Effect of 3T3 plasma membranes on cells exposed to epidermal growth factor

Epidermal growth factor (EGF) induced DNA synthesis in non-confluent, G₀-arrested Swiss 3T3 fibroblasts is partially blocked by plasma membranes isolated from the EGF receptor deficient NR-6 Swiss 3T3 cell line. This inhibition could be due to either a steric block of the receptor by the membranes, a membrane induced down regulation of the EGF receptor, or a signal generated by membrane binding which is antagonistic towards the mitogenic signal generated by EGF. Binding measurements utilizing ¹²⁵I-labeled EGF demonstrated that membranes do not block either the EGF induced down regulation of the receptor or alter the number of receptors on the surface. These results suggest that the membranes exert their inhibitory effect via generation of a signal which is antagonistic to the EGF induced mitogenic signal, with the result expressed as a reduced mitogenic response.     

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.     

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G₁ protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.     

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.     

Role of serum components in density-dependent inhibition of growth of cells in culture. Platelet-derived growth factor is the major serum determinant of saturation density

The effects of platelet-derived growth factor and plasma components on saturation density in cultures of 3T3 cells were investigated. Both of these components of whole blood serum affect saturation density; however, when 3T3 cells become quiescent at high density in medium containing whole blood serum, only platelet-derived growth factor and fresh whole blood serum are capable of stimulating proliferation. Addition of fresh plasma- derived serum has little effect on cell growth. These results suggest that the platelet factor is the major determinant of saturation density in cultures of 3T3 cells maintained in medium supplemented with whole blood serum. Experiments were performed to investigate the mechanism by which platelet-derived growth factor regulates saturation density. We investigated the possibilities of inactivation of growth factors by proliferating cells, and the effects of cell density on the response of 3T3 cells to platelet-derived growth factor. The amount of platelet- derived growth factor required to initiated DNA synthesis increases with increasing cell density. Some inactivation of growth factors by growing cells was detected, but this depletion was only evident at high cell density. We propose that density-dependent inhibition in cultured 3T3 cells is the result both of an increased requirement for the platelet- derived growth factor as the cultures become more crowded and of inactivation of growth factor activity by growing cells.     

Effect of imposed serum deprivation on growth of the mouse 3T3 cell. Dissociation from changes in potassium ion transport as measured from [36Rb]rubidium ion uptake

Decreased serum concentrations that substantially alter the growth of normal 3T3 cells alter neither the active and non-active components of unidirectional ⁸⁶Rb⁺ influx nor the intracellular K⁺ content when compared with cells in exponential growth. Thus the changes in K⁺ transport (measured with ⁸⁶Rb⁺ as an analogue for K⁺ movements) that occur on density-dependent growth inhibition of the mouse 3T3 cell are not mimicked by serum deprivation of the cells before density inhibition.     

Binding of isolated 3T3 surface membranes to growing 3T3 cells and their effect on cell growth

We have quantitated by autoradiography the binding of [¹²⁵I]labeled 3T3 plasma membrane fragments to 3T3 cells growing on the surface of plastic dishes; ie, the same conditions in which these membranes specifically arrest the growth of 3T3 cells early in the G₁ phase of the cell cycle. We have been able to demonstrate that binding of membranes to cells is coincidental with the expression of the growth inhibitory activity of protein(s) present in the membrane fragments. Treatments that reduce binding (heat denaturation of the membranes or culture in the presence of high scrum) also reduce growth inhibitory activity. [¹²⁵I]labeled membranes bound to cells are located primarily on the cell surface (as determined by electron microscope autoradiography) and are exchangeable with unlabeled membranes. We conclude that binding of membranes to cells is necessary but may not be sufficient for the expression of the growth inhibitory activity of these membranes. This approach provides information not only on the average level of binding of membranes to cells, but also provides a quantitative assessment of the variation of the level of membrane to cell binding between different cells in the population.     

