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1.
This report describes investigations of plasma membrane transferrin receptors on a variety of lymphoid cell lines and normal peripheral blood lymphocytes during activation and cell growth cycles. Transformed lymphoid cell lines have as many as 1,000 times the number of receptors found on normal resting lymphocytes. The number of iron transferrin receptors on continuous cell lines as well as normal human fibroblasts is down-regulated during the transition from log-phase growth to stationary plateau growth. When normal lymphocytes are transformed by mixed lymphocyte culture or mitogens, they rapidly express a 50-fold increase in the number of transferrin binding sites. This appearance of iron transferrin receptors anticipates nuclear changes during cell activation and subsequent mitosis of normal cells. 相似文献
2.
Reduced binding of epidermal growth factor by avian sarcoma virus-transformed rat cells 总被引:1,自引:0,他引:1
S Decker 《Biochemical and biophysical research communications》1983,113(2):678-686
Rat cells transformed by Rous sarcoma virus and Fujinami sarcoma virus bound 5-10% of the amount of epidermal growth factor (EGF) bound by normal cells. Scatchard plot analysis indicated that the reduction in binding by transformed cells was due to a decreased number of receptors rather than to altered binding affinity. In experiments with temperature sensitive mutants of Rous sarcoma virus and Fujinami sarcoma virus significant loss of EGF binding occurred within one hour of shift from non-permissive to permissive temperature. Conditioned media from various normal and transformed cell lines were examined for the ability to inhibit EGF binding to normal cells or to cause "down regulation" of EGF receptors. No activity of either type was found. EGF-dependent phosphorylation in isolated membrane preparations was also examined. Membranes from normal cells displayed EGF-dependent phosphorylation of a Mr 180,000 protein presumed to be the EGF receptor. This activity was absent in membranes from transformed cells. The data suggest a close correlation between activation of avian sarcoma virus transforming gene products and modulation of the EGF growth regulatory system. 相似文献
3.
The effects of the tumor promotor, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), on the intra- and extracellular distribution of transferrin receptors and rates of iron uptake were studied in normal developing myogenic cells and myogenic cells transformed with a temperature-sensitive strain of the Rous sarcoma virus. In normal developing cells PMA was found to increase the rate of iron uptake by 15-30%. There was, however, no effect on transferrin receptor distribution, suggesting that the increase in iron uptake was due to stimulation of the rate of receptor cycling. In contrast, in transformed myogenic cells, PMA had no effect even at concentrations 10 times those effective in normal myogenic cells. The specificity of PMA was demonstrated by comparison with 4 alpha-phorbol which had no effect compared with the control cells which were incubated with dimethyl sulfoxide, the solvent used to dissolve the phorbols. These results indicate a functional difference in the transferrin receptor between normal and transformed myogenic cells. The data for normal myogenic cells are similar to those previously reported for normal erythroid cells, but differ from those for some transformed cell lines in which phorbol esters were shown to cause internalization of transferrin receptors. 相似文献
4.
Treatment of two human leukemia cell lines with 1.25% dimethyl sulfoxide at 37 degrees C results in a rapid increase in the number of transferrin receptors on the cell surface detected by fluorescein-labeled anti-transferrin receptor antibodies. Both HL-60 cells, a human myeloid cell line, and K562 cells, a human erythroid-myeloid cell line, showed a 25-65% increase in cell surface transferrin binding in parallel experiments. Scatchard plot analysis of the data indicates that the number of receptors increases while the affinity of transferrin for the receptor remains the same. This rapid increase in the number of receptors at the cell surface appears to be due to a slowing of endocytosis rather than an increase in externalization of the receptor. 相似文献
5.
