Cyclic AMP does not mediate inhibition of DNA synthesis by interferon in mouse Swiss 3T3 cells

The purpose of this study was to determine whether cyclic AMP (cAMP) plays any direct or indirect role in the antiproliferative effect of mouse L-cell interferon in Swiss 3T3 cells. Firstly, we found that interferon did not affect intracellular levels of cAMP in these cells in the absence or the presence of cAMP-elevating agents. Secondly, we examined the effect of interferon on the stimulation of DNA synthesis of quiescent 3T3 cells by a range of cyclic AMP-elevating agents, including cholera toxin, cAMP derivatives, and prostaglandin E, added in the presence of insulin or vasopressin. Interferon inhibited cyclic AMP-stimulated DNA synthesis as measured by incorporation of radioactive thymidine into acid-insoluble material and autoradiographic analysis of the fraction of labelled cells. Dose-response curves and kinetics of inhibition were identical to those obtained in cultures stimulated by combinations of growth factors that do not increase the intracellular level of cAMP. The inhibition by interferon of cAMP-stimulated DNA synthesis was also observed in secondary cultures of mouse embryo fibroblasts, where cAMP-elevating agents provide a mitogenic signal in the absence of other added growth factors. These results show that the inhibitory effect of interferon on DNA synthesis in Swiss 3T3 cells is not mediated by cyclic AMP.     

Regulation of cell proliferation inhibitory and stimulatory factors diffused by 3T3 cultured cells

The growth rate of normal cells multiplied in vitro decreases as the cell density of the culture increases. Previous results suggested that this density-dependent inhibition of growth in nontransformed cells was due to the diffusion of growth inhibitory substances in the medium of dense cultures. In this paper, we demonstrate that dense cultures of 3T3 cells secrete inhibitory and stimulatory factors. Macromolecules of conditioned medium were fractionated on Biogel P150 and the different fractions were tested on quiescent cultures of 3T3 cells stimulated or not to proliferate by addition of alpha globulin. When target cells were not stimulated to proliferate by addition of exocrine growth factors, we observed the inhibitory activity of a large molecular weight inhibitor (IDF45) and the stimulatory activity of autocrine growth factors (fraction about 35 and 10 K molecular weight), on the incorporation of 14C inosine into nucleotide pool and RNA. However, DNA synthesis was significantly stimulated with fraction 10 K only. This discrepancy between the stimulation of RNA and DNA synthesis may be explained by the presence, simultaneously, of inhibitory and stimulatory factors in fraction 35 and 10 K molecular weight. The presence of inhibitory factor was demonstrated when the fractions were tested on target cells stimulated to proliferate by alpha globulin addition and labeled with 14C thymidine. In these conditions, the stimulatory activity of autocrine growth factors was not observable, and only the inhibitory activity on DNA synthesis of fractions 35 and 10 K appeared. It is tempting to assume that the regulation of in vitro cell proliferation is determined by the balance between these antagonist stimulatory and inhibitory autocrine growth factors.     

Ligand-receptor interactions involved in the stimulation of Swiss 3T3 fibroblasts by insulin-like growth factors and insulin.

1. The binding of 125I-labelled insulin-like growth factor 1 (125I-IGF-1) to Swiss mouse 3T3 fibroblasts was time- and concentration-dependent. Unlabelled IGF-1 had a slightly higher potency than multiplication-stimulating activity (MSA) in inhibiting the binding of 125I-IGF-1, and insulin gave a parallel inhibition curve at 300-1000-fold lower potency. Chemical cross-linking of bound 125I-IGF-1 to its receptors, followed by polyacrylamide-gel electrophoresis under reducing conditions, revealed a major band of Mr 130,000, the labelling of which was inhibited by IGF-1 or high concentrations of insulin. 2. The binding of 125I-IGF-1 was not affected by either co-incubation or preincubation of the cells with a range of heterologous growth factors and mitogens. However, IGF-1 and MSA each induced down-regulation of 125I-IGF-1 binding sites. 3. The maximal stimulations of DNA synthesis induced by IGF-1, MSA and insulin, in the presence of a synergizing mitogen, were similar. The dose-response curve for insulin was not parallel to those for IGF-1 and MSA; in particular, low concentrations of insulin induced a greater stimulation than expected on the basis of its potency in the inhibition or down-regulation of 125I-IGF-1 binding. 4. The preincubation of 125I-IGF-1 with Swiss 3T3 cells at 37 degrees C decreased its ability to bind to a second batch of cells. This inactivation did not occur when the preincubation was performed at 4 degrees C or in the presence of cycloheximide. Chemical cross-linking revealed that the cells released an IGF-binding protein, giving a complex of Mr about 48,000. 5. It is concluded that type I IGF receptors mediate the stimulation of Swiss 3T3 cells by insulin-like mitogens, but that insulin probably stimulates the cells through insulin receptors. The cells can modulate the amount of ligand binding, both by down-regulation of the receptors and by the secretion of an IGF-binding protein.     

