RESUMO
L1 cell adhesion molecule (L1-CAM) is a transmembrane cell adhesion molecule involved in cell migration and axon guidance in the developing nervous system. L1 is also overexpressed in ovarian and endometrial carcinomas and is associated with a bad prognosis. In carcinoma cell lines, L1 overexpression augments cell motility, tumor growth in mice and induces expression of Erk-dependent genes. Here, we show that a mutation in the cytoplasmic portion of L1 (T1247A, S1248A) abrogates Erk activation, blocks cell migration on extracellular matrix proteins and did not augment tumor growth in non-obese diabetic/severe combined immuno-deficient mice. In cells expressing mutant L1, the induction of Erk-dependent genes such as beta3-integrin, cathepsin-B and several transcription factors is eliminated and the invasive phenotype is abrogated. L1 antibodies showed similar effects. They prevented Erk activation and interfered with the Erk-dependent gene expression pattern. These findings provide a rationale for the mode of action of L1 antibodies and suggest that interference with L1 function could become a valuable target for therapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Proliferação de Células , Citoplasma/fisiologia , Regulação Neoplásica da Expressão Gênica , Neoplasias/imunologia , Neoplasias/terapia , Molécula L1 de Adesão de Célula Nervosa/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Citoplasma/química , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/patologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Molécula L1 de Adesão de Célula Nervosa/química , Estrutura Terciária de ProteínaRESUMO
A method for the large scale expression and purification of rat betacellulin (BTC) from Escherichia coli has been developed using a cleavable fusion protein strategy. Insoluble fusion protein collected as inclusion bodies was dissolved in urea under reducing conditions, re-folded, and purified by gel filtration chromatography and C(4) RP-HPLC. Authentic rat BTC was obtained after proteolytic cleavage of the fusion protein with Factor Xa. Factor Xa cleaved an additional site within the BTC protein, generating a truncated isoform separable from full-length BTC by heparin-affinity chromatography. Recombinant rat BTC stimulated the proliferation of mouse Balb/c 3T3 fibroblasts and competed for binding to the ErbB1 receptor in a dose-dependent manner analogous to that of BTC purified from natural sources.