Several reports have suggested that variations of albumin concentration in the incubation medium can modulate the magnitude of transferrin binding to the cells. We have investigated this problem further using K562 cells. In the absence of human serum albumin, transferrin binding demonstrated a non-saturable curve which, upon Scatchard analysis, showed two components with high and low affinities. In the presence of 0.5% human serum albumin, the low-affinity but not the high-affinity component was totally inhibited and, thus, the binding showed a saturation plateau at transferrin concentration of 6 micrograms/ml. Increasing concentrations of human serum albumin in the incubation medium led to progressive inhibition of transferrin binding, reaching a plateau at 0.2% human serum albumin. At this concentration transferrin binding was about 12 ng/10(6) cells, corresponding to the saturation plateau for high-affinity binding. Low-affinity transferrin binding in the absence of human serum albumin could readily be displaced by subsequent addition of albumin. Similar inhibition was obtained by another serum protein, ceruloplasmin, suggesting that this inhibition is not unique to albumin and may be a common property of all proteins. Incubation at 37 degrees C with 59Fe-labeled transferrin indicated that all iron uptake occurs through high-affinity binding. We conclude that the reported variations in magnitude of transferrin binding by the cell due to variations in albumin concentration are the result of inhibition of low-affinity binding of transferrin by albumin. 相似文献
6.
All dividing cells require transferrin as a growth factor. During in vitro culture of human lymphocytes, transferrin is usually supplied in the form of serum, either synergic or xenogenic (usually fetal bovine serum (FBS)). In the present work the growth of certain human T-cell lines was examined; these lines were derived from the synovium of rheumatoid arthritis patients and maintained in 10% FBS and 1% synovial fluid. Their growth especially at limiting dilutions was found to be strongly dependent on the presence of synovial fluid at low concentration (0.05-0.1%) in culture medium containing 10% FBS. Further studies indicated that this effect of synovial fluid was duplicated by human serum or plasma, and was due to the presence of human transferrin. A significant effect on T-cell growth was observed using 2 micrograms/ml human transferrin with optimal growth at 10-20 micrograms/ml. This requirement for human transferrin was not a peculiarity of the synovium-derived T-cell lines, but was observed with all T-cell lines tested irrespective of phenotype or function. These observations suggest that bovine transferrin is inadequate for T-cell growth, and that the growth enhancing properties of FBS do not primarily reflect the provision of transferrin. Since some T cells have recently been shown to be capable of secreting transferrin upon activation, endogenous synthesis of transferrin may be an important factor in the in vitro growth of T cells so that such cells would be selected when FBS is the source of serum used to grow human T-cell lines or clones. 相似文献
7.
This study systematically examined the characteristics of specific binding of adult diferric transferrin to its receptor using a Triton X-100 solubilized preparation from human placentas as the receptor source. The following information was obtained. The ionic strength for maximal binding is in the range of 0.1-0.3 M NaCl. The pH optimum for specific binding extends over the range, from pH 6.0-10.0. Specific binding of diferric transferrin is not affected by 2.5 approximately 50 mM CaCl2 or by 10 mM EDTA. Triton X-100 in the concentration range of 0.02-3.0% does not affect specific binding. Specific binding is saturated within 10 min at 25 or 37 degrees C in the presence of excess amounts of diferric transferrin. The binding is reversible and the dissociation of diferric transferrin from the transferrin receptor is complete within 40 min at 25 degrees C. Apotransferrin, both adult and fetal, showed less binding than the holotransferrin species by competitive binding assay in the presence of 10 mM EDTA independent of up to 20 mM CaCl2. A 1500-fold molar excess of adult and fetal apotransferrin is required to give 40% inhibition for 125I-labeled diferric transferrin binding. Since calcium ion is not a factor, and since apotransferrin has such high binding affinity for iron (Ka = 1 X 10(24], this experiment suggests that the EDTA was necessary to prevent conversion of apotransferrin to holotransferrin from available iron in the reaction system. The specificity of the transferrin receptor for transferrin was examined by competitive binding studies in which 125I-diferric transferrin binding was measured in the presence of a series of other proteins. The proteins tested in the competitive binding studies were classified into three groups; in the first group were human serum albumin and ovalbumin; in the second group were proteins containing iron ions, such as hemoglobin, hemoglobin-haptoglobin complex, heme-hemopexin complex, ferritin, and diferric lactoferrin; in the third group were the metal-binding serum proteins, ceruloplasmin and metallothionein. None of these proteins except ferritin showed inhibition of diferric transferrin binding to the receptor. The effect of ferritin was small since a 700- to 1500-fold molar excess of ferritin is required for 50% inhibition of binding of diferric transferrin to the receptor. 相似文献
8.