Effect of plasma membranes on solute transport in 3T3 cells.

Sparse cultures of Swiss 3T3 cells are arrested early in the G1 phase of growth by the addition of a plasma membrane fraction obtained from confluent 3T3 cells. We have examined whether the changes in solute transport which are usually associated with cessation of growth at confluency also take place when cell growth is arrested by the addition of plasma membranes. We find that the rate of uptake of alpha-aminoisobutyric acid and uridine is decreased after the addition of plasma membranes to 3T3 cells, but the rate of uptake of 2-deoxyglucose and phosphate is not. We conclude from these observations that uptake of uridine and alpha-aminoisobutyric acid are related to contact inhibition of growth, while the decline in the rate of uptake of 2-deoxyglucose and phosphate observed at high cell density must be due to changes other than cell to cell contact.     

Aluminum ions stimulate DNA synthesis in quiescent cultures of Swiss 3T3 and 3T6 cells

Micromolar concentrations of AI3+ are shown to be strongly mitogenic for quiescent cultures of Swiss 3T3 and 3T6 cells. AI3+ caused a striking shift in the dose-response curve for the effect of fetal bovine serum on 3H-thymidine incorporation. In the absence of serum the mitogenic effect of aluminum was greatly potentiated by insulin or cholera toxin, but not epidermal growth factor or 12-0-tetradecanoyl-phorbol-13-acetate. The stimulation of DNA synthesis was maximal by 15-20 microM AI3+ X AI3+ at 100 microM had no inhibitory effect on DNA synthesis. AI3+ had no significant effect on cellular cyclic adenosine monophosphate in the presence or absence of insulin or an inhibitor of cyclic nucleotide phosphodiesterases.     

Inhibition of sugar uptake in adenosine-treated 3T3 cells

Addition of 5 to 250 micromolar adenosine to the culture medium resulted in a 30–80% inhibition of the rate of uptake of 2-deoxyglucose or 3–0-methylglucose by sparse or confluent 3T3 cells within three hours. The inhibition of deoxyglucose uptake could be reversed partially by changing the cells to medium without adenosine for two hours and could be prevented completely by the addition of persantin, an inhibitor of nucleoside uptake. The adenosine effect is not due to inhibition of pyrimidine synthesis, since it is not prevented by uridine. It is not seen in 3T6 cells lacking adenosine kinase. The inhibition could be observed on confluent cells whose deoxyglucose uptake was stimulated by insulin, epidermal growth factor (EGF), calf serum or calcium phosphate. Although the percentage stimulation over control by these factors varied, the percentage inhibition by addition of adenosine of the stimulated rates, as well as the unstimulated rate, was relatively constant. EGF, insulin and calcium phosphate caused little or no stimulation of deoxyglucose uptake by sparse cells, whether adenosine treated or untreated. The results suggest that adenosine acts intracellularly after phosphorylation to regulate sugar uptake through a mechanism which is independent of the regulation by hormones and cell density.     

Density dependent proliferation of human glia cells stimulated by epidermal growth factor.

Epidermal growth factor (EGF) isolated from mouse salivary glands, enhanced the multiplication and [³H]TdR incorporation of human normal glia cells in serum-free medium supplemented with human serum albumin. Optimal dose was 2 ng/ml for both dense and sparse cultures but dense cultures were stimulated by EGF to a much less extent than sparse cultures. Data are presented that make the possibility unlikely that the density dependent inhibition of the EGF response is due to depletion of EGF in the medium or a local, juxtacellular starvation for the factor.     

