Akt/Protein Kinase B plays an important role in many biological responses including cell survival, proliferation, and nutrient metabolism. Activation of Akt/PKB by growth factors involves PIP3 binding, membrane translocation and the phosphorylation at Thr308 and Ser473 residues by PDK1 and PDK2, respectively. Dephosphorylation and AKT binding proteins were two inhibitory mechanisms for AKT. Carboxyl-terminal modulator protein (CTMP) has been demonstrated to bind to carboxyl-terminus of Akt and as a regulator on plasma membrane. However, the interaction region of CTMP to Akt is still unknown. On the other hand, contradict results of positive and negative effects to AKT activity were reported. In this study, we firstly demonstrated the CTMP protein level shows positive correlation with Akt phosphorylation and cell proliferation in human breast cancer cell. The interaction of CTMP and phosphorylation Akt by co-immunoprecipitation and immunofluorescence experiments shows that CTMP may be involved in regulating Akt phosphrylation. The results of GST pull down assay demonstrated that the direct binding and revealed more then one interaction region of CTMP to Akt. Interestingly, in HeLa cells, transiently or stably expressing CTMP can directly promote Akt phosphorylation even in basal state and significantly increase the phosphorylation at both Thr308 and Ser473 of Akt upon the insulin stimulation in the pre-starvation condition. When compared the cell growth of HeLa cells in 10% serum condition using cell proliferation and soft-agar colony forming assay, rapid cell proliferation and colony number increase were observed in cells stably expressing CTMP suggesting that CTMP may be involved in the regulation of Akt-mediated cell growth. The analogous results were also obtained from colony formation assay with different condition from 10% to 1% serum. The result of cell cycle distribution shows that the increasment of cell proliferation rate may due to the increasment of cell cycle progression in CTMP stable clones. Furthermore, CTMP stable clone promote tumor growth and metastasis in xenograft animal model. Together, these results indicated that the binding of CTMP positively modulate Akt and thus increase insulin sensitivity in HeLa cells.

As the origin of a ¡§life-and-death¡¨ signal that reflects central cardiovascular regulatory failure during brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate to evaluate the cellular mechanism of this fateful phenomenon. Based on a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult, this study evaluated two hypotheses. First, transcriptional upregulation of nitric oxide synthase I or II (NOS I or II) gene expression by nuclear factor-£eB (NF-£eB) on activation of phosphoinositide 3-kinases (PI3K)/Akt/phosphatase and tensin homologue deleted on chromosome ten (PTEN) cascade in the RVLM underlies brain stem death. Second, muscarinic receptor-independent activation of cyclic adenosine monophosphate-dependent protein kinase A (PKA) in the RVLM is involved in the cardiovascular responses exhibited during Mev intoxication. In Sprague-Dawley rats, our results showed that microinjection bilaterally of Mev (10 nmol) into RVLM induced a progressive augmentation in NF-£eB, PI3K, Akt or PTEN activity that paralleled the increase in NOS II or peroxynitrite level in RVLM. Loss-of-function manipulations that included pharmacological blockade, gene knockdown, or immunoneutralization of NF-£eB, PI3K or Akt in RVLM significantly potentiated and prolonged the initial increase in ¡§life-and-death¡¨ signal, reversed the cardiovascular depression, and blunted the augmented expression of NOS II or nitrotyrosine on induced by Mev. Blockade of PI3K or Akt in RVLM also significantly blunted the Mev-induced activation of NF-£eB in the RVLM. However, immunoneutralization of PTEN in RVLM significantly diminished the increase in ¡§life-and-death¡¨ signal and potentiated the increase in Akt activity. We conclude that the PI3K/Akt cascade plays a ¡§pro-death¡¨ role in our Mev intoxication model of brain stem death by upregulating NF-£eB/NOS II/peroxynitrite in the RVLM, subject to antagonism by PTEN in this process. Microinjection bilaterally of Mev (10 nmol) into the RVLM induced a significantly augmentation in PKA activity in ventrolateral medulla that was not antagonized by coadministration of a nonselevtive or selective muscarinic receptor inhibitor. However, pharmacological blockade PKA in RVLM significantly blunted the initial increase in ¡§life-and-death¡¨ signal and the accompanying augmentation of NOS I expression in the ventrolateral medulla exhibited during Mev intoxication. We conclude that a muscarinic receptor-independent activation of PKA plays a ¡§pro-life¡¨ role in our Mev intoxication model of brain stem death by up regulating NOS I/PKG in the RVLM. According to this study, we proved that Mev stimulates different mechanism, muscarinic receptor-independent/PKA and PI3K/Akt/NF-£eB, to regulate NOS I and NOS II expression respectively, and induces cardiovascular responses during ¡§pro-life¡¨ and ¡§pro-death¡¨ phases. This information should provide further insights on the cellular mechanism of central cardiovascular regulation during the progression towards brain stem death, and offer news vistas in our search for therapeutic remedies or management strategies against fatal organophosphate poisoning and brain stem death.

