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.     

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).     

Secreted proteins induced by epidermal growth factor and transforming growth factor beta in EL2 rat fibroblasts. Role in the mitogenic response

Most growth active hormones and peptides are mitogenic only in the presence of other growth factors [e.g., Platelet Derived Growth Factor (PDGF) and Epidermal Growth Factor (EGF) in "competence-progression" fibroblast model]. We have previously described that EGF alone is able to induce the signals which appear necessary for the mitogenic stimulation of EL2 rat embryo fibroblast line. Recently, we have demonstrated that Transforming Growth Factor beta (TGF beta) slightly stimulates the mitogenic response in EL2 cells. Here, we show that in EGF-treated EL2 cells the induction of at least four inducible-secreted proteins (ISPs, range from 29,000 to 68,000 Mr) is accompanied by a marked increase in DNA synthesis. In contrast, TGF beta or different concentrations of EGF induce a slow increase of the ISPs proportional to slow induction in DNA synthesis. Our results suggest that the mitogenic response in EL2 cell line may be connected with the qualitative and quantitative induction of these secreted proteins.     

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 rat fibroblast cell proliferation at specific cell cycle stages by cocaine

We have analyzed the role of cocaine in the control of the rat fibroblast (EL2) cell proliferation. Our data show a dose-related effect on the inhibition of DNA synthesis and cell growth when cocaine is added with serum or with a pure growth factor [Epidermal Growth Factor (EGF)]. Pretreatment by drug did not appreciably enhance the inhibition of S-phase entry above that obtained when cocaine and mitogen were added simultaneously. On the contrary, exposure of quiescent EL2 cells to cocaine has little or no effect on DNA synthesis, when drug is removed before the mitogenic stimulus. Moreover, even when cocaine is added after EGF, an exposure only within 1–8 hours is required in order to inhibit stimulation of DNA synthesis. Cocaine also suppressed the general increase in protein synthesis that occurs during the first hour after EGF addition. The combined data suggest that cocaine inhibits the traverse of mitogen-stimulated quiescent EL2 cells from Go to S phase by acting on processes that take place during the initial phase of the cell cycle.     

Epidermal growth factor stimulates proliferation and DNA synthesis in ciliate cells

We studied the effect of murine epidermal growth factor on cell proliferation and DNA synthesis in macronuclei of ciliate Tetrahymena pyriformis G1. Mitogenic effect of epidermal growth factor on proliferation-induced tetrahymena cells has been revealed. This effect is due to the induced progression of cells at G1 and, consequently, their earlier entering DNA synthesis phase of the first cell cycle. Epidermal growth factor had no mitogenic effect on the resting cells from stationary culture (G0 phase) whose development is independent of the growth factors in the medium.     

Thrombin, epidermal growth factor, and phorbol myristate acetate stimulate tubulin polymerization in quiescent cells: a potential link to mitogenesis.

Previous studies suggest that alterations in the microtubule (MT)-tubulin equilibrium during G0/G1 affect mitogenesis. To determine the effect of growth factors on the MT-tubulin equilibrium, we developed a radioactive monoclonal antibody binding assay (Ball et al.: J. Cell. Biol. 103:1033-1041, 1986). With this assay, 3H-Ab 1-1.1 binding to cytoskeletons in confluent populations of cultured cells is proportional to the number of tubulin subunits polymerized into MTs. We now show that purified alpha-thrombin increases 3H-Ab 1-1.1 binding to cytoskeletons of serum-arrested mouse embryo (ME) fibroblasts from 1.5- to 3-fold. This stimulation is dose-dependent and correlates with concentrations of thrombin required for initiation of DNA synthesis. Other mitogenic factors, epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA), also stimulate MT polymerization. Addition of colchicine (0.3 microM) eight hours after growth factor addition, blocks stimulation of 3H-thymidine incorporation by thrombin, EGF, or PMA, suggesting that tubulin polymerization or subsequent events triggered by MT polymerization are required for cells to enter a proliferative cycle. Consistent with models for autoregulation of tubulin synthesis, thrombin, EGF, and PMA all increase tubulin synthesis 9 to 15 hr after growth factor addition, raising the possibility that the decrease in free tubulin and subsequent stimulation of tubulin synthesis is linked to progression of cells into a proliferative cycle. Colchicine addition to these cells also stimulates DNA synthesis, but colchicine-stimulated cells enter S phase 6 to 8 hr later than those stimulated by growth factors. This delayed stimulation may be related to the time required for degradation of tubulin-colchicine complexes below a critical level. These data suggest that regulation of cell proliferation may be linked to increased MT polymerization and the resulting decrease in free tubulin pools.     

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.     

