Platelet derived growth factor induces ornithine decarboxylase activity in NIH 3T3 cells

Incubation with highly purified human Platelet Derived Growth Factor induced ornithine decarboxylase activity in quiescent NIH 3T3 cells concomitantly with mitogenic stimulation. Pretreatment of cells with a specific ornithine decarboxylase inhibitor, DL-alpha-difluoromethyl-ornithine significantly inhibited the effect of the mitogen on DNA synthesis. These experiments suggest that the mitogenic activity of Platelet Derived Growth Factor, similarly to that of other serum growth factors or tumor promoters, is mediated through rise in polyamine levels.     

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.     

Control of K+ influx in 3T3 cells transformed by a conditional mutant of Rous sarcoma virus

Mouse 3T3 cells transformed by a conditional mutant of Rous sarcoma virus (LA90) can assume either a normal or a transformed phenotype, depending on the temperature of cultivation. These cells (LA90) were arrested at the G0/G1 phase of the cell cycle by starvation for serum growth factors at the nonpermissive temperature (39 degrees C). Release from the G0/G1 phase by serum growth factors resulted in a rapid stimulation of Rb+ influx. To investigate whether the stimulation of Rb+ influx is obligatory for cell proliferation, the cultures were released from the G0/G1 phase by a temperature decrease in the absence of serum. A temperature decrease from 39 to 32 degrees C activated the viral pp60src gene mitogenic activity. Under these conditions, no rapid stimulation of Rb+ influx was observed. These results suggest that the rapid stimulation of Rb+ influx induced by serum growth factors is not an essential signal for cell release from the G0/G1 phase. However, a delayed increase in Rb+ influx concomitant with an increase in the cell content of K+ was observed in the cultures released from the G0/G1 phase by temperature decrease in the absence of serum growth factors. We found that the LA90 cells incubated at the permissive temperature (32 degrees C) secreted a mitogenic activity into the medium. Moreover, the conditioned medium from cultures incubated at 32 degrees C, but not at 39 degrees C, stimulate Rb+ influx in G0/G1 cells. These results indicate that Rous sarcoma virus pp60src induces a slow autocrine secretion of a mitogenic activity. This mitogenic activity slowly modulates the K+ content. Therefore, the slow elevation in cellular content of K+ is proposed to be an obligatory event for proliferation in normal and transformed cells.     

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

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

Sulfation and mitogenic activities of growth factors in human serum: importance of compounds of molecular weight lower than 1,000 in the global expression of activity of this serum on 3 different cellular types

A human serum ultrafiltrate contains compounds needed for a maximal expression of sulfation and mitogenic activities of the corresponding retentate. These low molecular weight (less than 1,000) molecules have no effect, by their own, on 35SO4(2-) uptake in chick embryo cartilage, but show a significant synergistic effect with serum growth polypeptides. Tested alone, they display a slight mitogenic activity as measured by 3H-thymidine incorporation in chick embryo fibroblasts or in lymphocytes activated by phytohemagglutinin. Here they also act in a synergistic way on mitogenic activities of growth factors from human retentate. A fraction (P2B), partially purified from human plasma ultrafiltrates, produces the same synergistic response with human retentate in the three cellular systems (cartilages, fibroblasts, activated lymphocytes). However, concentrations which lead to optimum responses are very different according to the cell type studied. These results suggest the existence of several compounds which can each act specifically on a cellular system.     

