Cadmium produces a delayed mitogenic response and modulates the EGF response in quiescent NRK cells

Recent studies have shown that cadmium, at subtoxic levels, may induce a response characteristic of that elicited by a type of growth factor that supports the anchorage independent growth of cells that are not fully transformed. That is, Cd⁺⁺ was found to replace transforming growth factor beta in supporting soft agar growth of NRK-49F cells. To tes the extent to which Cd⁺⁺ further mimics transforming growth factor beta in its effects and to establish response patterns that suggest possible molecular mechnisms of action, we have determined the effects of Cd⁺⁺ and/or epidermal growth factor (EGF) on DNA synthesis in quiescent NRK-49F cells. We found that subtoxic doses of Cd⁺⁺ modulate EGF-induced DNA synthesis in a dose-dependent fashion. Although Cd⁺⁺ effects on early (16–24 hr) EGF-induced DNA synthesis are primarily inhibitory, later effects involve stimulation as well. Subtoxic doses of Cd⁺⁺ did not stimulate DNA synthesis in quiescent cells within 24 hr of addition. At later times (40 or 64 hr), however, an increase in DNA synthesis of up to threefold was induced by 0.25 M Cd⁺⁺. This pattern of mitogenic response, involving inhibition of early growth-factor induced DNA synthesis and stimulation of late DNA synthesis, is consistent with that reported to be effected in some instances by transforming growth factor beta. Because a defined pattern of gene expression also is associated with the mitogenic responses to transforming growth factor beta, future studies at the molecular level can definitively test the degree to which Cd⁺⁺ and transforming growth factor beta effects are common.Abbreviations CFE colony forming efficiency - EGF epidermal growth factor - MT metallothionein - PGDF paltelet derived growth factor - TGF transforming growth factor     

Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin

Summary Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [³H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10⁻⁹ M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells. This work was supported by grants from the Medical Research Council of Canada; the Department of Medicine, University of Toronto; and the National Cancer Institute of Canada. J. E. E. is a C.H. Best Foundation and Department of Medicine postdoctoral fellow.     

Effects of medium and substratum conditions on the rates of DNA synthesis in primary cultures of bile ductular epithelial cells

Summary Select medium and substratum conditions were investigated for their effects on semiconservative DNA synthesis in essentially pure primary cultures of bile ductular epithelial cells that were initially isolated from cholestatic rat livers at 6 to 10 wk after bile duct ligation. DNA synthesis in these cultured cells was serum-dependent, being at its highest level when the concentration of fetal bovine serum present in the medium was maintained at 10%. This serum-dependent DNA synthesis was completely inhibited when 10 mM hydroxyurea was also included in the medium, and bile ductular cells cultured in the continued presence of 1.0% fetal bovine serum showed only marginal DNA synthesis during 8 to 10 d of primary culture when compared with no-serum controls. Maximum rates of serum-dependent DNA synthesis were obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with either fibronectin from bovine plasma or type I rat-tail collagen. Cells cultured on plastic coated with basement membrane Matrigel exhibited the lowest levels of DNA synthesis, whereas those on plastic alone had intermediate amounts. Furthermore, the addition of epidermal growth factor (50 ng·ml⁻¹·d⁻¹) to medium supplemented with 1.0% fetal bovine serum greatly enhanced the rate of DNA synthesis in bile ductular cells after 6 d in primary culture on type I collagen-coated plastic over that measured in solvent control cultures. These findings indicate that our bile ductular epithelial cell culture model is potentially useful in the study of biliary cell growth regulation and carcinogenesis. This investigation was supported by USPHS grant RO1 CA 39225 to A. E. Sirica by the National Cancer Institute, Department of Health and Human Services, Bethesda, MD. During the period of this study, G. A. Mathis was a recipient of a Fellowship from the Fund for Academic Career Development of the State of Zurich, Switzerland.     

