Apparent heterogeneity in the response of quiescent swiss 3T3 cells to serum growth factors: implications for the transition probability model and parallels with "cellular senescence" and "competence"

When subconfluent, Swiss 3T3 cells made quiescent by serum deprivation are stimulated with low concentrations of serum (ca. 1%), only a proportion of them (roughly 50%) enter S phase despite daily replacement with fresh, low-serum medium. The cells that fail to enter S phase are not incapable of doing so, since most of them initiate DNA synthesis after transfer to 10% serum. It would appear that individual cells vary in their growth factor requirements. Using time-lapse cinemicroscopy a few of the cells that respond to low serum were seen to give rise to several generations of progeny, while the majority of cells failed to divide at all, or divided once at most. Despite this, differences between cells in growth factor requirements do not seem to be heritable in the long term, since attempts to enrich for responding cells by prolonged culture in 1% serum have been unsuccessful. Rather, it would appear that the capacity to respond to low serum is an unstable property lost after a few generations in low serum. The loss of responsiveness shows parallels with "cellular senescence" and could conceivably result from decay of the platelet-derived growth factor-induced state of "competence." But regardless of why some cells respond to low serum while others do not, it is clear that the kinetics of entry into S phase after serum stimulation of quiescent 3T3 cells are not strictly first-order, since the labelling index plateaus after roughly 3 days at values substantially below 100%. As such, the kinetics, though not contradicting the transition probability model, cannot be taken to support it as was previously thought.     

Epidermal growth factor and the control of proliferation of Balb 3T3 and benzo[a]pyrene-transformed Balb 3T3 cells.

Benzo[a]pyrene-transformed Balb 3T3 cells (BP3T3) exhibit "normal" growth controls at low concentrations of serum. Epidermal growth factor (EGF) stimulates DNA synthesis and cell division in both Balb 3T3 and BP3T3 cells at physiological concentrations. The growth response of BP3T3 cells to EGF is qualitatively the same as that of 3T3 cells, however, the transformed cells have a lower quantitative requirement. Both 3T3 and BP3T3 cells show a density-dependent response to EGF, but the shift in the dose response curve for BP3T3 cells at high cell density is smaller than that seen for 3T3 cells. One cause of the restricted growth of 3T3 cells at high cell density compared with BP3T3 cells is the increased concentration of growth factor needed for stimulation of 3T3 cells at higher cell densities. A lower rate of depletion of other growth factory by BP3T3 cells may also explain the smaller effect of cell density on the EGF response of these cells.     

Apparent desensitization of Swiss 3T3 cells to the mitogens FGF and vasopressin

The growth factors FGF and vasopressin were found to have only a transient effect on confluent quiescent monolayers of Swiss 3T3 cells. Whether measured as cumulative entry into S-phase by autoradiography, or as cell division by time-lapse filming, the elevated rate of cell proliferation was maintained only over 10-15 hr. Several trivial or artifactual explanations for this transience were ruled out, including toxicity of 3H-thymidine; exhaustion or degradation of medium components, nutrients or growth factors (although some medium depletion was observed); and the generation during quiescence of cells incapable of division. We have also eliminated heritable variation in the capacity to respond to individual growth factors. However, unstable phenotypic heterogeneity in growth factor requirements between cells may play some part, as found elsewhere for the response to low concentrations of serum (Brooks et al, 1984). Cell populations that had ceased to respond to vasopressin recovered their sensitivity after 2-3 days' incubation in conditioned medium lacking vasopressin. The phenomenon thus resembles the mitogen-induced desensitization described by Collins and Rosengurt (1982, 1983). However, in our case, the loss of sensitivity was not selective for vasopressin but applied also to epidermal growth factor (EGF) and to prostaglandin F2 alpha. Furthermore, changes in responsiveness to vasopressin with time were associated with changes in cell density. Although some element of selective desensitization has not been ruled out, the transient response in our experiments can be accounted for in terms of unstable heterogeneity in growth factor requirements and/or in terms of density-dependent regulation of growth.     

