RESUMEN
Fibronectin molecules are dimers composed of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor-derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts. Furthermore, we observed that 45-65% of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparin-binding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Fibronectinas/metabolismo , Neoplasias/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Fibronectinas/genética , Fibronectinas/inmunología , Humanos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Empalme del ARN , Sialoglicoproteínas/metabolismo , Virus 40 de los Simios , Termolisina/metabolismoRESUMEN
Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered.
Asunto(s)
Anticuerpos Monoclonales , Transformación Celular Neoplásica , Fibronectinas/genética , Precursores de Ácido Nucleico/genética , Empalme del ARN , ARN Mensajero/genética , Secuencia de Aminoácidos , Línea Celular , Fibronectinas/análisis , Humanos , Peso Molecular , Neoplasias , Fragmentos de Péptidos/análisis , Precursores del ARNRESUMEN
Fibronectins isolated from human plasma (pFN) and from the conditioned media of normal (N-cFN) and tumor (T-cFN) human cells were compared by cathepsin D digestion followed by immunostaining of released fragments with the monoclonal antibody 3E3, specific for the cell binding site. Two different staining patterns were obtained, one specific for pFN and N-cFN, the second common to fibronectins from the 3 different kinds of tumors studied. This indicates structural differences between N-cFN and T-cFN in the cell binding region of the fibronectin molecule.
Asunto(s)
Fibronectinas/metabolismo , Neoplasias/metabolismo , Anticuerpos Monoclonales , Sitios de Unión , Catepsina D/metabolismo , Línea Celular , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Fibrosarcoma/metabolismo , Humanos , Técnicas Inmunológicas , Melanoma/metabolismo , Fragmentos de Péptidos/metabolismo , Rabdomiosarcoma/metabolismoRESUMEN
Vitamin A is known to be able to modulate cell growth and differentiation and to act as an inhibitor of the process of carcinogenesis in some experimental models. Here we have studied the effect of different concentrations of vitamin A on chemotactic and chemoinvasive behaviour of a metastatic osteosarcoma cell line. The cell proliferation was partially inhibited in the presence of 10(-5) M retinol after 4 days of incubation. Retinol effect on chemotactic and chemoinvasive activity of osteosarcoma cells seemed to be dose-dependent. The highest retinol concentration used (10(-5) M) had an inhibitory effect on migratory and invasive cell response. Lower retinol concentrations seemed to be able to enhance (10(-8) M) both chemotactic and chemoinvasive activity of osteosarcoma cells. Chemotaxis and chemoinvasion assays provide rapid and quantitative tools to study the "in vitro" behaviour of metastatic cells. Furthermore, they represent a mean to screen for drugs, hormones and other substances able to alter the metastatic phenotype.