Synthesis of lactosaminoglycan-containing glycoproteins by vascular endothelial cells

Human vascular endothelial cells synthesize lactosaminoglycan-type glycoproteins which are found both associated with cells and secreted into the culture medium. Pronase-derived glycopeptides prepared from [³H]glucosamine-labeled glycoproteins were found to contain about 10% of the labeled products as a large size (M_r > 5000) ³H-labeled glycopeptide. Digestion of these ³H-labeled glycopeptides with endo-β-galactosidase resulted in the release of smaller size saccharides, which were characterized as having the structure sialic acid → Gal → GlcNAc → Gal. Treatment of [³H]glucosamine-labeled cells with melittin caused ³H-labeled glycoconjugates to be released from the cells. Separation of released glycoproteins from proteoglycans by DEAE-cellulose chromatography indicated that melittin had released 25% of the total ³H-labeled glycoproteins from the cell and 3% of the ³H-labeled proteoglycans. The ³H-labeled glycoproteins were digested with Pronase and the resulting ³H-labeled glycopeptides were fractionated on Sephadex G-50. The large size fraction (M_r > 5000) now comprised about 30% of these released ³H-labeled glycopeptides. These high molecular weight ³H-labeled glycopeptides were degraded with endo-β-galactosidase but not with testicular hyaluronidase. Analysis of the released ³H-labeled glycoproteins indicated a preferential release of glycoproteins of 70–90 kDa enriched in lactosaminoglycan-type oligosaccharides.     

Mutually Independent Cyclic AMP and Sodium Responses to Nerve Growth Factor in Embryonic Chick Dorsal Root Ganglia

Abstract: Chick embryo dorsal root ganglia display a rapid and transient rise in their cyclic AMP content when presented with nerve growth factor. These ganglia also depend on nerve growth factor for control of their intracellular Na⁺ and K⁺ levels. A sequential relationship between the cyclic AMP and Na⁺ responses is not readily apparent. Incubation of chick sensory ganglia in a sodium-free medium does not prevent the cyclic AMP response to nerve growth factor from occurring. When ganglia are first incubated with ouabain for 6 h, presentation of nerve growth factor elicits a cyclic AMP response, but no Na⁺ response. The cyclic AMP response therefore does not depend on the Na⁺ environment. An initial presentation of nerve growth factor to the ganglia for 30 min, followed by its withdrawal and subsequent re-administration at different intervals over several hours failed to result in a second cyclic AMP response. Nevertheless, the expected Na⁺ behaviors were still observed. Dibutyryl cyclic AMP is capable of eliciting a cyclic AMP response in chick sensory ganglia after 6 h of nerve growth factor deprivation. When both agents were presented simultaneously to the ganglia, only a single cyclic AMP response was obtained, corresponding in time to the response elicited by dibutyryl cyclic AMP alone-indicating that this drug acts on the NGF-sensitive cells. At the same time dibutyryl cyclic AMP alone failed to result in a Na⁺ response, leading one to conclude that the cyclic AMP response to nerve growth factor is truly not mediating the Na⁺ response. Additional support for the mutual independence of these two short-latency responses is provided by the apparent inability of nerve growth factor to cause a cyclic AMP response in chick embryo sympathetic ganglia, another traditional target for the factor, which is capable of displaying a Na⁺ response.     

Epidermal growth factor stimulates collagen synthesis in liver-derived epithelial clone cells

The effect of epidermal growth factor (EGF) on collagen fiber formation in clone RLC-18(4) epithelial cells obtained from rat liver was investigated by silver impregnation and assay of hydroxyproline content. EGF caused dose-related stimulation of collagen fiber formation and was effective at as low as concentration as 0.5 ng/ml. Actinomycin D suppressed collagen fiber formation increased by EGF, suggesting that this factor stimulates de novo collagen synthesis in the cells.     