Elevated cAMP is required for stimulation of eicosanoid synthesis by interleukin 1 and bradykinin in BALB/c 3T3 fibroblasts.

In Swiss 3T3 murine fibroblasts, interleukin 1 (IL-1) and bradykinin stimulate prostaglandin E2 (PGE2) synthesis. However, in the present study, we found that neither agonist stimulated PGE2 synthesis in BALB/c 3T3 murine fibroblasts, this in spite of expression of similar numbers of receptors for each agonist compared to Swiss 3T3 cells. When BALB/c 3T3 cells were preincubated with cAMP analogs, both IL-1 and bradykinin stimulated PGE2 synthesis to levels similar to those observed in Swiss 3T3 cells. Similarly, when the cells were preincubated with forskolin, which activates the catalytic subunit of adenylate cyclase directly, or NECA, which stimulates cellular cAMP accumulation by activating adenosine receptors, IL-1 and bradykinin stimulated PGE2 synthesis. Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase, blocked the ability of cAMP or NECA to render cells responsive to IL-1 and bradykinin. In basal BALB/c 3T3 cells, bradykinin and IL-1 stimulated arachidonate release in the absence of cAMP, but little conversion of released arachidonate to PGE2 occurred. cAMP, forskolin, and NECA all increased cyclooxygenase activity in the cells. SV-T2 is a clonal line originating from BALB/c 3T3 transformed with SV-40. In these cells, IL-1 and bradykinin stimulated PGE2 synthesis despite basal intracellular cAMP concentrations similar to BALB/c, and cAMP only modestly potentiated the response. In summary, cyclooxygenase expression appears to be regulated by cAMP in BALB/c 3T3 cells, and SV-40 transformation results in increased cyclooxygenase expression, apparently independent of cAMP.     

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.     

Independent regulation of the nuclear and mitochondrial DNA synthesis induced by either Simian virus 40 or serum in mouse fibroblast 3T3 cells.

Resting cultures of 3T3 cells (an established line of mouse fibroblasts) were released from density inhibition by either infection with Simian virus 40 or addition of serum. The increased rate of thymidine incorporation into DNA, induced by these two agents, was measured in the presence and in the absence of three inhibitory conditions (cycloheximide or dibutyryladenosine 3':5'-monophosphate added to the medium, or lack of anchorage). The inhibition was found to be quite similar in cultures stimulated by virus or serum; under the same conditions, however, the incorporation into mitochondrial DNA was much less inhibited than that into nuclear DNA. The experiments also suggest that new protein synthesis may not be necessary, for either virus or serum, to start the inductive mechanism.     

Effects of S-adenosyl-1,8-diamino-3-thiooctane on polyamine metabolism

Exposure of mammalian cells (transformed mouse fibroblasts or rat hepatoma cells) to S-adenosyl-1,8-diamino-3-thiooctane produced profound changes in the intracellular polyamine content. Putrescine was increased and spermidine was decreased, consistent with the inhibition of spermidine synthase by this compound, which is a potent and specific "transition-state analogue inhibitor" of the isolated enzyme in vitro. The spermine content of the cells was increased by exposure to this drug presumably since spermine synthase was able to use a greater proportion of the available decarboxylated S-adenosylmethionine when spermidine synthase was inhibited. The decarboxylated S-adenosylmethionine content rose substantially because the activity of S-adenosylmethionine decarboxylase was increased in response to the decline in spermidine. These results indicate that S-adenosyl-1,8-diamino-3-thiooctane is taken up by mammalian cells and is an effective inhibitor of spermidine synthase in vivo and that S-adenosylmethionine decarboxylase is regulated by the content of spermidine, but not of spermine. The growth of SV-3T3 cells was substantially reduced in the presence of S-adenosyl-1,8-diamino-3-thiooctane at concentrations of 50 microM or greater. Such inhibition was reversed by the addition of spermidine but not by putrescine. When SV-3T3 cells were exposed to 5 mM alpha-(difluoromethyl)ornithine and 50 microM S-adenosyl-1,8-diamino-3-thiooctane, the content of all polyamines was reduced. Putrescine and spermidine declined by more than 90% and spermine by 80%. Such cells grew very slowly unless spermidine was added.     

