Simian Virus 40-transformed 3T3 Research Articles

Antitumor activity of (10 E, 12 Z)-9-hydroxy-10,12-octadecadienoic acid from rice bran

Two types of hydroxy acids, (10 E, 12 Z)-9-hydroxy-10,12-octadecadienoic acid [1] and (9 Z, 11 E)-13-hydroxy-9,11-octadecadienoic acid [2], were obtained as cytotoxic compounds from a water extract of rice bran. The cytotoxicity of acid 1 was stronger than that of acid 2 in vitro, against P388 mouse leukemia cells. Acid 1 slightly inhibited the growth of a Balb c mouse 3T3 fibroblast cells ( BALB c 3T3), but strongly inhibited that of simian virus 40-transformed 3T3 cells (SV-T2); 1 was therefore, found to have cytotoxicity against cancer cells. On the other hand, 2 inhibited neither normals cells nor transformed cells. Intraperitoneal injection of acid 1 effectively prolonged the survival time of C3H HeJ mice after intraperitoneal transplantation of MM46 mammary tumor cells. The difference in antitumor activity between 1 and 2 was also observed in vivo.

Selective cytotoxic effects of 5-fluorouridine 5′-diphosphate sugars on virus-transformed malignant cells of mouse fibroblast

The cytosolic fraction from 3T3 cells of Balb/c mouse fibroblast showed weak nucleotide sugar pyrophosphatase activity with 5-fluorouridine 5′-diphosphate α-d-galactose (FUDPGal). Unlike the membrane fraction from 3T3 cells, which exhibited no such activity with FUDPGal or 5-fluorouridine 5′-diphosphate N-acetyl-α-d-glucosamine (FUDPGlcNAc), the membrane fraction from simian virus 40-transformed 3T3 (3T3SV) cells showed higher hydrolytic activities with these nucleotide sugar analogs than the cytosolic fraction. Both FUDPGal and FUDPGlcNAc exhibited potent cytotoxic effects comparable to that of 5-fluorouridine (FUR) on 3T3SV cells but did not affect the viability of the native 3T3 cells even at 1 mM. FUR was detected only in the culture of 3T3SV cells grown with FUDPGal or FUDPGlcNAc. These results revealed the critical role of membrane-associated nucleotide sugar pyrophosphatase in the selective cytotoxic effects of FUDPGal and FUDPGlcNAc.

Cell density- and membrane region-dependent mobility of anionic residue-containing molecules in 3T3 and transformed 3T3 cell surfaces.

Mobility of anionic residue-containing molecules on 3T3 and simian virus 40-transformed 3T3 (SV40-3T3) cell surfaces was ultrastructurally examined using polycationic ferritin (CF) particles in vitro. CF-binding experiments at 4 degrees C showed continuous distribution of CF particles on entire surfaces of both cells. When 3T3 cells were incubated with CF at 37 degrees C, cell membrane-bound CF particles were rearranged to form clusters in sparse cultures. Almost all CFs on the ventral cell surface were present in clusters. Clustering rate and size were smaller on the dorso-lateral surface in high cell density than on the same region in low density. On the ventral in high density, CFs distributed in clusters or singly at broader zone than in sparse cultures. In the case of sparse cultures on dishes from which confluent 3T3 cells had been removed, the CF distribution pattern was similar to that in high density. On the other hand, its density-dependent change was hardly found in both surfaces of transformed counterparts. The clustering was remarkable on all cell surface areas and tended to differ regionally. The distribution pattern was similar to those in sparse 3T3 cell cultures. These results show cell density-, membrane region- and extracellular matrix-dependent mobility of anionic residue-containing molecules on the 3T3 cell membrane, and remarkably high, a little region-dependent, and density- and matrix-independent mobility in SV40-3T3 cells, suggesting relation of the mobility to cell-cell communication.

Suppression of tumorigenicity in simian virus 40-transformed 3T3 cells transfected with alpha-actinin cDNA.

Human cytoskeletal alpha-actinin cDNA was transfected into highly malignant simian virus 40-transformed BALB/c 3T3 (SVT2) cells that express 6-fold lower levels of alpha-actinin than nontransformed BALB/c 3T3 cells. SVT2 clones expressing various levels of alpha-actinin were isolated and their structure and tumorigenic properties were determined. Transfected SVT2 clones expressing alpha-actinin at levels found in nontumorigenic 3T3 cells displayed a flatter phenotype, a decreased ability to grow in suspension culture in soft agar, and a marked reduction in their ability to form tumors in syngeneic BALB/c mice and in athymic nude mice. Clones overexpressing alpha-actinin at the highest level (about 2-fold higher than 3T3 cells) were completely suppressed in their ability to form tumors in syngeneic BALB/c mice. The results suggest that alpha-actinin, an actin-crosslinking protein that is also localized in cell junctions, may have an effective suppressive ability on the transformed phenotype.