Cyclic AMP stimulation of Na-K pump activity in quiescent swiss 3T3 cells

Recently, we have found that an increase in the intracellular level of cAMP acts as a mitogenic signal for Swiss 3T3 cells (Rozengurt et al., Proc. Natl. Acad, Sci. USA, 78:4392, 1981). The results presented in this paper demonstrate that addition of cAMP-elevating agents to confluent and quiescent cultures of Swiss 3T# causes a marked increase in the rate of 86Rb+ uptake but has no effect on the rate of cation efflux. The stimulation of ion uptake is mediated by the Na-K pump as shown by the ouabain sensitivity of the 86Rb+ fluxes. The increase in Na-K pump activity occurs whether cAMP is generated endogenously by stimulation of adenylate cyclase activity by cholera toxin, adenosine agonists, or PGE1 or added exogenously as 8BrcAMP. The stimulatory effect of these compounds on 86Rb+ uptake is potentiated by inhibitors of cyclic nucleotide phosphodiesterase activity. Cholera toxin stimulates the Na-K pump in a dose-dependent manner; half-maximal effect is achieved at 0.7 ng/ml. The stimulation of ouabain-sensitive 86Rb+ uptake by cAMP-elevating agents reaches a maximum after 2-3 h of incubation. This contrasts with the rapid (within minutes) stimulation of the Na-K pump caused by serum and other mitogenic agents. Further, cAMP-elevating agents fail to increase Na+ influx into 3T3 cells whereas serum causes a marked increase in Na+ influx, under identical experimental conditions. These findings suggest that the stimulation of Na-K pump activity caused by increased cAMP levels contrasts mechanistically with the rapid control of pump activity by serum which is primarily mediated by increased Na+ entry into the cells.     

The antiproliferative effect of interferon and the mitogenic activity of growth factors are independent cell cycle events : Studies with vascular smooth muscle cells and endothelial cells

We studied the antagonistic effects of interferon (IFN) and growth factors in G0/G1-arrested normal bovine aortic smooth muscle cells (SMC) which were stimulated by serum, or purified platelet derived growth factor (PDGF), supplemented with plasma-derived serum (PDS). The growth response, measured as [3H]thymidine incorporation into DNA, was dependent on the concentration of the mitogen. Human IFN alpha, recombinant human IFN alpha 2, or a crude bovine-IFN preparation prepared from virus-infected bovine aortic endothelial cells, inhibited SMC growth induced by either serum or PDGF with PDS. The extent of IFN inhibition was inversely related to the concentration of the mitogenic stimulus. We also investigated whether IFN inhibited the early events in G1 phase, stimulated by the competence factor PDGF, or the progression of the cell into the S phase induced by PDS. The results indicated that IFN inhibited these two stages of the G1 phase independently. In addition, we investigated the antiproliferative effect of IFN on bovine aortic endothelial cells (BAEC), which do not respond to PDGF but to the mitogenic activity of fibroblast growth factor (FGF). IFN inhibited the mitogenic activity of FGF in a dose-dependent manner. The results indicate that the anti-proliferative activity of IFN and the mitogenic effects of different growth factors are independent.     

Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line

The regulation of creatine phosphokinase (CPK) expression by polypeptide growth factors has been examined in the clonal mouse muscle BC3H1 cell line. After arrest of cell growth by exposure to low concentrations of serum, BC3H1 cells accumulate high levels of muscle-specific proteins including CPK. The induction of this enzyme is reversible in the presence of high concentrations of fetal calf serum, which cause quiescent, differentiated cells to reenter the cell cycle. Under these conditions, the rate of CPK synthesis is drastically reduced. We show in the present communication that either pituitary-derived fibroblast growth factor (FGF) or brain-derived FGF are as effective as serum in repressing the synthesis of CPK when added to quiescent, differentiated cells. The decrease in the rate of synthesis of CPK occurs within 22 h after the addition of pituitary FGF to the cells. Pituitary FGF had very little effect, if any, on the rate CPK degradation. The overall rate of protein synthesis and the pattern of synthesis of the major polypeptides made by these cells was not altered by the addition of FGF. Although pituitary FGF was mitogenic for BC3H1 cells, the rate of cell growth was not absolutely correlated with the extent of repression of CPK. Brain-derived FGF fully repressed CPK induction under conditions where it showed no significant mitogenic activity. These results show that the expression of a muscle-specific protein, CPK, can be controlled by a single defined polypeptide growth factor in fully differentiated cultures, and that initiation of cell division is not required for their regulation to take place.     