Zugl.: Marburg, Univ., Diss., 2007

Zugl.: Marburg, Univ., Diss., 2007.

Orientador: Sara Teresinha Ollala Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-05T23:02:24Z (GMT). No. of bitstreams: 1 Favaro_PatriciaMariaBergamo_D.pdf: 1970664 bytes, checksum: 6c1818859a4b90ebac61f9c01b4f62cc (MD5) Previous issue date: 2006 / Resumo: Forminas são proteínas relacionadas com o controle de morfogênese, diferenciação embrionária, citocinese, polaridade e sobrevivência celular. São conservadas entre várias espécies, incluindo fungos, plantas, moscas de fruta, camundongos e humanos, apresentando múltiplos domínios. Com o objetivo de descrever novas proteínas, através das informações geradas pelo projeto Genoma do Câncer Humano, uma parceria entre a Fapesp e o Instituto Ludwig, duas EST humanas (expression sequence tags) foram identificadas com homologia aos genes da família das forminas. O objetivo do presente estudo foi obter a seqüência completa do RNAm deste novo gene e a estrutura primária da proteína para estudos de localização, interações com outras proteínas e estudos funcionais da mesma. Para obter a seqüência completa do RNAm, foram realizadas buscas no banco de dados genômico do NCBI (National Center for Biotechnology lnformation) e usadas técnicas de biologia molecular. Assim, após a obtenção da seqüência completa do novo RNAm foi possível o depósito do mesmo no banco de dados do portal NCBI, com o nome de Formina Leucocitária Humana (número de acesso AY278319). Posteriormente, baseando-se na nomenclatura de novos genes e proteínas, aprovada pelo Gene Nomenclature Committee RUGO, o novo gene foi renomeado formin-like 1, doravante FMNL 1. A produção de um anticorpo policlonal específico para a proteína codificada pelo FMNL1, permitiu o estudo de sua expressão em diversos tecidos humanos. Através da técnica de Western Blotting, foi observada expressão preferencial em células neoplásicas, especialmente linfóides. Dentre os tecidos normais, foi detectada expressão apenas em células mononucleares de sangue periférico, em linfonodo e preferencialmente em células CD19- quando comparadas com células CD19+ de tonsilas normais. Dentre as linhagens celulares neoplásicas, a expressão foi maior em MOLT-4 e Jurkat, quando comparadas com outras linhagens mielóides, indicando expressão preferencial da proteína em neoplasias linfóides. Além disso, a alta expressão da proteína foi observada em células mononucleares de sangue periférico de 20 pacientes com. LLC (leucemia linfóide crônica), quando comparadas com células mononucleares de sangue periférico de indivíduos normais. Quando a expressão da proteína FMNL1 foi estudada em diferentes tipos histológicos de Linfomas não-Hodgkin (LNH) e em linfonodos reativos, novamente foi observada a expressão preferencial no tecido neoplásico em relação ao tecido normal, sendo os LNH- T aqueles com expressão mais significativa. Esta nova proteína FMNL1 localiza-se no citoplasma, conforme verificado pela microscopia confocal e confirmado através da técnica de separação de frações. Das interações desta proteína, foi detectada a associação de FMNL 1 com AKT, sendo que essa associação é independente da ativação de AKT. Também foi observado que a ativação da via das caspases em duas linhagens celulares neoplásicas levava à diminuição da expressão da proteína FMNL 1. Em síntese, a expressão preferencial da proteína FMNL1 em tecidos neoplásicos línfóides e a sua associação com AKT, sugere a sua participação na sobrevivência dessas células e, possivelmente, na fisiopatologia das neoplasias hematológicas / Abstract: Formins are proteins that are involved in processes such as morphogenesis, embryonic differentiation, cytokinesis, cell polarity and survival. The formins are multidomain proteins that are conserved from plants to fungi and vertebrates. A search of the ORESTES database, generated from the Human Cancer Genome Project, identified 2 ESTs that were similar to the Formin-related proteins. The aim of this study was to obtain the full-Iength sequence ofthis new gene, the primary structure ofthe protein coded by this gene, its subcellular localization and to characterize its possible interaction with other proteins in addition to functional studies. In an attempt to obtain the full-Iength sequence of this rnRNA, searches in the human genome database at NCBI (National Center for Biotechnology Information) and molecular biology techniques were performed. The whole coding sequence ofthis gene was named Human Leukocyte Formin and was deposited at the human genome database at NCBI (GenBank Accession No. AY278319). Later, based on the HUGO Gene Nomenc1ature Committee, the new gene was renamed formin-like 1 and the symbol FMNLl will herein be used. The production of a polyc1onal antibody specific for the protein FMNLl allowed the study of the protein's expression in different human tissues. Using the Westem Blotting technique, a preferential expression of this protein in lymphoid malignancies was observed. Among normal tissues, a low expression in lymph node, peripheral blood mononuClear cells and in CD19- cells (when compared with CD19+ cells from normal tonsils) was observed. Among the malignant celllines, a high expression of the protein was detected in MOLT-4 and the Jurkat cell line, when compared with myeloid cancer cell lines, suggesting a preferential expression of FMNLl in lymphoid malignancies. Moreover, a high expression of the protein in peripheral blood mononuc1ear cells from 20 chronic lymphocytic leukemia patients was observed when compared with peripheral blood mononuc1ear cells of normal donors. When the expression of FMNLl protein was compared among different histological types of non-Hodgkin's lymphoma (NHL) and reactive lymph nodes, again, a preferential expression of the protein in the malignant tissue was detected and the T cell NHL presented the highest expression. This new protein is located in the cytoplasm of the cells, as observed by confocal microscopy and confirmed by separation of the cellular ftaction. With regard to the interactions ofthis new protein, an association ofFMNLI with AKT was detected, and this association was not dependent on AKT activation. A decrease in FMNLI expression in 2 cancer celllines after caspase activation was also detected. ln conclusion, the preferential expression of FMNLI in lymphoid malignancies and its association with AKT, suggests the role ofFMNLI in cell survival and a possible role in the pathophysiology ofhematological malignancies / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