Differential adaptive response to hyperosmolarity of 3T3 and transformed SV3T3 cells

Both 3T3 and simian virus 40-transformed 3T3 (SV3T3) cells were used to investigate differences in population kinetics, protein synthesis, monovalent ion levels, and amino acid accumulations between normal and transformed cells exposed to hyperosmolarity at 0.5 Osm. Under similar culture conditions, SV3T3 cells were found to be more sensitive in their proliferative response than normal cells to the hyperosmolar treatment. In the normal 3T3 cells, the increase in transport of amino acids was less sustained and was associated with higher levels of accumulated amino acids. The equilibrium distribution of intracellular monovalent cations and the rate of protein synthesis also returned faster to baseline values in the normal cells than in the transformed cells. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis revealed the induction of a 69-kDa polypeptide in the 3T3 cells but not in the SV3T3 cells after exposure to hyperosmolarity. On electrofocusing and relative mass analysis, this polypeptide closely migrated with the 70-kDa heat shock protein (hsp) family, although it was unrelated immunologically to the inducible 72-kDa hsp.

Nonterminal differentiation represses the neoplastic phenotype in spontaneously and simian virus 40-transformed cells.

The potential of nonterminal cellular differentiation to stably repress the expression of the neoplastic phenotype of transformed cells is established. Nonterminal differentiation induces spontaneously transformed 3T3 T cells to revert to a nontransformed state and induces the revertant cell clones to become resistant to retransformation by UV irradiation or 4-nitroquinoline oxide treatment. Nonterminal differentiation also induces simian virus 40-transformed 3T3 T cells to repress expression of the large tumor antigen and to revert to a nontransformed state. Although the molecular mechanisms that mediate these and other forms of anticancer activity have not been definitively established, data are presented which suggest that differentiation-induced repression of large tumor antigen expression can be regulated at the level of transcription and/or RNA processing.

Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells.

We observed six major tryptic phosphopeptides in p53 from simian virus 40-transformed and normal NIH 3T3 cells. Analyses of the phosphopeptides indicated that serines 37, 310 and/or 312, 389 and one or more of serines 7, 9, 12, 18, and 23 were phosphorylated. Phosphorylation of serines 310 and/or 312 was twofold higher in the simian virus 40-transformed cells as compared with that in normal NIH 3T3 cells.

Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells.

We observed six major tryptic phosphopeptides in p53 from simian virus 40-transformed and normal NIH 3T3 cells. Analyses of the phosphopeptides indicated that serines 37, 310 and/or 312, 389 and one or more of serines 7, 9, 12, 18, and 23 were phosphorylated. Phosphorylation of serines 310 and/or 312 was twofold higher in the simian virus 40-transformed cells as compared with that in normal NIH 3T3 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.

Hyaluronate‐binding protein of simian virus 40‐transformed 3T3 cells: Membrane distribution and reconstitution into lipid vesicles

Hyaluronate-binding protein (HABP) has been extracted in detergent from the membranes of simian virus 40-transformed 3T3 (SV-3T3) cells (Underhill et al, J Biol Chem 258:8086-8091, 1983). When SV-3T3 cells were treated with trypsin prior to isolation and dissolution of the membranes, no hyaluronate-binding activity could be detected. This indicates that all of the detectable HABP of SV-3T3 cells is located on the external surface of the plasma membrane rather than on internal membranes, which would be inaccessible to the trypsin. The detergent-extracted HABP from SV-3T3 membranes was reconstituted into the membrane of lipid vesicles, which were formed by addition of exogenous phosphatidylcholine and cholic acid to the extracts followed by removal of detergent by dialysis against 0.02 M Tris pH 8.0 in the presence of protease inhibitors. Reconstitution was assessed by sedimentation in a discontinuous sucrose gradient and by gel filtration on Sepharose 4B in the presence and absence of detergent. The characteristics of binding of hyaluronate to the reconstituted HABP were then compared with those studied previously for the original membrane-bound HABP and the detergent-extracted HABP (Underhill et al, J Biol Chem 258:8086-8091, 1983). It was observed previously that binding of hyaluronate to HABP in the cell membranes was of higher affinity and specificity than to HABP in the detergent extracts of these membranes. It was found here that reconstitution of the extracted HABP into the membranes of lipid vesicles led to restoration of affinity of binding to the level observed in the original cell membranes. However, whereas chondroitin sulfate does not compete significantly for binding of hyaluronate to cell membrane-bound HABP, partial competition was observed for the reconstituted HABP as well as for detergent-extracted HABP. Thus, it is concluded that the high affinity of binding of hyaluronate to the plasma membrane of SV-3T3 cells is in part dependent on insertion of the HABP in the membrane, but that other interactions, not duplicated in our reconstitution experiments, must be necessary for the specificity of the HABP.

