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.     

Enhanced rates of fluid pinocytosis during exponential growth and monolayer regeneration by cultured arterial endothelial cells

Rates of fluid pinocytosis by bovine aortic endothelial cells were measured during various manipulations of growth status in vitro. Sparsely seeded cultures grew exponentially until a confluent monolayer was formed, at which time growth slowed. This change in growth rate coincided with a decline in the rate of pinocytosis to about one-third that in the growing cultures. During the subsequent attainment of maximal cell density in the confluent monolayer, the pinocytic rate remained constant. There was close correlation between ³H-thymidine labelling indices, as measured by autoradiography, and the rates of pinocytosis. Mechanical “wounding” of the confluent monolayer resulted in cell migration and proliferation. Twenty-four hours after “wounding,” rates of pinocytosis per mg. cell protein were significantly enhanced. When regeneration of the monolayer was blocked by cytochalasin B, pinocytosis remained at the same rate as in the uninjured, confluent monolayer. These experiments support, and extend to endothelium, earlier observations that in growing cells pinocytosis proceeds at a higher rate than in non-growing, quiescent cells. Furthermore, they raise the possibility that the transendothelial transport of macromolecules such as lipoproteins by receptor-in-dependent fluid pinocytosis in vivo may be altered by the growth status of the endothelium.     

Collagen synthesis by monkey arterial smooth muscle cells during proliferation and quiescence in culture

Monkey arterial smooth muscle cells (SMC) which are stimulated to proliferate in the presence of 5% monkey blood serum (MBS) and which remain quiescent in 5% monkey platelet-poor plasma serum (MPPPS) were examined for their ability to synthesize collagen in each of these conditions in culture. Collagen synthesis was measured by determining amounts of newly formed labeled hydroxyproline, following labelling in the presence of [³H]proline and ascorbic acid. Ascorbate requirements of SMC were examined to assure maximal hydroxylation. SMC synthesize the same amount of collagen/cell in 5% whole blood serum (MBS) during the early phase of rapid proliferation as during slow growth in later phases in culture. SMC grown in the presence of serum-lacking platelet factors synthesize 60–90% less collagen and 60–90% less non-collagen protein (per cell or per mg protein) than cells grown in MBS. Non-collagen protein synthesis was measured as incorporation of both [³H]proline and of [³H]leucine, determined as trichloroacetic acid (TCA)-precipitable material. Previous studies indicate that a factor derived from platelets is the principal mitogen present in whole blood serum for diploid cells such as SMC and fibroblasts in culture. Similarly derived factors are potent stimulators of both collagen and non-collagen protein synthesis by SMC. SMC, quiescent in medium lacking platelet derived material (MPPPS), is being used to investigate factors important in SMC proliferation since this is a significant event in atherogenesis in vivo. An increased deposition of collagen also occurs during atherogenesis. Consequently it will be useful to employ similar cultures of quiescent SMC to examine agents which affect production of this connective tissue matrix protein.     

Effect of cultivation conditions on the growth properties of Swiss 3T3 cells

A possibility of using Swiss 3T3 cells, adapted to the growth in the Eagle basal medium and bovine serum, in studies of cell proliferation and quiescent state was shown on the basis of their growth characteristics. Proliferative activity of cultures was estimated by measuring the intensity of DNA synthesis (incorporation of labeled thymidine and flow cytofluorometric analysis), mitotic index and cell number counts. Growth rate and saturation density of the culture were analyzed depending on serum concentration, substrate quality and medium changes both in growth and quiescent states. In spite of repeated medium changes such adapted cells had saturation density within 4.10(4)--7.10(4) cells/cm2, standard for this line. Besides, a distinct inhibition of cell proliferation at confluence or after incubation with low serum (0.5%) and a possibility of the following stimulation of cell divisions by adding a fresh medium containing different concentrations of serum were demonstrated. The increased rate of adipose conversion was detected in resting confluent 3T3 cells cultivated in closed vessels, as compared to cells growing in tissue culture dishes in the CO2 incubator.     

