• JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
 
  Bookmark and Share
 
 
Thèse de Doctorat
DOI
10.11606/T.5.2006.tde-07102014-112203
Document
Auteur
Nom complet
Fabiana Henriques Machado de Melo
Adresse Mail
Unité de l'USP
Domain de Connaissance
Date de Soutenance
Editeur
São Paulo, 2006
Directeur
Jury
Chammas, Roger (Président)
Barreira, Maria Cristina Roque Antunes
Brentani, Maria Mitzi
Martins, Vilma Regina
Soriano, Francisco Garcia
Titre en portugais
Exploração funcional do processo de glicosilação aberrante em tumores: mecanismos envolvidos na atividade pró-migratória de galectina-3
Mots-clés en portugais
Biotecnologia
Galectina-3
Glicosilação
Movimento celular
Sarcoma experimental
Resumé en portugais
Ao longo do processo de progressão tumoral, se observa alteração na expressão de glicoconjugados contendo oligossacarídeos N-ligados. Uma das formas mais comuns de glicosilação aberrante observada em células transformadas e em tumores humanos é representada por (poli)lactosaminas presentes em oligossacarídeos N-ligados. Estes glicanos são ligantes de galectina-3. Com o objetivo de identificar a expressão e distribuição dos ligantes de galectina-3 associados a processos fisiopatológicos, como a transformação maligna, desenvolvemos uma proteína quimérica, a galectina-3 conjugada a fosfatase alcalina (Gal-3/FA). Observamos que a Gal-3/FA possui a mesma especificidade de galectina-3 e que pode ser usada como sonda em ensaios de overlay e ensaios de imunoistoquímica. Entre os ligantes de galectina-3 identificamos a ?1 integrina, mediador de processos biológicos dependentes da interação célula-matriz como a migração celular. Linhagens de células de origem mesenquimal derivadas de tumores induzidos com metilcolantreno de animais selvagens (linhagens S11 e S12) e nulizigoto (linhagem ?12) para o gene da galectina-3 foram estabelecidas. Avaliamos a capacidade migratória dessas células e os nossos resultados mostraram que células que expressam galectina-3 são mais migratórias em superfícies de laminina-1. Este dado sugere que a galectina-3 seja um modulador positivo do processo de migração celular em superfícies de laminina-1. No entanto, o mecanismo pelo qual a galectina-3 medeia esse processo não é conhecido. Células que possuem fenótipo mais migratório apresentam um estado intermediário de adesão. Nós observamos que a galectina-3 se encontra nos complexos focais. Na presença de galectina-3 observamos diminuição de FAK fosforilado e recrutamento da fosfatase SHP-2 para os complexos focais. A diminuição de FAK fosforilado no lamelipódio leva ao turnover dos complexos focais e ao aumento da migração celular. Analisamos também a via de sinalização e observamos que a galectina-3 não ativa PAK. Contudo, o inibidor de PI3quinase, wortmanina, inibiu o efeito pró-migratório de galectina-3. Esses dados reforçam a noção do papel de galectina-3 na modulação do processo de migração de fibroblastos transformados, funcionando como uma molécula
Titre en anglais
Exploiting the functional significance of aberrant glycosylation in tumors: mechanisms involved in the promigratory activity of galectin-3
Mots-clés en anglais
Biotechnology
Cell movement
Experimental sarcoma
Galectin-3
Glycosylation
Resumé en anglais
Altered expression of cell surface N-linked oligosaccharides are often associated with malignant transformation of cells. One of the most common forms of aberrant glycosylation in transformed cells and human tumors is the highly elevated ?1,6 branching of N-linked oligosaccharides caused by increased expression of N-acetylglucosaminytransferase V (Mgat5). Galectin-3, a ?