Cell-cycle-dependent expression of human ornithine decarboxylase

A human ornithine decarboxylase (ODC) gene probe has been isolated from a Jurkat T-cell cDNA expression library, sequenced, and used to analyze ODC mRNA levels in untransformed human lymphocytes and fibroblasts stimulated to proliferate by various mitogens. The partial cDNA sequence is 86% homologous to the mouse ODC cDNA, and Northern blots indicate that the human and mouse mRNA species are similar in size. ODC mRNA is barely detectable in quiescent human T lymphocytes and undetectable in density-arrested W138 fibroblasts. Following stimulation of T-lymphocyte proliferation with phytohemagglutinin, the ODC mRNA level rises to a peak around mid G1 phase and decreases as the cells enter S phase. Serum stimulation of density-arrested fibroblasts results in an elevation of the ODC mRNA level which persists throughout the cell cycle. Epidermal growth factor (20 ng/ml) but not insulin (10 mg/ml) or dexamethasone (55 ng/ml) stimulates ODC expression in quiescent W138 fibroblasts. Southern blots suggest that human cells have a single copy of the ODC gene.     

Increased glutathione synthesis associated with platelet-derived growth factor stimulation of NIH3T3 fibroblasts

Previous data show a relation between GSH content and proliferation of normal and tumour cells. We recently demonstrated a specific involvement of GSH in the autophosphorylation activity of the platelet-derived growth factor (PDGF) receptor in NIH3T3 fibroblasts. In this study we demonstrate that the stimulation by PDGF of serum-starved NIH3T3 cells increases cellular GSH content, while no change in oxidized GSH content was measured. Experiments performed with actinomycin, cycloheximide and buthionine sulfoximide, a specific inhibitor of the rate-limiting enzyme of the de novo synthesis of GSH gamma-glutamylcysteine synthetase (gamma-GCS), confirm PDGF induction of GSH synthesis. These results provide the first demonstration that PDGF mediated transduction signals seem strictly related to mechanisms involved in the increase of gamma-GCS activity associated with increased gamma-GCS heavy subunit mRNA levels. In fact, serum and epidermal growth factor (EGF) stimulation of quiescent NIH3T3 and NIH3T3, which overexpress EGF receptor, does not affect GSH content or its synthesis. These data may be related to a possible GSH role in the redox regulation of cell proliferation mediated by PDGF.     

Elevation of intracellular glutathione content associated with mitogenic stimulation of quiescent fibroblasts

The relationship between total glutathione (GSH) content and cell growth was examined in 3T3 fibroblasts. The intracellular GSH level of actively growing cultures gradually decreases as these cells become quiescent by either serum deprivation or high cell density. Upon mitogenic stimulation of sparse, quiescent (G0/G1) cultures with serum, there is a rapid 2.3-fold elevation in intracellular GSH levels which is maximal by 1 h and returns to baseline by 2 h. This is followed by a more gradual increase in GSH content as cells enter the S phase. In addition, the elevation in GSH content is required for maximum induction of DNA synthesis. Treatments that prevent the early increase in intracellular GSH levels do not affect protein synthesis but result in a reversible dose-dependent decrease in the percent of cells capable of entering S phase. These results indicate that GSH may be important in the regulation of cellular proliferation.     

Uridine transport and phosphorylation in mouse cells in culture: effect of growth-promoting factors, cell cycle transit and oncogenic transformation

The rapid increase in uridine uptake produced by the addition of serum to quiescent cultures of fibroblasts is primarily caused by an enhanced rate of nucleoside phosphorylation. While quiescent and serum-stimulated cells display identical initial rates of transport, they show a considerable change in the composition of the acid-soluble pools labelled with [3H] uridine for five seconds. The radioactivity recovered in the phosphorylated pools increases 2-, 3-, 4- and 6-fold after addition of serum to cultures of Swiss 3T3 cells, tertiary mouse embryo fibroblasts, Swiss 3T6 and Balb 3T3, cells respectively. Furthermore, insulin, a growth factor isolated from medium conditioned by SV40 BHK cells (FDGF) and epidermal growth factor (EGF) also stimulate uridine phosphorylation within minutes. The initial rate of uridine uptake is 2- to 3-fold faster in rapidly growing normal and Simian virus 40 or polyoma virus transformed 3T3 cells as compared to untransformed 3T3 cells in the quiescent state. When quiescent cultures of 3T3 or mouse embryo cells are stimulated to leave G1 and enter into DNA synthesis, transport increases several hours after addition of serum and apparently coincides with the S phase of the cell cycle. The results demonstrate that an increase in uridine phosphorylation is a rapid metabolic response elicited by growth-promoting agents in a variety of cell types and that uridine transport and phosphorylation are independently regulated.     