Heparin inhibition of smooth muscle cell proliferation: a cellular site of action

The potential of a given amount of heparin to inhibit smooth muscle cell (SMC) proliferation can be increased more than 13 fold if quiescent cultures are pretreated with this mucopolysaccharide for 48 h. The large increase in antiproliferative activity was attributable to a 74% inhibition of the first cell cycle traverse of SMC after serum addition. If the mucopolysaccharide was added to SMC coincident with serum, the initial cell cycle traverse was only suppressed by 27%. In both heparin pretreated and nonpretreated SMC cultures, 48 to 72 h elapsed before substantial inhibition was observed. The inhibitory effects of heparin were reversible and inversely proportional to the starting cell density of the cultures. The effects of known heparin binding proteins on the inhibitory capability of heparin were examined. Neither platelet-derived growth factor (PDGF), low density lipoprotein (LDL), nor platelet factor 4 (PF4) were able to reduce the antiproliferative effects. Heparin retained full biological activity in medium containing serum depleted of all heparin binding proteins by heparin-Sepharose chromatography. These results indicate that heparin does not inhibit growth by preventing serum mitogens or nutrients from interacting with SMC. Rather, our data suggest that heparin is slowly internalized by SMC following binding to specific, non-PF4 dissociable sites. Heparin may accumulate intracellularly and block a crucial point in the proliferative machinery of SMC.     

Demonstration of stimulatory effects of platelet-derived growth factor on cultivated rat arterial smooth muscle cells : Differences between cells from young and adult animals

The effects of platelet-derived growth factor (PDGF) on DNA synthesis and proliferation in cultures of arterial smooth muscle cells obtained from young and adult rats, respectively, were measured. Addition of 10-20 ng/ml of PDGF to medium MCDB 104 induced DNA synthesis in quiescent cultures of cells from young animals to a similar extent as 10-20% whole blood serum (WBS). PDGF further stimulated proliferation of the cells in medium MCDB 104, although less markedly than 10% WBS. Antibodies against PDGF partially inhibited the growth response after stimulation with serum. This shows that PDGF is a major growth factor in serum for these cells and that PDGF can promote entrance into and passage through S phase and mitosis independent o plasma factors. Cells from adult animals were also found to respond to PDGF, although a higher concentration (25 ng/ml) was required to obtain a maximum effect. These cells, however, responded better than cells from young animals to stimulation with serum. Further, antibodies against PDGF did not inhibit the growth-stimulatory effect of serum to any appreciable extent. Thus, serum contains growth factors other than PDGF that stimulate preferentiaLly the proliferation of smooth muscle cells from adult animals.     

Effects of collagen gel configuration on behavior of vascular smooth muscle cells in vitro: Association with vascular morphogenesis

Summary The growth, behavior, and contractile protein expression of rabbit aortic smooth muscle cells (SMC) grown on, between layers, or within a collagen gel was investigated by confocal laser scanning fluorescence microscopy and Western analysis. SMC grown on collagen gel behaved similarly to those on conventional culture dishes. However, when a second layer of collagen was overlaid, cells underwent an elongated quiescent phase before onset of proliferation and a more than threefold lower logarithmic growth rate was observed. These cells self-organized into a network with ring-like structures. With increasing culture time, some of the rings developed into funnel-like, incomplete or complete tubular structures. If a tubular template preexisted within the gel, the SMC established a cylinder-shaped tube with several circularly arranged muscular layers (similar to an artery wall). This behavior mimicked endothelial cells during angiogenesis in vitro. A similar phenomenon occurred in cultures in which SMC were randomly mixed in a collagen gel, but here their behavior and morphology varied with their position within the gel. Western blot analysis showed that the SMC differentiation marker, smooth muscle myosin heavy chain-2 (SM-2), rapidly decreased, disappearing by day 10 in SMC grown on collagen, but was still detectable until day 25 in cells cultured between or within the same gel. These findings indicate that like endothelial cells, vascular SMC can display blood vessel formation behavior in vitro when an appropriate three-dimensional matrix environment is provided to keep them in a relatively higher-differentiated and low-proliferative state.     

A high level of cell surface phosphatidylinositol-specific phospholipase C activity is characteristic of growth-arrested 3T3 fibroblasts but not of transformed variants.

