Colchicine acts as a progression factor to initiate DNA synthesis in quiescent Balb/c 3T3 cells

In quiescent Balb/c 3T3 cells, competence factors such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and platelet-derived growth factor (PDGF) synergize with progression factors such as insulin to initiate DNA synthesis. In this study, we found that colchicine, a microtubule-disrupting agent, acted synergistically with TPA, but not with insulin, to induce the maximal stimulation of DNA synthesis. Colchicine also synergized with PDGF in the presence of epidermal growth factor to elicit nearly the optimal induction of DNA synthesis. Moreover, it acted synergistically with fibroblast growth factor, another competence factor. These results suggest that colchicine acts as a progression factor like insulin in quiescent Balb/c 3T3 cells.     

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.     

Immediate induction of a 45 K secreted glycoprotein by serum and growth factors in quiescent mouse 3T3 cells : Two-dimensional gel analysis

Stimulation of quiescent 3T3 cells by serum dramatically induces the synthesis of a group of secreted polypeptides with a molecular weight (MW) of 45 K (p45 A, B, C, D). The synthesis of these polypeptides increases 10-fold during the first 2 h. Cycloheximide superinduces the 45 K polypeptides and actinomycin D (actD) blocks completely their induction by serum. Peptide mapping analysis and pulse-chase experiments revealed that p45 A is a precursor of polypeptides p45 B, C, D. Tunicamycin treatment inhibits the synthesis of all four polypeptides but a new related protein appears, p-p45, the unglycosylated precursor. In the presence of tunicamycin, p-p45 is also found in the medium, demonstrating that glycosylation is not essential for the secretion. In vitro translation experiments show that the levels of p45 mRNA present in stimulated cells are severalfold higher than that of non-stimulated cells. Purified growth factors, such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) induce the synthesis of p45 in quiescent cells.     

Obligatory action of polypeptide growth factors for the IL-1-mediated prostaglandin E2 production in fibroblasts. Potential role of growth factors in modulation of tissue response to IL-1

Our studies show that in connective tissue cells, induction of PGE2 synthesis in response to IL-1 requires costimulation with platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF). In cells incubated in medium containing fresh serum, IL-1 induced a dose-dependent synthesis of PGE2. However, when the cells were incubated in medium containing low serum or platelet poor plasma (lacking PDGF), IL-1 alone failed to induce PGE2 synthesis. PGE2 synthesis was restored when platelet poor plasma was supplemented with PDGF. Addition of PDGF or FGF together with IL-1 resulted in a 14- and 66-fold stimulation of PGE2 synthesis, respectively. Stimulation was dependent on the concentration of both IL-1 and the growth factor. PGE2 synthesis was also dependent on the synthesis of new proteins. In cells simultaneously treated with IL-1 and PDGF, PGE2 synthesis was initiated after a lag of 2 to 3 h, proceeded first with a rapid rate for 6 h, and then with a slower rate through 24 h. PGE2 synthesis during the latter, slower phase was greatly enhanced by pretreatment with PDGF, but not by pretreatment with IL-1. PDGF pretreatment also resulted in maintenance of 10- to 12-fold higher cell surface IL-1-binding during this phase. These data provide evidence for potentially novel interactions between PDGF and IL-1 activities, one of which is the modulation of IL-1 receptors by PDGF. Furthermore, these studies suggest that by virtue of their effect on IL-1 activities, PDGF and FGF may play additional roles in connective tissues, including an indirect role in inflammatory processes.     

