Epidermal growth factor (EGF) is required only during the traverse of early G1 in PDGF stimulated density-arrested BALB/c-3T3 cells

Density-arrested BALB/c-3T3 cells that had received a transient exposure to PDGF and were then transferred to medium containing only EGF and somatomedin C (Sm-C) began DNA synthesis after the G0/G1 lag. Supraphysiological concentrations of insulin could be employed to replace the Sm-C requirement. This G0/G1 lag phase was bisected by the requirement for the exogenous presence of EGF. Our data indicated that EGF was required during the traverse of only the first half of G0/G1 phase (6 h) and not during the traverse of late G1. Subphysiological serum concentrations of Sm-C were also necessary to be present with EGF for progression through early G0/G1; however, traverse of the final half of G0/G1 and commitment to DNA synthesis required the presence of Sm-C. It was found that physiological concentrations of Sm-C were required for the traverse late G1. The requirement for Sm-C for G0/G1 traverse of BALB/c-3T3 cells as opposed to human fibroblasts or glial cells may be due to a difference in endogenous synthesis of an insulin-like growth factor. Our data are in close agreement with previous reports that EGF is only required for approximately the first 8 h during traverse of the G0/G1 phase. The requirement for EGF to be present for the first 6 h of G0/G1 could result from a continued or repetitious event or by more than one distinct EGF-requiring event.     

Platelet-derived growth factor stimulation of [3H]-glucosamine incorporation in density-arrested BALB/c-3T3 cells

G0/G1 traverse in density-arrested BALB/c-3T3 cells is controlled by multiple serum-derived growth factors. Platelet-derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G0/G1. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. We have identified the stimulation of a biochemical process specific to competence formation in BALB/c-3T3 cells. PDGF treated BALB/c-3T3 cells incorporated 5-10-fold more [3H]-glucosamine (GlcN) into acid-insoluble material as compared to platelet-poor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density-arrested BALB/c-3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca3 (PO4)2 and was not due to cell-cycle traverse. Stimulation of [3H]-GlcN incorporation by PDGF was time dependent, and increased incorporation of [3H]-GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.     

Elevated intracellular concentrations of cyclic AMP inhibited serum-stimulated, density-arrested BALB/c-3T3 cells in mid G1

The stimulation of DNA synthesis in quiescent, density-arrested BALB/c-3T3 cells by platelet-derived growth factor in plasma-supplemented medium was inhibited by the presence of isobutylmethylxanthine (IBMX) and cholera toxin, although neither IBMX or cholera toxin when used alone inhibited the stimulation of DNA synthesis. The cells were reversibly inhibited in mid G1 at a point 6 hr prior to the initiation of DNA synthesis. The inhibition of cell cycle traverse was associated with a 10-15 fold increase in cellular cyclic AMP concentration over basal levels. The reversal of this inhibition by removal of IBMX was correlated with a dramatic decrease in cyclic AMP levels. The traverse of G1 and the initiation of DNA synthesis after release from the cholera toxin and IBMX inhibition was dependent on the presence of plasma in the medium. Either somatomedin C (10-20 ng/ml) or insulin (10(-6)-10(-5) M) completely replaced the plasma requirement for late G1 progression and entry into S phase. Once the inhibited cells were released from the IBMX and cholera toxin block a subsequent increase in cyclic AMP did not prevent entry into S phase. The presence of cholera toxin alone inhibited the stimulation of human dermal fibroblasts. The elevation of intracellular cyclic AMP levels in the human dermal fibroblasts by cholera toxin was two to three fold greater than that found in the BALB/c-3T3 cells in the presence of cholera toxin and the IBMX.     

Control of the Balb/c-3T3 cell cycle by nutrients and serum factors: analysis using platelet-derived growth factor and platelet-poor plasma.

Much controversy regarding the relationship between nutrients and serum in regulation of cell growth can be reconciled by recognizing that serum contains multiple factors which regulate different events in the cell cycle. Serum was fractionated into a platelet-derived growth factor (PDGF), which induces cells to become competent to synthesize DNA, and plasma which allows competent cells to traverse G0/G1 and enter the S phase. Nutrients are not required for the cellular response to PDGF; however amino acids are required for plasma to promote the entry of PDGF-treated, competent cells into S phase. The nutrient independent, PDGF-modulated, growth regulatory event (competence) is located 12 hours prior to the G1/S phase boundary in quiescent, density-arrested Balb/c-3T3 cells. The nutrient dependent, plasma-modulated event is located six hours prior to the G1/S phase boundary and corresponds in concentration of amino acids required for DNA synthesis. Infection of density-arrested Balb/c3T3 cells with SV40 overrides both the nutrient independent and the nutrient dependent growth regulatory events.     