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.     

Extracellular ATP shows synergistic enhancement of DNA synthesis when combined with agents that are active in wound healing or as neurotransmitters

The polypeptides PDGF, TGF alpha, and EGF have previously been shown by others to stimulate proliferation of fibroblasts and keratinocytes in the process of wound healing. Here we demonstrate that extracellular ATP, ADP or AMPPNP caused synergistic enhancement of DNA synthesis in 3T6 mouse fibroblasts and BALB/MK keratinocytes when combined with any of the above polypeptides. TGF beta showed synergistic stimulation with ATP in fibroblasts but it inhibited keratinocytes. ATP acted as a mitogen for NIE-115 neuroblastoma cultures. In 3T6 cells, ATP stimulated thymidine incorporation in combination with carbachol or norepinephrine. The effect of carbachol was sensitive to atropine. We suggest that extracellular ATP and ADP may play a physiological role in wound healing and as a mitogenic neurotransmitter in the nervous system.     

Epidermal growth factor-induced topoisomerase(s). Intracellular translocation and relation to DNA synthesis

We have used epidermal growth factor (EGF) to investigate the relationship between eukaryotic topoisomerases and DNA synthesis. We found that EGF stimulates topoisomerase activity in human fibroblasts and Swiss/3T3 mouse fibroblasts. The first increase is seen in the cytoplasm, followed by increased activity in the nucleus. The nuclear increases correspond to increases in DNA synthesis. A type II topoisomerase is stimulated as indicated by the ATP dependence of the relaxing reaction and by the formation of catenanes. We have also found that the topoisomerase activity in the cytoplasm is sedimentable indicating that it is either membrane-associated or in a supramolecular complex. The stimulation of topoisomerase activity by EGF may represent a key step in the process by which EGF induces DNA synthesis and cell division.     

Growth factor-independent proliferation of normal human neonatal keratinocytes: production of autocrine- and paracrine-acting mitogenic factors

When normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 x 10(3)/cells cm2, the cells proliferated continuously and the addition of IGF-I, EGF, TGF alpha, bFGF, or aFGF did not significantly alter growth rate. Heparin sulfate, TGF beta, or suramin inhibited keratinocyte growth factor-independent proliferation. The addition of EGF, TGF alpha, or aFGF reversed heparin-induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte-derived conditioned medium (CM) indicated the presence of TGF alpha peptide at a concentration of approximately 235 pg/ml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte-derived CM replaced EGF in stimulating keratinocyte clonal growth, and an anti-EGF receptor mAb inhibited CM-induced keratinocyte clonal growth. In addition to its effect on keratinocytes, keratinocyte-derived CM stimulated the incorporation of [3H]thymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR-2B cells, and EGF-receptorless mouse NR6 cells. CM-stimulated [3H]thymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti-EGF receptor mAb. Heparin sulfate partially inhibited CM-induced keratinocyte clonal growth and [3H]thymidine incorporation into quiescent AKR-2B cells. We hypothesize from these data that autocrine and paracrine-acting factors produced by keratinocytes mediated their effect through the activation of both EGF receptor-dependent and EGF receptor-independent mitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin.     

