Protein and lipid lateral diffusion in normal and Rous sarcoma virus transformed chick embryo fibroblasts (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Biomembranes, 1980
The intramembrane particles of freeze-fractured chick embryo fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (TS68) are distributed differently at the permissive and non-permissive temperatures if, and only if, the cells are treated with glycerol before fixation. Few aggregates of intramembrane particles are present in glycerol-treated cells grown at the permissive temperature for transformation (36 ° C), while numerous large aggregates of particles are present at the non-permissive temperature (41°C). Changes in the distribution of particles after cells are shifted from 36 to 41°C are observed after 20 min, while a temperature shift from 41 to 36°C causes changes in glycerol-induced redistributions after 1 h. The changes observed in temperature shifts from 36 to 41°C and from 41 to 36°C do not require protein synthesis or RNA synthesis.
Tumori Journal, 1988
Cloning efficiency in hard agar (0.6%) and high chemotactic migration toward fibroblast conditioned medium have been shown to characterize metastatic tumor cells. We studied growth in 0.6% agar and chemotaxis of two lines of Rous Sarcoma virus-transformed Balb/ c3T3 cells, B77/3T3 (low metastatic) and AA12 (high metastatic), and compared them to their non-transformed counterpart, in order to verify whether these properties were maintained during several subcultivations. Cells were cryopreserved at early passages and thawed for experiments. Both assays were performed on freshly thawed cells (4-6 weeks in culture) and on cells which had been cultured 15-20 weeks after thawing. B77/3T3, which are tumorigenic but low metastatic and which have a very low cloning efficiency in hard agar (0.1-1%), showed a chemotactic response to Balb/c3T3 conditioned medium about two-fold higher than control Balb/c3T3. This response did not change with time in culture. AA12 cells, a genetic unstable varia...
Biochemical Medicine and Metabolic Biology, 1987
The 3T3 cells in culture exhibit density-dependent inhibition of growth (1) in opposition to the simian virus-transformed 3T3 (SV3T3) cells which have the ability to grow to much higher densities (2). The density-dependent inhibition of growth is associated with decreases in the uptake of amino acid (3,4), phosphate (5-S), and glucose (9-12), while in the virus-transformed cells the uptake of these nutrients has been shown to be consistently high (3,5,13-l@. Since the transport of amino acid, glucose, and phosphate is a Na-dependent process (19-21), the maintenance of the sodium gradient across the cell membrane which is largely regulated by the Na-K pump is of importance in regulating cell growth. The activity of the Na-K pump in cultured cells has been shown by Aiton and Lamb (22) to be affected by serum concentration in the growth media. We have shown that sera types can also affect the activity of the Na-K pump in 3T3 cells (23) and also of several other membrane-bound enzymes (24). Since the 3T3 cells when first established were grown in medium supplemented with fetal bovine serum (l), while the SV3T3 cells were established in medium supplemented with newborn calf serum (2), we thought that the use of these two cell lines for comparing normal and malignant growth is only meaningful if the SV3T3 cells are grown in the same serum as that used in establishing the 3T3 cells. In this paper we report the effects of cell population density on the activity of Na-K pump and their roles in growth regulation in 3T3 and SV3T3 cells grown in medium supplemented with fetal bovine serum. Materials MATERIALS AND METHODS Swiss 3T3 and SV3T3 cells and materials needed for the growth of the cells which includes Dulbecco's modification of Eagle's minimal essential medium, fetal bovine serum, and trypsin were obtained from Flow Laboratories (Irvive,
Experimental Cell Research, 1978
The fluorescent probes pyrene, pyrene butyric acid and N-phenyl I-naphthylamine were used to study membranes of normal cells, RSV-transformed cells, cells treated with a proteolytic enzyme, and cells persistently infected with lymphocytic choriomeningitis virus. The lifetimes of excited pyrene and pyrene butyric acid showed only minor changes when these probes were in normal, transformed, trypsinized or persistently infected cells. However, pyrene, but not pyrene butyric acid, lifetimes are shorter in cell membranes than in homogeneous solvents. The quenching of exctted pyrene in cells by quencher molecules was slower than corresponding reactions in homogeneous solutions indicating that the probe was screened from the quenchers by the membrane. However, quenching reactions with the pyrene butyric acid probe were similar in cells and homogeneous solvents. This indicates that