Cachexia or muscle atrophy is a condition that is associated with a variety of diseases such as chronic heart failure and cancer. In North America, Europe and Japan, more than 8 million patients suffer from cachexia, and it is estimated that cachexia is the cause of death in 30% of cancer patients. Unfortunately, there is no available treatment for cachexia. Bexarotene, a retinoid X receptor (RXR) agonist, is a FDA approved drug used to treat cancer and is able to induce myogenic differentiation in embryonic stem cells. In this study, we investigated the mechanism by which bexarotene enhances myogenic differentiation. The Akt signaling pathway is required for myogenesis and thus we examined its involvement in bexarotene-enhanced myogenic differentiation. We showed that bexarotene, through the activation of RXR signaling, regulates Akt2 expression to enhance myoblast differentiation and fusion. Additionally, we showed that Akt2, but neither Akt1 nor Akt3, is required for bexarotene-enhanced differentiation. Furthermore, we showed that the activation of RXR signaling by bexarotene correlates with a specific histone acetylation mark at the Akt2 locus. More importantly, we demonstrated that bexarotene is able to rescue myoblast differentiation in an in vitro cachexia system. Taken together, our data revealed the significance of Akt2 in bexarotene-enhanced myogenic differentiation and the potential of using bexarotene as a treatment for cachexia.

No description available.