Cell surface and other morphological changes accompanying growth inhibition in simian virus 40-transformed 3T3 mouse fibroblasts cells induced by glucocorticoids§

A number of morphological changes accompany the G2 blockage caused by glucocorticosteroids in simian virus 40-transformed 3T3 mouse fibroblasts cells. Under phase contrast microscopy dexamethasone-treated cells have darkened and raised nuclear regions with ‘lines’ running over their cytoplasmic areas. They are more resistant to trypsinization and smaller in volume. Since inhibitors of DNA and protein synthesis prevent this ‘glucocorticoid morphology’, the ‘darkening’ may be due to the accumulation of macromolecules within G2-blocked cells and the induction of a protein(s) may be needed for the morphological changes. Colchicine and cytochalasin B do not bring about the glucocorticoid morphology, suggesting that it is not due to a general de-polymerization of microtubules or microfilaments. With scanning electron microscopy treated cells show a great reduction in the amount and a re-organization of microvilli and microplicae. Granules of lead precipitate at the periphery are also clearly evident in transmission electron micrographs. These observations reveal profound morphological alterations, including cell surface changes, induced by glucocorticosteroids.

Mapping of phosphomonoester and apparent phosphodiester bonds of the oncogene product p53 from simian virus 40-transformed 3T3 cells.

The oncogene product p53, isolated from SV3T3 cells where it forms a complex with simian virus 40 large tumor antigen (T antigen) in the nucleus, has been found to be phosphorylated at at least four distinct sites on the 390 amino acid protein. Separation of tryptic phosphopeptides has permitted identification of two sites as Ser-312 and Ser-389, and permitted analysis of the types of phosphate bonds. The peptide containing Ser-312 separates electrophoretically into three charged forms; two are resistant to dephosphorylation by both alkaline phosphatase and alkaline hydrolysis, suggesting a phosphodiester. The carboxyl-terminal phosphopeptide containing Ser-389 was alkaline phosphatase-resistant and liberated four ribonucleoside monophosphates upon base or RNase hydrolysis, suggesting that Ser-389 may be covalently linked to RNA. Phosphorylation of Ser-389 decreased markedly at the nonpermissive temperature in simian virus 40 tsA58-transformed cells, indicating a dependence on native T antigen function and a possible role in transformation by T antigen. Two additional phosphorylation sites, one involving serine and one involving threonine, probably reside in the amino-terminal segment of p53 and appear to be peptide-phosphate monoesters.

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 M r 46 000 and M r 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.

Endogenous hyaluronate-cell surface interactions in 3T3 and simian virus-transformed 3T3 cells.

3T3 cells have a large, pericellular coat which contains 30 times more hyaluronate than the amount of cell surface hyaluronate associated with simian virus 40-transformed 3T3 (SV-3T3) cells. On the other hand, SV-3T3 cells have high affinity binding sites for exogenously added hyaluronate, whereas 3T3 cells have much lower affinity sites. Removal of cell surface hyaluronate from SV-3T3 cells by treatment with hyaluronidase caused a reproducible increase in their maximum binding capacity for exogenous hyaluronate but no significant change in binding affinity or specificity. For 3T3 cells, however, the maximum amount of binding decreased and the affinity of binding increased after hyaluronidase treatment. When endogenous cell surface hyaluronate was labeled metabolically and then the cells incubated in the presence of exogenous unlabeled hyaluronate, the labeled cell surface hyaluronate was quantitatively displaced from the SV-3T3 cells but was not displaced from the 3T3 cells. Chondroitin sulfate and heparin did not displace cell surface hyaluronate from either cell type. Membranes isolated from SV-3T3 cells bound hyaluronate specifically and with high affinity, whereas membranes from 3T3 cells did not consistently bind a significant amount of hyaluronate. We conclude from these studies that the retention of endogenous hyaluronate on the surface of SV-3T3 cells is mediated by binding sites similar to those detected by the addition of exogenous hyaluronate, and the mechanism of retention of endogenous hyaluronate on the surface of 3T3 cells differs from SV-3T3 cells.

Analysis of the reduced growth factor dependency of simian virus 40-transformed 3T3 cells.

