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1.
Immunohistochemical localization of basic fibroblast growth factor in A431 human epidermoid carcinoma cells. 总被引:2,自引:0,他引:2
The intracellular location of basic fibroblast growth factor (bFGF) was determined in A431 human epidermoid carcinoma cells both on immunofluorescence and on immunoelectron microscopy using a monoclonal anti-bFGF antibody. The immunofluorescence was located in the cytoplasm in quiescent cells. Following the addition of FCS to the culture medium of quiescent sparse cells the growth factor was translocated to and accumulated in the nucleolus. Immunogold particles were dense near the ribosomes, but were not recognized in the cytoplasmic structures concerned with the usual secretory pathway such as the rough endoplasmic reticulum, the Golgi apparatus, and secretory granules. These results suggest that endogenous bFGF undergoes intracellular sorting and enters the nucleoli in A431 cells according to an extracellular growth signal. 相似文献
2.
A M Arnautov I P Arnautova N N Zheleznova E S Kornilova N N Nikol'ski? 《Tsitologiia》1999,41(5):380-385
Intracellular distribution of p42/p44 MAP-kinases in HER14 and A431 cell lines was investigated. Using subcellular fractionation and immunofluorescence approaches we have shown that in quiescent cells of both types MAP-kinases are associated with endoplasmic reticulum. Moreover, ER-localized MAP-kinases were shown to exist only in a nonphosphorylated form. In HER14 cells the epidermal growth factor (EGF) elevates the level of the ER-associated MAP-kinases. In contrast, exposure of A431 cells to EGF leads to a significant decrease in the observed association. The physiological role of this association is discussed. 相似文献
3.
Basic fibroblast growth factor accumulates in the nuclei of various bFGF-producing cell types 总被引:7,自引:0,他引:7
The intracellular localization of basic fibroblast growth factor (bFGF) was studied in BHK-21 cells transfected with an expression vector containing the complementary DNA (cDNA) of the human bFGF gene (pbFGF). The intracellular location of bFGF was determined using indirect immunofluorescence. The antibodies used were polyclonal antibodies directed against either recombinant human bFGF or recombinant Xenopus bFGF. The nuclei of transfected cells that produce bFGF, but not the nuclei of untransfected cells, were labeled strongly by the antibodies. The nuclear staining was totally abolished when anti-bFGF antibodies preadsorbed with bFGF were used. Several types of endothelial cells known to produce bFGF were also stained in their nuclei by the antibodies. Nuclear extracts prepared from transfected cells were found to contain bFGF as determined using heparin-sepharose affinity chromatography, followed by Western blot analysis of fractions, which stimulated the proliferation BHK-21 cells. The mitogenic activity associated with the nuclei was not destroyed when isolated cell nuclei were digested by trypsin. It is therefore likely that the nucleus associated bFGF is intranuclear. These findings suggest that some biological activities of bFGF may be mediated by nuclear bFGF binding proteins or by the direct binding of bFGF to DNA. 相似文献
4.
5.
Wolfgang Hagmann Xiang Gao Joszef Timar Yong Q. Chen Anja-Rose Strohmaier Chris Fahrenkopf David Kagawa Miu Lee Alex Zacharek Kenneth V. Honn 《Experimental cell research》1996,228(2):197
Human A431 epidermoid carcinoma cells express 12-lipoxygenase enzymatic activity. However, the isoform identity based on cDNA sequence data is not known. Further, the simultaneous characterization of the intracellular distribution of 12-lipoxygenase protein and activity is lacking. Here we report that the cDNA sequence from RT-PCR-amplified 12-lipoxygenase mRNA is identical with the platelet-type 12-lipoxygenase isoform, and the leukocyte-type isoform of 12-lipoxygenase is not expressed in A431 cells. The predominant amount (78%) of 12-lipoxygenase protein resides in the cytosol. In contrast, the predominant (98%) 12-lipoxygenase activity is localized in the membrane fraction. Western blot and immunofluorescence data demonstrate that epidermal growth factor increases total cellular 12-lipoxygenase protein and enhances the association of 12-lipoxygenase protein with perinuclear or nuclear membrane sites. In addition, epidermal growth factor stimulates 12-lipoxygenase activity resulting in generation of 12(S)-hydroxyeicosatetraenoic acid from cellular arachidonate. In contrast, both 12-lipoxygenase protein and activity decrease approximately 80% within 24 h during serum starvation. The recovery of 12-lipoxygenase expression in serum-deprived cells can be induced by readdition of epidermal growth factor or serum. Further, the basal expression of 12-lipoxygenase depends on signal pathways requiring protein tyrosine kinase activity, since genistein, herbimycin A, and tyrphostin 25 reduce the expression of 12-lipoxygenase protein in A431 cells. 相似文献
6.