Confluent monolayers of four contact-inhibited mouse fibroblast lines (Swiss 3T3, Balb/c 3T3, NIH 3T3, and C3H10T1/2) were found to have substantial levels of a cell surface phosphatidylinositol-specific phospholipase C (ecto-PLC). In contrast, confluent cultures of virally, chemically, or spontaneously transformed variants derived from these cell lines expressed undetectable or negligible levels of this enzyme activity. A simple and rapid assay, using lysophosphatidylinositol radio-labeled in the inositol group ([3H]-lysoPI) as the substrate was developed to provide a quantitative measure of the phospholipase C activity present at the external cell surface. For cells testing positive for ecto-PLC activity, rapid uptake of [3H]-lysoPI is accompanied by the simultaneous appearance of [3H]-inositol phosphate in the external medium. Confluent monolayers of the four mouse fibroblast lines exhibiting density-dependent growth inhibition had levels of ecto-PLC activity in the range of 50-800 pmol/min/10(6) cells (i.e., about 20-50 times greater than the activity observed for the transformed variants). The expression of ecto-PLC activity at the cell surface of the Swiss or Balb/c cells was dependent on the state of cell proliferation. Cultures which had become quiescent through attainment of confluence displayed a tenfold increased activity over that of subconfluent, growing cultures of these cells. Similarly, subconfluent Swiss 3T3 cells which had become quiescent following exposure to low serum conditions also showed increased activity. These results indicate that there may exist a correlation between the control of cell proliferation in contact-inhibited mouse fibroblasts and the expression of inositol phospholipid-specific phospholipase C activity at the external cell surface.     

Regulation of human amnion cell growth and morphology by sera,plasma, and growth factors

Summary The requirements of human epithelial cells derived from the amnion membrane for serum factors were investigated. The growth promoting effects of human whole blood serum (WBS), platelet-poor defibrinogenated plasma, and plasma-derived serum (PDS) were examined in primary cultures of these ectodermal cells. The numbers of population doublings recorded after 10 days in the presence of 10% WBS, defibrinogenated plasma, or PDS were 2.3, 2.0 or 1.5, respectively. Although dialysis of sera or plasma had little effect on growth promotion, it markedly decreased the capacity of plasma to maintain cells in culture beyond 10 days. The differences in growth activities could not be attributed to the presence of anticoagulant in plasma and PDS or to the presence of excess calcium in PDS. Platelet lysates and purified platelet-derived growth factor had no effect on growth. Amnion cell growth was enhanced by epidermal growth factor (EGF) or hydrocortisone, but the glucocorticoid did not condition cells to respond to growth factors. Insulin and fibroblast growth factor singly or in combination had no effect on cell replication. Giant cell formation accompanied maintenance in hydrocortisone with defibrinogenated plasma and PDS. Discrete regions of dense population appeared in the presence of hydrocortisone, EGF, and undialyzed supplements.Supported in part by ACS grant PDT-140     

Uridine transport and phosphorylation in mouse cells in culture: effect of growth-promoting factors, cell cycle transit and oncogenic transformation

The rapid increase in uridine uptake produced by the addition of serum to quiescent cultures of fibroblasts is primarily caused by an enhanced rate of nucleoside phosphorylation. While quiescent and serum-stimulated cells display identical initial rates of transport, they show a considerable change in the composition of the acid-soluble pools labelled with [3H] uridine for five seconds. The radioactivity recovered in the phosphorylated pools increases 2-, 3-, 4- and 6-fold after addition of serum to cultures of Swiss 3T3 cells, tertiary mouse embryo fibroblasts, Swiss 3T6 and Balb 3T3, cells respectively. Furthermore, insulin, a growth factor isolated from medium conditioned by SV40 BHK cells (FDGF) and epidermal growth factor (EGF) also stimulate uridine phosphorylation within minutes. The initial rate of uridine uptake is 2- to 3-fold faster in rapidly growing normal and Simian virus 40 or polyoma virus transformed 3T3 cells as compared to untransformed 3T3 cells in the quiescent state. When quiescent cultures of 3T3 or mouse embryo cells are stimulated to leave G1 and enter into DNA synthesis, transport increases several hours after addition of serum and apparently coincides with the S phase of the cell cycle. The results demonstrate that an increase in uridine phosphorylation is a rapid metabolic response elicited by growth-promoting agents in a variety of cell types and that uridine transport and phosphorylation are independently regulated.     

Stimulation of human vascular endothelial cell growth by a platelet-derived growth factor and thrombin

Repair of a vascular wound is mediated by migration and subsequent replication of the endothelial cells that form the inner lining of blood vessels. We have measured the growth response of human umbilical vein endothelial cells (HuE) to two polypeptides that are transiently produced in high concentrations at the site of a wound; the platelet-derived growth factor (PDGF) and the protease thrombin. When 10⁴ HuE cells are seeded as a dense island (2-mm diameter) in the center of a 16-mm tissue culture well in medium containing 20% human serum derived from platelet-poor plasma (PDS), no increase in cell number or colony size is observed. With the addition of 0.5 ng/ml partially purified PDGF, colony size increases and the number of cells after 8 days is 4.8 × 10⁴. When human thrombin (1 μg/ml) is added along with the PDGF, the cell number rises to 9.2 × 10⁴. Thrombin alone stimulates no increase in cell number. Although partially purified PDGF stimulates endothelial cells maintained in PDS as well as those maintained in whole blood serum (WBS), pure PDGF is active only when assayed in medium that contains WBS and is supplemented with thrombin. These results suggest the existence of a second class of platelet-derived factors that enable HuE cells to respond to the mitogenic activity of the purified platelet mitogen and thrombin.     