Density-dependent inhibition of growth: Inhibitorydiffusible factors from 3T3- and rous sarcoma virus (RSV)-transformed 3T3 cells

We recently fractionated, from the culture medium of 3T3 cells, a thermolabile inhibitory diffusible factor (IDF_N) with a molecular weight of about 40,000 daltons, which decreased nucleic acids synthesis of stimulated target 3T3 cells. In the present publication the inhibitory activities of IDF_N (produced by 3T3 cells) and IDF_T (produced by RSV-transformed 3T3 [3T3 SRA/H] cells) on 3T3 and 3T3 SRA/H cells have been compared. The inhibitory activity of IDF_N decreased (by a mean of 57%) when it was tested on transformed instead of 3T3 cells. On the other hand IDF_T was able to decrease ¹⁴C-inosine incorporation in target 3T3 cells. However, the inhibitory activity of IDF_T decreased (by mean 50%) when tested on 3T3 SRA/H instead of 3T3 cells. Therefore, transformed cells produced an inhibitory factor but were less sensitive than 3T3 cells to its inhibitory activity. The inhibitory activity of IDF_T on 3T3 SRA/H cells was only 20% of the inhibitory activity of IDF_N on 3T3 cells. This appreciable difference is of particular interest, since it could explain the release of density-dependent inhibition of growth (DDI) in transformed 3T3 SRA/H cells. Furthermore, it provides more evidence for the hypothesis that, in 3T3 cells, DDI of growth is due to the release of an inhibitory molecule into the medium, and that IDF_N is in fact, the inhibitory molecule involved in this phenomenon.     

Cell-cell contact and growth regulation of pinocytosis in 3T3 cells

In sub-confluent cultures of Balb/c-3T3 cells, pinocytosis rates were increased after exposure to specific growth factors (serum; platelet-derived growth factor, PDGF; epidermal growth factor, EGF). Conversely, as cells became growth-inhibited with increasing culture density, there was a corresponding decline in pinocytosis rate per cell. In order to test whether density-inhibition of pinocytosis was influenced either by the growth cycle or by cell contact independently of growth, cells were induced into a quiescent state at a range of subconfluent and confluent densities. Under such conditions, cell density did not significantly inhibit pinocytosis rate. When confluent quiescent cultures in 2.5% serum were exposed to 10% serum, the resulting round of DNA synthesis was accompanied by enhanced pinocytosis per cell, even though the cells were incontact with one another. Furthermore, in a SV40-viral transformed 3T3 cell line, both the growth fraction and the pinocytosis rate per cell remained unchanged over a wide range of culture densities. These studies indicate that density-dependent inhibition of pinocytosis in 3T3 cells appears to be secondary to growth-inhibition rather than to any direct physical effects of cell–cell contact.     

Inhibition of phorbol ester-receptor binding by a factor from human serum.

The inhibition of receptor binding of [3H]phorbol-12,13-dibutyrate (PDBu) by a factor from human serum was characterized. The serum factor inhibited [3H]PDBu binding in intact monolayer cultures of the rat embryo cell line CREF N and in a subcellular system containing membranes from these cells. Inhibition occurred at both 37 and 4 degrees C and was rapid and reversible. An analysis of [3H]PDBu binding in the presence of the serum factor indicated that inhibition of [3H]PDBu binding by the serum factor was noncompetitive. Using gel filtration to separate the serum factor from free [3H]PDBu, we obtained evidence that the serum factor does not act by binding or trapping the [3H]PDBu. Unlike the phorbol ester tumor promoters, the serum factor alone did not stimulate the release of choline or arachidonic acid from cellular phospholipids, nor did it inhibit the binding of 125I-labeled epidermal growth factor to cellular receptors. The factor did, however, antagonize the inhibition of epidermal growth factor binding induced by PDBu. Sera from pregnant women were, in general, more inhibitory of [3H]PDBu binding than were those from nonpregnant women, which were more inhibitory than those from men. During these studies we found that CREF N cells responded to being grown in the presence of PDBu by partial down regulation of the phorboid receptor. The 50% effective dose for down regulation was 8 nM PDBu, and the maximum effect occurred after 6 h. Taken together, our results indicate that the serum factor inhibits [3H]PDBu binding by a direct physical effect at the level of the phorboid receptors or their associated membranes. It would appear that if this factor acts in vivo, then it might antagonize certain effects of this class of tumor promoters.     