pyrene and pyrene butyric acid reside in different lipid regions of the membrane. Transformed and trypsinized cells showed increased membrane fluidity compared to normal and persistently infected cells. Membrane fluidity was determined from the excimer/monomer fluorescence ratios of pyrene, and by the polarization of N-phenyl l-naphthylamine fluorescence. Several techniques distinguished between normal and transformed or trypsinized cells; however, the only parameter unique to viral transformation was a blue shift of the fluorescence maxima of N-phenyl I-naphthylamine. This shift reflected a less polar environment for N-phenyl I-naphthylamine in virus-transformed cells. Department of Energy. This is ERDA Document No. creased amounts of sialic acid, variable ~~~~-1743. Exp Cell Res 116 (1978) 292 Burleson et al. may be more important than membrane pany the transformation of mammalian composition in cellular regulatory mechancells. isms [ 10, Ill. Fluorescent probes have been used extensively to study micellar systems METHOD [12-171, phospholipid dispersions [18], pro-Pure grade pyrene from Kodak was further purified by passage through a silica gel column in cyclohexane
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1980
The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37°C) had a 2-fold higher rate of 2-deoxy-D-glucose uptake than the same cells cultured at the non-permissive temperature (41°C). However, both the non-transformed and transformed cells had comparable rates of a-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41°C or 37°C, displayed carrier-mediated, intravesicular uptake of D-glucose and a-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37°C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41 ° C. The two types of membrane vesicle had similar uptake rates of a-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. Km values for stereospecific D-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37°C were similar, but the V value was Abbreviations: CEF, chicken embryo fibroblasts; TS-68, temperature-sensitive mutant of the Rous sarcoma virus; Glc, glucose; Gal, galactose; 3-OMeGlc, 3-O-methyl-D-glucose; 2-dGlc, 2-deoxy-D-glucose; Hepes, N-2-hydroxyethylpiperazine.NW.2.ethanesulfonic acid. greater for the membrane vesicles from TS-68-infected cells cultured at 37°C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virallytransformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.
Turnover of cellular carbohydrates in normal and Rous sarcoma virus-transformed cells
PubMed, 1978
We have analyzed the distribution of glucosamine-labeled polymers on the cell surface, in the growth medium, and inside the cell and the net turnover of these polymers during the process of malignant transformation of chick embryo fibroblasts by Rous sarcoma virus. The distribution of label and the turnover kinetics for hyaluronic acid, total glycoproteins, and chondroitins were found to be identical in both normal and transforming cultures. We conclude that nonspecific degradative processes are probably not involved in causing transformation-related alterations in cell surface carbohydrates, althougn degradation of specific macromolecules is not excluded.
Turnover of Cellular Carbohydrates in Normal and Rous Sarcoma Virus transformed Cells1
2000
We have analyzed the distribution of glucosamine-la- beled polymerson the cell surface, in the growthmedium, and inside the cell and the net turnover of these polymers during the process of malignant transformation of chick embryo fibroblasts by Rous sarcoma virus. The distribu tion of label and the turnover kinetics for hyaluronicacid, total glycoproteins, and chondroitins were found to be identical
Different cyclic changes in the surface membrane of normal and malignant transformed cells
Experimental Cell Research, 1974
Transformed fibroblasts in interphase and normal fibroblasts in mitosis were agglutinated by Con A and the lectin from wheat germ, whereas normal fibrob!asts in interphase and transformed fibroblasts in mitosis were not agglutinated by these lectins. The percentage of fluorescent cells at non-saturation concentrations of fluorescent ConA was also higher with transformed interphase and normal mitotic cells, than with normal interphase and transformed mitotic cells. Under the same conditions, a similar number of radioactively labeled ConA molecules were bound to normal and transformed cells in interphase and mitosis. Our results indicate different cyclic changes in the surface membrane of normal and transformed fibroblasts, so that regarding interaction with these lectins, normal mitotic cells resemble transformed interphase cells and transformed mitotic resemble normal interphase cells. The data suggest that there is a reversed cyclic change in the mobility of specific surface membrane sites in normal and transformed cells.