Enhanced binding of fibronectin-coated latex beads to quiescent 3T3-L1 cells is correlated with escape from growth arrest

The possible involvement of fibronectin receptors in growth stimulation was investigated by an analysis of fibronectin-coated latex bead binding to 3T3-L1 cells under various conditions. 3T3-L1 cells, growth-arrested in a medium with a low concentration of calf serum, bound few fibronectin-coated beads. After addition of serum at concentrations of 1.0% or higher, there was a rapid and transient increase in the number of cells with bound beads and a subsequent increase in the incorporation of bromodeoxyuridine (BrdU) into cell nuclei. Incorporation of BrdU was observed in about 60% of the cells with bound beads. Fibroblast growth factor and platelet-derived growth factor at concentrations of 5 ng/ml or higher also enhanced binding of fibronectin-coated beads to cells. Stimulation of bead binding by epidermal growth factor and insulin was weak. Fibroblast growth factor, but not epidermal growth factor, increased the incorporation of BrdU into nuclei. These results indicate a relationship between stimulation of cell proliferation in quiescent cells and increased binding by cells of fibronectin-coated latex beads.     

CD44 protein levels and its biological activity are regulated in Balb/c 3T3 fibroblasts by serum factors and by transformation with the ras but not with the sis Oncogene

CD44s (standard isoform) levels and hyaluronan-binding activity were investigated in Balb/c 3T3 cells and their derivatives transformed with ras or sis oncogenes as a function of serum concentration in the medium. 3T3 cells contained low levels of CD44 and did not bind hyaluronan when grown in medium containing 0.5 or 10% serum. In 5% serum, however, the cells had much higher levels of CD44 and were able to bind hyaluronan. CD44 levels also increased in 3T3 cells restimulated with either 5 or 10% serum after prior maintenance in low serum. In cells restimulated with 5% serum, high levels of CD44 were sustained for at least 72 hr. In cells restimulated with 10% serum, however, the increase in CD44 levels reverted by 48 hr. Transformation of 3T3 cells with ras (but not with sis) oncogene rendered CD44 levels insensitive to serum modulation: ras-transformed cells contained high levels of CD44 and bound hyaluronan at all serum concentrations and at all time points tested. Sis-transformed cells behaved like 3T3 cells in these modulatory changes. Platelet-derived growth factor (PDGF), when supplementing 0.5% serum, mimicked the effects of serum on the levels and hyaluronan-binding capacity of CD44 in 3T3 cells and the CD44-upregulating activity of serum was neutralized by incubation with anti-PDGF antibodies. These data demonstrate that serum factors, specifically PDGF, mediate regulation of CD44 levels in Balb/c 3T3 cells and that transformation of 3T3 cells by ras renders CD44 expression insensitive to the modulating effects of serum in vitro. These results correlate with the metastatic capacity of these cells in vivo. © 1996 Wiley-Liss, Inc.     

Glycolysis in quiescent cultures of 3T3 cells. Stimulation by serum, epidermal growth factor, and insulin in intact cells and persistence of the stimulation after cell homogenization.

Addition of serum to quiescent cultures of 3T3 cells rapidly increases lactic acid formation and subsequently stimulates cell division. The stimulation of lactic acid production is seen at high, saturating concentrations of extra-cellular glucose. It is dependent on the time of exposure and on the dose of serum and is not blocked by the addition of cycloheximide, puromycin, or actinomycin D. In contrast, serum only marginally affects glycolysis by rapidly growing 3T6 or SV40-3T3 cells. In addition to serum, epidermal growth factor (0.1 to 10 ng/ml) and insulin (10 to 500 ng/ml) cause a striking stimulation of glycolysis in quiescent 3T3 cells. Neither exogenous cyclic nucleotides nor ouabain effect the glycolytic response, but the presence of Ca2+ markedly influences the activation of glycolysis by epidermal growth factor and by insulin. A novel finding in this study is that homogenates prepared from quiescent cells treated with serum, epidermal growth factor, or insulin show increased glycolysis as compared with homogenates from nonstimulated cultures. This finding will allow further experimental analysis of the cause of increased glycolysis in rapidly proliferating cells.     

Reduction of lactic acid secreted from SV3T3 cells by a serum factor and biotin and its relationship to cell growth

Supplementation of media containing a low concentration (0.15–0.30% $\text{v}\text{>v}$) of calf serum with biotin or a low molecular weight serum growth factor (Peak III) reduces the amount of lactic acid secreted by simian virus 40-transformed 3T3 cells. While biotin and Peak III (which has been tentatively identified as biotin) can stimulate “stationary phase” cells to resume viable cell division, this growth promotion is not due to an alleviation of lactic acid toxicity per se. This conclusion is based on the finding that, although higher concentrations of lactic acid are cytotoxic, lactic acid added at concentrations found during “stationary phase” to cells plated in fresh medium is not growth inhibitory. These results suggest, instead, a possible major role for biotin and Peak III in energy production.     