Induction of fibronectin mRNA by urokinase- and tissue-type plasminogen activator in human skin fibroblasts: differential role of u-PA and t-PA at the fibronectin protein level

Plasminogen activators of the urokinase- and tissue-type and fetal calf serum (u-PA, t-PA, FCS) exert their mitogenic effect on quiescent human dermal fibroblasts and modulate the mRNA expression of cell-cycle related genes. The present study deals with the effects of PAs on the expression of fibronectin (FN), a heterodimeric extracellular matrix (ECM) protein that can be modulated in different ways by various mitogens. The kinetics of FN gene response was examined in quiescent fibroblasts upon PA stimulation (30 min -24 h). The results obtained evidenced that: (i) all mitogens tested (u-PA, t-PA and FCS) led to an increase of FN mRNA expression in early G1, as shown by the analysis of two sequences, III-9, common to all FN mRNAs, and EDA+, present only in the EDA+FN isoform; (ii) the kinetic profiles of FN mRNA stimulation were comparable for the three mitogens, although the effects on the FN-ECM assembly were distinct; (iii) t-PA and FCS led to FN assembly in the ECM, which was absent or decreased in u-PA-treated cultures. Immunobiochemical analysis of total FN and EDA+ FN showed that FN induced by t-PA was mainly dimeric (450-500 kDa), whereas FN induced by u-PA was mainly monomeric (230-250 kDa). These differences are probably due to the differential enzymatic action of t-PA and u-PA on FN, which might be related to a differential role of the two PAs in several physiopathological conditions.     

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.     

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.     

Transforming growth factor-beta 1 stimulates glucose uptake and the expression of glucose transporter mRNA in quiescent Swiss mouse 3T3 cells

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional polypeptide that regulates the proliferation and differentiation of various types of animal cells. TGF-beta 1 stimulated glucose uptake and the expression of a brain-type glucose transporter (GLUT1) mRNA in quiescent mouse 3T3 cells. TGF-beta 1 also synergistically stimulated these activities when given together with calf serum, phorbol ester, fibroblast growth factor, or epidermal growth factor. The increases in glucose uptake and the GLUT1 mRNA level were induced by picomolar concentrations of TGF-beta 1 within 3 h of stimulation, reached a peak between 6 and 9 h, and then decreased gradually to basal levels before an increase in DNA synthesis. The stimulation of GLUT1 mRNA expression was completely abolished by actinomycin D, but was not affected by cycloheximide, suggesting that new protein synthesis was not required for the expression of GLUT1 mRNA. TGF-beta 1 had little mitogenic activity and did not affect serum-induced DNA synthesis in quiescent 3T3 cells. However, it stimulated DNA synthesis synergistically when given with fibroblast growth factor, epidermal growth factor, phorbol ester, or insulin. These results suggest that TGF-beta 1 mediates the stimulation of glucose uptake, GLUT1 mRNA expression, and DNA synthesis via a pathway(s) and cellular components distinct from those for other growth factors. The possible role of the TGF-beta 1-induced stimulation of glucose transport activity in the control of mouse fibroblast proliferation is also discussed.     

Production of Second Messengers Following Chemotactic and Mitogenic Urokinase-Receptor Interaction in Human Fibroblasts and Mouse Fibroblasts Transfected with Human Urokinase Receptor

We studied urokinase-type plasminogen activator (u-PA)-dependent chemotaxis and DNA synthesis in both human fibroblasts and LB6 mouse fibroblasts transferred with human u-PA receptor (u-PAR) gene (LB6 clone 19). Both cell lines have receptors for the amino-terminal fragment of u-PA (u-PA-ATF). We observed that u-PA and u-PA-ATF stimulated chemotactic migration of both LB6 clone 19 cells and human fibroblasts, which could be impaired by down-regulation of protein kinase C (PKC) with phorbol myristate acetate (PMA). While LB6 clone 19 cells were unable to undergo mitosis following exposure to either u-PA or u-PA-ATF, human fibroblasts were stimulated to mitosis by exogenous addition of native u-PA, and u-PA-ATF was ineffective. The mitogenic activity of u-PA on human fibroblasts could also be impaired by down-regulation of PKC with PMA. We studied second messenger formation following u-PAR stimulation. Neither inositol lipid metabolism nor intracellular Ca²⁺ content were affected, while an increase of diacylglycerol (DAG) generation was observed. Such DAG formation was related to de novo synthesis from glucose and was dependent on ligand-receptor interaction. Both u-PA-ATF and the native u-PA molecule were able to stimulate DAG formation, u-PA being from three to fourfold more efficient than ATF. These data suggest that u-PAR stimulation per se is sufficient to trigger DAG formation. The native molecule confers on the cell an additional stimulus, possibly related with the activation of a u-PA-catalytic site-dependent substrate. Such stimulation allows the cell to reach the DAG treshold level required to trigger DNA synthesis.     