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

Les travaux de la thèse portent sur l'implication de la voie de signalisation Akt (sérine/thréonine kinase Akt) et mTOR (mammalian target of rapamycin) dans la physiopathologie de l'hypertension artérielle pulmonaire (HTAP) expérimentale. L'HTAP résulte d'une prolifération exagérée des cellules constitutives des vaisseaux pulmonaires, principalement les cellules musculaires lisses artérielles pulmonaires (CML-AP). De nombreux effecteurs biologiques et physiques préalablement identifiés agissent sur les CML-AP et participent à l'hyperplasie de celles-ci. Nous montrons que ces nombreux effecteurs convergent vers une voie de signalisation intracellulaire commune, la voie Akt/mTOR, qui de fait représente une cible thérapeutique pour le traitement de l'HTAP, et pourrait conditionner l'hyperplasie des CML-AP. mTOR est présent dans la cellule sous forme de deux complexes, mTORC1 et mTORC2, qui phosphorylent des substrats variés contrôlant la prolifération cellulaire. Les effecteurs de mTORC1 incluent les S6 kinases (S6K1 and S6K2) et les "eIF4E-binding proteins" (4EBP) alors que mTORC2 active la sérine/thréonine kinase Akt et parmi les kinases sous-jacentes, la kinase GSK3. La première étude est consacrée à l'évaluation des effets des inhibiteurs de protéases du VIH (ritonavir, amprenavir, nelfinavir) sur la progression de HTAP expérimentale, induite par la monocrotaline ou l'hypoxie. Nous montrons que ces deux formes d'HTAP sont associées à une activation de la voie Akt/mTOR dans les artères pulmonaires. Les traitements respectifs par les trois inhibiteurs des protéases du VIH durant 3 semaines induisent une réversibilité de l'HTAP, de l'hypertrophie ventriculaire droite et du remodelage des vaisseaux pulmonaires, de même qu'une inhibition de la phosphorylation d'Akt, de S6K et de GSK3. La prolifération des CML-AP induite par le PDGF ou le SVF 5%, associée à une augmentation de p-Akt et p-GSK3, est également bloquée par les inhibiteurs des protéases, de façon similaire et non additive à celle d'inhibiteurs spécifiques de la PI3 kinase et de GSK3. La conclusion est que ces traitements antirétroviraux inihibent la progression de l'HTAP en inhibant la voir Akt/mTOR dans les CML-AP. Cette proposition permettrait d'expliquer l'effet suspecté en clinique des traitements antirétroviraux sur l'HTAP compliquant l'infection par le VIH.Dans la seconde étude, nous montrons que les CML-AP en culture prélevées à partir de rats ayant développé une HTAP induite par la monocrotaline proliférent de façon exagérée en comparaison avec les cellules de rats témoins. Ce phénotype prolifératif est observé en présence de nombreux facteurs mitogènes parmi lesquels le SVF 5%, le PDGF, la sérotonine ou 5-HT, l'IGF1 ou l'IL1-beta, et est associé à une activation des substrats de mTORC1 et mTORC2. Le traitement in vitro par la rapamycine des CML-AP de rats avec HTAP établie permet d'inhiber la prolifération de ces cellules et de bloquer à la fois mTORC1 et mTORC2. De même, le traitement par la rapamycine de rats porteurs de l'HTAP préétablie pendant une semaine permet de normaliser la prolifération des CML-AP in vitro et in vivo et d'inhiber mTORC1 et mTORC2, effets non observés par l'Imatinib ou la Fluoxetine. De plus, le traitement des rats par la rapamycine prévient ou corrige l'HTAP induite par la monocrotaline de façon plus importante que l'Imatinib ou la Fluoxétine.Ces résultats indiquent donc que l'activation de la voie Akt/mTOR, très étroitement associée au développement de l'HTAP expérimentale, pourrait expliquer le phénotype prolifératif anormal des CML-AP inhérent à la pathologie, et ainsi représenter une cible thérapeutique de choix pour le traitement de l'HTAP humaine. / The major objectives of research described in this thesis is focused on the cell signaling pathway of Akt (serine/threonine kinase Akt) and mTOR (mammalian target of rapamycin) in the patho-physiology of experimental pulmonary arterial hypertension (PAH). PAH occurs as a result ofhyperplasia of the components of pulmonary vessels, principally the pulmonary arterial smooth muscle cells (PA-SMCs). Numerous previously identified biological and physical effectors act on the PA-SMCs and participate in PA-SMC hyperplasia. Here we show studied that these different effectors converge into a common intracellular signaling pathway, Akt/mTOR signaling pathway, which represents actually a therapeutic target for PAH treatment, and could be involved in the hyperplasia of PA-SMCs. In cells mTOR, is presented in the form of two complexes, mTORC1 and mTORC2, which phosphorylate various substrates controlling the cellular proliferation. The effectors of mTORC1 include the S6 kinases (S6K1 and S6K2) and eIF4E-binding proteins (4EBP), meanwhile mTORC2 activates the serine/threonine kinase Akt and the underlying kinases, e.g. GSK3 kinase.The first study is devoted to evaluate the effects of the protease inhibitors of HIV (ritonavir, amprenavir, nelfinavir) on experimental PAH development induced by monocrotaline or hypoxia. We studied that the two forms of PAH are associated with an activation of Akt/mTOR signaling pathway in pulmonary arteries. The treatment by the three protease inhibitors of HIV during 3 weeks causes reversibility in experimental PAH with decreased right ventricular hypertrophy and pulmonary vascular remodeling as well as inhibition of phosphorylation of Akt, S6K and GSK3. The proliferation of PA-SMCs stimulated by PDGF or FCS 5%, which is associated with an increased p-Akt and p-GSK3, is also blocked by the proteases inhibitors, in a similar and non additive way like the specific inhibitors of PI3 kinase and GSK3. We conclude that the antiretroviral treatments significantly inhibits PAH development by inhibiting Akt/mTOR signaling pathway in PA-SMCs. This proposition allows explaining the effect of antiretroviral treatments of PAH accompanied with HIV in patients.In the second study, we studied that the cultured PA-SMCs extracted from the rats with monocrotaline induced-PAH(MCT-PAH) proliferates faster as compared to control. This proliferative phenotype is observed in the presence of different mitogenic factors including FCS 5%, PDGF, 5-HT, IGF1 or IL-1β, and is associated with an activation of the substrates of mTORC1 and mTORC2. Treatment with rapamycin in the PA-SMCs extracted from the rats with PAH in vitro inhibits the proliferation and also blocks the activation of mTORC1 and mTORC2. The treatment by rapamycin in the rats with PAH during one week allows normalizing the proliferation of PA-SMCs in vitro and inhibiting the activation of mTORC1 and mTORC2 in vivo. These effects were not observed when treated with imatinib or fluoxetine. Moreover, treatment with rapamycin prevents or reverse MCT induced PAH more significantly than that by imatinib or fluoxetine.These results indicate that the activation of Akt/mTOR signaling pathway isclosely related to experimental PAH development, which can explain the abnormal proliferative phenotype of PA-SMCs involved in the patho-physiology of PAH, and represent a therapeutic target for the treatment of PAH in human.