Growth factor- and dexamethasone-induced proteins in Swiss 3T3 cells. Relationship to DNA synthesis

Dexamethasone synergistically enhances the stimulation of DNA synthesis in quiescent Swiss 3T3 cells by cartilage-derived growth factor (CDGF) while having no consistent effect when added with platelet-derived growth factor (PDGF) or serum. We examined the hypothesis that this difference might be attributed to selective synthesis of individual proteins early in the G1 phase of the cell cycle. Swiss 3T3 cells were treated with CDGF, PDGF, and fetal bovine serum for 3 h, with or without dexamethasone, and [35S]methionine-labeled proteins were separated by two-dimensional electrophoresis on giant gels. Over 3300 proteins could be distinguished; 34 of these were consistently induced more than 3-fold by all three factors, while an additional 30 inductions were variably present. Dexamethasone by itself induced 8 other proteins, and at least 9 growth factor inductions were synergistically enhanced by addition of the hormone. To identify proteins intimately associated with growth control, we looked for inductions that reflected the dexamethasone synergy with CDGF on DNA synthesis and lack of such an effect with PDGF. The induction of only one group of proteins, the Band 1 isoforms (44-46 kDa, pI 6.1-5.9) displayed such selective synergy. The majority of the other growth factor inductions were inhibited by dexamethasone, even in the context of maximal DNA synthesis, implying that their increased synthesis is not required for growth. When 3T3 cells were treated with increasing doses of CDGF with and without dexamethasone, autoradiographic densities of induced proteins varied in a dose-responsive fashion. However, only levels of the Band 1 proteins bore a constant linear relationship to DNA synthesis, suggesting that they play an important role in early control of the cell cycle.     

Superinduction by cycloheximide of mitogen-induced secreted proteins produced by Balb/c 3T3 cells

We describe here some of the characteristics of the regulation of a group of secretory proteins whose secreted levels rise within 2-4 h of adding fibroblast growth factor (FGF), epidermal growth factor (EGF), or serum to quiescent Balb/c 3T3 cells. The levels of these secretory proteins are regulated similarly to the interferons. When cycloheximide is present during the induction period, the amounts of [35S]methionine incorporated into five of these proteins that we have called "superinducible proteins" (SIPs) is increased 2-5-fold. Superinduction of the SIPs is seen also in response to polyribol-polyriboC, the classical inducer of interferons. None of the SIPs, however, are immuno-precipitated by anti-beta-interferon antibody. Induction and superinduction of the SIPs is inhibited by actinomycin D. Superinduction occurs at concentrations of cycloheximide that inhibit protein synthesis by at least 85%. The SIPs are not major intracellular proteins; they are barely detectable in cellular fractions. Their induction is, however, correlated with the ability of the polypeptide growth factor to stimulate DNA synthesis; EGF, FGF, and serum induce the SIPs, whereas insulin does not, and insulin alone weakly stimulates DNA synthesis in these cells. Because FGF, EGF, and serum cause the SIPs to be produced at concentrations of cycloheximide that inhibit 85% of bulk protein and DNA synthesis, it follows that the SIPs are produced directly from the action of the growth factor and not as a consequence of increased growth. Although probably not interferons, in analogy to the lymphokines, the SIPs could be a set of autocrine or paracrine factors that rapidly convey the growth or differentiation signal between cells.     

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.     

Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors

Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.     

Alteration of the chemotactic response of NIH/3T3 cells to PDGF by growth factors, transformation, and tumor promoters

The platelet-derived growth factor (PDGF) is a potent chemoattractant for cells that respond to PDGF as a mitogen. The chemotactic response of these cells to PDGF is inversely related to their rate of proliferation, with quiescent cells exhibiting a 25-fold greater chemotactic response than exponentially growing cells. Factors that stimulate the growth of quiescent cells (EGF, FGF, PDGF, and serum) decrease the cells' migratory response to PDGF but not to fibronectin, suggesting that the decreased migration is not due to a general paralysis of cell motility. Transformed lines of NIH/3T3 cells lose their ability to respond to PDGF as a chemoattractant but can still migrate in response to fibronectin. Similarly, after treatment of 3T3 cells with the tumor-promoter phorbol myristate acetate, which induces a transformation-like phenotype, the cells no longer respond to PDGF as a chemoattractant but retain their migratory response to fibronectin. Thus it appears that the growth state of the cells can alter their migratory response to PDGF. These data suggest that growth factors, transformation, and tumor promoters specifically alter the cells' ability to respond to the PDGF-mediated chemotactic signal. It appears that both transformation and tumor promoters accomplish this by altering PDGF-binding to the cell surface.     