The hyaluronan-binding protease (HABP) is a serine protease in human plasma which is structurally related to plasminogen activators, coagulation factor XII and hepathocyte growth factor activator. It can in vitro activate the coagulation factor FVII, kininogen and plasminogen activators. The present study was initiated to gain a more complete picture of the cell-associated activities of this fibrinolysis-related protease. Treatment of lung fibroblasts with HABP lead to a rapid activation of signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway with c-Raf, MEK and ERK1/2. Additionally the activation of the PI3K/Akt pathway and of several translation-related proteins was found. Proliferation assays confirmed the assumption of a strong growth-stimulating effect of HABP on human lung and skin fibroblasts. Intracellular signalling and growth stimulation were strongly dependent on the proteolytic activity of HABP. Stimulation of signalling and proliferation by HABP involved the fibroblast growth factor receptor 1 (FGFR-1). HABP-stimulated proliferation of lung fibroblasts MRC-5 was accompanied by a significant intracellular increase in basic fibroblast growth factor (bFGF), the major ligand of FGFR-1; bFGF could however not be identified in the supernatant of HABP-treated cells. Though, the conditioned medium from HABP-treated cells showed a strong growth-promoting activity on quiescent fibroblasts, indicating the release of a yet unknown growth factor amplifying the initial growth stimulus. In a two-dimensional wound model HABP stimulated the invasion of fibroblasts into a scratch area, adding a strong pro-migratory activity to this plasma protease. In summary, HABP exhibits a significant growth factor-like activity on quiescent human lung and dermal fibroblasts. Our findings suggest that this fibrinolysis-related plasma protease may participate in physiologic or pathologic processes where cell proliferation and migration are pivotal, like tissue repair, vascular remodelling, wound healing or tumor development. 相似文献
7.
Production and characterization of antibody blocking epidermal growth factor:receptor interactions 总被引:13,自引:0,他引:13
Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 . 10(6) 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 immunoglobulin 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 degrees 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-A431 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. 相似文献
8.
几种扩血管多肽对bFGF促血管平滑肌细胞增殖作用的影响 总被引:3,自引:0,他引:3
姜志胜 《中国应用生理学杂志》2000,16(2):161-164
目的和方法:研究肾上腺髓质素(Adm)、降钙素基因相关肽(CGRP)及C-型心房利太(CNP)对碱性成纤维细胞生长因子(bFGF)促血管平滑细胞(VSMC)增殖作用的影响及其机制。结果:孵育24h后,bFGF刺激VSMC增殖较对照组增加2.1倍(P〈0.01),细胞内蛋白磷酸化程度增加1.4倍(P〈0.01),PKC及MAPK活性分别增加1.5和2.5倍(P〈0.010;Adm.CGRPt CNP 相似文献
9.
10.