Cellular microenvironment influences the ability of mammary epithelia to undergo cell cycle

The use of cell culture models is a principal and fundamental technology used in understanding how mammalian cells work. However, for some cell types such as mammary epithelia, the lines selected for extended culture are often transformed or have chromosomal abnormalities, while primary cultures have such a curtailed lifespan that their use is restricted. For example, mammary luminal epithelial cells (MECs) are used to study mechanisms of breast cancer, but the proliferation of primary cell cultures is highly limited. Here we describe the establishment of a new culture system to allow extended analysis of cultures of primary mouse MECs. In 2D monolayer culture, primary MECs showed a burst of proliferation 2-3 days post isolation, after which cell cycle decreased substantially. Addition of mammary epithelial growth factors, such as Epidermal Growth Factor, Fibroblast Growth Factor-2, Hepatocyte Growth Factor, and Receptor Activator for Nuclear Factor κB Ligand, or extracellular matrix proteins did not maintain their proliferation potential, neither did replating the cells to increase the mitogenic response. However, culturing MECs directly after tissue extraction in a 3D microenvironment consisting of basement membrane proteins, extended the time in culture in which the cells could proliferate. Our data reveal that the cellular microenvironment has profound effects on the proliferative properties of the mammary epithelia and is dominant over growth factors. Moreover, manipulating the cellular environment using this novel method can maintain the proliferative potential of primary MECs, thus enabling cell cycle to be studied as an endpoint after gene transfer or gene deletion experiments.     

Cycloheximide or puromycin can substitute for PDGF in inducing cellular DNA synthesis in quiescent 3T3 cells

A brief exposure of quiescent (Go) Swiss 3T3 mouse fibroblasts to inhibitors of protein synthesis can replace platelet-derived growth factor in the stimulation of cellular DNA synthesis. When 3T3 cells, after a 6 hr exposure to either cycloheximide or puromycin, are incubated with platelet-poor plasma, a significant percentage of cells enters DNA synthesis. Either inhibition of protein synthesis, or platelet poor plasma by themselves are totally ineffective. A possible mechanism by which inhibitors of protein synthesis may initiate cell cycle progression is through the activation of the c-myc gene.     

Stimulation of DNA synthesis in quiescent rabbit chondrocytes in culture by limited exposure to somatomedin-like growth factors

Cartilage-derived factor (CDF), extracted from fetal bovine cartilage, and multiplication-stimulating activity (MSA) stimulated DNA synthesis in quiescent rabbit costal chondrocytes in culture under serum-free conditions. As described previously, when added in the presence of fibroblast growth factor (FGF) or epidermal growth factor (EGF) a somatomedin-like growth factor, CDF or MSA, synergistically stimulated DNA synthesis in the cultured chondrocytes. The present study showed that exposure of the cells to MSA or CDF for only the initial 5 h was sufficient for transmission of their full stimulatory effect. Furthermore, the limited exposure did not alter the time course of stimulation of DNA synthesis: [3H]thymidine incorporation into DNA began to increase after 16 h and reached a maximum after 24 h. In contrast to the somatomedin-like growth factors, FGF and EGF were required continuously in the culture medium during traverse of the entire G1 phase for stimulation of DNA synthesis, and the mitogenic effects of FGF and EGF in cultured chondrocytes were stronger than those of CDF and MSA. Synergistic stimulation of DNA synthesis by CDF or MSA in the presence of FGF or EGF could be observed as long as FGF or EGF was continuously present, even when CDF or MSA was withdrawn after the first 5 h of culture. These findings suggest that, in contrast to FGF and EGF, somatomedin-like growth factors affect an early distinct stage in the G1 phase of chondrocytes.     

Transplantation of adult neural progenitor cells transfected with vascular endothelial growth factor rescues grafted cells in the rat brain

Growth factors are currently evaluated as therapeutics in stroke and neurodegeneration. Besides direct neurotrophic effects, they promote proliferation, survival, and differentiation of both transplanted and endogenous neural precursor cells (NPCs). In the current study, we investigated whether NPCs expressing Vascular Endothelial Growth Factor VEGF-A165 are a useful vehicle for growth factor delivery after transplantation into the caudate putamen of the rat brain. We found an increased survival of adenovirally transfected NPCs after 11 days, but not after 24 hours or 4 days. Additional brain immunohistochemistry revealed increased expression of the endothelial cell marker PECAM-1 (CD31) after 24 hours, 4 day, and 11 days after transplantation. In conclusion, we show that the graft itself is a useful vehicle for growth factor delivery, promoting the survival of NPCs. Moreover, transplantation of VEGF-expressing NPCs supports angiogenesis in the brain, which may contribute to potential brain repair.     

Control of proliferation of bovine vascular endothelial cells.