G1 phase heterogeneity in exponentially growing Swiss 3T3 mouse fibroblasts

The growth rate of normal cultured Swiss 3T3 fibroblasts is function of serum concentration and the fraction of G1 cells, and hence the average residence time in G1, increases with the generation time. Serum contains two sets of factors: competence factors, essentially platelet-derived growth factor (PDGF), which induces competence in quiescent fibroblasts and prevents replicating cells from entering G0, and plasma, which allows progression. The increase in the duplication time and the duration of Gl at low serum concentration could hence be due to the fact that competence factors become limiting. The fraction of non-competent cells, operationally defined as those G1 cells unable to leave G1 in the presence of plasma alone, was determined in populations exponentially growing at serum concentrations between 5 and 20%. To do so exponentially growing cultures were shifted to plasma plus colcemid: one part of the cell population progressed through the cycle and accumulated with a G2 DNA content, whereas non-competent cells remained in G1. Analysis of the DNA distributions performed 24 h after the shift showed that as serum concentration was lowered more cells were found in the non-competent state: they were less than 5% in 20% serum and almost 50% in 5% serum. The non-competent cells constitute a dynamic fraction of the population, since in the presence of serum they can leave Gl and progress in the cycle. These data indicate that one of the steps limiting exponential growth is the acquisition of competence and that this event gives rise to heterogeneity within the G1 population.     

Effects of bovine platelet-poor plasma on the proliferation of normal and transformed BALB/c 3T3 cells

Summary Platelet-poor plasma, as well as autologous platelet-rich serum, was prepared from freshly-drawn bovine whole blood. Bovine platelet-poor plasma had properties similar to those previously decribed for human platelet-poor plasma; e. g., it would (a) support the growth of virally transformed but not normal BALB/ c 3T3 cells, (b) act synergistically with either partially purified platelet-derived growth factor or fibroblast growth factor to initiate cell replication in quiescent 3T3 cells, and (c) act sequentially with platelet-derived growth factor to initiate 3T3 replication. It appears that bovine serum contains both competence and progression factors and that stimulation of fibroblasts with bovine serum involves at least two sequential stages analogous to those described for stimulation with human serum.     

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.     

Survival of 3T3 cells expressing or co-expressing bFGF and/or IGF-I and/or IGF-II in low serum and serum free media

The aim of this study was to improve the survival ofNIH3T3 cells in low serum or serum-free media by theendogenous expression of recombinant bFGF (basicfibroblast growth factor) and/or IGF (insulin-likegrowth factor) -I and II. Expression was detected byWestern blotting, and the growth characteristics ofdifferent transfected cell lines investigated in bothserum-free and low serum conditions and also in softagar. Morphological changes and the growth rate werecompared with growth in normal serum-containingmedium. The experimental data suggested that theexpression of either bFGF alone, or the co-expressionof bFGF and either IGF-I or II could improve thesurvival of NIH3T3 cells in low serum or serum-freemedia. The use of such lines could decrease the use ofserum in cell culture and thus both reduce the costsinvolved in this technique and simplify thedown-stream purification procedure in protein harvest.Hence, such lines may be of value in both experimentaland industrial applications.     

Growth effects of lithium chloride in BALB/c 3T3 fibroblasts and madin-darby canine kidney epithelial cells

The ability of LiCl to initiate DNA synthesis was studied in Madin-Darby canine kidney (MDCK) cells, and mouse BALB/c 3T3 fibroblasts. In a defined culture medium lacking serum, LiCl increased DNA synthesis in BALB/c 3T3 cells 100–200% over control values. Maximum DNA synthesis was observed with concentrations of LiCl between 10 and 25 mM and increases from 40–50% over control were observed with concentrations as low as 1 mM. Exposure of BALB/c 3T3 cultures to LiCl resulted in an increase in the percentage of cells initiating DNA synthesis, total DNA content and cell number. Lithium chloride, in combination with insulin or epidermal growth factor (EGF), had either an additive or synergistic effect upon the growth of BALB/c 3T3 fibroblasts. MDCK cells proved refractory to the growth actions of LiCl, although they responded to EGF and insulin with increased DNA synthesis. Lithium chloride appears to have a direct effect on cell proliferation in some but not all cell types.     

A partially purified serum fraction synergistically enhances the mitogenic activity of epidermal growth factor and insulin in quiescent cultures of 3T3 cells.