Tumor necrosis factor stimulates proliferation of human osteosarcoma cells and accumulation of c-myc messenger RNA

The objective of this study was to establish whether human recombinant tumor necrosis factor (TNF) can significantly stimulate the proliferation of some tumor cells. Treatment with TNF had little or no effect on the growth of human tumor cells and murine NIH/3T3 cells cultured in medium with high serum concentration. Two tumor lines, SK-MEL-109 melanoma and HOS osteosarcoma cells, were adapted to grow in medium supplemented with 0.5% serum. The growth of these SK-MEL-109 cells was inhibited by TNF, but that of the HOS cells was greatly stimulated by TNF in a dose-dependent way. Treatment with 10 ng/ml of TNF resulted in a two-fold increase in the rate of cell division. This effect of TNF was also shown by measuring DNA and protein synthesis. The continuous presence of TNF was not required for its mitogenic activity on HOS cells cultured with 0.5% serum, since treatment for only one day with TNF resulted in prolonged growth stimulation. The failure of TNF to promote division of cells cultured in medium with 10% serum may possibly be explained by the presence of saturating amounts of growth factors in serum. Interferons abolished the mitogenic activity of TNF on HOS cells. Furthermore, TNF did not show synergism with insulin or epidermal growth factor in stimulating growth of these cells. The level of c-myc mRNA was increased five-fold after 30 minutes of treatment with TNF. This shows that TNF is a growth factor for HOS cells and that it induces accumulation of c-myc mRNA.     

Factors in the rat submaxillary gland that stimulate growth of cultured glioma cells: identification and partial characterization

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum-free culture has been examined. Extracts (10-15 microgram/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum-deprived C6 cells and, in combination with insulin, transferrin, and NIH-LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat-labile, acid-stable, and partially inactivated by protease and 2-mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth-enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF-binding protein and gamma-subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations. The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G-100, preparative isoelectric focusing in a pH gradient of 3-10, or adsorption to DEAE-cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c-3T3 cell growth in serum-free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83:96a) is also discussed.     

Mitogenic factors for BALB/c 3T3 cells isolated from the serum of horses by affinity chromatography on a column using fetal calf serum as the ligand

Fetal calf serum (SVF), insolubilized on a sepharose matrix, retains mitogenic factors (FM) from horse serum (SC) which can be eluted by molar acetic acid. The FM can: 1 degree initiate the synthesis of DNA in serum starved, contact inhibited 3T3 BALB/c fibroblasts and 2 degrees allow at least three generations to take place. The FM have no antitryptic activity and do not allow cell attachment. Weight to weight, this fraction is 10 to 50 times more active than horse serum. Polyacrylamide gel electrophoresis shows that various proteins are present, particularly immunoglobulins. The intervention of epidermal growth factor (EGF), fibroblast growth factor (FGF) or the platelet-derived growth factor (PDGF) have been excluded.     

Epidermal growth factor induces rapid centrosomal separation in HeLa and 3T3 cells

Using indirect immunofluorescence, we have found that epidermal growth factor (EGF), at 100 ng/ml, induces centrosomal separation within 20 min in HeLa and 3T3 cells. The effect was evident both in unsynchronized cultures and in HeLa cells blocked in early S phase by hydroxyurea. EGF also induced centrosomal separation in quiescent 3T3 cells blocked in G0/G1 by serum deprivation, indicating that DNA replication is not necessary for this effect. The mechanism of this rapid centrosomal separation and its role in the mitogenic effects of EGF remains to be determined.     

Stimulatory effect of serum albumin on the proliferation of serum-free SV40-transformed Balb/c 3T3 cells

Commercial serum albumins have been found to be able to stimulate the proliferation of Balb/c 3T3 cells transformed by SV40, but not that of the normal counterpart. The effect is most pronounced with crystalline samples of albumin depleted of both globulin and fatty acid components, and depends on conditions used for the attachment and on seeding density. Physical and chemical treatments aimed to remove tightly bound impurities do not abolish the activity of fatty acid free serum albumin, thus supporting the idea that albumin per se is mitogenic towards these cells.     