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

Growth factor contents of autologous human sera prepared by different production methods and their biological effects on chondrocytes

To discuss the autologous serum production for cartilage tissue engineering, we compared three kinds of sera: whole blood-derived serum (WBS), platelet-containing plasma-derived serum (PCS), and plasma-derived serum (PDS), on the growth factor contents and their biological effects on human auricular chondrocytes. EGF, VEGF and PDGF levels were highest in WBS, while PCS and PDS followed WBS. The proliferation effects of WBS were the most pronounced, followed by that of PCS, both of which realized a 1000-fold-increase in chondrocyte numbers at the third passage, whereas PDS reached it after passage 4. No significant differences were observed in histology or cartilaginous matrix measurements of tissue-engineered cartilage produced from chondrocytes cultured under different serum conditions. WBS would be clinically useful because of its potent proliferation effects, while PCS, which possibly saves the red cell concentrate, may be an option in cases where there are elevated risks of blood loss.     

Growth-mediated, density-dependent inhibition of endocytosis in cultured arterial smooth muscle cells

The effects of cell density and growth upon fluid phase endocytosis were investigated in quiescent and growing cultures of monkey arterial smooth muscle cells. Cells were maintained in a quiescent state of growth in 5% plasma-derived serum. Subsequent exposure of subconfluent cultures to the specific mitogens, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), or to whole blood serum, resulted in up to 4-fold increases in the rate of fluid endocytosis/cell. The changes began several hours after entry into G1 phase of the cell cycle and continued through S. The fraction of cells entering the growth cycle was variable (PDGF=FGF>EGF) and a close correlation existed between the rate of endocytosis and the fraction of [³H]thymidine-labelled cells (r = 0.929, p<0.01). At a range of cell densities, the rate of fluid endocytosis/cell was similar in sparse, confluent and post-confluent cultures of quiescent cells; in contrast, in growing cells there was density-dependent inhibition of endocytosis. Furthermore, when quiescent cells were in contact with each other and were then exposed to mitogens, the growth response was diminished and there was only a 25–50% increase in the rate of endocytosis, even in the presence of high concentrations of growth factors.These studies indicate that the influence of cell density upon fluid endocytosis in arterial smooth muscle cells is indirect in that it represents a secondary effect of decreased mitogenic response to specific growth factors.     

Different Growth Responses of Hamster Dermal Fibroblasts and Chondrocytes to Platelet–derived Growth Facter in Cell Culture

The growth responses of dermal fibroblasts and chondrocytes obtained from 1-week-old hamsters to a growth factor from platelets [platelet-derived growth factor (PDGF)] were compared. Autoradiography showed that most of the whole nuclei of dermal fibroblasts were labeled with ³H-thymidine in 5% whole blood serum (WBS), which contains platelet releasate, but only a few percent were labeled in 5% plasma-derived serum (PDS), which is free of platelet releasate. Addition of platelet releasate to PDS restored the growth stimulatory activity of the serum for dermal fibroblasts. In contrast, most of the nuclei of chondrocytes were labeled with ³H-thymidine both in 5% WBS and 5% PDS. Dermal fibroblasts grew in 5% WBS, but not in 5% PDS, whereas chondrocytes grew in both 5% WBS and 5% PDS. Partially purified PDGF added to the medium with 0.5% fetal calf serum stimulated DNA synthesis in dermal fibroblasts, but not in chondrocytes.     