We have measured in a defined serum-free medium the platelet-derived growth factor (PDGF) and insulin requirements of normal Swiss 3T3 cells, simian virus 40-transformed 3T3 cells, and partial revertants of simian virus 40-transformed 3T3 cells. Swiss 3T3 cells displayed strong requirements for both PDGF and insulin. Both of these requirements were significantly diminished in simian virus 40-transformed 3T3 cells. Analysis of the PDGF and insulin requirements of the revertants indicated that the loss of either of these two growth factor requirements was not necessarily linked to the other; rather, the growth factor requirements were specifically associated with other parameters of transformation. The reacquisition of a PDGF requirement cosegregated with reversion to density-dependent growth inhibition, whereas reacquisition of a normal insulin requirement cosegregated with reversion to a normal growth dependence on calf serum. Anchorage dependence was dissociable from both growth factor requirements. The relationship between the PDGF requirement and density-dependent growth inhibition was further analyzed in normal 3T3 cells by measuring the PDGF requirement at different cell densities. At high cell densities, the requirement for PDGF became significantly greater. We suggest that at least in part the ability of transformed cells to grow to high saturation densities results from their loss of a requirement for PDGF.

Analysis of the reduced growth factor dependency of simian virus 40-transformed 3T3 cells.

We have measured in a defined serum-free medium the platelet-derived growth factor (PDGF) and insulin requirements of normal Swiss 3T3 cells, simian virus 40-transformed 3T3 cells, and partial revertants of simian virus 40-transformed 3T3 cells. Swiss 3T3 cells displayed strong requirements for both PDGF and insulin. Both of these requirements were significantly diminished in simian virus 40-transformed 3T3 cells. Analysis of the PDGF and insulin requirements of the revertants indicated that the loss of either of these two growth factor requirements was not necessarily linked to the other; rather, the growth factor requirements were specifically associated with other parameters of transformation. The reacquisition of a PDGF requirement cosegregated with reversion to density-dependent growth inhibition, whereas reacquisition of a normal insulin requirement cosegregated with reversion to a normal growth dependence on calf serum. Anchorage dependence was dissociable from both growth factor requirements. The relationship between the PDGF requirement and density-dependent growth inhibition was further analyzed in normal 3T3 cells by measuring the PDGF requirement at different cell densities. At high cell densities, the requirement for PDGF became significantly greater. We suggest that at least in part the ability of transformed cells to grow to high saturation densities results from their loss of a requirement for PDGF.

Evidence for a transforming growth factor(s) in the serum-free conditioned medium of SV40-transformed 3T3 fibroblasts.

Unlike untransformed 3T3 fibroblasts, Simian Virus 40-transformed 3T3 cells (SV3T3) do not require the presence of exogenous polypeptide mitogens in order to become competent for the initiation of DNA synthesis. As an explanation for this state of perpetual competence, growth activity for 3T3 cells was found in the serum-free conditioned medium of SV3T3 cells. This activity, which is abolished by incubation with pepsin, but withstands to a fair degree denaturants (8 M urea and 4 M guanidinium HCl) and a reducing agent (65 mM dithioerythritol), enhances the growth of Swiss and BALB/c 3T3 cells in 1% (v/v) calf serum medium and ensures the complete survival of Swiss 3T3 cells for several days in the total absence of serum. The growth activity appears to be derived from the SV3T3 cells, since it is not found in the non-conditioned medium (and the insulin and transferrin components of this serum-free medium have only weak growth activity alone) and the chances of it being due to residual serum growth factors are slight because of the method of growing the cells and the collection of the conditioned medium. Moreover, in a separate experiment to test the possible involvement of cell attachment factors, rat fibronectin was found not to affect 3T3 growth in the standard growth assay. The conditioned medium growth activity apparently has the biological capabilities of transforming growth factors. After concentration by ammonium sulfate precipitation, it can stimulate DNA synthesis in confluent, quiescent Swiss 3T3 cells, in both the absence and presence of 10% depleted calf serum. It also permits the growth of large colonies of Swiss 3T3 cells in soft agar. As part of a preliminary characterization of the conditioned medium growth activity, gel filtration in 0.15 M NaCl, 0.001 M HCl resolves the activity into high (mol. wt. greater than or equal to 100,000) and low (mol. wt. 5,000-6,000) molecular weight factors. Since re-chromatography of the high molecular weight factor, which may still be in an aggregated form, does not generate the smaller, the latter is not a dissociation product of the former. The precise relationship of these SV3T3-conditioned medium growth factors to other growth factors, including known transforming growth factors, has not yet been determined.

Monoclonal antibodies that prevent adhesion of B 16 melanoma cells and reduce metastases in mice: crossreaction with human tumor cells.

