as proteínas E6 e E7 do HPV-16 (células TC-1). A partir das evidências geradas, desenvolvemos um novo vetor vacinal que codifica as proteínas E7, E6 e E5 do HPV-16 (pgD-E7E6E5). Em ensaios em modelo murino, apenas uma dose da vacina foi capaz de gerar ativação de células T CD8+ específicas e 70% dos camundongos previamente desafiados com células TC-1 e inoculados com 3 doses da vacina mantiveram-se livres de tumores. Como tentativa de potencializar o efeito protetor terapêutico encontrado, adotamos duas medidas: a co-administração de plasmídeos que codificam citocinas e a otimização de códons da sequência gênica que codifica a proteína quimérica. A combinação das vacinas pgDE7 ou pgD-E7E6E5 com plasmídeos que carregam os genes das citocinas IL-2, IL-12 ou GM-CSF foi capaz de aumentar a proteção terapêutica para 100% em regime vacinal de dose única. A adequação da sequência antigênica ao sistema de expressão humano, aumentou em cerca de 5 vezes o potencial terapêutico do vetor vacinal pgDE7. Em conjunto, os dados apresentados nesta tese demonstram a evolução do desenvolvimento de uma estratégia vacinal contra tumores induzidos por HPV-16 e encorajam seu potencial para uso em futuros ensaios clínicos. / The development of immunotherapeutic strategies against human papillomavirus (HPV) is a priority for the control of HPV-induced lesions and cervical cancer. In this study, we developed DNA vaccines encoding HPV-16 proteins fused to glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) as an approach to control HPV-16 induced tumors. The vaccine encoding HPV-16 E7 fused to HSV-1 gD (pgDE7), when administered in a four doses vaccine regimen, was able to generate significant activation of E7-specific CD8+ T cells and showed 40% of therapeutic anti-tumor effect in mice previously challenged with tumor cells expressing HPV-16 E6 and E7 proteins (TC-1 cells). Following these evidences, we developed a new vaccine vector encoding HPV-16 E7, E6, E5 proteins fused to HSV-1 gD (pgD-E7E6E5). Only one vaccine dose generated antigen-specific CD8+ T cell responses and three doses conferred 70% protection to mice previously challenged with TC-1 cells. As an attempt to enhance the observed therapeutic anti-tumor effects, we tested two approaches: co-administration of cytokine-expressing plasmids and codon optimization of the gene encoding the chimeric protein. The combination of the vaccines pgDE7 or pgD-E7E6E5 with plasmids encoding the cytokines IL-2, IL-12 or GM-CSF increased the therapeutic protection to 100% of the vaccinated animals following a single dose. The gene sequence adaptation increased by a factor of 5 the therapeutic potential of the pgDE7 vaccine. In summary, the data presented in this thesis demonstrated the development of a vaccine strategy against HPV-16 induced tumors and reinforces its potential use in future clinical trials.

