Dartsch P C, Ritter M, Häussinger D, Lang F
Physiologisches Institut (I), Universität Tübingen, Germany.
Eur J Cell Biol. 1994 Apr;63(2):316-25.
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.
Ha-ras癌基因在NIH 3T3成纤维细胞中的表达导致细胞体积调节的设定点偏移，并通过激活Na⁺/H⁺交换和Na⁺、K⁺、2Cl⁻协同转运使细胞体积增加。由于这两种离子转运系统都被认为受细胞骨架调控，本研究的目的是检测由于癌基因表达导致的生长特性和细胞骨架组织的改变。实验在转染了转化型Ha-ras MMTV-LTR构建体并在低血清培养基和1 μmol/L地塞米松处理后表达癌基因的NIH 3T3成纤维细胞（+ras细胞）上进行。未表达癌基因的转染细胞（-ras细胞）用低血清培养基处理，但不添加地塞米松，作为对照。检测生长特性，并使用特异性抗体和荧光染料通过间接免疫荧光显微镜观察细胞骨架结构。ras癌基因的表达伴随着增殖活性显著且不依赖血清的增加，无论培养皿是否用附着因子（聚-L-赖氨酸、I型胶原）包被。-ras和+ras细胞在聚-L-赖氨酸包被的底物上增殖均比在组织培养塑料或I型胶原上慢。ras癌基因的表达还导致细胞体积显著增加，且与底物无关。与 -ras细胞相比，+ras细胞变得更加细长，表现出长的细胞质突起，并且易于脱离。对+ras和 -ras细胞的细胞骨架结构检查发现了明显差异，如应力纤维网络解聚为强荧光的“焦点”以及不含纽蛋白的附着斑（粘着斑）缺失，+ras细胞中非肌肉肌球蛋白和细胞表面纤连蛋白的紊乱。此外，在+ras细胞中还观察到微管和波形蛋白丝向核周区域的回缩。作为对照，未转染ras癌基因的NIH 3T3成纤维细胞（0ras细胞）也接受上述实验条件（低血清培养基+/-地塞米松）处理，未表现出如+ras细胞所观察到的细胞骨架改变。结果表明，ras癌基因的表达不仅导致增殖活性、细胞体积和细胞形态的深刻改变，还导致细胞骨架结构的显著重组，这可能参与了细胞膜中体积调节离子转运体的调节改变。
