Abstract. Recent results showing that a singlefibronectin 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. F IBRONECT1NS (FNs) ~ are high-molecular-mass, adhesive glycoproteins present in the soluble form in plasma and other body fluids and in insoluble form in the extracellular matrices and basement membranes. FN molecules act as bridges between the cell surface and extracellular material. In fact, the FN molecules contain a cellbinding site and binding sites for collagen, heparin, gangliosides, and fibrin. Because of their multiple interactions, FNs play an important role in diverse biological phenomena, including cell adhesion, cell migration, hemostasis and thrombosis, wound healing and the ability to induce a more normal phenotype in transformed cells (for reviews on distribution, structure, and biological functions, see references 1,7,8,18,24).It has been demonstrated that FN polymorphism may be at least partially due to alternative splicing schemes in two regions (ED and IIICS) because as many as 10 different mRNAs may originate from the primary transcript of a single gene (9, 12-15, 25, 26, 29) localized on chromosome 2 (11, 34). In fact, Schwarzbauer et al. (25) have shown that an antiserum specific for the rat fibronectin IIICS sequence recognizes the larger subunit of rat plasma FN (plFN), but not the smaller one.Here we report the characterization of mAbs for the ED 1. Abbreviations used in this paper: cFN and plFN, fibronectin from cultured cell media and plasma, respectively. fragment of fibronectin. Using these mAbs in a quantitative assay, we have demonstrated that this sequence is not present in plFN and that tumor-derived and SV-40-transformed human cells release a population of FN molecules in which the percentage of subunits containing the ED sequence is about 10 times higher than in the FN released by normal human fibroblasts.

Abstract. 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 heparinbinding 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.F IBRONECTINS (FNs) ~ are high molecular weight adhesive glycoproteins present in soluble form in plasma and other body fluids and in insoluble form in the extracellular matrices and basement membranes. FN molecules act as bridges between the cell surface and extracellular material. In fact, the FN molecules contain a cell-binding site and binding sites for collagen, heparin, ganglioside, and fibrin. Due to their multiple interactions FNs play an important role in diverse biological phenomena including cell adhesion, cell migration, hemostasis and thrombosis, wound healing, and the ability to induce a more normal phenotype in transformed cells (for reviews on distribution, structure, and biological functions see references 1, 3, 4, 13, and 16).Recently it has been demonstrated that the FN polymorphism may be due to alternative splicing schemes since many different mRNAs may originate from the primary transcript of a single gene (5, 7-10, 17, 18, 22) localized on chromosome two (6, 27).Here, using the domain-specific monoclonal antibodies IST-7 and IST-2 we have studied the presence of the IIICS sequence in FN from plasma and from the conditioned media of normal and tumor-derived human cells. Materials and Methods Cell Lines and Monoclonal AntibodiesCultured normal human fibroblast cell lines (LZ, from adult human skin; GM-3651-C, from adult human skin; GM-5386, from embryonic human 1. Abbreviations used in this paper: FN, fibronectin; RIA, radioimmunoassay; SV-40, simian virus 40.skin; WI-38, from embryonic human lung) and transformed cell lines (HT-1080, from a human fbrosarcoma; RD, from an embryonic human rhabdomyosarcoma; IgR3, from a human melanoma; and WI-38VA13, simian virus 40 lSV-40]-transformed WI-38 cells) were grown in Eagle's minimum essential medium supplemented with 10% fetal calf serum (Flow Laboratories, Irvine, Scotland) that had been depleted of bovine FN by passage through a large capacity gelatin-Sepharose column.Monoclonal antibodies to human plasma FN were prepared as previously described (25). Partial ...

Fibronectinsisolated from human plasma @FN) 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. Fibronectin Tumor cellCell binding site Immunoblotting