Regulation of transferrin receptor expression in concanavalin A stimulated and Gross virus transformed rat lymphoblasts 总被引:9,自引:0,他引:9
T A Hamilton 《Journal of cellular physiology》1982,113(1):40-46
Expression of the cell surface receptor for the serum glycoprotein transferrin has been correlated with cellular proliferation in normal lymphocytes undergoing mitogen or antigen induced proliferative responses. In the present study, the expression of transferrin receptor in Concanavalin A stimulated rat lymphocytes or Gross virus transformed lymphoma cells has been examined with respect to the following questions: (1) is expression of receptor activity related to blastogenesis or to the subsequent IL-2 dependent DNA synthetic activity, and (2) is transferrin receptor expression regulated in similar fashion in both normal and malignant lymphoblasts? Scatchard analysis of saturation binding data illustrated that binding site number increased and subsequently decreased during the response while the receptor affinity for transferrin remained constant. These findings were confirmed by SDS-polyacrylamide gel electrophoretic analysis of radiolabeled cell surface proteins which specifically interact with transferrin. Examination of nonproliferating normal lymphoblasts (96 hr post Con A stimulation) compared with the same population of cells stimulated to reinitiate DNA synthesis with a partially purified preparation of Interleukin 2 (IL-2) showed that transferrin receptor expression was tightly linked to the IL-2 dependent stimulation of DNA replication. This coordinate regulation of receptor expression was markedly less stringent in retrovirus transformed thymic lymphoma cells. 相似文献
9.
Albumin prevents nonspecific transferrin binding and iron uptake by isolated hepatocytes 总被引:5,自引:0,他引:5
Bovine serum albumin inhibits binding of transferrin by hepatocytes in suspension by 60-70%. Iron uptake is inhibited by less than 20%. A Scatchard analysis of the transferrin-binding data reveals a biphasic plot in the absence of bovine serum albumin, but a monophasic plot in the presence of bovine serum albumin. Bovine serum albumin inhibits low-affinity binding of transferrin (125000 molecules/cell), but has no effect on high-affinity binding (38000 molecules/cell). In pronase-treated cells, transferrin binding is reduced by 40%, and when bovine serum albumin is added, the binding is reduced by a further 40%. Corresponding figures for iron uptake are 70 and 10%, respectively. The results are strong evidence that the major part of iron uptake by hepatocytes occurs from transferrin bound to the plasma membrane transferrin receptor. 相似文献
10.
体外低钾培养肾细胞能刺激细胞膜钠-钾ATP酶。本研究利用Madin Darby狗肾细胞能在无血清培养液中健康生存48h这一特征,研究体外低钾刺激细胞膜钠-钾ATP酶所依赖的血清中的活性因子,观察了表皮生长因子(EGF)、胰岛素样生长因子(IGF1)、前列腺素1(PGE1)和转铁蛋白(tranderrin)在这一过程中的作用。结果表明,在无血清培养液中低钾并不能刺激细胞膜钠—钾ATP酶,而添加转铁蛋白可模拟血清的作用。转铁蛋白能剂量依赖性地增加ouabain结合位点,对细胞膜钠-钾ATP酶作用呈良好的时间效应关系。在低钾无血清培养液中,细胞膜钠-钾ATP酶α1亚基启动子活性增强,α1与β1亚基蛋白质表达的增加依赖于转铁蛋白的存在。进一步研究结果表明,低钾在转铁蛋白的无血清培养液环境中能增加细胞对铁的摄取(^59Fe),该作用可被铁螯合剂(deferoxamine,DFO;35 μmol/L)所阻断。DFO也可阻断转铁蛋白依赖性低钾刺激细胞膜钠-钾ATP酶数目的增多,α1亚基启动子活性增强,α1与β1亚基蛋白质表达增加。以上结果表明,低钾对细胞膜钠-钾ATP酶活性的刺激作用依赖于转铁蛋白所调节的铁的摄取。 相似文献
11.