Imiquimod é uma imidazoquinolina, agonista do TLR-7, ativador de células dendríticas (DC) e indutor da transcrição de diversas citocinas. O objetivo deste estudo é avaliar o crescimento tumoral, a formação de metástases em órgãos internos, a atividade do sistema imune e o tipo de morte celular induzido devido ao tratamento tópico. Camundongos foram inoculados com células de melanoma murino B16F10. O fármaco, na dose de 5mg, foi aplicado no local da inoculação uma vez por dia até o fim do experimento. Foram estudados quatro grupos experimentais, dois inoculado com células tumorais, sendo um tratado e outro não, e outros dois grupos, um recebeu somente o tratamento com o fármaco e o outro não recebeu tratamento. Os animais foram pesados e seus tumores medidos periodicamente. Exemplares dos grupos foram sacrificados em quatro tempos diferentes, imediatamente submetidos à necropsia para a análise de massas tumorais por citometria de fluxo com caspase-3 e anexina-v para investigar a indução de apoptose e/ ou necrose tumoral, e remoção do tumor, pele, Baço, linfonodo e pulmão, que foram fixados, inclusos em parafina, cortados e corados com HE, para análise histológica. / Imiquimod is a TLR-7 agonist and activator of dendritic cells (DC), and induces transcription of several cytokines. This study`s objective is to evaluate tumor growth, development of metastasis within internal organs and the immune system`s function, due to the topical treatment. Two groups of C57BL/6J mice were inoculated with B16F10 melanoma cells subcutaneously, and only one of the groups was treated, applied topically at the injection site once a day, followed by a second round of two groups, one treated and the other as a healthy group. Every two tumors and weight were measured. In order to observe the tumor progression and treatment effects, groups of animals were sacrificed on four days apart and immediately submitted to necropsy for a tumor analysis by flow cytometry, to evaluate the induction of apoptosis and necrosis, and to macroscopically observe the tumor development within internal organs, vascularization and posterior inclusion of the organs in paraffin for histological analysis. Tumor, skin, spleen, limph node and lung were colored with HE for histological analysis of migrating immune system cells and tumoral cells in the organs and describing its integrity.

Carcinoma adenóide cístico é uma neoplasia maligna de glândula salivar com alto grau de recorrência e metástase. Células tumorais que invadem tecidos subjacentes formam invadopódios, protrusões de membrana ricas em actina, cortactina e MT1-MMP capazes de degradar a matriz extracelular (MEC). Proteólise de moléculas da MEC, como a laminina, promove liberação de fragmentos e peptídeos bioativos. Nesse trabalho, estudamos o papel dos peptídeos AG73 e C16, da laminina-111, na atividade de invadopódios de células derivadas de carcinoma adenóide cístico (CAC2). Nossos resultados mostram que AG73 e C16 regulam atividade de invadopódio e aumentam a expressão de MT1-MMP em células CAC2. Silenciamento da integrina <font face=\"Symbol\">b1 e inibição da via ERK diminuem a atividade de invadopódio induzida por esses peptídeos. Já a inibição da via Rac diminui a atividade de invadopódios induzida apenas por AG73. Propomos que os peptídeos AG73 e C16 regulariam a atividade de invadopódios através da integrina <font face=\"Symbol\">b1 e da via ERK 1/2. Já a via Rac1 transduziria sinais gerados apenas pelo peptídeo AG73. / Adenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm with high level of recurrence and metastasis. Tumor cells that invade surrounding tissues rely on invadopodia to degrade extracellular matrix (ECM). Invadopodia are actin and cortactin-rich membrane protrusions that localize MT1-MMP required for ECM degradation. Breakdown of ECM molecules, such as laminins, releases fragments and bioactive peptides. Here we addressed the role of laminin-111 peptides AG73 and C16 in invadopodia activity of cells derived from human adenoid cystic carcinoma (CAC2). Our results show that AG73 and C16 increase invadopodia activity and MT1-MMP expression in CAC2 cells. Silencing of <font face=\"Symbol\">b1 integrin and ERK pathway inhibition decrease AG73 and C16-induced invadopodia. Rac1 pathway inhibition decreases only AG73-induced invadopodia formation. We propose that AG73 and C16 increase invadopodia activity in CAC2 cells through <font face=\"Symbol\">b1 integrin. ERK1/2 pathway would transduce signals generated by both peptides, while Rac1 pathway is related to AG73 signaling.