-galactoside binding protein, binds preferentially to poly-N-acetyllactosamines, which are the products of Mgat5. In order to exploit this hallmark of cancer cells, we have developed a tool for in situ identification of these tumors associated glycoconjugates. Human galectin-3 was fused to bacterial alkaline phosphatase, generating a hybrid molecule displaying both the carbohydrate binding properties of galectin-3 and enzymatic activity of alkaline phosphatase (Gal-3/FA). Gal-3/FA has the same fine of galectin-3 which was confirmed in direct binding assays. The tool presented herein was therefore useful for several immunoenzymatic assays, and will allow to establish whether the expression pattern of galectin-3 ligands have any physiological or clinical significance. We have identified ?1 integrin as a galectin-3 ligand. ?1 integrins are the actual effector of cell adhesion and migration. We have established cell lines from methylcholantrene-induced sarcomas from both wild type and galectin-3 null mice. In this system, galectin-3 null cells were less migratory than control cells in laminin-1. When galectin-3 was transiently expressed in galectin-3 null sarcoma cells, it inhibited cell adhesion to laminin-1 and stimulate the migratory response to laminin-1. The addition of exogenous galectin-3 also enhanced the migratory capacity of ?12 cells in a carbohydrate dependent way. Galectin-3 was found in focal contacts of ?12 cells where it may interact with many glycoproteins containing polyllactosamines on the cell surface. Here we showed that ?1 integrins are among them. Exogenously added galectin-3 led to a decrease in phosphorylated-FAK in lamellipodia and increased the recruitment of Shp-2 phosphatase of migrating cells. The effect of galectin-3 in migration was not dependent on the activation of the p21-activated kinase (PAK). Wortmannin inhibited the increased migration elicited by galectin-3, suggesting the involvement of the PI3-kinase signaling in the galectin-3 pathway. We propose that extracellular galectin-3 bound ?1integrins and disrupted the focal adhesion plaque, thus favoring cell migration.
 
AVERTISSEMENT - Regarde ce document est soumise à votre acceptation des conditions d'utilisation suivantes:
Ce document est uniquement à des fins privées pour la recherche et l'enseignement. Reproduction à des fins commerciales est interdite. Cette droits couvrent l'ensemble des données sur ce document ainsi que son contenu. Toute utilisation ou de copie de ce document, en totalité ou en partie, doit inclure le nom de l'auteur.
Date de Publication
2014-10-07
 
AVERTISSEMENT: Le matériau se réfère à des documents provenant de cette thèse ou mémoire. Le contenu de ces documents est la responsabilité de l'auteur de la thèse ou mémoire.
  • CHAMMAS, R., et al. Biological applications of a novel chimeric molecule for the assessment of galectin-3 ligands [doi:10.1369/jhc.7a7174.2007]. Journal of Histochemistry and Cytochemistry [online], 2007, vol. 55, p. 1015-1026.
  • Melo, Fabiana H. M., et al. The Promigratory Activity of the Matricellular Protein Galectin-3 Depends on the Activation of PI-3 Kinase [doi:10.1371/journal.pone.0029313]. Plos One [online], 2011, vol. 6, p. e29313.
  • VERBISCK, N. V., et al. ADAM23 negatively modulates avb3 integrin activation during metastasis [doi:10.1158/0008-5472.CAN-08-2976]. Cancer Research [online], 2009, vol. 69, p. 5546-5552.
  • MELO, F H M, et al. A novel tool for the assessment of galectin-3 ligands. In SIMEC2004-Simposio Internacional sobre Matriz Extracelular, Angra dos Reis, 2004. SIMEC-2004., 2004. Abstract.
  • CHAMMAS, R., et al. Galectin-3 modulates the migratory response of fibroblasts to laminin: a possible function of galectin-3 in sarcoma tumor progression. In 93rd Annual Meeting of the American Association for Cancer Research, San Francisco, 2002. Proceedings of the 93rd AACR Annual Meeting., 2002. Abstract.
  • CHAMMAS, R., et al. Papel dos glicoconjugados na modulação funcional de integrinas ao longo da progressão tumoral. In XI Congresso da Sociedade Brasileira de Biologia Celular, Porto Alegre, 2002. Avanços em Biologia Celular.Porto Alegre : EDIPUCRS, 2002.
  • CHAMMAS, R., et al. Role of tumor-associated glycoconjugates in cell migration. In XXXI Reunião Anual da Sociedade Brasileira de Bioquímica e Biologia Molecular, Caxambu, 2002. Programa e Resumos da XXXI Reunião Anual., 2002. Abstract.
