Inhibition of DNA synthesis in SV3T3 cultures by isolated 3T3 plasma membranes

3T3 plasma membranes were added to subconfluent cultures of SV3T3 cells in the presence of fusogens. If this protocol results in the introduction into the SV3T3 cell membrane of 3T3 plasma membrane components responsible for density-dependent inhibition of growth, then the SV3T3 cell cultures would be expected to show decreased rates of DNA synthesis as they approach confluence. Results of these experiments indicate that rates of DNA synthesis in SV3T3 cultures so treated were as much as 63% less than in untreated controls. This effect could not be attributed to the fusogens or to the 3T3 plasma membranes alone. This growth-inhibitory effect is specific for 3T3 membranes and is not observed when SV3T3 plasma membranes are fused with SV3T3 cell cultures. These data support the hypothesis that one aspect of the loss of density-dependent inhibition of growth in SV3T3 cells is a deletion or alteration in plasma membrane components and, further, that density- dependent inhibition of growth can be in part restored to SV3T3 cell cultures by fusing the cells with 3T3 plasma membranes.     

Analysis of hexose transport in untransformed and sarcoma virus-transformed mouse 3T3 cells by photoaffinity binding of cytochalasin B

The effect of simian virus 40 transformation on the hexose transport system in mouse embryo fibroblast Swiss 3T3 cells was examined. The concentration of hexose transporters was estimated by measuring D-glucose-inhibitable cytochalasin B binding. The binding of cytochalasin B to the plasma membranes of simian virus 40-transformed mouse 3T3 cells (SV3T3 cells) was significantly greater than that of 3T3 cells. On the other hand, cytochalasin B binding to the microsomal membranes of SV3T3 cells was decreased, and the total amount of binding to plasma and microsomal membranes was not significantly changed in both cell lines. The electrophoretic analysis demonstrated that both hexose-transporter components of Mr 46 000 and Mr 58 000 affinity labeled were responsible for an increase in the hexose transport by viral transformation. These results suggested that the higher hexose-transport activity of transformed cells is caused by a redistribution of transporter from intracellular membranes to plasma membranes.     

Protein synthesis in 3T3 and SV40-transformed 3T3 cells. Activity of ribosomes and cytosol proteins in cell-free protein synthesis.

The activity of specific components involved in protein synthesis in 3T3 cells and its SV40-transformed derivative, SV3T3, were examined in a cell-free protein synthetic system, and the results correlated with previous studies, indicating that a decreasing rate of protein synthesis does not accompany the stationary phase of growth. We found that 3T3 and SV3T3 polysome preparations containing endogenous mRNA were equally efficient in supporting cell-free protein synthesis in this system. Further, the net protein synthesis observed was not altered by an increase in the population density of the cellular polysome source. The activity of the aminoacyl-tRNA synthetase enzymes from 3T3 and SV3T3 cells was examined in vitro after isolation by pH 5 precipitation and by ammonium sulfate fractionation. The activity of these preparations from stationary phase 3T3 and nonexponential phase SV3T3 cells was found to be approximately 3 times higher than the activity of fractions from the homologous exponential phase cell. However, at both growth stages, the SV3T3 preparations were 30 to 40 times more active than the 3T3 preparations. These findings may have implications for the different growth properties observed in the two cell types.     

Hyaluronate degradation in 3T3 and simian virus-transformed 3T3 cells

The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.     

Lipids of whole cells and plasma membrane fractions from Balb/c3T3, SV3T3, and concanavalin A-selected revertant cells.

The lipid composition of Balb/c3T3, SV3T3, and the concanavalin A-selected SV3T3 revertant cells has been analyzed at the whole cell and plasma membrane levels. In comparison to untransformed 3T3 whole cells, SV3T3 cells showed an unchanged content of triacylglycerols, free fatty acids, and glycerylether diesters but a lower concentration of total phospholipids, while no significant difference was found in the phospholipid composition. Whole SV3T3 revertant cells exhibited a lipid composition similar to that in untransformed 3T3 cells with the exception of a higher proportion of sphingomyelin. Analysis of isolated plasma membranes did not reveal any significant differences in the cholesterol to phospholipid molar ratio between 3T3 and SV3T3 or SV3T3 revertant cells. The major changes in the acyl chain pattern SV3T3 compared with whole 3T3 cells consisted of an increase of oleic and palmitoleic acids coupled with a decrease of C20 and C22 polyunsaturated acids in phosphatidylethanolamine and phosphatidylcholine; an increase of oleic acid was also evident in SV3T3 phosphatidylinositol plus phosphatidylserine. An increase of palmitoleic and oleic acids together with a decrease of arachidonic acid was also found in phosphatidylethanolamine of SV3T3 plasma membranes; the only change in SV3T3 plasma membrane phosphatidylcholine was an increase of oleic acid. An increase of monoenoic acids together with a decrease of arachidonic acid was also found in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol plus phosphatidylserine of SV3T3 revertant cells at the level of both whole cells and plasma membranes.     