ES936 stimulates DNA synthesis in HeLa cells independently on NAD(P)H:quinone oxidoreductase 1 inhibition,through a mechanism involving p38 MAPK

The indolequinone ES936 (5-methoxy-1,2-dimethyl-3-[(4-nitrophenol)methyl]-indole-4,7-dione) is a potent mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1). Here, we report that ES936 significantly stimulated thymidine incorporation in sparse cultures of human adenocarcinoma HeLa cells, but was without effect in dense cultures. Stimulation of DNA synthesis was not related with a DNA repair response because an increase in thymidine incorporation was not observed in cells treated with 2,5 bis-[1-aziridyl]-1,4 benzoquinone, a well-established antitumor quinone that causes DNA damage. Conversely, it was related with an increase of cell growth. NQO1 inhibition was not involved in ES936 stimulation of DNA synthesis, because the same response was observed in cells where NQO1 expression had been knocked down by small interfering RNA. Stimulation of DNA synthesis was reverted by treatment with ambroxol, a SOD mimetic, and by pyruvate, an efficient peroxide scavenger, supporting the involvement of alterations in cellular redox state. Pharmacological inhibition of p38 with either SB203580 or PD169316 completely abolished ES936-stimulated DNA synthesis, indicating the requirement of p38 activity. This is the first report that demonstrates the existence of an ES936-sensitive system which is separate from NQO1, modulating the redox state and cell growth in HeLa cells through a p38-dependent mechanism. Our results show that the effect ES936 exerts on DNA synthesis may be either positive or negative depending on the cellular context and growth conditions.     

Studies of Long-Range Density Effects on the Proliferation of 3T3 and RLCW Cells in Recirculated Medium

Swiss mouse 3T3 cells and rat liver-derived RLCW cells were grown in monolayers and perfused with culture medium. A flow-rate dependent increase in the growth rate was observed both by ³H-thymidine uptake and by a rise in cell numbers. The characteristics of the response were dependent on the recirculating volume and on whether serum was present in the culture medium. In RLC cultures perfused with serum-supplemented medium the growth promoting effect decreased with increasing density of the cells. In the absence of serum, recirculation of NCTC medium had no effect on RLCs but increased growth was observed in recirculated MEM. In 3T3 cultures, a linear response was observed over a limited density range in the presence of 10% serum-supplemented medium indicating that substances present in the serum substantially modify the behaviour of the monolayer to perfusion. In serum-free medium the effect of perfusion on 3T3 cultures was confined to a small density range and was consistent with the more rapid removal of a diffusible inhibitor from the pericellular environment by recirculating the medium. Treatment of the perfusing medium with immobilised proteinases (trypsin, chymotrypsin, protease) did not alter the response except in the presence of putrescine.     

Studies of long-range density effects on the proliferation of 3T3 and RLCW cells in recirculated medium.

Swiss mouse 3T3 cells and rat liver-derived RLCW cells were grown in monolayers and perfused with culture medium. A flow-rate dependent increase in the growth rate was observed both by 3H-thymidine uptake and by a rise in cell numbers. The characteristics of the response were dependent on the recirculating volume and on whether serum was present in the culture medium. In RLC cultures perfused with serum-supplemented medium the growth promoting effect decreased with increasing density of the cells. In the absence of serum, recirculation of NCTC medium had no effect on RLCs but increased growth was observed in recirculated MEM. In 3T3 cultures, a linear response was observed over a limited density range in the presence of 10% serum-supplemented medium indicating that substances present in the serum substantially modify the behaviour of the monolayer to perfusion. In serum-free medium the effect of perfusion on 3T3 cultures was confined to a small density range and was consistent with the more rapid removal of a diffusible inhibitor from the pericellular environment by recirculating the medium. Treatment of the perfusing medium with immobilised proteinases (trypsin, chymotrypsin, protease) did not alter the response except in the presence of putrescine.     

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.     