Effect of ouabain on growth regulation by serum components in Balb/c-3T3 cells: inhibition of entry into S phase by decreased protein synthesis

The effect of inhibition of the cell membrane Na+-K+ pump on the Balb/c-3T3 cell growth cycle was studied. Inhibition of the Na+-K+ pump resulted in a dose-dependent reduction of intracellular K+ concentration ((K+)i). However, inhibition of protein synthesis in Go/G1 and of subsequent entry into S phase occurred only after (K+)i fell below a critical threshold (50-60 mmoles/liter). Thus, when the (K+)i falls below a critical threshold, protein synthesis is inhibited, preventing cells from entering the S phase. The platelet-derived growth factor (PDGF) induces cells to become "competent" to traverse the cell cycle; the platelet-poor plasma component of serum allows competent cells to progress through G0/G1 and enter S phase. Inhibition of the Na+-K+ pump did not prevent the induction of competence by PDGF, but it did reversibly inhibit plasma-mediated events in early G0/G1. Similarly, cycloheximide inhibited plasma-mediated events but did not prevent PDGF-induced competence. Thus, protein synthesis may not be required for induction of competence; alternatively, the induction of the competent state may occur in these cells after removal of PDGF and protein synthesis inhibitor. Protein synthesis is required for subsequent plasma-mediated events in G0/G1.     

Rapid induction of competence formation is PDGF-isoform specific.

Platelet-derived growth factor (PDGF) stimulates the expression of a number of genes associated with entry of quiescent Balb/c-3T3 fibroblasts into the cell cycle. We determined that two of these genes, c-myc and c-fos, are induced equivalently in medium supplemented with platelet-poor plasma (PPP) and either PDGF-BB or PDGF-AA. The rate at which fibroblasts entered S phase was also similar in PDGF-BB- and AA-treated cells as was the expression of the late G1 gene, thymidine kinase (TK). However, PDGF-AA must be present for a period of 16 h to stimulate the proliferation of 90% of the cells, whereas PDGF-BB was required for only 4 h. Exposure of cells to PDGF-AA for 4 h, a time during which maximum expression of c-fos and c-myc occurred, only induced 20% of the cells in a quiescent population to enter the cell cycle. Therefore, PDGF-AA-mediated expression of the immediate early genes c-fos and c-myc may be necessary but is not sufficient to rapidly stimulate density-arrested Balb/c-3T3 fibroblasts into the competent state. Thus, these data suggest that PDGF-AA and PDGF-BB initiate traverse of the cell cycle by distinct mechanisms.     

Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.     

Conditional responsiveness of a chemically transformed cell line to growth factor stimulated DNA synthesis

BP3T3, a clonal benzo(a)pyrene-transformed BALB/c-3T3 cell line, is conditionally responsive to growth factor stimulation. Density arrested cell populations deprived of growth factors by pretreatment with 0.5% platelet-poor plasma synthesized DNA both in response to ng/ml concentrations of PDGF, EGF, and somatomedin C, and in response to insulin, plasma, and serum. The above agents acted singly to induce DNA synthesis, but synergism is suggested because a higher percentage of cells were stimulated to enter the S phase when the growth factors were added in combination. Desensitization to growth factors occurred when cultures were pretreated with the high concentration of growth factors present in 10% serum (or plasma). In desensitized cultures none of the above agents, added singly or in combination, stimulated DNA synthesis. This effect appears to be global because pretreatment with one growth factor (e.g., insulin) inhibited the action of another (e.g., PDGF). Cell density appears to play a critical role in regulating DNA synthesis. Unlike nontransformed BALB/c-3T3 cells whose density is regulated by the serum concentration, the density of BP3T3 cells reached a plateau when cultures were grown in a serum (or plasma) concentration of 3% or greater. Such density arrested cultures were growth factor unresponsive; however, the cells rapidly responded to growth factors by synthesizing DNA and replicating when reseeded at a lower cell density. Thus the growth of BP3T3 cells is regulated by both growth factors and cell density.     

Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells

Exposure of BALB/c-3T3 cells (clone A31) to platelet-derived growth factor (PDGF) results in a rapid time- and dose-dependent alteration in the distribution of vinculin and actin. PDGF treatment (6-50 ng/ml) causes vinculin to disappear from adhesion plaques (within 2.5 min after PDGF exposure) and is followed by an accumulation of vinculin in punctate spots in the perinuclear region of the cell. This alteration in vinculin distribution is followed by a disruption of actin-containing stress fibers (within 5 to 10 min after PDGF exposure). Vinculin reappears in adhesion plaques by 60 min after PDGF addition while stress fiber staining is nondetectable at this time. PDGF treatment had no effect on talin, vimentin, or microtubule distribution in BALB/c-3T3 cells; in addition, exposure of cells to 5% platelet-poor plasma (PPP), 0.1% PPP, 30 ng/ml epidermal growth factor (EGF), 30 ng/ml somatomedin C, or 10 microM insulin also had no effect on vinculin or actin distribution. Other competence-inducing factors (fibroblast growth factor, calcium phosphate, and choleragen) and tumor growth factor produced similar alterations in vinculin and actin distribution as did PDGF, though not to the same extent. PDGF treatment of cells for 60 min followed by exposure to EGF (0.1-30 ng/ml for as long as 8 h after PDGF removal), or 5% PPP resulted in the nontransient disappearance of vinculin staining within 10 min after EGF or PPP additions; PDGF followed by 0.1% PPP or 10 microM insulin had no effect. Treatment of cells with low doses of PDGF (3.25 ng/ml), which did not affect vinculin or actin organization in cells, followed by EGF (10 ng/ml), resulted in the disappearance of vinculin staining in adhesion plaques, thus demonstrating the synergistic nature of PDGF and EGF. These data suggest that PDGF-induced competence and stimulation of cell growth in quiescent fibroblasts are associated with specific rapid alterations in the cellular organization of vinculin and actin.     

Resumption of cell cycle in BALB/c-3T3 fibroblasts arrested by polyamine depletion: relation with "competence" gene expression

Serum deprivation arrests BALB/c-3T3 fibroblasts (clone A31) in G0 phase, where resumption of the cell division cycle can be induced by addition of serum or of specific growth factors in a defined sequence: PDGF (inducer of a state of "competence," characterized by the expression of a family of genes including c-myc), epidermal growth factor EGF and IGF1 (Leof et al., 1982, 1983). When exponentially growing A31 cells are placed for greater than or equal to 2 days in a medium containing the alpha-difluoromethylornithine (alpha DFMO), an irreversible inhibitor of ornithine decarboxylase, they become arrested in G1 phase as a consequence of polyamine depletion (Medrano et al., 1983). In the alpha DFMO-arrested cells, addition of putrescine (60 microM) in a culture medium containing 6% fetal calf serum (FCS), but not in serum-free medium, is sufficient to induce G1 progression and entry into S phase (as determined by 3H-thymidine incorporation). The level of "competence" mRNAs is high in alpha DFMO-arrested cells. After addition of putrescine in FCS-containing medium, these mRNAs continue to be present for at least 3 h. A large proportion of alpha DFMO-arrested cells can be induced to progress to S phase by insulin (1 microM, acting via IGF1 receptor) plus putrescine in a serum-free medium (greater than or equal to 50% of FCS effect). In this case, the levels of "competence" mRNAs become low or undetectable within 3 h, EGF (10 nM) plus insulin had only slightly greater effect than insulin alone on the progression of alpha DFMO-arrested cells. When the alpha DFMO-arrested cells are subsequently incubated during 3 days in a low-serum-containing medium (0.25% FCS), they do not replenish their supply of polyamines, and then continue to express the c-myc gene. The recruitment of the polyamine-depleted, serum-deprived cells into the cell division cycle does not require PDGF and can be induced by addition of EGF and insulin plus putrescine. These data indicate that alpha DFMO arrests majority of the cells at a point situated beyond the PDGF- and EGF-dependent portion of G1 phase. During the subsequent serum deprivation, the alpha DFMO-arrested cells remain "competent" (PDGF-independent), probably as a consequence of their continued expression of c-myc (and possibly other PDGF-inducible genes).     