Altered growth factor sensitivity in EL2 rat fibroblasts: influence of this biological characteristic on cell growth

Extensive evidence indicate that platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) play a key role in the stimulation of the 3T3 fibroblast replication: in this connection, PDGF and EGF act as a competence and a progression factor, respectively. We have previously demonstrated that EGF alone leads density-arrested EL2 rat fibroblasts to synthesize DNA and proliferate in serum-free cultures. Here, we have analyzed the role of EGF in the control of EL2 cell proliferation. Our data show a dose-related effect of EGF on DNA synthesis and cell growth, with maximal stimulation for both parameters at 20 ng/ml. On the other hand, autocrine production of PDGF or PDGF-like substances by EL2 cells is seemingly excluded by experiments with anti-PDGF serum or medium conditioned by EL2 fibroblasts. EGF binding studies show that EL2 cells possess high affinity EGF receptors, at a density level 3 to 4-fold higher than other fibroblastic lines. In addition, EL2 cells show a normal down-regulation of EGF receptors, following exposure to EGF, but PDGF, fibroblast growth factor (FGF), transforming growth factor beta (TGF beta) and bombesin have not decreased the affinity of EGF receptor for its ligand. Moreover, in EL2 cells, the EGF is able to induce the synthesis of putative intracellular regulatory proteins that govern the PDGF-induced competence in 3T3 cells. Our data indicate that EGF in EL2 cells may act as both a competence and a progression factor, via induction of the mechanisms, regulated in other cell lines by cooperation between different growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The relative effects of different types of growth factors on DNA replication, mitosis, and cellular enlargement

It was recently demonstrated that growth in cell size can be dissociated from DNA synthesis and mitosis. 3T3 cells starved to quiescence in low serum concentration can be stimulated to undergo DNA synthesis and one cell division without growing in size (unbalanced growth) (42-44). We report here that in cells stimulated to undergo unbalanced growth, the cell nucleus undergoes balanced growth, i.e., nearly doubles in size prior to mitosis. The reduced ability to grow in cell size under unbalanced growth conditions is thus mainly ascribable to the cytoplasm. Furthermore, the extent to which cells grow in size prior to mitosis is dependent on the serum concentration in the tissue culture medium (44). This data suggests that some macromolecular factor or factors in serum are required for growth in cell size prior to mitosis. We report in this study that epidermal growth factor (EGF) alone exerts a small but significant stimulatory influence on DNA synthesis and mitosis but does not affect cellular enlargement. In contrast, insulin added at supraphysiological concentrations does not stimulate quiescent cells to enter S phase but instead stimulates growth in cell size in the small fraction of dividing cells. Furthermore, cells stimulated to proliferate by EGF could be induced to undergo balanced growth when insulin was added concomitantly. Finally, platelet-derived growth factor (PDGF) stimulates quiescent sparse 3T3 cells to undergo DNA synthesis and mitosis. PDGF also exerts a limited but significant effect on cellular enlargement. However, PDGF alone could not induce a complete balanced growth, i.e., a doubling in cell size prior to mitosis.     

Effects of mitogens on ornithine decarboxylase activity and messenger RNA levels in normal and protein kinase C-deficient NIH-3T3 fibroblasts