Epidermal growth factor and insulin need a low concentration of serum to effectively stimulate quiescent 3T3 cells into DNA synthesis. We partially purify a polypeptide component of serum which has no activity by itself but which acts synergistically with epidermal growth factor and insulin to stimulate cultures of 3T3 cells into DNA synthesis as effectively as whole serum. The active fraction is separated from serum by gel chromatography on Sephadex G-100, under acid dissociating conditions, and chromatographs with a molecular weight of 18,000 daltons.We suggest as a general technique, the use of pure growth factors to assay for growth promoting fractions from serum or other sources. Fractions which are not mitogenic by themselves can be detected when assayed together with their complementary pure factors.     

Binding, internalization, and degradation of epidermal growth factor by balb 3T3 and BP3T3 cells: relationship to cell density and the stimulation of cell proliferation.

Epidermal growth factor (EGF) stimulates the growth of both benzo[a]pyrene-transformed Balb 3T3 cells (BP3T3) and untransformed Balb 3T3 cells. We describe here the binding, internalization, and degradation of [125I]-EGF by BP3T3 cells and 3T3 cells. Binding of [125I]-EGF reaches a maximum after 45 to 90 minutes incubation at 37 degrees C. In both BP3T3 and 3T3 cells the extent of EGF binding required to stimulate DNA synthesis is density dependent; sparse cultures require a 15-30% occupancy to elicit a maximal response whereas dense cultures require a 70-85% occupancy. At physiological concentrations the total binding of [125I]-EGF to 3T3 cells is higher than to BP3T3 cells, and this difference increases at higher cell densities. The rate of degradation of [125I]-EGF is directly proportional to the total [125I]-EGF binding in each cell type. This supports the hypothesis that one cause of the diminished serum requirement of BP3T3 cells is a reduced rate of utilization of serum growth factors.     

Dihydrocytochalasin B disorganizes actin cytoarchitecture and inhibits initiation of DNA synthesis in 3T3 cells

Dihydrocytochalasin B (H2CB) disrupts the actin structure of Swiss/3T3 mouse fibroblasts and inhibits the ability of serum growth factors to stimulate DNA synthesis in quiescent cultures. Low doses of H2CB (2-10 X 10(-7) M) added to serum-arrested cells reversibly block initiation of DNA synthesis by serum; by epidermal growth factor and insulin; or by epidermal growth factor, fibroblast growth factor and insulin. H2CB is effective only when added to cells within 8-10 hr after stimulation. Low doses of H2CB cause cell rounding and a loss of actin microfilament bundles, but they do not interfere with glucose or thymidine transport. These results suggest that stimulation of 3T3 cells involves at least one obligatory actin-mediated step. Transformed cells appear to obviate this step, for H2CB does not inhibit the entry into S phase of SV40-transformed or Moloney murine sarcoma virus-transformed 3T3 cells synchronized by mitotic shake-off.     

The suppression of cellular proliferation in SV40-transformed 3T3 cells by glucocorticoids

Glucocorticosteroids, when added two hours after cell plating to SV40-transformed, 3T3 mouse fibroblasts in low serum (0.3% v/v), biotin-supplemented medium, suppress cellular proliferation by 24 hours. While some cell death probably occurs, the growth inhibition is not primarily due to cytotoxicity and cytolysis. This conclusion is supported by the following: 1) both dead and viable cell numbers are suppressed, 2) little cell debris is evident in the medium, and 3) very high concentrations of glucocorticoids do not cause an increase in the dead cell count. Furthermore, this growth suppression, which is specific for glucocorticoids since several non-glucocorticoid steroids have no inhibitory effect, is not permanent nor irreversible. Removal of the glucocorticoid and replacement with 10% serum restore rapid proliferation. Although higher concentrations (1% and 10%) of serum afford some protection against glucocorticoid inhibition, this protection is not simply a consequence of faster growth rates. SV3T3 cells can be grown in serum-free medium supplemented with biotin, transferrin, insulin, and epidermal growth factor (EGF). Under these conditions growth rates are comparable to high serum media, yet glucocorticoids are still powerful inhibitors. However, the omission of insulin from serum-free, glucocorticoid cultures does result in observable cell death and lysis. Flow microfluorometry and autoradiographic studies have determined that glucocorticoid-inhibited cells are partially blocked in G1. The proportions of S phase and G2 + M cells are greatly reduced with an accompanying accumulation of G1 cells. These results suggest that glucocorticoids regulate a biochemical step(s) in G1 which is critical for DNA initiation.     