Regulation of the Balb/c-3T3 cell cycle-effects of growth factors

The platelet-derived growth factor (PDGF), which is found in serum but not in plasma, has been purified to homogeneity; it stimulates replication at a concentration of 10^?10M. Brief treatment with PDGF causes densityinhibited Balb/c-3T3 cells to become competent to synthesize DNA; pituitary fibroblast growth factor (FGF) or precipitates of calcium phosphate also induce competence. Continuous treatment with plasma allows competent, but not incompetent, cells to synthesize DNA. A critical component of plasma is somatomedin, a group of hormones with insulin-like activity; multiplication-stimulating activity (MSA) or insulin replace plasma somatomedin in promoting DNA synthesis. We have studied the molecular correlates of competence and the role of SV40 gene A products in regulating DNA synthesis. Treatment of quiescent cells with pure PDGF or FGF causes the preferential synthesis of five cytoplasmic proteins (approximate molecular weight 29,000, 35,000, 45,000, 60,000, and 72,000 detected by SDS-PAGE under reducing conditions). Two of these competence-associated proteins (29,000 and 35,000 daltons) are found within 40 min of PDGF addition; they are not induced by plasma, insulin, or epidermal growth factor (EGF), PDGF, FGF, or calcium phosphate induce an ultrastructure change within the centriole of 3T3 cells; this ultrastructural modification of the centriole is detectable by immunofluorescence within 2 h of PDGF treatment. Plasma, EGF, or MSA do not modify the centriole. SV40 induces replicative DNA synthesis in growth-arrested 3T3 cells but does not cause this alteration in centriole structure. Gene A variants of SV40, including a mutant with temperature-sensitive (ts) T-antigen (ts A209), a deletion in t-antigen (dl 884), and several ts A209 strains containing t-antigen deletions were used to induce DNA synthesis in Balb/c-3T3 cells. Like wild type SV40, all strains induced DNA synthesis equally well under permissive or nonpermissive conditions. Addition of PDGF or plasma had little effect on SV40-induced DNA synthesis. Thus, the viral function that induces replicative DNA synthesis in Balb/c-3T3 cells is not t and is not temperature sensitive. This SV40 gene function overrides the cellular requirement for hormonal growth factors. It does not induce transient centriole deciliation, a hormonally regulated event.     

Control of cytolysis of BALB/c-3T3 cells by platelet-derived growth factor: a model system for analyzing cell death

In culture medium supplemented with 10% clotted blood serum, the saturation density of BALB/c-3T3 cells is determined jointly by cell replication and cell loss. By prelabelling cellular DNA with ³H-thymidine and also by time lapse photography, we studied cell loss independently of replication. Cell loss was accelerated when BALB/c-3T3 cells were transferred from serum-supplemented medium, which contains the platelet derived growth factor (PDGF), to medium supplemented with platelet-poor plasma which lacks it. Loss occurred via the disintegration of cell attached to the surface of the tissue culture dish. Cytolysis of individual cells occurred rapidly; less than 15 minutes transpired between the first indication of a perturbance (by phase contrast microscopy) and fragmentation of the cell cytoplasm. Kinetic analysis was consistent with random cell death rather than a fixed lifetime. The percentage of cells undergoing cytolysis was governed by the cell density; at high densities, such as are present in confluent cultures, a higher percentage of cell loss was noted than at low density. Cell death was antagonized by partially purified or electrophoretically homogenous preparations of-PDGF. Pure PDGF stimulated cell survivial at ng/ml in a concentration dependent fashion. The process of cell replication was not necessary for survival because PDGF prevented cytolysis in the presence of methotrexate, an inhibitor of DNA synthesis. A brief (4 hour) treatment with PDGF prevented cell death; such PDGF treated cells displayed increased survival after being taken up with trypsin and planted onto a fresh surface in plasma supplemented medium. Pituitary fibroblast growth factor, a functional analogue of PDGF for induc of DNA synthesis in BALB/c-3T3 cells, also functioned as an anticytolytic agent. By contrast, epidermal growth factor and insulin did not. Cytolysis of SV40-transformed cells occurred at a constitutively low rate and was insensitive to PDGF.     