Histiocytic sarcoma (HS) is an exceptionally rare malignant neoplasm derived from dendritic cells and histiocytes, with no available effective treatment options. Akt signaling and proteasome dysfunction have been implicated in the pathogenesis of the disease, both in humans and dogs. Our work aims to investigate the importance of the Akt signaling pathway and evaluate the potential of Akt-targeted therapy in a canine model of histiocytic sarcoma. We demonstrated Akt signaling to be active in 9 out of 10 canine HS tumor samples, regardless the presence of PTEN. Moreover, the Akt signaling pathway appears to be constitutively active in DH82 cells — a cell line model of canine HS, when compared to control canine dendritic cells. Pharmacologic Akt inhibition resulted in significant decrease in Akt S473 phosphorylation, GSK-3β S9 phosphorylation, Akt activity, cell viability, increased apoptosis, and resulted in sensitization to proteasome inhibition-depended cell death in a synergistic manner. Proteasome inhibition using carfilzomib, an irreversible proteasome inhibitor, induced dose-depended/caspase-3 independent cell death, at clinically relevant drug concentrations. The therapeutic effect of Akt inhibition was validated in vivo using a DH82 xenograft murine model. Akt inhibition lead to reduced tumor growth, prolonged overall survival, and ameliorated splenomegaly, but not affected the lung metastasis. Moreover, the therapeutic effect of Akt inhibition was potentiated in combination with carfilzomib. In conclusion, targeting Akt signaling may represent an attractive potential therapeutic target for the HS. Future studies are required to examine the clinical efficacy of Akt-targeted therapy in dogs with HS using novel selective Akt inhibitors. / Ph. D. / Histiocytic sarcoma (HS) is an exceptionally rare cancer of the immune system, with no effective treatment options available. Canine histiocytic sarcoma (cHS) is an aggressive tumor of the same cellular lineage, identified at increased relative frequency in specific dog breeds, with significant translational value. Akt signaling and proteasome dysfunction have been implicated in the pathogenesis of the disease, both in humans and dogs. Our study aims to investigate the importance of the Akt signaling pathway in the dog model of the disease and evaluate the potential of Akt-targeted therapy in a translational of histiocytic sarcoma. The work presented here demonstrates that Akt signaling appears aberrantly and constitutively activated in the canine model of HS. Importantly, Akt inhibition significantly reduced the tumor growth and prolonged the overall survival of the experimental animals. Moreover, Akt inhibition potentiated the anti-cancer activities of other anticancer drugs. Collectively, these findings provide an attractive therapeutic approach for the treatment of HS.