Cyclic AMP specifically blocks proliferation of rat 3T3 cells transformed by polyomavirus.

Elevated exogenous and intracellular levels of cyclic AMP could totally block proliferation of polyomavirus (PyV) transformants derived from rat 3T3 cells without affecting proliferation of normal cells or simian virus 40 (SV40)-induced transformants. Concanavalin A (ConA) had the opposite effect; it could totally block proliferation of both normal cells and SV40 transformants but reduced proliferation of PyV transformants only twofold. Adenylate cyclase was threefold less active in membranes of PyV transformants, and the number of ConA receptors was similar to that of normal cells. Proliferating PyV transformants contained threefold less cyclic AMP than did proliferating SV40 transformants. The sensitivity to cyclic AMP did not correlate with the degree of transformation: cells transformed by Rous sarcoma virus and tumor cells derived from SV40 transformants were not sensitive to cyclic AMP. The differential effect of cyclic AMP and ConA on proliferation was probably due to the activity of an intact middle t protein. The presence of both large T and small t together with middle t was also required for cyclic AMP sensitivity.     

Transport of amino acids in intact 3T3 and SV3T3 cells. Binding activity for leucine in membrane preparations of ehrlich ascites tumor cells

Transport of amino acids into 3T3 and SV3T3 (SV40 virus-transformed 3T3) cells was measured on glass cover slips. The 3T3 and SV3T3 cells contain both A (alanine preferring) and L (leucine preferring) systems for neutral amino acid transport. Initial rates of uptake of amino acids are about twofold higher in SV3T3 than in 3T3 cells. Other parameters measured, however, do not indicate marked differences in the transport of amino acids by the two cell types. L-system amino acids, such as leucine, are subject to trans-stimulation in both cell lines, whereas A-system amino acids, such as alanine and glycine, are not. Leucine was transported to higher levels in confluent cells than in nonconfluent cells. Glycine, however, shows distinctly less transport activity as the cells become confluent. Ehrlich ascites cell plasma membranes were prepared and assayed for amino acid-binding activity. Leucine-binding activity was detected by equilibrium dialysis in Triton X-100-treated membrane preparations.     

Cholesterol levels and plasma membrane fluidity in 3T3 and SV101-3T3 cells

Polyene antibiotics such as filipin selectively inhibit wheat germ agglutinin-induced agglutination of transformed and malignant cells compared to normal cells (Hatten ME, Burger MM: Biochemistry 18:739, 1979). Since filipin binds specifically to cholesterol, we measured cholesterol levels in 3T3 cells and SV101-3T3 cells. SV101-3T3 cells contained 50-100% more cholesterol per cell than 3T3 cells. Both cell types were starved for cholesterol by growth in lipid-depleted medium plus 25-hydroxycholesterol. The cholesterol level of SV101-3T3 cells decreased by 30-50%, while the level in 3T3 cells remained constant. Filipin-stained SV101–3T3 cells revealed bright patches of filipin under fluorescence microscopy. These patches were absent in 3T3 cells and in SV101–3T3 and 3T3 cells starved for cholesterol. We selectively labeled plasma membranes of these cells with a spin label analog of phosphatidylcholine. The spin label indicated differences in plasma membrane fluidity that may be related to the different cholesterol levels in 3T3 and SV101–3T3 cells.     

Identification of a PDGF-like mitoattractant produced by NIH/3T3 cells after transformation with SV40

It has previously been shown that fibroblastic cells transformed by SV40 exhibit a reduced requirement for PDGF for growth. In addition, NIH/3T3 cells lose both their chemotactic response to PDGF and specific cell surface binding of PDGF after transformation with SV40. We have now examined whether the SV40 transformed NIH/3T3 cells are producing a factor which acts similarly to PDGF. Our studies indicate that NIH/3T3 cells transformed with SV 40 produce a factor which shares many biological properties with PDGF. We were unable to detect this activity in conditioned media from nontransformed NIH/3T3 cells. The SV40/NIH/3T3 derived factor appears to possess both chemotactic and mitogenic activity for connective tissue cells but not endothelial or epithelial cells. Furthermore, in preliminary studies, this activity competes with 125I-PDGF for binding to smooth muscle cells. The biochemical properties of the SV40/NIH/3T3 derived factor are different from those of PDGF. The SV40 activity appears to reside in a heat labile acidic protein (pI less than 7.0) of MW less than 30,000 whereas PDGF is a heat stable basic protein (pI9.8) of 30,000 MW. Production of this factor may play a role in the decreased serum requirement for cell replication exhibited by SV40-transformed NIH/3T3 cells by supplying the cells with their own PDGF-like growth factor.     