Increases in cyclic AMP potentiate competence formation in BALB/c-3T3 cells

The ability of platelet-derived growth factor and fibroblast growth factor to stimulate the initiation of DNA synthesis in quiescent BALB/c-3T3 cells was enhanced by cholera toxin. However, the addition of cholera toxin to unsupplemented medium was not mitogenic, nor did cholera toxin increase the mitogenic potential of mediuum supplemented with platelet-poor plasma. The enhancement of serum-induced DNA synthesis by cholera toxin was due to a specific effect on competence formation and not plasma-controlled progression. Cholera toxin increased the rate of competence formation during a transient exposure of quiescent cells to platelet-derived growth factor; this rate was further increased by the addition of isobutylmethylxanthine, a cyclic nucleotide phosphodiesterase inhibitor. Intracellular cyclic AMP concentrations in quiescent BALB/c-3T3 cells were increased 2- to 3-fold after the addition of cholera toxin. The addition of cholera toxin plus 30 m?M isobutylmethylxanthine caused an even greater (7- to 8-fold) increase in the cellular levels of cyclic AMP. That these increases in cyclic AMP concentrations mediated at least part of the increased sensitivity of quiescent cells to competence factors was substantiated by the observation that 0.01 to 1 mM monobutrylcyclic AMP or 8-bromocyclic AMP also caused a concentration-dependent potentiation of competence formation in quiescent cells during a transient exposure to platelet-derived growth factor.     

Uncoupling of RNA and DNA synthesis after plasma stimulation of G0-arrested BALB/C-3T3 cells.

The addition of whole serum to G0-arrested, confluent Balb/c-3T3 cells induces them to progress through G1 and synthesize DNA after a 12-h lag period. Prior to the onset of DNA synthesis, RNA is synthesized and RNA content increases. Serum has been fractionated into two sets of growth factors: a platelet-derived growth factor present in heat-treated (100 degrees C) platelet extracts and platelet-poor plasma. Addition of whole serum, platelet-derived growth factor or platelet-poor plasma induces quiescent cells to increase their cytoplasmic RNA content, but the cells treated with platelet-poor plasma do not synthesize DNA. Messenger RNA content increases within 2 h after stimulation with whole serum or platelet-poor plasma, and after 18 h, mRNA has accumulated to a greater degree than rRNA.     

Mitogenic activity elaborated by macrophage-like cell lines acts as competence factor(s) for BALB/c 3T3 cells

The culture medium from several murine macrophage-like cell lines contained a mitogenic activity that functioned synergistically with platelet-poor plasma to induce DNA synthesis in quiescent density-inhibited BALB/c 3T3 fibroblasts. This mitogenic activity was generated by P388D₁ (and other established lines of) macrophage-like cells that were cultured either in medium alone or in medium supplemented with platelet-poor plasma. The amount of mitogenic activity produced was directly related to the length of time the macrophage-like cells were maintained in the medium. Serum-free medium conditioned by macrophage-cells did not stimulate DNA synthesis in density-inhibited 3T3 cells in the absence of plasma; however, a transient (4-hr) exposure to serum-free macrophage-conditioned medium allowed quiescent cells to respond to plasma-derived progression factors. The addition of plasma to 3T3 cells that had been treated with the macrophage-conditioned medium brought about DNA synthesis after a 12-hr lag. The mitogenic activity that was in macrophage-conditioned medium bound to DEAE-Sephadex and eluted in a single peak using a linear NaCl gradient. This macrophage-derived competence factor was not mitogenic for lymphocytes and was clearly separated by DEAE-Sephadex chromatography from the major peak of the previously described mitogenic monokine, Interleukin-I (lymphocyte activating factor).     

Centriole deciliation associated with the early response of 3T3 cells to growth factors but not to SV40.

BALB/c-3T3 cells which are growth-arrested by high cell density or low serum have ciliated, unduplicated centrioles. Stimulation of these quiescent cells by serum is associated with a rapid (within 1–2 hr) deciliation of the centriole, followed by reciliation within 6–10 hr. This transient deciliation of the centriole is induced by the platelet-derived growth factor (PDGF) component of serum. The cells treated with PDGF became competent to replicate their DNA; most PDGF treated cells, however, did not progress from Go toward S phase unless they were incubated with the platelet-poor plasma component of serum. Addition of CaCl₂ or Fibroblast Growth Factor to the media mimicked PDGF by producing both centriole deciliation and competence to replicate DNA. In fact, over a range of concentrations of each of these factors, only doses which produced centriole deciliation were capable of producing competence for DNA synthesis. Plasma alone or factors such as Multiplication Stimulating Activity produced neither centriole deciliation nor competence; these agents were, however, required for the optimum progression of competent cells into DNA synthesis. In contrast, infection with SV40 induced host cell DNA synthesis without an initial transient deciliation of the centriole. Thus while growth factors may have to induce centriole deciliation for 3T3 cells to synthesize DNA, abortive transformation by SV40 overrides this requirement.     