Calcium requirements for mitogenic stimulation of 3T3-cells in low and high serum concentration

The calcium requirements during mitogenic stimulation of quiescent 3T3-cells in low or in high serum concentration was investigated. It was found that calcium played an equally important role for growth stimulation in low as in high serum concentration. Calcium was not required during the first 6 hours after mitogenic stimulation. However, calcium had to be present thereafter for the cells to initiate DNA-synthesis. The absence of calcium during the first six hours after onset of stimulation did not delay the initiation of DNA-synthesis.     

Effect of TGF-Alpha on growth of normal human breast epithelial cells in serum-free primary culture using 3-dimensional collagen gels.

The mitogenic effect of TGF-alpha, acidic-FGF, basic-FGF and lithium on normal human breast epithelial cells was studied in a collagen gel culture system using a serum-free 1:1 mixture of Ham's F12 and DME medium containing insulin, cholera toxin and bovine serum albumin. TGF-alpha elicited a strong mitogenic response in a dose dependent manner. Addition of cortisol to TGF-alpha stimulated growth over and above that achieved with TGF-alpha alone. A consistent observation has been the effect of a combination of TGF-alpha and cortisol on growth stimulation of normal human breast epithelial cells resulting in 3-12 fold growth after 11-13 days in culture. Acidic-FGF, basic-FGF and lithium were not growth promoting.     

Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells.

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.     

Mitogenic action of factors secreted by avian erythroblastosis virus transformed cells

We describe here the capacity of erythroid LSCC HD3 cells, transformed with a ts mutant of avian erythroblastosis virus, to grow in a chemically defined medium without serum at 36 degrees C, but not at 41 degrees C. At this latter temperature the activity of v-erbB oncogene is suppressed. However, cell growth at 41 degrees C could take place either by addition of the medium derived from LSCC HD3 cells grown at 36 degrees C (conditioned medium), or by addition of fetal calf serum. These results show that LSCC HD3 cells, maintained under conditions in which the v-erbB oncogene is active, secrete growth factor(s) which exhibit a mitogenic effect similar to that observed with calf serum.     

Mitogenic activity of chicken chorioallantoic fluid is temporally correlated to vascular growth in the chorioallantoic membrane and related to fibroblast growth factors

The chorioallantoic membrane (CAM) is one of the most vascularized tissues in the chicken embryo. Capillary growth proceeds until day 10 of development and thereafter abruptly regresses. As it is generally accepted that the formation of new blood vessel is regulated by growth factors, we have investigated the presence of angiogenic and mitogenic factors in the chicken chorioallantois. In the present study, we show that chorioallantoic fluid (CAF) contains angiogenic substances that are probably synthesized in the CAM or the embryonic kidney. When applied in the chorioallantoic membrane assay, CAF from 9 day chicken embryos elicits a strong angiogenic response. This angiogenic activity of CAF is associated with pronounced mitogenic effects in vitro. Comparison of different embryonic fluids reveals that mitogenic activity is particularly evident in the CAF but not detectable in embryonic serum and amnion fluid. Expression of mitogenic activity is found to be temporally correlated with vascular growth in the CAM. High activity is detected in CAF prior to day 10 and then sharply decreases, thus preceding termination of capillary growth by one day. Heparin-sepharose affinity chromatography suggests that the biological activities of CAF probably correspond to the presence of acidic and basic fibroblast growth factor (aFGF and bFGF). In Western blot analyses of CAF, an immunoreactive bFGF-like protein of about 17 x 10(3) Mr is recognized by a monospecific anti-bFGF antiserum. This protein elutes at 2.4 M NaCl from the heparin-sepharose. The mitogenic activity of the CAF can be specifically blocked by the anti-bFGF antibody indicating bFGF to be the active mitogenic principle of the CAF. These results strongly suggest that basic and probably acidic FGF play an important role in the regulation of chorioallantoic vascular growth.