Gliomas são tumores do sistema nervoso central e o glioblastoma multiforme (GBM) é sua forma mais comum e de pior prognóstico. A proliferação desordenada, migração estão entre os processos biológicos que conferem ao GBM um comportamento altamente agressivo. O objetivo deste estudo foi analisar in vitro o papel de PGE2 na proliferação, migração e apoptose da linhagem de glioma humano T98G através de tratamento com ácido gama-linolênico (GLA), Ibuprofeno (IBU) e adição de prostaglandina E1 (PGE1) e prostaglandina E2 (PGE2) exógeno. Nossos resultados demonstraram através uma diminuição nas taxas de proliferação e de migração e uma indução de apoptose nas células T98G tratadas com GLA ou IBU. Por outro lado, a adição exógeno de PGE1 ou PGE2 resultou num aumentou da proliferação e da migração e numa inibição da apoptose. Esses resultados demonstram a influência de PGE2 na linhagem T98G. / Gliomas are tumors of the central nervous system and glioblastoma multiforme (GBM) is the most common form with the worst prognosis. Uncontrolled cell proliferation, migration are among the biological processes that give GBM a highly aggressive behavior. The aim of this study was to analyze in vitro the role of PGE2 the proliferation, migration and apoptosis of the T98G human glioma cell line through treatment with gamma-linolenic acid (GLA), ibuprofen (IBU) and the addition of exogenous prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2). Our results demonstrated through various tests a decrease in the rates of proliferation and migration and an induction of apoptosis in T98G cells treated with GLA or IBU. On the other hand, the addition of exogenous PGE1 or PGE2 resulted in increased proliferation and migration and inhibition of apoptosis. These results demonstrate the influence of PGE2 in the T98G cell line.

O mecanismo de formação e origem das alterações nucleares ainda é pouco conhecido, sendo o micronúcleo a mais estudada. As células tumorais geralmente apresentam vários tipos de alterações nucleares que estariam associadas à instabilidade genética. O objetivo deste trabalho foi analisar as possíveis associações entre alterações nucleares e citoesqueleto em células HK2 e A549, derivadas de carcinoma de pulmão humano. Otimizamos a metodologia de uso do MCVL para redefinir as alterações nucleares e caracterizar os principais filamentos do citoesqueleto em preparações coradas por Feulgen ou imunofluorescência. As células da linhagem HK2 apresentaram fibras de actina dispostas concentricamente e em "clusters" e os filamentos de tubulina apareceram de forma radial, enquanto que o padrão de distribuição em A549 foi mais semelhante ao das células normais (BRL3A). Os filamentos de lamina B foram os mais importantes para evidenciar as alterações nucleares, porém essas alterações não puderam ser relacionadas com alterações do citoesqueleto. / The origin and mechanism of formation of the nuclear alterations is largely unknown, with the micronucleus being the most well studied alteration. Tumor cells generally present various types of nuclear alterations witch can be associated with genetic instability. The propose of this study was to analyze the possible association between nuclear alterations and the cytoskeleton in the human lung carcinoma cells HK2 and 549. The method of LSM was optimized to redefine the nuclear alterations and to characterize the principal cytoskeletal filaments in preparations stained with Feulgen’s reagent or submitted to imunofluorescent methods. The HK2 cells presented actin fibres arranged either concentrically or in clusters and tubulin filaments arranged radially, while in the A549 cells the distribution pattern was similar to that of normal cells (BRL3A). The lamin B filaments were the most important to identify nuclear alterations, as these alterations could not be related to cytoskeletal alterations.