Growth control of A431 cells in protein-free medium: Secretory products do not affect cell growth 总被引:4,自引:0,他引:4
Yoshishige Masuda Yoshino Yoshitake Katsuzo Nishikawa 《In vitro cellular & developmental biology. Plant》1988,24(9):893-899
Summary A431 cells grew at similar rates in protein-free Coon's modified Ham's F12 medium (PF-C-F12) with and without added bovine
calf serum. The cells secreted a heparin-binding growth factor and a type-β transforming growth factor, but their growth in
PF-C-F12 was not affected by these factors, or by DNA synthesis factor from Rhodamine fibrosarcoma, basic fibroblast growth
factor, insulin, human transferrin, bovine serum albumin, and their combinations. Growth of A431 cells in PF-C-F12 was not
density dependent and was not affected by either addition of conditioned medium or replacement of conditioned medium by fresh
medium. These results indicate that A431 cells have an intracellular mechanism for autonomous growth, and that their growth
is not affected by factors that they secrete or by exogenous growth factors.
This work was supported by a grant-in-aid for cancer research from the Ministry of Education, Science, and Culture of Japan,
and a grant from Hokkoku Cancer Research Foundation. 相似文献
11.
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. 相似文献
12.
F J Ballard 《Experimental cell research》1984,151(1):268-272
Addition of epidermal growth factor (EGF) to serum-free or serum-supplemented cultures of A431 cells stimulates protein breakdown without affecting rates of protein synthesis. These effects are atypical because in other cell lines, including AG2804-transformed human fibroblasts examined for comparison, EGF inhibits protein breakdown and stimulates protein synthesis. The response to EGF in A431 cells does not reflect a general post-receptor modification in growth factor action, since addition of insulin to the cells leads to the normal inhibition of protein breakdown. These findings indicate that the unusual growth inhibition produced by EGF in A431 cells can be explained by an increased rate of intracellular protein breakdown. 相似文献
13.
Inhibition of epidermal growth factor-induced DNA synthesis by tyrosine kinase inhibitors 总被引:10,自引:0,他引:10
Kazuo Umezawa Takashi Hori Hirohisa Tajima Masaya Imoto Kunio Isshiki Tomio Takeuchi 《FEBS letters》1990,260(2):201-205
We prepared methyl 2,5-dihydroxycinnamate as a stable analogue of erbstatin, a tyrosine kinase inhibitor. This analogue was about 4 times more stable than erbstatin in calf serum. It inhibited epidermal growth factor receptor-associated tyrosine kinase in vitro with an IC50 of 0.15 μg/ml. It also inhibited in situ autophosphorylation of epidermal growth factor receptor in A431 cells. Methyl 2,5-dihydroxycinnamate was shown to delay the S-phase induction by epidermal growth factor in quiescent normal rat kidney cells, without affecting the total amount ofDNA synthesis. The effect of erbstatin on S-phase induction was smaller, possibly because of its shorter life time. 相似文献
14.
Basic fibroblast growth factor (bFGF) is one of the most potent angiogenic factors. Unlike many other growth factors, bFGF
lacks a classic peptide sequence for its secretion. Recent studies suggest that there is an unconventional secretory pathway
for this growth factor. The aim of this study was to identify the specific location of bFGF in endothelial cells and to find
morphologic evidences concerning its synthesis, storage and release from endothelial cells. The capillaries in hippocampus,
adrenal gland, kidney, peripheral nerves as well as the vessels in connective tissues were analysed by using immunogold labeling
techniques at electron microscope level. Results show that endogenous bFGF is mainly located in the nuclei of endothelial
cells. Slight immunoreactivity is found in the cytoplasm. Immunolabeling is notably absent in pinocytotic vesicles, Golgi
complexes, endoplasmic reticulum, nuclear membrane and intercellular junctions. These results provide morphologic evidence
suggesting that endothelial cells might export bFGF via unique cellular pathways that are clearly distinct from classical
signal peptide mediated secretion and/or release of this protein could be directly through mechanically induced disruptions
of these cells. The current study support the recent hypothesis related with unconventional secretory pathway for bFGF as
some other “cargo” proteins. 相似文献
15.
