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Doctoral Thesis
DOI
10.11606/T.9.2009.tde-26012011-141733
Document
Author
Full name
Tatiana Caroline Silveira Corrêa
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2009
Supervisor
Committee
Maria-Engler, Silvya Stuchi Maria (President)
Cella, Nathalie
Hirata, Mario Hiroyuki
Moreno, Fernando Salvador
Taboga, Sebastião Roberto
Title in Portuguese
Efeito da super-expressão do gene RECK na reversão do processo invasivo de glioma humano
Keywords in Portuguese
Gene RECK
Glioma
Invasão e migração celular
Invasão tumoral
Metaloproteinases
Abstract in Portuguese
Os gliomas são o tipo de tumor primário cerebral mais comum em adultos. A sobrevida média dos pacientes é de cerca de um ano para pacientes de glioblastoma multiforme, o maior grau de malignidade. As terapias disponíveis atualmente, que incluem cirurgia, radioterapia e quimioterapia, não têm sido eficientes devido a vários fatores, em particular a capacidade invasiva das células tumorais. RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) é um importante gene supressor de tumor cuja atividade anti-tumoral têm sido associada à sua atividade inibitória sobre algumas MMPs. A perda da função de RECK compromete a integridade tecidual, em parte causada pela atividade aumentada das MMPs. A linhagem celular T98G, derivada de glioblastoma multiforme (GBM), apresenta grande capacidade invasiva e níveis elevados de MMP-2 e -9. A fim de avaliar o efeito de RECK na contenção da invasão no modelo de glioma humano, foi feita a superexpressão de RECK nesta linhagem. A expressão de RECK, MMP-2, MMP-9 a MT1-MMP foram avaliadas por qPCR e por western blotting nas células T98G/RECK+ (células T98G superexpressando RECK após transfecção estável utilizando o vetor pCXN2). O potencial invasivo e migratório das células T98G/RECK+foi inibido, verificado por ensaio transwell. Foram observadas nas células T98G/RECK+ alterações importantes no arranjo do citoesqueleto, mas não nas células controle. Arranjos de actina na forma de "stress fibers" presentes no clone positivo podem ser responsáveis pela alteração observada na migração. A distribuição de FAK foi avaliada por imunocitoquímica e sua expressão por Western blot, mostrando que RECK só altera a distribuição desta proteína no citoesqueleto celular. Quanto ao potencial de inibição de MMPs, observou-se uma diminuição significativa dos níveis gênicos de MMP-9, mas não em termos protéicos ou de atividade de MMPs. Assim, este trabalho contribui para a discussão do papel de RECK na migração de células do modelo de glioma humano, uma das características responsáveis pela ausência de terapia efetiva deste tipo de tumor.
Title in English
RECK gene forced expression effect on the invasiveness in human glioma
Keywords in English
Glioma
Invasiveness
Metalloproteinases
RECK gene
Abstract in English
Malignant gliomas are the most common type of primary brain tumors in adults. Patient survival is less than one year in average for glioblastoma, the most malignant glioma. Therapies available today, which include surgery, radiotherapy and chemotherapy, have not been successful due to several factors, specially the invasiveness of the tumor. RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) is an important tumor suppressor gene whose anti-tumoral activity is associated to its anti-MMPs activity. Lack of functional RECK compromises tissue integrity, in part due to elevated MMPs activity. T98G cells, derived from a human multiform glioblastoma (GBM), were described as a highly invasive glioma cell line, which displays high levels of MMPs 2 and 9. In order to evaluate the effect of RECK in restraining glioma invasion, we overexpressed RECK in these invasive cell line derived from GBM. The expression of RECK, MMP-2, MMP-9 and MT1-MMP was evaluated by qPCR and by western blotting in T98G/RECK+ (T98G cells overexpressing RECK cells, where RECK gene was cloned into the pCXN2 vector generating a stable transfection). The invasion and migration capacity of T98G/RECK+ cells was inhibited in transwell assay. Important cytoskeleton modifications were also observed in T98G/RECK+ cells but not on the control cells. Actin arrangements representing stress fibers on the positive clones may be responsible for motility alteration patterns observed. FAK distribution was assessed through imunocytochemical staining, and its expression evaluated by western blot analyses, showing that RECK forced expression changed the distribution pattern but not FAK expression. Concerning MMPs inhibition, a significant inhibition of MMP-9 gene expression was observed in T98G/RECK+, but neither protein levels nor protein activity were affected. Thus, the present study improves the discussion about RECK role in the migration of glioma cells, an important feature for the failure of current therapies for this kind of tumor.
 
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2013-04-11
 
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