Cytoskeletal reorganization in NIH 3T3 fibroblasts expressing the ras oncogene.
Expression of the Ha-ras oncogene in NIH 3T3 fibroblasts leads to a set point shift of cell volume regulation and causes an increase in cell volume by activation of Na+/H+ exchange and Na+, K+, 2Cl- cotransport. 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 expression of the oncogene. The experiments were performed on NIH 3T3 fibroblasts transfected with a transforming Ha-ras MMTV-LTR construct and expressing the oncogene after treatment with low serum medium and 1 mumol/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 characteristics were examined and the cytoskeletal architecture was visualized by indirect immunofluorescence microscopy using specific antibodies and fluorescent dyes. Expression of the ras oncogene was accompanied by a significant and serum-independent increase in proliferative activity irrespective from the coating of the dishes with attachment factors (poly-L-lysine, collagen type I). Both, -ras and +ras cells, proliferated slower on substrates coated with poly-L-lysine than on tissue culture plastic or collagen type I. Expression of the ras oncogene also resulted in a significant increase in cell volume which was independent from the substrate. +ras Cells became more elongated, exhibited long cytoplasmic protrusions and tended to detach when compared with -ras cells. Examination of the cytoskeletal architecture in +ras and -ras cells revealed marked differences such as a depolymerization of the stress fiber network to strongly fluorescent "focals" as well as the absence of vinculin-containing attachment plaques (focal contacts), a disorganization of non-muscle myosin and of cell surface fibronectin in +ras cells. In addition, a retraction of microtubules and vimentin filaments to the perinuclear region was also observed in +ras cells. For comparison, NIH 3T3 fibroblasts which were not transfected with the ras oncogene (0ras cells) and which were also subjected to the experimental conditions described above (low serum medium +/- dexamethasone), did not exhibit the cytoskeletal alterations as observed for +ras cells. The results demonstrate that the expression of ras oncogene causes not only profound alterations in the proliferative activity, cell volume and cell morphology, but also a marked reorganization of cytoskeletal architecture, which may participate in the altered regulation of volume-regulatory ion transporters in the cell membrane.
Dartsch
P C
PC
Physiologisches Institut (I), Universität Tübingen, Germany.
Ritter
M
M
Häussinger
D
D
Lang
F
F
D016475:3T3 Cells / Q000378:metabolism / Q000648:ultrastructure*; D000199:Actins / Q000032:analysis; D000818:Animals; D002455:Cell Division; D048429:Cell Size; D003599:Cytoskeleton / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D011905:Genes, ras; D051379:Mice; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014746:Vimentin / Q000032:analysis
D000199:Actins; D011994:Recombinant Proteins; D014746:Vimentin; D016283:Proto-Oncogene Proteins p21(ras)