Ornithine decarboxylase activity was assessed in serum-deprived quiescent NIH-3T3 murine fibroblasts after exposure to a variety of growth-promoting factors. Ornithine decarboxylase activity increased after treatment with phorbol 12-myristate 13-acetate (PMA), fetal calf serum, bovine pituitary fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and the synthetic diacyglycerol sn-1,2-dioctanolyglycerol but not after treatment with epidermal growth factor, insulin, 4 alpha-phorbol 12,13-didecanoate, sn-1,2-dibutyrylglycerol, or the calcium ionophore A23187. Activity peaked at 3-4 h and returned to basal levels after 8 h. To determine the importance of protein kinase C in this increase, cells were pretreated with PMA for 16 h to make the cells effectively deficient in protein kinase C; this deficiency was documented by direct measurement of enzyme activity and immunoreactivity. The ornithine decarboxylase response to each mitogen was then compared in cells pretreated with PMA or control conditions. PMA pretreatment abolished the increase in ornithine decarboxylase activity due to additional PMA and decreased but did not eliminate the ability of serum, FGF, and PDGF to cause increases in ornithine decarboxylase activity. Similarly, pretreatment with PMA abolished the ability of additional PMA to increase ornithine decarboxylase mRNA levels but did not prevent the increases in these mRNA levels caused by FGF or serum. These data suggest that the increases in ornithine decarboxylase activity and mRNA levels that occur in quiescent fibroblasts in response to serum, FGF, or PDGF are due to activation of at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C.     

Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.     

Post-transcriptional control of protein synthesis in Balb/c-3T3 cells by platelet-derived growth factor and platelet-poor plasma

Platelet-derived growth factor (PDGF) and platelet-poor plasma, which lacks PDGF, both induce a rapid increase in the rate of total protein synthesis within quiescent, density-arrested Balb/c-3T3 cells. This stimulation of protein synthesis is associated with an increased aggregation of ribosomes into polyribosomes. Nuclear functions are not required for this response, as demonstrated by the observation that this stimulation of protein synthesis occurs in cells pretreated with actinomycin D and in enucleated cells (cytoplasts). The response to PDGF persists even after PDGF has been removed from the culture medium, but in contrast, when plasma is removed from the medium, polysomes disaggregate and protein synthesis declines. PDGF and plasma do not function synergistically to increase protein synthesis, whereas they do to induce optimum DNA synthesis. Thus stimulation of the translational apparatus may be necessary for the mitogenic response of Balb/c-3T3 cells to growth factors, but it is not by itself sufficient.     

Multiplication of Swiss 3T3 cells in a serum-free medium

Gently trypsinized Swiss 3T3 cells inoculated into medium MCDB 402 attach readily to polylysine-coated surfaces and remain viable for several days in the absence of exogenously added protein. Short-term multiplication under defined conditions can be obtained by supplementing the MCDB 402 with fibroblast growth factor (FGF), insulin (INS), and dexamethasone (DEX). Addition of bovine plasma fibronectin further improves attachment and viability. This system does not require initial plating in serum or the addition of poorly defined extracts for cellular attachment or for multiplication. In the complete system minus FGF, cells plated at a low density attach to the culture surface and become quiescent. The addition of FGF or PDGF 48–72 h after plating stimulates a high level of DNA synthesis during the following 24 h. EGF also stimulates DNA synthesis in these cells, but to a lesser extent. Insulin and dexamethasone are not needed for the initial DNA synthesis response to FGF, but are needed for continuing multiplication over a period of several days. This system provides a means for studying the effects of specific mitogens on Swiss 3T3 cells in the absence of undefined supplements, and without complications due to density-dependent inhibition.     

A transient rise in cytosolic calcium follows stimulation of quiescent cells with growth factors and is inhibitable with phorbol myristate acetate