Specific inhibition of endothelial cell proliferation by isolated endothelial plasma membranes

Cultures of human vascular endothelial cells were used to study the phenomenon of density-dependent inhibition of cell growth. Endothelial cells were disrupted by nitrogen cavitation, and a plasma membrane-enriched fraction was prepared by differential centrifugation followed in some cases by sucrose density gradient fractionation. Membrane suspension was added to low-density early-passage endothelial cultures grown in microwells. Hemocytometer cell counts and 6 hr 3H-thymidine pulses were performed in triplicate wells at varying intervals. Plasma membranes suppressed cell proliferation in a reversible, dose-dependent fashion. Increasing the ambient concentration of endothelial cell growth factor did not alter the inhibitory effect. The antiproliferative effect was sensitive to heat and trypsin and to incubation with 0.1 M sodium carbonate, pH 11.5. Membrane vesicles selectively derived from the apical cell surface also suppressed proliferation. This phenomenon showed at least some specificity for cell type and species in both human and bovine models. Therefore, cell-cell contact is capable of regulating endothelial cell proliferation in vitro despite the presence of available growth surfaces and of optimally supportive culture medium.     

Density-dependent inhibition of cell growth is correlated with the activity of a cell surface phosphatidylinositol-specific phospholipase C.

We previously identified a novel phospatidylinositol-specific phospholipase C (PI-PLC) present at the surface of Swiss 3T3 cells using a fluorescent analog of PI and showed that this cell surface PI-PLC (csPI-PLC) activity increases with increasing cell density (J. Biol. Chem. 265, 5337-5340 (1990)). Here, we find that csPI-PLC activity also increased in cultures in which growth was inhibited due to serum deprivation. csPI-PLC was inhibited by agents that inhibit other mammalian PI-PLCs, but not by treatments which inhibit glycosyl PI-PLCs (GPI-PLCs). We also extended our studies using fluorescent PI to other cell types and found that csPI-PLC activity was present only in cell lines that exhibit growth inhibition upon reaching confluency (Swiss 3T3, 3T3-L1, BALB/c 3T3, and normal rat kidney (NRK) fibroblasts), but not in cell lines that are tumorigenic and/or do not exhibit growth inhibition in a density-dependent manner (Chinese hamster ovary (CHO), mouse L, SV-40 transformed BALB/c 3T3 (SV-T2), baby hamster kidney (BHK), and Chinese hamster lung (V79) fibroblasts). These results support the hypothesis that csPI-PLC plays a role in the density-dependent inhibition of cell growth.     

Oncogenic Ras-transformed human fibroblasts exhibit differential changes in contraction and migration in 3D collagen matrices

Tractional force exerted by tissue cells in 3D collagen matrices can be utilized for matrix remodeling or cell migration. The interrelationship between these motile processes is not well understood. The current studies were carried out to test the consequences of oncogenic Ras (H-Ras^V12) transformation on human fibroblast contraction and migration in 3D collagen matrices. Beginning with hTERT-immortalized cells, we prepared fibroblasts stably transformed with E6/E7 and with the combination HPV16 E6/E7 and H-Ras^V12. Oncogenic Ras-transformed cells lost contact inhibition of cell growth, formed colonies in soft agar and were unable to make adherens junctions. We observed no changes in the extent or growth factor dependence of collagen matrix contraction (floating or stress-relaxation) by oncogenic Ras-transformed cells. On the other hand, transformed cells in nested collagen matrices lost not only growth factor selectivity, but also cell-matrix density-dependent inhibition of migration. These findings demonstrate differential regulation of collagen matrix contraction and cell migration in 3D collagen matrices.     