The effects of Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF) on the proliferation of bovine vascular endothelial cells has been examined. FGF induces the initiation of DNA synthesis and cell proliferation in cloned endothelial cells of fetal and adult origin at concentrations as low as 1 ng/ml and is saturating at 50 ng/ml. EGF had no effect over the same range of concentrations. The mitogenic effect of FGF is blocked by a crude extract of cartilage. Platelet extract is also mitogenic for vascular endothelial cells although to a lesser extent than the purified FGF. In contrast to vascular endothelial cells, both EGF and FGF are mitogenic for vascular smooth muscle cells although EGF is less mitogenic than FGF at 100 ng/ml. The mitogenic effect of EGF and FGF on vascular smooth muscle is not blocked by the addition of a crude extract of cartilage, thus demonstrating the specificity of the chalone like effect of cartilage crude extract for endothelial cells.     

The effect of butyrate on the cell cycle in root meristems of Pisum sativum

Sodium butyrate at 5 mM in aerated White's medium reduced the mitotic index in root meristems of seedlings of Pisum sativum to < 1% after 12 h. This effect was lessened as the butyrate concentrations were lowered. The fraction of the root meristem nuclei in G2 increased to ~ 70% after 12 h in butyrate. After 12 h exposure to butyrate, seedlings transferred lo medium without butyrate gradually re-established their normal root meristem mitotic pattern, with a burst of mitosis at 10 h after the transfer. Even a brief exposure to butyrate inhibited DNA synthesis, and nuclei released from butyrate exposure were still unable to resume normal DNA synthesis even after 12 h. This information suggests that butyrate halts progression through the cell cycle by arresting meristem nuclei in G2 and inhibiting DNA synthesis.     

Stimulation and inhibition of cardiac myocyte proliferation in vitro

Summary We have examined the effect of crude cardiac tissue extracts as well as that of several growth factors and triiodothyronin (T3) on DNA synthesis of cardiac myocytes in culture. Extracts from embryonic and adult cardiac tissue stimulated DNA synthesis of myocytes. Atrial myocytes exhibited overall higher degree of stimulation than their ventricular counterparts and extracts from adult atrial tissue had the highest apparent mitogenic activity for atrial myocytes. We have shown that adult heart contains basic fibroblast growth factor (bFGF), especially in the atria [1]. Transforming growth factor (TGF) and insulin-like growth factors (IGFs) are also accumulated in cardiac tissues [2, 3]. We found that bFGF and the IGFs stimulate myocyte cell proliferation and DNA synthesis. These factors also stimulate cardiac non-muscle proliferation, especially in the presence of serum. TGF inhibited proliferation and DNA synthesis and cancelled the effect of bFGF or IGFs on the myocytes. T3 also diminished the bFGF-induced mitogenic stimulation of cardiomyocytes. Our data suggest that these factors may be involved in the regulation of cardiomyocyte proliferation in vivo.Abbreviations bFGF basic Fibroblast Growth Factor - BSA Bovine Serum Albumin - DM Defined Medium - Fes Fetal calf serum - FITC Fluorescein - IGF Insulin-like Growth Factor - IgG Immunoglobulin - LI Labeling Index - PBS Phosphate Buffered Saline - T3 Triiodothyronine - TGF Transforming Growth Factor      

改构型酸性成纤维细胞生长因子对细胞增殖活性的影响

目的:研究aFGF和MaFGF对正常的肾小管上皮细胞及胃癌细胞增殖的影响。方法:用不同浓度的aFGF和MaFGF分别作用于肾小管上皮细胞及胃癌细胞,48h后采用WST-8法测定aFGF和MaFGF对两种细胞的促增殖活性。结果:在各浓度下,MaFGF组对肾小管上皮细胞和胃癌细胞的促增殖作用都显著低于aFGF组。结论:MaFGF对肾小管上皮细胞及胃癌细胞的促分裂活性较aFGF明显下降。     

Activation of coagulation releases endothelial cell mitogens

Recent studies have indicated that endothelial cell function includes elaboration of growth factors and regulation of coagulation. In this paper we demonstrate that activated coagulation Factor X (Factor Xa), a product of the coagulation mechanism generated before thrombin, induces enhanced release of endothelial cell mitogens, linking these two functions. Mitogenic activity generated by cultured bovine aortic endothelial cells in response to Factor Xa included platelet-derived growth-factor-like molecules based on a radioreceptor assay. Effective induction of mitogens by Factor Xa required the integrity of the enzyme's active center and the presence of the gamma-carboxyglutamic acid-containing domain of the molecule. Factor Xa-induced release of mitogens from endothelium occurred in serum-free medium and was not altered by hirudin or antibody to Factor V, indicating that it was a direct effect of Factor Xa and was not mediated by thrombin. Elaboration of mitogenic activity required only brief contact between Factor Xa and endothelium, and occurred in a time-dependent manner. Generation of enhanced mitogenic activity in response to Factor Xa was unaffected by the presence of actinomycin D and was not associated with increased hybridization of RNA from treated cells to a v-sis probe. Release of mitogenic activity was dependent on the dose of Factor Xa, being half-maximal at 0.5 nM and reaching a maximum by 5 nM. Radioligand binding studies demonstrated a class of endothelial cell sites half-maximally occupied at a Factor Xa concentration of 0.8 nM. The close correspondence between the parameters of Factor Xa-induced mitogen release and Factor Xa binding suggests these sites may be related. When Factor X was activated on the endothelial cell surface by Factors IXa and VIII, the Factor Xa formed resulted in the induction of enhanced release of mitogenic activity. These data suggest a mechanism by which the coagulation system can locally regulate endothelial cell function and vessel wall biology before thrombin-induced release of growth factors from platelets.     