Cell density and amino acid transport in 3T3, SV3T3, and SV3T3 revertant cells

The transport of selected neutral and cationic amino acids has been studied in Balb/c 3T3, SV3T3, and SV3T3 revertant cell lines. After properly timed preincubations to control the size of internal amino acid pools, the activity of systems A, ASC, L, and Ly⁺ has been discriminated by measurements of amino acid uptake (initial entry rate) in the presence and absence of sodium and of transportspecific model substrates. L-Proline, 2-aminoisobutyric acid, and glycine were primarily taken up by system A; L-alanine and L-serine by system ASC; L-phenylalanine by system L; and L-lysine by system Ly⁺ in SV3T3 cells. L-Proline and L-serine were also preferential substrates of systems A and ASC, respectively, in 3T3 and SV3T3 revertant cells. Transport activity of the Na⁺-dependent systems A and ASC decreased markedly with the increase of cell density, whereas the activity of the Na⁺-independent systems L and Ly⁺remained substantially unchanged. The density-dependent change in activity of system A occurred through a mechanism affecting transport maximum (V_max) rather than substrate concentration for half-maximal velocity (K_m). Transport activity of systems A and ASC was severalfold higher in transformed SV3T3 cells than in 3T3 parental cells at all the culture densities that could be compared. In SV3T3 revertant cells, transport activity by these systems remained substantially similar to that observed in transformed SV3T3 cells. The results presented here add cell density as a regulatory factor of the activity of systems A and ASC, and show that this control mechanism of amino acid transport is maintained in SV40 virus-transformed 3T3 cells that have lost density-dependent inhibition of growth, as well as in SV3T3 revertant cells that have resumed it.     

The binding and processing of plasminogen by Balb/c 3T3 and SV3T3 cells

The binding and processing of plasminogen by Balb/c 3T3 and SV3T3 cells was studied using ¹²⁵I-labeled canine plasminogen. Throughout a 3-day period, ¹²⁵I-plasminogen in the incubation medium bound to the cells and was degraded, first to intermediate-sized macromolecules that were the same size as the large (74,600-dalton) and small (25,000-dalton) chains of active plasmin, and to smaller fragments including 3-iodo-L-tyrosine. Binding to SV3T3 cells was independent of the protease-dependent morphological change (PDMC)¹ characteristic of these and many other transformed cells. The SV3T3, and to a somewhat lesser extent, the 3T3 cells, both accumulated and released into the incubation medium 3-iodo-L-tyrosine, a terminal lysozymal digestion product. The results of a sublethal cell-surface trypsinization assay suggest that the cell-associated plasminogen was primarily bound to the surfaces of the 3T3 and SV3T3 cells while the macromolecular degradation products including active plasmin were inside the cells. The rate of ¹²⁵I plasminogen degradation exhibited by SV3T3 cells was approximately two times greater than that of 3T3 cells, which presumably reflects differences in endocytosis or lysosomal hydrolysis, or both. The rates were unaffected by addition of pancreatic or soybean trypsin inhibitor sufficient to inhibit PDMC. In the incubation medium, plasminogen was activated to plasmin by SV3T3, but not by 3T3 cells. However, 95–100% of plasmin covalently bound to a 47,000-dalton canine serum component, which could be dissociated from plasmin by hydroxylamine: 95–100% of the plasmin was inactive to reaction with DF³²P. Thus the serum component is a plasmin inhibitor. The plasmin-containing complex in the medium had an apparent molecular weight of 212,000. Under denaturing conditions, the complex dissociated into two covalently modified plasmin-containing species of 153,000 and 127,000 daltons. In addition to forming a complex with a serum component, the plasmin is cleaved into two small fragments (～10,000 and 12,000 daltons) by as-yet uncharacterized serum factors.     