Protein kinase D potentiates DNA synthesis and cell proliferation induced by bombesin, vasopressin, or phorbol esters in Swiss 3T3 cells

We examined whether protein kinase D (PKD) overexpression in Swiss 3T3 cells potentiates the proliferative response to either the G protein-coupled receptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu). In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with retrovirus encoding murine PKD and green fluorescent protein (GFP) expressed as two separate proteins translated from the same mRNA. GFP was used as a marker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 cells was dramatically activated by cell treatment with bombesin or PDBu as judged by in vitro kinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, these stimuli induced PKD phosphorylation at Ser(744), Ser(748), and Ser(916). PKD activation and phosphorylation were prevented by exposure of the cells to protein kinase C-specific inhibitors. Addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in DNA synthesis and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulation of DNA synthesis in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results demonstrate that overexpression of PKD selectively potentiates mitogenesis induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells.     

Growth inhibition of mitogen-stimulated fibroblasts induced by double-stranded RNA depends on cell density

Polyinosinic-polycytidylic acid [poly(I:C)], a synthetic double-stranded RNA, is an inhibitor of mitogen-induced proliferation of normal fibroblasts. We show that this inhibition depends strongly on cell density. While cultures with densities at or above confluence are completely inhibited by poly(I:C) in their proliferative response to epidermal growth factor (EGF), the proliferation of sparse (subconfluent) cultures is only delayed. Conditioned medium from dense fibroblasts exposed to poly(I:C) inhibits EGF stimulation of sparse cells, indicating that the inhibition is, at least in part, mediated by a factor released from the cells. Preincubation of quiescent cultures with poly(I:C) renders the cells refractory to the inhibitory effects of poly(I:C). This desensitization correlates with a decreased production of the inhibitor. Since the inhibition of mitogenic stimulation by poly(I:C) is completely overcome by antisera recognizing interferon-beta (IFN-beta) and interleukin-6 (IL-6), we tested the effect of IL-6 and IFN-beta on EGF mitogenicity. None of the available IL-6 preparations had any effect on cell cycle entry. IFN-beta caused a dose-dependent delay of cell division but did not affect the density-dependent proportion of cells entering the cell cycle in response to EGF. Thus, IFN-beta cannot be the sole mediator of the poly(I:C)-induced inhibition. In the presence of dexamethasone, poly(I:C) did not inhibit EGF mitogenis. Indeed, the combined presence of poly(I:C) and dexamethasone did more than just restore the density-dependent control levels of EGF stimulation; most cells entered the cell cycle even at extremely high cell densities. Thus, poly(I:C) in combination with dexamethasone could deactivate the cell density-dependent negative control of 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.     

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.     

Inhibition of DNA synthesis and cell division by a cell surface sialoglycopeptide

We have isolated and purified a cell surface sialoglycopeptide (SGP) from bovine cerebral cortex cells that previously was shown to be a potent inhibitor of cellular protein synthesis. The following studies were carried out to characterize the potential ability of the SGP to inhibit DNA synthesis and to arrest cell division. Treatment of exponentially proliferating Swiss 3T3 cells with the SGP inhibitor resulted in a marked inhibition of thymidine incorporation within 24 h. When the SGP was removed from inhibited cultures, a sharp rise in 3H-thymidine incorporation followed within 3-4 h that peaked well above that measured in exponentially growing cultures, suggesting that the inhibitory action of the SGP was reversible and that a significant proportion of the arrested cells was synchronized in the mitotic cycle. In addition to DNA synthesis, the inhibitory action of the SGP was monitored by direct measurement of cell number. Consistent with the thymidine incorporation data, the SGP completely inhibited 3T3 cell division 20 h after its addition to exponentially growing cultures. Upon reversal there was a delay of 15 h before cell division resumed, when the arrested cells quickly doubled. Most, if not all, of the growth-arrested cells appeared to have been synchronized by the SGP. The SGP inhibited DNA synthesis in a surprisingly wide variety of target cells, and the relative degree of their sensitivity to the inhibitor was remarkably similar. Cells sensitive to the SGP ranged from vertebrate to invertebrate cells, fibroblast and epitheliallike cells, primary cells and established cell cultures, as well as a wide range of transformed cell lines.