Defined medium for normal adult human prostatic stromal cells

Summary Stromal-epithelial interactions are pivotal in many aspects of prostatic biology. A defined culture system is critical for the investigation of factors that regulate the growth and differentiation of human prostatic stromal cells. We have identified conditions which promote stromal cell attachment and proliferation in serum-free medium. MCDB 201, originally developed for the clonal growth of chick embryo fibroblasts, proved to be a superior basal medium of those that we tested. Supplementation of MCDB 201 with basic fibroblast growth factor (FGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) permitted attachment and exponential growth of cells throughout a 7-d period with an initial inoculum as low as 10³ cells per well of a 96-well microtiter dish. Using these assay conditions, we subsequently verified that basic FGF and IGF, but not PDGF, were required for optimal growth. No activity was found for heparin, transferrin, or the androgen R1881. Epidermal growth factor (EGF) didn’t stimulate growth when added to medium containing basic FGF and IGF, but was moderately stimulatory when added to basal medium alone. Cholera toxin inhibited growth. This simple and efficient culture medium provides a suitable assay system for more extensive studies of growth regulation and differentiation of human prostatic stromal cells, and will provide the basis for future development of a defined medium that supports clonal growth. Characterization of stromal-epithelial interactions will be facilitated by the use of this defined culture system for stromal cells in conjunction with the serum-free culture systems previously developed for human prostatic epithelial cells.     

Effects of Chronic Basic Fibroblast Growth Factor Administration to Rats with Partial Flmbrial Transections on Presynaptic Cholinergic Parameters and Muscarinic Receptors in the Hippocampus: Comparison with Nerve Growth Factor

Abstract: The present study compares the effects of chronic administration of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on various hippocampal cholinergic parameters in rats with partial unilateral fimbrial transections. Lesions resulted in marked reductions of several presynaptic cholinergic parameters: choline acetyltransferase (ChAT) activity (by 50%), [³H]-acetylcholine ([³H]ACh) synthesis (by 59%), basal and ve-ratridine (1 μM)-evoked [³H]ACh release (by 44 and 57%, respectively), and [³H]vesamicol binding site densities (by 35%). In addition, [³H]AF-DX 116/muscarinic M₂ binding site densities were also modestly decreased (by 23%). In contrast, [³H]pirenzepine/muscarinic M₁ and [³H]AF-DX 384/muscarinic M₂/M₄ binding site densities were not altered by the lesions, nor were they affected by any of the treatments. Intracerebroventricular administration of bFGF (10 ng, every other day, for 21 days) partially prevented the lesion-induced deficit in hippocampal ChAT activity, an effect that was not markedly different from that measured in the NGF-treated (1 μg intracerebroventricularly, every other day, for 21 days) rats. In rats treated with a combination of bFGF and NGF, ChAT activity was not different from that in rats treated with the individual factors alone. In contrast, the lesion-induced deficits in the other cholinergic parameters were not attenuated by bFGF treatment, although they were at least partially prevented by NGF administration. To determine whether higher concentrations of bFGF are necessary to affect cholinergic parameters other than hippocampal ChAT activity, rats were treated with 1 μg (every other day, 21 days) of the growth factor. In this group of rats, detrimental effects of bFGF, manifested by an increased death rate (46%), and marked reductions in body weight of the survivors, were observed. In addition, this concentration of bFGF appeared to exacerbate the lesion-induced reduction in [³H]ACh synthesis by hippocampal slices; [³H]ACh synthesis in lesioned hippocampi represented 36 and 52% of that in contralateral unlesioned hippocampi for the bFGF-treated and control groups, respectively. In conclusion, although bFGF administration attenuates the deficit in hippocampal ChAT activity induced by partial fimbrial transections, this does not appear to translate into enhanced functional capacity of the cholinergic terminals. This is clearly in contrast to NGF, which enhances not only hippocampal ChAT activity, but also other parameters indicative of increased function in the cholinergic terminals.     

Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site

Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 10⁴ receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.     

Production and characterization of antibody blocking epidermal growth factor: Receptor interactions

Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 · 10⁶ epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes.The immonoglobulin G fraction of this immune sera inhibited ¹²⁵I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of ¹²⁵I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5°C).Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A-431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.     