Inhibition by 1-beta-D-arabinofuranosylcytosine of the incorporation of N-acetylneuraminic acid into glycolipids and glycoproteins of hamster embryo cells

1-β-D-Arabinofuranosylcytosine which interferes with DNA synthesis in bacteria and mammalian cells and brings about transformation of hamster embryo fibroblasts, has been found to inhibit the incorporation of N-Acetylneuraminic acid into glycolipids and glycoproteins of both normal and transformed hamster embryo cells in tissue culture. Three hours after commencement of treatment (10^?3M ara-C), incorporation of [¹⁴C] thymidine into DNA was inhibited by 95 per cent, while incorporation of [³H] D-glycosamine (precursor of sialic acid) into glycolipids and glycoproteins was inhibited by 85 per cent. At 24 hours, the inhibition of incorporation of the two labelled components was 83 and 80 per cent respectively. In homogenates of both cell types, incorporation of [¹⁴C] N-acetylneuraminic acid was competitively inhibited by ara-CMP. Ara-C was found to have no effect on the incorporation of [¹⁴C] choline into phospholipids of cells grown in tissue culture. These results suggest that interference with DNA synthesis by ara-C may not be the only factor involved in cell transformation by this substance.     

Possible involvement of a GTP-binding protein, the substrate of islet-activating protein, in receptor-mediated signaling responsible for cell proliferation

Serum-induced DNA synthesis, as measured by increases in [3H]thymidine incorporation, in Swiss mouse 3T3 fibroblasts was markedly inhibited by exposure of the cells to islet-activating protein (IAP), pertussis toxin. The inhibition was well correlated with the toxin-induced ADP-ribosylation of a membrane GTP-binding protein with Mr = 41,000. The IAP-induced inhibition of cell growth was characterized by the following two features. First, the inhibition was selective to certain growth factors. DNA synthesis in 3T3 cells was supported by a combination of one of the competence factors and a progression factor such as insulin or epidermal growth factor. IAP was inhibitory when thrombin, fibroblast growth factor, prostaglandin F2 alpha, or phosphatidic acid was employed as a competence factor, but was not inhibitory when DNA synthesis was induced by combined addition of cholera toxin or phorbol ester with insulin. Second, IAP-induced inhibition was still observed when the toxin was added to cell culture 1-6 h later than the addition of the IAP-sensitive competence factors, which triggered rapid cellular responses such as adenylate cyclase inhibition, releases of inositol trisphosphate and arachidonic acid, and 45Ca influx within several minutes (Murayama, T., and Ui, M. (1985) J. Biol. Chem. 260, 7226-7233; Murayama, T., and Ui, M. (1987) J. Biol. Chem. 262, 5522-5529). Thus, IAP substrate GTP-binding protein(s) appears to be involved in the duration of rapid signals or the occurrence of new slow signals which are responsible for growth factor-induced cell proliferation. The site of the involvement may be proximal to protein phosphorylation by phorbol ester-activated and cAMP-dependent kinases.     

Short-chain fatty-acid-induced effects on the cell cycle in root meristems of Pisum sativum

Propionic acid and valeric acid at 1 m M reduced the mitotic index of root meristem cells of Pisum sativum to <1% after 12 h in aerated White's medium. After 12 h exposure to either acid, seedlings transferred to fresh medium resumed their normal mitotic index 12 h after transfer, with a burst of mitosis at 8 h. Exposure times of 8 h to either acid inhibited DNA synthesis, and nuclei released from either propionic or valeric acid inhibition were still unable to resume normal DNA synthesis after 12 h. Neither acid significantly altered the distribution of meristematic cells in G1 and G2 after 12 h. Propionic acid at 1 m M reduced the uptake of [¹⁴C]-leucin but conversion rates to protein were constant regardless of whether any acid was present. Another longer fatty acid, caprylic acid, at 1 m M did not significantly reduce the mitotic index nor did 1 m M benzoic acid, another organic acid. This information suggests that only the short-chain fatty acids, propionic acid and valeric acid, limit progression through the cell cycle by inhibiting DNA synthesis and arresting cells in G1 and G2 in a manner similar to butyric acid, a known cell arresting agent.