Cyclic AMP-dependent protein kinases from Balb 3T3 cells and other 3T3 derived lines

Cyclic AMP-dependent protein kinase and 3H-cAMP-binding activities were determined in normal Balb 3T3 cells and compared with the same preparations from SV40, chemical, and spontaneous transformants of 3T3 cells. The cytosolic protein kinase activities and protein kinase activity ratios were similar in all cell lines, although when the normal 3T3 cytosol was prepared by homogenization it contained less 3H-cAMP binding activity than the transformed 3T3 cytosols. The Triton X-100 treated particulate fractions from the normal and transformed 3T3 cells contained similar protein kinase and binding activities. The isozymic profile of cAMP-dependent protein kinases was examined by DEAE-chromatography. The 3T3 cells contained only type II isozyme in either cytosolic or membrane fractions. All transformants of the 3T3 cells contained both type I and type II isozymes. Other cell cultures, including chicken embryo fibroblasts, rat kidney cells, and human or calf endothelial cells contained type I and type II isozymes. Binding of the photoaffinity analogue of cAMP, 8-N3 cAMP, to the regulatory subunits of protein kinases in sonicates obtained from Balb 3T3 and SV 3T3 cells followed by separation on SDS polyacrylamide electrophoresis showed that the amount of RII subunit was approximately equal in the two cell lines. RI in Balb 3T3 cells was detectable but in a much lower quantity than in SV 3T3 cells. The cyclic AMP dependent-protein kinases from Balb 3T3 cells appears to be different from SV 3T3 cells by three criteria: 3H-cAMP binding in homogenates, DEAE chromatographic separation of isozymes, and 8-N3 cAMP binding.     

Purified plasma membranes inhibit polypeptide growth factor-induced DNA synthesis in subconfluent 3T3 cells

Plasma membranes derived from NR-6 cells, a variant line of Swiss mouse 3T3 cells that does not have cell surface receptors for epidermal growth factor (EGF), inhibited EGF-induced stimulation of DNA synthesis by 50% in serum-starved, subconfluent 3T3 cells. Membranes derived from SV3T3 cells were much less effective in inhibiting EGF-induced DNA synthesis. This inhibition on DNA synthesis by NR-6 membranes was not a direct effect of membranes on EGF, nor could it be overcome by high concentrations of EGF. NR-6 membranes were most effective when added 3 h before EGF addition and had little effect when added 2 h or more after EGF. NR-6 membranes also reduced the stimulation of DNA synthesis induced by platelet-derived growth factor or fibroblast growth factor in serum-starved 3T3 cells. These findings indicate that membrane- membrane interactions between nontransformed cells may diminish their ability to proliferate in response to serum polypeptide growth factors.     

The effects of newborn calf serum and foetal bovine serum on the Na+ + K+-activated adenosine triphosphatase activity in 3T3 and SV3T3 cells grown to confluency

The effects of cell density and growth in 10% foetal bovine serum and 10% newborn calf serum on the activity of the enzyme (Na+ + K+)-ATPase were studied in 3T3 and SV3T3 cells. The enzyme activity decreases in 3T3 cells grown in foetal bovine serum as the cells approach confluency while in those grown in newborn calf serum the enzyme activity increases. The (Na+ + K+)-ATPase activity does not change with increase in cell density in SV3T3 cells grown in foetal bovine serum while the enzyme activity in those grown in newborn calf serum increases with increase in cells density up to about 1.35 x 10(5) cells/sq. cm. and then decreases with further increase in cell number. At confluency it was found that the enzyme activity is higher in the SV3T3 as compared to the 3T3 cells when the cells were grown in 10% foetal bovine serum, whereas in those grown in 10% newborn calf serum the enzyme activity is higher in the 3T3 as compared to the SV3T3 cells.     

Correlation of anionic residue mobility at the filopodial plasma membranes with multilayer formation in transformed 3T3 cells

Simian virus 40-transformed 3T3 (SV40-3T3) cells formed multilayers on a Falcon dish and had numerous filopodial projections, some of which intertwined with those of adjacent cells, in contrast to the few projections of their nontransformed counterparts. When these cells were incubated with polycationic ferritin (0.5 mg/ml), ferritin particles, representative of anionic sites, were spread widely on their surfaces at 4 degrees C, while they formed clusters at 37 degrees C, especially on filopodial surface areas opposing adjacent projections in SV40-3T3 cells. These findings demonstrate an increase in the mobility of molecules with anionic residues on filopodial plasma membranes in SV40-3T3 cells, thus suggesting a role for these projections in the formation of multilayered cell aggregates.     

Dependence of intracellular alkali-ion concentrations of 3T3 and SV 40-3T3 cells on growth density.