The monoclonal antibody to the epidermal growth factor (EGF) receptor was generated after fusion of PAI myeloma cells with immunized BALB/c mouse spleen cells, using intact A431 epidermoid carcinoma cells as an immunogen. The antibody, denoted 5A9, is an IgG, which recognizes a protein with molecular mass 170 kDa during immunoblot analysis, immunoprecipitates phosphoprotein with molecular mass 170 kDa from the membrane preparations of A431 cells, and, according to immunofluorescence experiments, is distributed in the cell similar to the EGF-rhodamine conjugate. It is concluded that the produced antibodies are specific to EGF-receptor. At the same time the 5A9 (50 nM) do not compete with EGF for binding with high and low affinity receptors. They fail to induce internalization of the EGF-receptor and do not exert influence on intracellular degradation of EGF-receptor. Monoclonal antibodies 5A9 are also unable to inhibit the EGF-induced protein kinase activity of the receptor and do not stimulate protein kinase activity by themselves. Thus, the prepared monoclonal antibodies can be used to register the EGF-receptor cellular localization without affecting biologic activity of the receptor. 相似文献
16.
G Labourdette T Janet P Laeng F Perraud D Lawrence B Pettmann 《Journal of cellular physiology》1990,144(3):473-484
In a search of the growth factors possibly involved in brain ontogenesis we have examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the growth and phenotypic expression of rat astroblasts in primary culture. Along TGF-beta 1 elicited only a slight negative effect on the growth of these cells. However, this factor was found to modulate the mitogenic effects of other growth factors. On quiescent cells it potentiates the mitogenic effect of basic fibroblast growth factor (bFGF) but not that of other growth factors, namely, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and thrombin. TGF-beta 1 did not modulate significantly the stimulatory effect of these growth factors on the activity of the enzyme glutamine synthetase (GS); but kinetic studies showed that TGF-beta 1 delays the stimulation of GS activity. DNA synthesis monitored by the incorporation of [125I]iododeoxyuridine (125I-dUrd) was maximum after 24-30 h of treatment with bFGF. With bFGF plus TGF-beta 1 the maximum was shifted to 30-36 h. This shift is compatible with the idea that TGF-beta 1 induces responsiveness in some cells which are otherwise unresponsive to the mitogenic action of bFGF, and that this induction requires some time. This hypothesis is sustained by the observation that in cells treated for only 12 h with bFGF, the treatment with TGF-beta 1 for the same 12 h or for longer time did not stimulate significantly the cell growth. Stimulation occurred only when the bFGF treatment was continued after 12 h. Potentiation of the mitogenic effect of bFGF and shift of the maximum 125I-dUrd incorporation towards 24 h was seen with cells pretreated with TGF-beta 1. This potentiation effect decreased with increasing time between the two treatments. The potentiation effect of TGF-beta 1 is not mediated by an induction of new bFGF membrane receptors as seen by binding studies. 相似文献
17.
P M van Bergen en Henegouwen J C den Hartigh P Romeyn A J Verkleij J Boonstra 《Experimental cell research》1992,199(1):90-97
In this paper we describe our investigations on the association of receptors for the epidermal growth factor (EGF) with the cytoskeleton of A431 cells. In order to determine which filamentous system the EGF receptors are associated to, the cytoskeletal fraction to which these receptors bind was isolated. Second, the possible colocalization of EGF receptors with different cytoskeletal elements was examined in A431 cells. By selective extractions of the A431 cytoskeletons, it is shown that more than 90% of the cytoskeleton-associated EGF receptors are removed from the cytoskeletons together with the actin filamentous system. During several cycles of poly- and depolymerization of actin isolated from A431 cells, the EGF receptor precipitates together with the actin containing filaments, indicating that EGF receptors are able to bind in vitro to actin filaments. With immunofluorescence studies we show that EGF receptors especially colocalize with actin filaments. These results demonstrate that the EGF receptor is associated specifically with actin filaments in A431 cells. 相似文献
18.
