FIBRONECTIN SPLICE VARIANTS ARE DIFFERENTIALLY INCORPORATED INTO THE EXTRACELLULAR-MATRIX OF TUMORIGENIC AND NONTUMORIGENIC HYBRIDS BETWEENNORMAL FIBROBLASTS AND SARCOMA-CELLS

Recent reports have described transformation- and tumour-specific expr ession of fibronectin isoforms generated by alternative splicing of th e fibronectin pre-mRNA. We have investigated the expression and distri bution of EDIIIA+ and EDIIIB+ fibronectin splice variants in tumorigen ic and non-tumorigenic somatic cell hybrids made by fusing fibrosarcom a-derived cells (HT1080) and normal fibroblasts (GM00097). Alternative splicing of EDIIIA and EDIIIB was assessed quantitatively by S1 nucle ase analyses. The levels of EDIIIA+ and EDIIIB+ fibronectin mRNAs were similar in the parental and hybrid cells. Domain-specific monoclonal antibodies were used in immunohistochemical studies to identify EDIIIA + and EDIIIB+ fibronectins in fixed cells. GM00097 and the non-tumorig enic hybrid (clone G3) showed high levels of both EDIIIA+ and EDIIIBfibronectin staining. The tumorigenic hybrid (clone C1) showed reduced amounts of EDIIIA+ fibronectin, but no detectable EDIIIB+ fibronectin . No fibronectin was detected on the surface of HT1080 cells. Western blots of protein extracted from culture supernatants and extracellular matrices revealed that GM00097 and G3 cells incorporated most of the EDIIIA+ and EDIIIB+ fibronectin into the extracellular matrix whereas C1 cells released a large proportion of the EDIIIA+ fibronectin, and a lmost all of the EDIIIB+ fibronectin, into the supernatant. We conclud e that there are differences in the presence of EDIIIA+ and EDIIIB+ FN s on the surface of tumorigenic and non-tumorigenic cells and that the se differences are due to differential incorporation of FN variants in to the ECM.