We have used aequorin as an indicator for the intracellular free calcium ion concentration [( Ca++]i) of Swiss 3T3 fibroblasts. Estimated [Ca++]i of serum-deprived, subconfluent fibroblasts was 89 (+/-20) nM, almost twofold higher than that of subconfluent cells growing in serum, whose [Ca++]i was 50 (+/-19) nM. Serum, partially purified platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) stimulated DNA synthesis by the serum-deprived cells, whereas epidermal growth factor (EGF) did not. Serum immediately and transiently elevated the [Ca++]i of serum-deprived cells, which reached a maximal value of 5.3 microM at 18 s poststimulation but returned to near prestimulatory levels within 3 min. Moreover, no further changes in [Ca++]i were observed during 12 subsequent h of continuous recording. PDGF produced a peak rise in [Ca++]i to approximately 1.4 microM at 115 s after stimulation, and FGF to approximately 1.2 microM at 135 s after stimulation. EGF caused no change in [Ca++]i. The primary source of calcium for these transients was intracellular, since the magnitude of the serum-induced rise in [Ca++]i was reduced by only 30% in the absence of exogenous calcium. Phorbol 12-myristate 13-acetate (PMA) had no effect on resting [Ca++]i. When, however, quiescent cells were treated for 30 min with 100 nM PMA, serum-induced rises in [Ca++]i were reduced by sevenfold. PMA did not inhibit growth factor-induced DNA synthesis and was by itself partially mitogenic. We suggest that if calcium is involved as a cytoplasmic signal for mitogenic activation of quiescent fibroblasts, its action is early, transient, and can be partially substituted for by PMA. Activated protein kinase C may regulate growth factor-induced increases in [Ca++]i.     

Optimal conditions for the study of growth control in BALB/c 3T3 fibroblasts

The effect of a Fibroblast Growth Factor (FGF) on the initiation of DNA synthesis in sparse populations of BALB/c 3T3 cells maintained quiescent in the presence of various serum concentrations has been investigated. The initiation of DNA synthesis, as measured by ³H-thymidine incorporation, is greatest in cultures maintained quiescent in the presence of 0.8% serum. Under these conditions, the cells are on the border between quiescence and growth. The minimal effective dose of FGF needed to increase DNA synthesis is 0.01 ng/ml and plateau values are obtained between 2.5 and 5 ng/ml. At plateau concentrations, FGF is 65% as effective as saturating concentrations of serum in the stimulation of DNA synthesis. When dexamethasone and insulin are present, FGF was 82% as effective. In contrast, cultures maintained in the presence of lower serum concentrations (0.2% and 0.4%) are much less responsive to the FGF. This can be attributed to the lack of supplemental factors which make the cells maximally responsive to growth stimulation and to degenerative changes that take place in the cells. Insulin and the glucocorticoid, dexamethasone, potentiated the response to FGF and delayed the degeneration of cells maintained in low serum.     

Platelet-derived growth factors and fibroblast growth factors are mitogens for rat Schwann cells

Rat sciatic nerve Schwann cells in culture respond to a limited range of mitogens, including glial growth factor, transforming growth factors beta-1 and beta-2 (TGF-beta 1, TGF-beta 2), some cell membrane-associated factors, and to agents such as cholera toxin and forskolin which raise intracellular levels of cAMP. These responses require the presence of FCS, which exhibits little or no mitogenic activity in the absence of other factors. However, we recently found that forskolin greatly potentiates the mitogenic signal from TGFs-beta 1 and beta 2, raising the possibility that cAMP might couple other factors to mitogenesis. We have therefore screened a range of candidate mitogens using DNA synthesis assays. Other than TGFs-beta and glial growth factor, none of the factors tested were mitogenic in the presence of 10% serum alone. With the addition of forskolin, however, porcine PDGF, human PDGF, acidic and basic FGF were potent mitogens for rat Schwann cells, stimulating DNA synthesis and increasing cell number. Cholera toxin and dibutyrylcyclicAMP, but not 1,9-dideoxyforskolin, can substitute for forskolin indicating that the mitogenic effect is mediated via adenylyl cyclase activation. Porcine PDGF gave half-maximal stimulation at 15 pM, and human PGDF an equivalent response at 1 nM. Basic FGF was half maximal at 5 pM, acidic FGF at 1 nM. The recognition of PDGFs and FGFs as mitogens for Schwann cells has many implications for the study of Schwann cell proliferation in the development and regeneration of nerves, and in Schwann cell tumorigenesis.