Fibroblast growth factor stimulates protein kinase C in quiescent 3T3 cells without Ca2+ mobilization or inositol phosphate accumulation 总被引:2,自引:0,他引:2
Eewa Nnberg Clive Morris Theresa Higgins Francisco Vara Enrique Rozengurt 《Journal of cellular physiology》1990,143(2):232-242
To elucidate the transmembrane signalling processes initiated by fibroblast growth factor (FGF), we have studied the effect of recombinant basic FGF (bFGF) on various early events associated with mitogenesis in Swiss 3T3 fibroblasts. bFGF, at mitogenic concentrations, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates. In contrast, bFGF stimulated the phosphorylation of the Mr 80,000 (80K) cellular protein which is a major substrate of protein kinase C. This effect was potentiated by the diacylglycerol kinase inhibitor R59022. Two-dimensional polyacrylamide gel electrophoresis and phosphopeptide mapping showed that the 80K phosphoproteins generated in response to bFGF, bombesin, and phorbol 12,13-dibutyrate were indistinguishable. Down-regulation of protein kinase C prevented bFGF stimulation of 80K phosphorylation. Other protein kinase C-dependent early events such as transmodulation of the epidermal growth factor receptor, cytoplasmic alkalinization, inhibition of vasopressin induced increase in cytosolic [Ca2+], and enhancement of cAMP accumulation in response to forskolin were also induced by bFGF. Similar results were obtained when bFGF was added to quiescent cultures of tertiary mouse embryo fibroblasts. We conclude that bFGF stimulates protein kinase C through a signal transduction pathway distinct from inositol phospholipid turnover and Ca2+ mobilization. 相似文献
19.
Stimulation and inhibition of growth by EGF in different A431 cell clones is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes 总被引:23,自引:1,他引:22
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Stimulation of quiescent fibroblasts to growth by polypeptide growth factors is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes. In contrast to fibroblasts, A431 cells respond to epidermal growth factor (EGF) with a decreased growth rate. Here we report that, in spite of its growth inhibitory effect, EGF rapidly induces transient expression of c-fos mRNA, followed by the synthesis of nuclear c-fos protein. In addition, EGF treatment resulted in elevated levels of c-myc expression. Practically identical results were obtained with variant A431 clones that are resistant to the inhibitory effect of EGF on cell proliferation. These observations suggest that in A431 cells c-fos and c-myc induction is a primary consequence of growth factor-receptor interaction. Indeed, efficient induction of both genes was also observed with cyanide bromide-cleaved EGF, which has previously been shown to be non-mitogenic but able to trigger early events induced by EGF. We observed strong induction of c-fos and to a lesser extent of c-myc also by TPA, and by the calcium ionophore A23187, indicating an important role for kinase C in proto-oncogene activation by growth factors. 相似文献
20.
We have investigated the genetic stability of NIH-3T3 cells transfected with sequences coding for basic fibroblast growth factor (bFGF) by determining drug resistance and gene amplification potential. Colony-forming experiments and fluctuation analyses showed that the frequency and rate of resistance to N -(phosphonacetyl)-L-aspartate (PALA) was dramatically elevated in cells transfected with either the normal bFGF coding sequence that lacks a known signal for secretion or a chimeric bFGF sequence that targets the growth factor to the secretory pathway. Basic FGF-transfected cells that grew in the presence of PALA were found to possess an amplification of the CAD gene, which codes for a multifunctional protein involved in pyrimidine biosynthesis and is the site of action for PALA. The observation that these alterations occur in cells transfected with a bFGF sequence, without a conventional signal sequence for secretion, suggests an intracrine as opposed to autocrine mechanism of action. The results describe a new function for this growth factor and suggest a novel role for aberrant expression of bFGF in mechanisms of tumor progression. 相似文献