Intracellular contents of potassium and of sodium are determined for 3T3 and SV 40-3T3 cells in dependence of growth density. In parallel, total cell volume and volume of intracellular water is determined for these cells suspended in physiological buffer. Intracellular potassium concentration thus evaluated for suspended 3T3 cells exhibits a sharp decrease at cellular growth densities which lead to density dependent inhibition of cell proliferation. In the case of SV 40-3T3 cells, this drop of potassium concentration with increasing cellular growth density is not observed, which correlates well with the absence of cell density dependent inhibition of cell growth in the transformed cell line. These results support the notion that processes of stimulation of quiescent 3T3 cells or of cell density dependent inhibition of their proliferation are mediated by processes including changes of potassium transport characteristics leading to increase or decrease respectively of their intracellular potassium concentration. Furthermore, these and other results suggest, that a difference between normal and transformed cells most relevant to their different proliferation behaviour might reside in different transport characteristics for potassium of the plasma membranes of these cells.     

Transformation-associated changes in nuclear-coded mitochondrial proteins in 3T3 cells and SV40-transformed 3T3 cells

Comparative two-dimensional gel electrophoretic studies were performed on mitochondrial proteins in nontransformed mouse 3T3 cells and in SV40-transformed 3T3 cells, SV-T2. Two polypeptides, of 58 and 40 kDa, were present in increased amounts in SV40-transformed cells. These polypeptides were demonstrated to be nuclear-coded mitochondrial proteins by their absence in mitochondrial preparations, when labeling was performed in the presence of a mitochondrial-specific inhibitor, Rhodamine 6G. Temperature-sensitive mutants for transformation were derived from 3T3 cells by transfection with cloned SV40 DNA containing the ts A58 mutation. Increased amounts of the 58 kDa protein were apparent in these cells at the permissive temperature (33 degrees C) compared to the restrictive temperature (39.5 degrees C).     

Effects of angiotensin II and angiotensin II antagonist saralasin on cell growth and renin in 3T3 and SV3T3 cells.

Components of the renin-angiotensin system were studied in established cell culture lines of 3T3 and SV3T3 mouse fibroblasts. The renin content in 3T3 cells was significantly higher than in virus-transformed SV3T3 cells. With time after infection, renin decreased in Simian virus 40 transformed cells, while it increased steadily in mock-infected 3T3 cells. In contrast to renin, angiotensinase activity was higher in SV3T3 cells. Angiotensin II stimulated cell proliferation in 3T3 mouse fibroblasts and decreased their renin content in a dose-related manner. In contrast, saralasin, an angiotensin receptor antagonist, inhibited cell growth in 3T3 and SV3T3 cells and caused an increase of cellular renin concentration. The angiotensin fragments angiotensin (2-8) heptapeptide and angiotensin (4-8) pentapeptide had no effect on cell growth. A significant negative correlation was found between cell proliferation and renin levels in 3T3 and SV3T3 cells irrespective of the treatment. Our results indicate (1) that angiotensin II may be involved in cell growth regulation, (2) that a negative feedback exist between angiotensin II added and intracellular renin content, and (3) that virus infection causes a decrease in intracellular renin synthesis, while non-specific angiotensinase activity is increased under this condition.     

The demonstration of acid phosphatase in cultured 3T3 mouse cells.

Acid phosphatase has been demonstrated ultrastructurally in 3T3 and SV40-3T3 mouse cells using sodium beta-glycerophosphate and p-nitrophenyl phosphate as substrate. The former substrate only demonstrates the enzyme in lysosomes and elements of the Golgi apparatus while the latter demonstrates it in the cisternae of the endoplasmic reticulum and in the cell surface as well as at lysosomal sites. The significance of surface acid phosphatase activity is discussed in terms of sublethal autolysis.     

Transport of 6-deoxy-D-glucose and D-xylose by untransformed and SV40-transformed 3T3 cells

Transport rates of the nonphosphorylated D-glucose analogs 6-deoxy-D-glucose and D-xylose were measured in quiescent and serum-stimulated cultures of mouse 3T3 cells, in SV40-transformed 3T3 cells (SV101), and in a density revertant cell line derived from SV101 (Fl-SV101). Initial rates of both entry and exit of 6-deoxy-D-glucose and D-xylose were more than threefold higher in serum-stimulated 3T3 and in SV101 cells than they were in quiescent 3T3 cells, but transport rates were not higher in the transformed cells (SV101) than they were in serum-stimulated 3T3. Confluent cultures of Fl-SV101 showed lower rates of transport than serum-stimulated Fl-SV101, but not as low as quiescent 3T3 cells. These data confirm previous findings of others with other analogs that glucose transport is one of the cell functions that is depressed when 3T3 cells enter the quiescent G₀ state, but emphasize that SV40-transformed 3T3 cells do not show higher activity of the D-glucose carrier than do actively growing 3T3 cells. Thus, enhanced glucose transport appears not to be a specific consequence of transformation, but a reflection of the active growth state of the cell.