  • JASIULIONIS, M G, et al. Cell-matrix interactions in tumor progression. In X Congresso da Sociedade Brasileira de Biologia Celular, Santos, 1999. Acta Microscopica., 1999. Abstract.
  • MELO, F H M, et al. Galectin-3 acts as a matricellular protein. In xii Congresso da Sociedade Brasileira de Biologia Celular e IX Congresso da Sociedade Iberoamericana de Biologia Celular, Campinas, 2004. Abstract.
  • MELO, F H M, SANTOS, M F, and CHAMMAS, R. Extracellular galectin-3 desestabilizes adhesion plaques by interacting with b1 integrin. In SIMEC 2004 Simpósio Internacional sobre Matriz Extracelular, Angra dos Reis, 2004. SIMEC-2004., 2004. Abstract.
  • MELO, F. H. M., et al. Extracellular galectin-3 acts as a de-adhesion molecule, promoting sarcoma cell migration. In I Simpósio Avanços em Pesquisas Médicas dos Laboratórios de Investigação Médica do HC-FMUSP, São Paulo, 2003. Resumo. Dispon?vel em: http://www.usp.br/medicina/direxlim.
  • MELO, F. H. M., et al. Extracellular Galectin-3 Destabilizes Adhesion Plaques by Interacting With B1 Integrin. In IV São Paulo Research Conference Cancer Today: From Molecular Biology to Treatment, São Paulo, 2005. Applied Cancer Researcg Supplement., 2005. Abstract.
  • MELO, F. H. M., et al. Extracellular galectin-3 modulates dysfunctional migration of fibroblasts on laminin-1 surfaces. In XXXIII Reunião Anual da Sociedade Brasileira de Bioquímica e Biologia Molecular, Caxambu, 2004. SBBq2004- Livro de resumos., 2004. Abstract.
  • MELO, F. H. M., et al. Galectin-3 is a modulator of fibroblast migration towards laminin-1. In SIMEC 2000- Simpósio Internacional sobre Matriz Extracelular, Angra dos Reis, 2000. SIMEC2000- Program and Abstract Book., 2000. Abstract.
  • MELO, F. H. M., et al. Involvement of galectin-3 in dysfunctional migration of fibroblasts on laminin-1 surfaces. In Conference on Mechanisms of Invasion and Metastasis, Madrid, 2002. CNIO Conference on Mechanisms of Invasion and Metastasis., 2002. Abstract.
  • MELO, F. H. M., et al. Involvement of galectin-3 in dysfunctional migration of fibroblasts on laminin-1. In SIMEC 2002- Simposio Internacional sobre Matriz Extracelular, Angra dos Reis, 2002. SIMEC2002- Program and Abstract Book., 2002. Abstract.
  • VERBISCK, N V, et al. Loss of ADAM23, a Putative New Metastasis Suppressor Gene, Leads to Enhanced Migration of the MDA-MB-435 Breast Cancer Cell Line Possibly by Modulating the Alfavbeta3 Integrin Activation. In IV São Paulo Research Conference Cancer Today: From Molecular Biology to Treatment, São Paulo, 2005. Applied Cancer Research Supplement., 2005. Abstract.
  • VERBISCK, N V, et al. Use of RNA interference to investigate ADAM23 function and its role in breast cancer progression. In SIMEC2004-Simposio Internacional sobre Matriz Extracelular, Angra dos Reis, 2004. SIMEC2004., 2004. Abstract.
  • CHAMMAS, R.. Molécula híbrida para diagnóstico e/ou prognóstico de doenças crônico-degenerativas, infecciosas e do desenvolvimento. B, S, . Produção Técnica, Patentes e registros, Patente, Diagnóstico, Fapesp. Brasil. Privilégio de Inovação 0501095-0. .
Tous droits de la thèse/dissertation appartiennent aux auteurs
Centro de Informática de São Carlos
Bibliothèque Numérique de Thèses et Mémoires de l'USP. Copyright © 2001-2021. Tous droits réservés.