Monoclonal antibodies raised against B 16 melanoma cells in syngeneic mice were functionally screened for their ability to inhibit cell adhesion in tissue culture. Three of these antibodies (16/43, 16/77, 16/82), when preinjected into C57BL/6 mice, markedly reduced the number of experimental lung metastases produced by B 16 cells, possibly by interference with their adhesion to the lung endothelia. We now report that these monoclonal antibodies block in vitro attachment of the majority of human melanoma cell lines tested and also of carcinoma, neuroblastoma, and glioblastoma cells from both mice and humans but untransformed cell lines such as 3T3 mouse or MRC-5 human fibroblasts are not affected. The antibodies also react with mouse teratocarcinoma stem cells (F9, PCC4) but not with differentiated teratocarcinoma lines (PYS-2, 944). Furthermore, the antiadhesion activity of the antibodies could be quantitatively absorbed by intact human and mouse tumor cells but not by untransformed cells, suggesting that the corresponding antigens may represent tumor-associated cell surface components. Correspondingly, the antigens were found on simian virus 40-transformed 3T3 mouse fibroblasts and are expressed in a temperature-sensitive fashion in chicken fibroblasts transformed with a temperature-sensitive Rous sarcoma virus. On "immunoblots" of NaDodSO4-containing gels the three selected antibodies (16/43, 16/82, 19/1) were absorbed by antigens with molecular weights of 40,000 and 50,000.

Effects of detergent solubilization on the hyaluronate-binding protein from membranes of simian virus 40-transformed 3T3 cells.

Simian virus 40-transformed 3T3 (SV-3T3) cells have been shown to possess on their surfaces binding sites for hyaluronate which mediate the divalent cation independent aggregation of these cells. To further characterize these binding sites, membranes were prepared from SV-3T3 cells and solubilized with the detergent, sodium deoxycholate. The binding activity present in the detergent solution was measured by the addition of [3H]hyaluronate followed by separation of free and bound ligand by (NH4)2SO4 precipitation. Using this assay, the soluble hyaluronate-binding protein was compared with the membrane-associated protein in situ. In both cases, binding was found to be saturable, linear with protein content, competitively inhibited by unlabeled hyaluronate and dependent on a minimum of 6 sugar residues of hyaluronate for recognition. However, the solubilized binding protein was found to differ from the membrane-associated protein in the following characteristics: a) the affinity of the interaction with hyaluronate was reduced (the Kd was higher), while the amount of ligand bound at saturation (Bmax) was increased; b) in competition experiments with unlabeled preparations of hyaluronate, the effect that the MWv of the hyaluronate had on its inhibitory potency was greatly reduced; and c) the binding was inhibited to a greater extent by chondroitin sulfate and dermatan sulfate. All of these differences can be accounted for by assuming that the detergent solubilization changes the nature of the hyaluronate-binding site interaction from one that is multivalent (i.e. one molecule of hyaluronate is attached to several sites) to one that is monovalent.

Interaction of hyaluronate with the surface of simian virus 40-transformed 3T3 cells: aggregation and binding studies.

Previous studies have shown that the divalent-cation independent aggregation of simian virus 40-transformed 3T3 cells (SV-3T3) is mediated by the interaction of endogenous hyaluronate with binding sites on the cell surface. In the present study, the nature of the interaction of hyaluronate with the binding sites was characterized further by examining both the aggregation of SV-3T3 cells and the binding of exogenously added [3H]hyaluronate. The aggregation of SV-3T3 cells was found to be: (1) optimal at pH 7; (2) only slightly affected by variations in temperature between 0 and 40 degrees C; (3) more potently inhibited by the addition of high molecular weight preparations of hyaluronate than by lower molecular weight preparations; and (4) enhanced by increasing the ionic strength of the medium. With the exception of the effects of temperature, these characteristics correspond closely to those previously described for binding of [3H]hyaluronate. The effect of ionic strength on the binding of [3H]hyaluronate by SV-3T3 cells was found to correspond closely to its effect on lowering the viscosity of hyaluronate. Both effects (i.e. binding and reduction in viscosity) had similar dose-response curves for NaCl and MgCl2. This close correspondence between binding and the reduction of viscosity suggested that the effect of ionic strength on binding was due to a change in the structure of hyaluronate. Lowering the temperature (40 to 0 degrees C) also enhanced the binding of [3H]hyaluronate to SV-3T3 cells. However, this effect was blocked if the cells were first fixed with glutaraldehyde, suggesting that temperature mediates its effect through the binding sites. Additional experiments showed that the binding of [3H]hyaluronate by SV-3T3 cells could be prevented by preincubating the cells with several types of lectins.