CYTOSKELETAL REORGANIZATION IN NIH 3T3 FIBROBLASTS EXPRESSING THE RASONCOGENE

Expression of the Ha-ras oncogene in NIH 3T3 fibroblasts leads to a se t point shift of cell volume regulation and causes an increase in cell volume by activation of Na+/H+ exchange and Na+, K+, 2Cl(-) cotranspo rt. Since both ion transport systems are thought to be governed by the cytoskeleton, the aim of this study was to examine the alterations in growth characteristics and cytoskeletal organization due to the expre ssion of the oncogene. The experiments were performed on NIH 3T3 fibro blasts transfected with a transforming Ha-ras MMTV-LTR construct and e xpressing the oncogene after treatment with low serum medium and 1 mu mol/l dexamethasone (+ras cells). Transfected cells not expressing the oncogene (-ras cells) and treated with low serum medium, but without the addition of dexamethasone, served as controls. The growth characte ristics were examined and the cytoskeletal architecture was visualized by indirect immunofluorescence microscopy using specific antibodies a nd fluorescent dyes. Expression of the ras oncogene was accompanied by a significant and serum independent increase in proliferative activit y irrespective from the coating of the dishes with attachment factors (poly-L-lysine, collagen type I). Both,-ras and +ras cells, proliferat ed slower on substrates coated with poly-L-lysine than on tissue cultu re plastic or collagen type I. Expression of the ras oncogene also res ulted in a significant increase in cell volume which was independent f rom the substrate. +ras Cells became more elongated, exhibited long cy toplasmic protrusions and tended to detach when compared with -ras cel ls. Examination of the cytoskeletal architecture in +ras and -ras cell s revealed marked differences such as a depolymerization of the stress fiber network to strongly fluorescent ''focals'' as well as the absen ce of vinculin-containing attachment plaques (focal contacts), a disor ganization of non-musle myosin and of cell surface fibronectin in +ras cells. In addition, a retraction of microtubules and vimentin filamen ts to the perinuclear region was also observed in +ras cells. For comp arison, NIH 3T3 fibroblasts which were not transfected with the ras on cogene (Oras cells) and which were also subjected to the experimental conditions described above (low serum medium +/- dexamethasone), did n ot exhibit the cytoskeletal alterations as observed for +ras cells. Th e results demonstrate that the expression of ras oncogene causes not o nly profound alterations in the proliferative activity, cell volume an d cell morphology, but also a marked reorganization of cytoskeletal ar chitecture, which may participate in the altered regulation of volume- regulatory ion transporters in the cell membrane.