Synergistic stimulation of S6 ribosomal protein phosphorylation and DNA synthesis by epidermal growth factor and insulin in quiescent 3T3 cells

Using an improved method to quantify the level of phosphorylation of the S6 ribosomal protein, we have analyzed the effect of growth stimuli on S6 phosphorylation in quiescent murine Swiss/3T3 cells to see if it can be dissociated from the later increase in DNA synthesis. Saturating concentrations of epidermal growth factor (EGF), insulin and serum each stimulate phosphorylation of the S6 ribosomal protein to the same maximal level; this is not so for DNA synthesis. Subsaturating concentrations of EGF and insulin act synergistically to stimulate both S6 phosphorylation and DNA synthesis, but qualitatively the two synergistic interactions are expressed differently. Insulin increases the maximal response of DNA synthesis to EGF, whereas it decreases the concentration of EGF required for half-maximal stimulation of S6 phosphorylation. We conclude that S6 phosphorylation is not a principal regulator of DNA synthesis, and that insulin and EGF regulate both S6 phosphorylation and DNA synthesis through different, but interacting, pathways of action.     

Hexose uptake and control of fibroblast proliferation

The role of glucose uptake in control of cell growth was studied by experimentally varying the rate of glucose uptake and examining the subsequent effect on initiation and cessation of cell proliferation. The rate of glucose uptake was varied by adjusting the concentration of glucose in the culture medium. This permitted analysis of two changes in rate of glucose uptake which are closely related to the regulation of cell growth: (1) the rapid increase in glucose uptake that can be detected within several minutes after mitogenic stimulation of quiescent fibroblasts and (2) the decrease in glucose uptake which accompanies growth to a quiescent state. Quiescent cultures of mouse 3T3, human diploid foreskin and secondary chick embryo cells were switched to fresh serum-containing medium with either the normal amount of glucose or a reduced level that lowered the rate of glucose uptake below the rate characteristic of quiescent control cells. The subsequent increases in cell number were equal in both media, demonstrating that the increase in glucose uptake, commonly observed after mitogenic stimulation, was not necessary for initiation of cell division. Measurements of intracellular D-glucose pools after serum stimulation of quiescent cells revealed that the increase in glucose uptake was not accompanied by a detectable change in the intracellular concentration of glucose. Nonconfluent growing cultures of mouse 3T3, human diploid foreskin and secondary chick embryo cells were switched to low glucose media, lowering the rate of glucose uptake below levels observed for quiescent cells. This did not affect rates of DNA synthesis or cell division over a several-day period. Thus, the decrease in glucose uptake, which usually occurs at about the same time as the decrease in DNA synthesis as cells grow to quiescence, does not cause the decline in cell proliferation. Experiments indicated that there was no set temporal relationship between the decline in glucose uptake and DNA synthesis as cells grew to quiescence. The sequence was variable and probably depended on the cell type as well as culture conditions. Measurements of intracellular D-glucose pools in secondary chick embryo cells demonstrated that the internal concentration of glucose in these cells did not significantly vary during growth to quiescence. Taken together, our results show that these fluctuations in the rate of glucose uptake do not lead to detectable changes in the intracellular concentration of glucose and that they do not control cell proliferation rates under usual culture conditions.     

鹿茸表皮生长因子

作者等通过应用一凝肢柱或DEAE凝胶柱和固定金属离子亲和凝腔柱(Lmmobilised Metal Affinity chromatography Column,IMAC)的组合,成功地从梅花鹿的鹿茸表皮层中分离出表皮生长因子(Epidermal Growth Factor, EGF)。此半纯化的EGF能刺激小鼠Balb/c 3T3的静态成纤维细胞对~3H-胸嘧啶的渗入,渗入量随EGF剂量的增加而升高。在鹿茸表皮层的组织中亦同时含有大量的EGF受体,而骨轴组织不舍有EGF,只含少量受体。