Hexose uptake and control of fibroblast proliferation

The role of glucose uptake in control of cell growth was studied by experimentally varying the rate of glucose uptake and examining the subsequent effect on initiation and cessation of cell proliferation. The rate of glucose uptake was varied by adjusting the concentration of glucose in the culture medium. This permitted analysis of two changes in rate of glucose uptake which are closely related to the regulation of cell growth: (1) the rapid increase in glucose uptake that can be detected within several minutes after mitogenic stimulation of quiescent fibroblasts and (2) the decrease in glucose uptake which accompanies growth to a quiescent state. Quiescent cultures of mouse 3T3, human diploid foreskin and secondary chick embryo cells were switched to fresh serum-containing medium with either the normal amount of glucose or a reduced level that lowered the rate of glucose uptake below the rate characteristic of quiescent control cells. The subsequent increases in cell number were equal in both media, demonstrating that the increase in glucose uptake, commonly observed after mitogenic stimulation, was not necessary for initiation of cell division. Measurements of intracellular D-glucose pools after serum stimulation of quiescent cells revealed that the increase in glucose uptake was not accompanied by a detectable change in the intracellular concentration of glucose. Nonconfluent growing cultures of mouse 3T3, human diploid foreskin and secondary chick embryo cells were switched to low glucose media, lowering the rate of glucose uptake below levels observed for quiescent cells. This did not affect rates of DNA synthesis or cell division over a several-day period. Thus, the decrease in glucose uptake, which usually occurs at about the same time as the decrease in DNA synthesis as cells grow to quiescence, does not cause the decline in cell proliferation. Experiments indicated that there was no set temporal relationship between the decline in glucose uptake and DNA synthesis as cells grew to quiescence. The sequence was variable and probably depended on the cell type as well as culture conditions. Measurements of intracellular D-glucose pools in secondary chick embryo cells demonstrated that the internal concentration of glucose in these cells did not significantly vary during growth to quiescence. Taken together, our results show that these fluctuations in the rate of glucose uptake do not lead to detectable changes in the intracellular concentration of glucose and that they do not control cell proliferation rates under usual culture conditions.     

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E2 (PGE2) on fibroblast proliferation was examined. The presence of PGE2 for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [3H]thymidine occurred at concentrations greater than 10(-7) M. The inhibitory effect of PGE2 was less potent in exponentially growing cells and was not the result of conversion of PGE2 to PGA2 during incubation in growth medium. The G1 phase was determined to be 12-14 h in untreated cultures. The extent of growth inhibition by PGE2 was similar with addition of PGE2 at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE2-induced growth inhibition. Short-term exposure to PGE2 (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE2 affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE2 for 24 h, indicated that release from PGE2 exposure is associated with prolongation of the G1 phase of the cell cycle.     

Variation in the length of the lag phase following serum restimulation of mouse 3T3 cells

We have investigated the length of the lag phase (time taken for the first cells to enter S phase) and the kinetics of entry into DNA synthesis after serum restimulation of Swiss mouse 3T3 cell cultures that were allowed to become quiescent under different conditions. Cells were allowed to reach quiescence as a confluent monolayer in medium containing 10% (v/v) calf serum. Alternatively, when serum was reduced to 1% (v/v), cultures became quiescent at about 30% confluency and there was little cell to cell contact. The results show that the lag, or prereplicative phase becomes longer as the time spent in the quiescent state increases. This is the case in both confluent and non-confluent cultures. The rate of entry of cells into the S phase, however, remains the same under all conditions.     

Amine inhibition of serum-induced DNA synthesis in 3T3 and 3T6 cell cultures

Several amines were shown to inhibit growth stimulation in quiescent confluent and sparse cultures of Swiss 3T3 and 3T6 cells by changing for a fresh medium containing 10-20% serum. Proliferation was inhibited by dansylcadaverine (0.1 mM), chloroquine (0.05 mM), 5-methoxytryptamine (0.1 mM), cystamine (0.1 mM), dimethylurea (100 mM), methylurea (100 mM), and in some experiments--by urea (100 mM). The inhibitory activity was not associated with a direct influence of amines on DNA synthesis or thymidine phosphorylation. The findings suggest that amines may influence the process of clustering of growth factor-receptor complexes, or the fusion of plasma membrane and intracellular vesicles containing some components required for cell proliferation.     

Early effect of growth factors (EGF + insulin) upon ATP turnover in 3T3 cells. Inhibition by cytochalasin B and D

We have demonstrated previously a rapid increase in ATP turnover soon after adding epidermal growth factor (EGF) and insulin to quiescent cultures of Swiss 3T3 cells. In the present work, we tried to determine whether this increase could be correlated with the early stimulation by growth factors of cell movements. We showed that cytochalasin B (CB), in complete or glucose-free medium, inhibited this early increase caused by growth factors, in phosphate incorporation in small organic acid-soluble compounds (Po). Cytochalasin D (CD) specifically inhibited the stimulation caused by growth factors of Po labelling and ATP turnover, but lacked all inhibitory effect on unstimulated cells. The inhibitory effect of CD was transient. We hypothesize that addition of EGF and insulin to quiescent 3T3 cell cultures induces a rapid and transient change in cell movements, which could be responsible for about half of the early increase in ATP degradation and turnover.