Inhibition of BALB/c-3T3 cells in late G1: Commitment to DNA synthesis controlled by somatomedin C

Methylglyoxal bis-(guanylhydrazone) (mGBG) blocked the stimulation of DNA synthesis in quiescent, density-inhibited BALB/c-3T3 cells treated with platelet-derived growth factor (PDGF) and platelet-poor plasma (PPP). Competence formation produced by a transient exposure to PDGF was not effected by mGBG. In contrast, mGBG effectively inhibited the PPP-stimulated progression of competent cells through the G₁ phase of the cell cycle, although maximal inhibition was observed when mGBG was present during both the exposure to PDGF- and PPP-supplemented media. When quiescent cells were treated with PDGF and PPP-supplemented media in the presence of mGBG for 12–18 hours and the mGBG was then removed, cells entered the S phase after a 4 hour lag. The rate of entry into the S phase, but not the time necessary for the cells to progress from the mGBG block into the S phase, was dependent on the concentration of PPP present after removal of the mGBG. Either somatomedin C or insulin, but not epidermal growth factor, fibroblast growth factor, or PDGF were able to substitute for PPP in allowing cells to enter the S phase after the cells were released from the mGBG block. A marked inhibition of (³H)-leucine incorporation in serum-stimulated cultures was produced at mGBG concentrations which caused no decrease in the amount of (³H)-uridine incorporated during a short (15 minute) pulse. The ability of hormones to allow cells to progress to the late G₁ phase and become committed to DNA synthesis after a mGBG inhibition was not related to their ability to restore the normal rate of protein synthesis as determined by (³H)-leucine incorporation.     

Plasma and platelet associated factors act in G1 to maintain proliferation and to stabilize arrested cells in a viable quiescent state : A temporal map of control points in the G1 phase

In medium supplemented with defibrinogenated, platelet-poor human plasma and a low molecular weight growth factor derived from human platelets (PDGF), Swiss 3T3 cells proliferate exponentially with the same cell cycle kinetics as cells cultured in medium supplemented with commercial calf serum. Removal of PDGF from the culture medium arrests proliferating cells in a stable, reversible G0/G1 quiescent state. This arrested state is similar to the known quiescent state induced by deprivation of calf serum in cell exit kinetics and cytoplasmic proteins synthesized. Cells are sensitive to PDGF deprivation only at the beginning of G1. Reduction of the plasma concentration in the culture medium also arrests cells in G1. The resulting arrested population is unstable and exhibits progressive cell death. Reduced levels of plasma block cellular transit through the cell cycle at a median time of approx. 2.1 h following mitosis, approx. 3.3 h prior to S phase initiation. In addition to being required by cycling cells, plasma associated factors are required to maintain G1 cells blocked by PDGF deprivation in a stable quiescent state. Establishment of a stable, viable G0/G1 growth-arrested state, therefore, apparently involves two distinct processes: arrest of cellular proliferation in G1 and stabilization of the arrested cells in a viable quiescent state. Together with previously reported findings on serum and isoleucine starvation, these results provide a temporal map of growth control points in the G1 phase.     

Molecular cloning of gene sequences regulated by platelet-derived growth factor

We have screened a cDNA library for gene sequences that are regulated by platelet-derived growth factor (PDGF) in BALB/c-3T3 cells. Of 8000 clones screened, less than 14 independent PDGF-inducible sequences were found. Two of these (KC and JE) were studied in detail. By hybrid-selection and translation the KC and JE mRNAs encode 10,000 and 19,000 dalton polypeptides, respectively. In the absence of PDGF, the JE and KC sequences correspond to low abundance mRNAs. One hour after addition of PDGF their abundance level can be increased 10- to 20-fold. Within 4 hr, a 60-fold induction of JE can be attained. Nanogram per ml quantities of pure PDGF regulate these sequences whereas μg/ml quantities of chemically unrelated mitogens (EGF, insulin, or platelet-poor plasma) have either a weak or an undetectable effect. Inhibitors of protein synthesis block the progression of quiescent 3T3 cells through G1 into S phase; however these drugs do not block the induction of KC and JE by PDGF. This result indicates that these sequences correspond to “early genes” which are not induced as a consequence of cell growth, but rather are directly regulated by PDGF.