Cell-density-dependent changes in cell-surface glycopeptides and in adhesion of cultured intestinal epithelial cells

We studied mannose-containing glycopeptides and glycoproteins of subconfluent and confluent intestinal epithelial cells in culture. Cells were labelled with d-[2-³H]mannose for 24h and treated with Pronase or trypsin to release cell-surface components. The cell-surface and cell-residue fractions were then exhaustively digested with Pronase and the resulting glycopeptides were fractionated on Bio-Gel P-6, before and after treatment with endo-β-N-acetylglucosaminidase H to distinguish between high-mannose and complex oligosaccharides. The cell-surface glycopeptides were enriched in complex oligosaccharides as compared with residue glycopeptides, which contained predominantly high-mannose oligosaccharides. Cell-surface glycopeptides of confluent cells contained a much higher proportion of complex oligosaccharides than did glycopeptides from subconfluent cells. The ability of the cells to bind [³H]concanavalin A decreased linearly with increasing cell density up to 5 days in culture and then remained constant. When growth of the cells was completely inhibited by either retinoic acid or cortisol, no significant difference was observed in the ratio of complex to high-mannose oligosaccharides in the cell-surface glycopeptides of subconfluent cells. Only minor differences were found in total mannose-labelled glycoproteins between subconfluent and confluent cells by two-dimensional gel analysis. The adhesion of the cells to the substratum was measured at different stages of growth and cell density. Subconfluent cells displayed a relatively weak adhesion, which markedly increased with increased cell density up to 6 days in culture. It is suggested that alterations in the structure of the carbohydrates of the cell-surface glycoproteins are dependent on cell density rather than on cell growth. These changes in the glycopeptides are correlated with the changes in adhesion of the cells to the substratum.     

Multiple interactions in juxtaposed monolayers of amphibian neuronal,epidermal, and mesodermal limb blastema cells

Summary We have utilized a novel cell culture system to probe nerve-epidermal-blastema cell interactions, in which the cellular participants are plated as compartmentalized juxtaposed monolayers. A mesoderm monolayer is attached to a culture plate insert and placed into a tissue culture well, the bottom of which is seeded with a second cell type(s); the two monolayers are separated by a distance of 1 mm. An examination of the multiple interactions occurring during blastemal growth can thus be accomplished by deletion of one or more of the cellular participants and employment of a replacement strategy utilizing substances with putative growth-promoting activity. [³H]Thymidine incorporation into DNA of urodele amphibian blastemal mesoderm cells was evaluated in response to the influence of juxtaposed brain or epidermal cell monolayers or both cultured in paired combinations. Brain cells were resolved by differential adhesive selectively into enriched neuronal and glial subpopulations. In the presence of enriched neuronal cells, radiolabel incorporation into blastemal mesoderm DNA was significantly greater than that achieved by coculturing with glial or unresolved brain cell populations. The effect of epidermal cells was not overtly expressed in the absence of neuronal cells. The effect of growth factors [fibroblast growth factor (FGF), epidermal growth factor (EGF), nerve growth factor (NGF)] on radiolabel incorporation into blastemal mesoderm DNA was also assessed in both the presence and absence of neuronal cells. Both EGF and FGF enhanced radiolabel incorporation into DNA when blastemal mesoderm was cultured in the absence of nerve, but the effect was significantly less pronounced than that achieved by neuronal cells alone. This system has provided us with an additional means of characterizing the dialogue between epidermis, nerve, and mesodermal blastema cells, and determining the identity of growth signals. This work was supported by National Sciences and Engineering Research Council of Canada grant A6933 to M. Globus.     

Long-term culture of human endothelial cells

Summary Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10³/cm²) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts. This research was supported by grants from the U.S. Public Health Service (AG 01732, HL 16387, and HL 07080), the Cystic Fibrosis Foundation, and the New York and American Heart Associations. Victor B. Hatcher is an Established Fellow of the New York Heart Association and a recipient of the Ann Weinberg Cystic Fibrosis Research Scholarship Award.     