Differential mitogenic effects of methyl isobutyl xanthine and a tumor growth factor on G1-arrested 3T3 T proadipocytes at the predifferentiation GD state and the growth-factor deficiency GS state

Two growth-states exist in the G1 phase of the 3T3 T proadipocyte cell cycle. GD is the arrest state at which proadipocytes must growth-arrest prior to differentiation. GS is the arrest state at which proadipocytes growth-arrest following deprivation of serum or growth factors. In an attempt to further distinguish these arrest states, we have compared the relative ability of a variety of mitogens to induce GD- and GS-arrested cells to initiate DNA synthesis. The data show that GD-arrested cells at both high and low densities can be induced to proliferate by methyl-isobutyl-xanthine (MIX), whereas high and low density GS-arrested cells are not. The data also show that a tumor growth factor can stimulate the proliferation of both high and low density GD- and GS-arrested cells, whereas other agents are poor mitogens for high density GD-arrested cells. We conclude that MIX and a tumor growth factor (TUGF) can serve as probes to study the characteristics of the GD arrest state.     

Role of serum in the developmental expression of alkaline phosphatase in MC3T3-E1 osteoblasts

MC3T3-E1 cells in culture exhibit a temporal sequence of development similar to in vivo bone formation. To examine whether the developmental expression of the osteoblast phenotype depends on serum derived factors, we compared the timedependent expression of alkaline phosphatase (ALP)-a marker of osteoblastic maturation- in MC3T3-E1 cells grown in the presence of fetal bovine serum (FBS) or resin/charcoal-stripped (AXC) serum. ALP was assessed by measuring enzyme activity, immunoblotting, and Northern analysis. Growth of MC3T3-E1 cells in FBS resulted in the programmed upregulation of alkaline phosphatase (ALP) post-proliferatively during osteoblast differentiation. In the presence of complete serum, actively proliferating cells during the initial culture period expressed low ALP levels consistent with their designation as pre-osteoblasts, whereas postmitotic cultures upregulated ALP protein, message, and enzyme activity. In addition, undifferentiated early cultures of MC3T3-E1 cells were refractory to forskolin (FSK) stimulation of ALP, but became forskolin responsive following prolonged culture in FBS containing media. In contrast, MC3T3-E1 cells grown in AXC serum displayed limited growth and failed to show a time-dependent increase in alkaline phosphatase. Neither the addition of IGF-I to AXC serum to augment cell number or plating at high density restored the time-dependent upregulation of alkaline phosphatase. Cells incubated in AXC serum for 14 days, however, though expressing low alkaline phosphatase levels, maintained the capacity to upregulate ALP after FBS re-addition or forskolin activation of cAMP-dependent pathways. Such time-dependent acquisition of FSK responsiveness and serum stimulation of ALP expression only in mature osteoblasts indicate the possible presence of differentiation switches that impart competency for a subset of osteoblast developmental events that require complete serum for maximal expression. © 1994 Wiley-Liss, Inc.     

The partial replacement of the serum growth factor requirement of SV3T3 cells by lactalbumin hydrolyzate.

Lactalbumin hydrolyzate (LH), a commercially prepared, enzymatic digest of a milk protein fraction, can partially replace the serum requirement of SV3T3 cells. In a low, but obligatory, background of calf serum (0.15% v/v), LH carses a large increase in the final cell density (5-10 x over the control) while modestly steimulating the actual growth rate. Lactalbumin hydrolyzate does not contain survival activity for SV3T3 cells and does not affect the growth of 3T3 cells. Since even prolonged exposure to pH 4 or 2 results in complete abolishment of the growth-rate stimulatory capability without affecting the capacity to increase the final cell density, it is possible that the LH growth activity may consist of two dissociable components. All of LH growth activity is water soluble, autoclavable, and resists proteolytic treatment. On Sephadex G15 growth activity appears as a single peak at the void volume but on G25 it is retained beyond the void volume as a broad, skewed peak. The relevant molecular weight range lies between 1,500 and 4,000. The putative low pH resistant material is strongly adsorbed to Dowex 1 x 2 and can be displaced from the column by a reduction in the pH. A comparison of the properties of the LH factors with those of known growth promoting agents isolated from serum and various enzymatic digests indicates that these new factors do not correspond to any of these agents.