Exossomos (Exo) originados de células dendríticas (DCs) carregam moléculas associadas à apresentação antigênica. Neste trabalho procurou-se estabelecer se Exo de DCs seriam capazes de conferir imunogenicidade às células tumorais. Os Exo isolados de culturas de DCs expressavam as moléculas HLA-ABC, HLA-DR, CD86, CD11c, CD81, CD54 e CD18. Estes foram então adicionados às células da linhagem humana de adenocarcinoma mamário, SK-BR-3, as quais passaram a expressar as moléculas HLA-DR, CD86 e CD11c. As células tumorais modificadas pelos Exo induziram a produção de IL-6 e IL-10, detectados no sobrenadante das co-culturas destas com linfócitos T. Estas células tumorais também induziram aumento do número de linfócitos produtores de IFN-<font face=\"Symbol\">g, pré-sensibilizados contra antígenos tumorais, e aumento da expressão de SOCS3 nestes. Em conclusão, nossos resultados mostram que, Exo de DCs alteram o fenótipo de células tumorais, modificando sua interação com linfócitos T, sem induzir nas mesmas capacidade de ativar respostas proliferativas ou citotóxicas de linfócitos T in vitro. / Exosomes (Exo) originated from dendritic cells (DCs) contain molecules involved in antigen presentation. The present work sought to determine if Exo from DCs would be able to transfer immunogenicity to tumor cells. Exo isolated from DCs cultures carried HLA-ABC, HLA-DR, CD86, CD11c, CD81, CD54 and CD18. These Exo were added to cultures of the human breast adenocarcinoma cell line, SK-BR-3, which gained expression of HLA-DR, CD86 and CD11c. Tumor cells modified by Exo induced IL-6 and IL-10 production, detected in the supernatant of their co-cultures with T lymphocytes. These tumor cells also induced an increase in the frequency of IFN-<font face=\"Symbol\">g-producing T lymphocytes, pre-sensitized against tumor antigens, and an increased expression of SOCS3. In conclusion, our results show that, Exo from DCs affect the phenotype of tumor cells, modifying their interaction with T lymphocytes, without inducing the ability to activate cytotoxic or T cell proliferative responses in vitro.

O fator esteroidogênico 1 (SF-1) é um fator de transcrição envolvido no desenvolvimento, na produção de esteroides e na proliferação do córtex adrenal. POD-1/TCF21 parece estar envolvido na regulação de SF-1, inibindo sua expressão e suas funções nas células esteroidogênicas. No entanto, a expressão e a relação entre SF-1 e POD-1 em células normais adrenocorticais não está esclarecida. O objetivo desse trabalho foi determinar se Pod-1 regula a expressão de Sf-1 através da hiperexpressão de Pod-1 em culturas primárias de células adrenocorticais de rato. Foi analisada a expressão endógena de Sf-1 e Pod-1, através da reação de RT-PCR quantitativo, em células glomerulosas (G) e células fasciculadas/reticulares (F/R), bem como em células transfectadas com pCMVMycPod-1. Os resultados mostraram que Sf-1 está mais expresso nas células F/R do que nas células G e ambos os tipos celulares apresentaram baixa expressão endógena de Pod-1. As células F/R transfectadas apresentaram um aumento estatisticamente significante da expressão de Sf-1, mas não as células G. Esses resultados sugerem um mecanismo de ação de Pod-1 no controle da expressão de Sf-1 em células adrenocorticais normais distinto do descrito em células de tumores adrenais e células esteroidogênicas. / The steroidogenic factor 1 (SF-1) is a transcription factor involved in the development, steroids production and adrenal cortex proliferation. There are evidences that suggest that POD-1/TCF21 may be involved in the inhibition of SF-1 and modulating the SF-1 function in steroidogenic cells. However, the expression and the relation between SF-1 and POD-1 in normal adrenocortical cells are still unclear. The aim of this study was to determine whether Pod-1 regulates the expression of Sf-1 by overexpression of Pod-1 in primary cell cultures of rat adrenal cortex. We analyzed the expression of endogenous Sf-1 and Pod-1 by RT-PCR quantitative in glomerulosa (G) and fasciculata/reticularis (F/R) cells and also in cells transfected with plasmid CMVMycPod-1. The results showed that the SF-1 expression is higher in F/R cells than the G cells. Moreover, both cell types showed low endogenous expression of Pod-1. Analysis of Sf-1 expression in transfected cells showed a statistically significant increase in the Sf-1 expression in F/R cells but not in G cells. These results suggest a mechanism of action of Pod-1 in the regulation of Sf-1 in normal cells distinct of described in adrenocortical tumor cells and other steroidogenic cells.