Sulfated glycoproteins and extracellular matrix of cultured human pulmonary endothelial cells

Endothelial cells derived from human pulmonary arteries incorporate (³H)-glucosamine and ³⁵SO₄ into glycosaminoglycans and into the carbohydrate side chains of glycoproteins. These ³H/³⁵S-carbohydrate chains were isolated from cells and culture medium after Pronase digestion. The ³H/³⁵S-glycosaminoglycans were separated from the ³H/³⁵S glycopeptides by chromatography on Sephadex G-50. The distribution of cellular glycosaminoglycans and glycopeptides indicated that 30–60% of the cellular ³⁵S-glycopeptides may be associated with the matrix components that are synthesized by the cell and attached to a plastic substratum. Human pulmonary arterial endothelial cells were grown on collagen or on a matrix derived from vascular smooth muscle cells in order to investigate how smooth muscle cell extracellular matrix components may regulate the synthesis of endothelial cell glycoconjugates. Endothelial cells grown on plastic release various proportions of the glycoconjugates they synthesize into the culture medium. However, these same cells, when grown on substratum composed of extracellular matrix materials, synthesized altered proportions of cell-associated glycosaminoglycans and reduced the levels of total glycosaminoglycans they released into the culture medium. Thus the growth of endothelial cells on a matrix of smooth muscle cell components indicates that the glycosaminoglycan materials released into the culture medium by cells grown on a plastic substratum may not be an accurate reflection of the levels or composition of extracellular matrix materials made by endothelial cells in vivo.     

The growth of WI-38 cells in a serum-free,growth factor-free,medium with elevated calcium concentrations

Summary The growth of WI-38 cells in serum-free growth medium with and without hormone supplementation in the presence of elevated Ca²⁺ concentrations was investigated. At 5 mM CaCl₂, WI-38 cells seeded at low density without serum or hormone supplementation showed up to a 12-fold increased in cell number at saturation density over that obtained at day 1. Saturation densities were comparable when either 5 mM CaCl₂ or epidermal growth factor (1 mM CaCl₂) was used in the presence of insulin, dexamethasone and transferrin. Combining suboptimal doses of epidermal growth factor and CaCl₂ resulted in an additive effect on saturation density. Thus, nornal human diploid cells are capable of substantial growth in serum-free, hormone-free growth medium. In contrast, confluent cultures refed with the same medium are not responsive to elevated Ca²⁺ concentrations. In fact, elevated Ca²⁺ concentrations inhibited the proliferative response of confluent cultures to epidermal growth factor, but enhanced their response to the combined treatment of insulin, transferrin and dexamethasone. This work was supported by the United States Public Health Society grants T-32, CA09171 and AG-00378. Editor's Statement This paper rigorously dissects the interplay among external Ca²⁺ concentration, cell density and specific growth factors on fibroblast growth in defined medium. Wallace L. McKeehan     

Modulation of Rabbit Corneal Epithelial Cell Proliferation by Growth Factor-regulated K<Superscript>+</Superscript> Channel Activity

We characterized the dependence of the mitogenic response by rabbit corneal epithelial (RCE) cells to serum containing growth factors on K⁺ channel activation. Using both cell-attached and nystatin-perforated patch-clamp configurations, a K⁺ channel was identified whose current-voltage relationship is linear with a single-channel conductance of 31 pS. Its activity was barely detectable following 24 h serum starvation. Exposure of starved cells to either 10% FBS, 5 ng/ml epidermal growth factor (EGF) or 2 nM endothelin-1 (ET-1) continuously increased its activity within 30 min by 40%, 54% and 29%, respectively. EGF and ET-1 in combination had additive effects on such activity. Application of 100 µM 4-aminopyridine (4-AP), a K⁺ channel blocker, inhibited serum-stimulated K⁺ channel activity by 85%. DNA synthesis was markedly stimulated by serum, whereas incubation with either 4-AP (200 µM) or Ba²⁺ (1 mM) suppressed this increase by 51% and 23%, respectively, whereas 5 mM tetra ethyl ammonium (TEA) had no effect. Taken together, growth factor-induced increases in proliferation are dependent on K⁺ channel stimulation. As the increases in K⁺ channel activity induced by ET-1 and EGF were additive, these mitogens may stimulate K⁺ channel activity through different signaling pathways linked to their cognate receptors.     