Identification and characterization of a soluble suppressor factor(s) in the serum of AKR mice bearing lymphocytic leukemia

Leukemia in AKR mice was found to be associated with the presence of a serum factor(s) termed AKR leukemic suppressor factor (AKR-LSF). Suppression was quantitated by measuring the inhibition of PHA-stimulated [³H]thymidine incorporation by normal AKR spleen cells at various dilutions of leukemic mouse serum (LMS). AKR-LSF activity was expressed as units per milliliter, which is the reciprocal of the LMS dilution that inhibited [³H]thymidine uptake by 50% with respect to fetal calf serum control cultures. The amount of activity in the serum directly correlated to the rate of tumor cell growth. Mice receiving 10⁷ BW5147 transplanted leukemia cells had 130 ± 12 units of AKR-LSF activity/ml of serum compared to 40 ± 8 units/ ml for mice with spontaneous leukemia. Normal mouse serum contained 33 ± 11 units/ml. The leukemic serum exhibited no strain specificity in either phytohemagglutinin or lipopolysaccharide assays, but was found to be twofold more inhibitory against mouse spleen cells than that against rat spleen cells. Human lymphocyte blastogenesis was not inhibited by the leukemic serum. LMS did not inhibit the growth of L929 fibroblasts or murine tumor cells in vitro. Further work is necessary to determine what role the suppressor factor may play in the regulation of antitumor cell immunity.     

Effect of liver cell coat acid mucopolysaccharide on the appearance of density-dependent inhibition in hepatoma cell growth.

Yoshida ascites hepatoma 66 (AH 66) cells grown in monolayer cultures show a lack of density-dependent inhibition of growth. When acid mucopolysaccharide (AMPS) isolated from rat liver cell coats is added to growing cultures at concentrations of 50–100 μg/ml, AH 66 cell cultures became markedly inhibited and exhibited density-dependent inhibition of growth at a cell density of 19 × 10⁴ cells/cm². Inhibition reached 84% below control levels. Inhibition is a density-related phenomenon since cells at densities below 19×10⁴ cells/cm² do not exhibit inhibition of growth. AH 66 cells inhibited in the plateau state are capable of resuming growth when AMPS is removed from the cultures. When AMPS is added at a concentration of 0.5 μg/ml, growth of tumor cells is promoted. Promotion reaches 78% above control levels. It is suggested that AMPS may play an important part in the regulation of cellular proliferation.     

Glucocorticoids inhibit the stimulatory effect of epidermal growth factor on the initiation of DNA synthesis

Confluent, quiescent Swiss 3T3 cells in culture can be stimulated to initiate DNA synthesis and divide by addition of growth factors to the culture medium. Here we show that hydrocortisone and other steroids which have glucocorticoid activity inhibit the stimulation of these cells by epidermal growth factor (EGF) in contrast to their reported enhancement of stimulation by fibroblast growth factor (FGF). Binding studies using [³H]-triamcinolone acetonide show that Swiss 3T3 cells contain a single class of glucocortioid receptor of uniform affinity (K_D = 2.0 nM), and about 34,000 receptor sites per cell. Those steroids which displace bound [³H]-triamcinolone acetonide are also effective in inhibiting the stimulation of DNA synthesis by EGF in the presence or absence of insulin, and the concentration of triamcinolone acetonide required for one-half maximal biological effect is in the same range as the K_D. A similar concentration is required for one-half maximal enhancement of the effect of FGF. These results suggest that both the inhibitory and stimulatory effects of glucocorticoids may be mediated via these receptors, the different effects thus being due to differences in the intracellular events triggered by each growth factor.     

Stimulation of the release of two glycoproteins from mouse 3T3 cells by growth factors and by agents that increase intralysosomal pH

Peptide growth factors selectively increase the amount of mitogen-regulated protein (MRP) and major excreted protein (MEP) released by mouse 3T3 cells. $\text{Balb}\text{c}$ 3T3 cells release mainly MEP and Swiss 3T3 cells release mainly MRP. Fibroblast growth factor, epidermal growth factor, nerve growth factor, serum, and concanavalin A increase the extracellular appearance of both MEP and MRP, but to different extents. Several agents that have been shown to, or would be expected to increase, intralysosomal pH also selectively increase the release of MEP and MRP from both $\text{Balb}\text{c}$ and Swiss 3T3 cells. The effective agents are monensin, nigericin, ammonium chloride, methylamine, chloroquine, and high extracellular pH.     

Production and characterization of antibody blocking epidermal growth factor: Receptor interactions

Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 · 10⁶ epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes.The immonoglobulin G fraction of this immune sera inhibited ¹²⁵I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of ¹²⁵I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5°C).Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A-431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.     

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.