Nerve Growth Factor Stimulates the Production of Inositol 1,3,4-and 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate in PC 12 Cells

Abstract: In PC12 cells, preincubated with [³H]inositol, nerve growth factor (NGF) stimulated an ～ 100% increase in the levels of [³H]inositol 1,3,4-trisphosphate {[³H]-Ins(1,3,4)P₃}, [³H]inositol 1,4,5-trisphosphate {[³H]lns(1,4,5)P₃}, and [³H]inositol 1,3,4,5-tetrakisphosphate {[³H]-Ins(1,3,4,5)P₄} as early as 5–15 s after addition of NGF. This NGF-mediated response was apparent only when the cells had been cultured in the absence of fetal bovine serum (FBS). PC12 cells cultured in FBS-containing medium did not display NGF-mediated increases in [³H]-Ins(1,3,4)P₃, [³H]-Ins(1,4,5)P₃, and [³H]-Ins(1,3,4,5)P₄ levels. Using cells cultured in the absence of FBS, epidermal growth factor (EGF) and fibroblast growth factor also stimulated production of [³H]lns(1,3,4)P₃, [³H]-Ins(1,4,5)P₃, and [³H]lns(1,3,4,5)P₄. Lavendustin A, a tyrosine kinase inhibitor, inhibited both the EGF-and NGF-stimulated increases in the levels of these tritiated inositol phosphates. These results suggest that NGF stimulates the production of lns(1,3,4)P₃, lns(1,4,5)P₃, and lns(1,3,4,5)P₄ and that this response is dependent on tyrosine kinase activity. Furthermore, although the production of lns(1,3,4)P₃, lns(1,4,5)P₃, and lns(1,3,4,5)P₄ may be a common response to factors stimulating neuronal differentiation, it is not sufficient for stimulation of neuronal differentiation.     

Influence of retinoids and EGF on growth of embryonic mouse palatal epithelia in culture

Summary Retinoids and growth factors seem to be important for normal mammalian reproduction and development. High levels of retinoic acid are teratogenic and induce cleft palate in the mouse. Little is known concerning the mechanisms through which retinoids induce cleft palate. Palatal epithelia from CD-1 embryonic mice on Day 12 of gestation were isolated from the mesenchyme and cultured in serum-free media, with all-trans retinoic acid or 13-cis retinoic acid, with or without epidermal growth factor (EGF). The epithelia attached and grew, and the cells differentiated over a 72-h culture period. Binding of [¹²⁵I]EGF was observed in all cultures in a pattern that correlated with thymidine (TdR) uptake by the epithelia. EGF enhanced growth and [³H]TdR incorporation of the oral cells, but nasal cells generally did not proliferate. In this culture system, both retinoids suppressed [³H]TdR incorporation in a concentration-dependent manner for epithelia cultured with or without EGF. Medial cells are important to normal palatogenesis as they play a role in fusion of opposing shelves and subsequently many of these cells undergo programmed cell death. Death of medial cells in vitro is prevented by EGF and by the retinoids, either with or without EGF. This response occurs in the absence of a mesenchymal interaction, suggesting that the medial cell response to EGF and retinoids is not mediated by or dependent on the mesenchymal tissues. The survival of medial cells may be responsible for the failure of opposing shelves to fuse.     

多肽生长因子受体的研究进展

多肽生长因子受体是介导多肽生长因子对细胞的调控作用的膜结合糖蛋白．它们在结构上都可分成胞外区、跨膜区和胞内区三个区段．根据这些区域的结构特点,特别是有无蛋白激酶结构域,提出了新的分类方法;并比较了各类生长因子受体信号转导的异同．     

Modulation of EGF binding and action by succinylated concanavalin A in fibroblast cell cultures

The role of the binding of succinylated concanavalin A to tissue culture cells in influencing epidermal growth factor (EGF)-mediated cell proliferation has been studied. Succinylated concanavalin A dramatically reduces the stimulation of 3T6 cells by EGF in Dulbecco's modified Eagle's medium (DME) containing insulin and vitamin B₁₂ as additional growth factors, but no serum. Furthermore, binding studies using ¹²⁵I-labeled EGF have shown that the binding of EGF to the cell surface is reduced upon addition of succinylated concanavalin A.