Mitogenic agents induce redistribution of transferrin receptors from internal pools to the cell surface. 总被引:2,自引:0,他引:2
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Incubation of serum-growth HeLa cells in serum-free medium causes a rapid (t1/2 3 min) 30-60% decrease in the binding of 125I-diferric transferrin to the cell surface. Addition of fetal bovine serum to cells in serum-free medium results in a rapid (t1/2 3 min) and concentration-dependent increase in binding activity. The loss or gain in ligand binding is a result of changes in surface receptor number rather than an alteration in ligand-receptor affinity. A variety of hormones (insulin, insulin-like growth factor, interleukin 1 and platelet-derived factor) were found to mimic the effect of serum on receptor number. The alteration in surface receptor number was found to be calcium-dependent. Changes in surface receptor number were independent of either receptor biosynthetic rate or the absolute cellular content of receptors. The effect of insulin or serum on Hela cell transferrin receptor distribution was unaffected by the presence of transferrin, demonstrating that receptor distribution in this cell type is ligand-independent. The ability of serum or insulin to modify surface transferrin receptor number was also observed in mouse L-cells, human skin fibroblasts, and J774 macrophage tumour cells. However, transferrin receptors on K562 and Epstein-Barr virus-transformed human lymphoblasts were unaltered by these agents. The quantities of receptors whose distribution is predominantly on the surface (i.e. epidermal growth factor or low density lipoprotein receptor) were unaltered by addition of the mitogenic agents. These results extend our previous studies [H.S. Wiley & J. Kaplan (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7456-7460] demonstrating that mitogenic agents can induce redistribution of receptor pools in selected cell types. 相似文献
12.
"Spontaneously" or SV40 virus transformed AL/N mouse cell lines were passed repeatedly through syngeneic mice. Cell lines were re-established in culture from minced pieces of tumors in the presence of concentrated fetal calf serum or from tumor cells dispersed by trypsin. The aim of this study was to compare the two cell lines in regard to the selection processes which operate during such procedures by characterization of the resulting cell lines. Measurements of growth in tissue culture on substratum showed no significant difference between any of the transformed cell lines. The SV40 transformed cells and its derivative cells had a low anchorage requirement for growth. The greatest anchorage requirement for growth was in the normal untransformed cells and in the derivative cells from the "spontaneously" transformed cells which were established from minced tumors. The spontaneously transformed cells and all derivative cells had high tumorigenicity (TD50 is less than 10-2). The SV40 transformed cells had no observable tumorigenicity (TD50 is greater than 10-8), except when injected into irradiated mice (TD50 = 1-5 X 10-5 in the immunocompetent mice, 5 X 10-4 in the irradiated mice). The SV40 transformed derivative cells maintained their SV40 specific T antigen and their susceptibility to lysis by specific antiserum. 相似文献
13.
M V Jesudason G Sridharan M Santhosh T J John 《Indian journal of experimental biology》1992,30(3):250-251
Antiserum to cell wall proteins of S. typhi showed immunoprecipitin lines of identity with homologous antigen, normal human serum and human transferrin. Also, anti-transferrin antibody showed immunoprecipitin band with S. typhi cell wall proteins and agglutinated S. typhi whole cells, but not Eschericia coli. It is speculated that transferrin or a transferrin-like protein present in the cell wall may play a role in iron-uptake by S. typhi. 相似文献
14.
Masahiro Oka Kouji Ogita Hideya Ando Tatsuya Horikawa Kazuhito Hayashibe Naoaki Saito Ushio Kikkawa Masamitsu Ichihashi 《Journal of cellular physiology》1996,167(3):406-412
It has been shown that tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulates the proliferation of normal human melanocytes, whereas it inhibits the growth of human melanoma cell lines. The expression of protein kinase C (PKC) subspecies, the major intracellular receptors for TPA, was examined in normal melanocytes and the four melanoma cell lines HM3KO, MeWo, HMV-1, and G361. PKC was partially purified and then separated into subspecies by column chromatography on Mono Q and hydroxyapatite successively, and finally subjected to immunoblot analysis using antibodies specific for the PKC subspecies. Of the PKC subspecies examined, δ-, ϵ-, and ζ-PKC were detected in both normal melanocytes and the four melanoma cell lines. In contrast, both α-PKC and β-PKC were expressed in normal melanocytes, whereas either α-PKC or β-PKC was detected in melanoma cells. Specifically, HM3KO, MeWo, and HMV-1 cells were shown to contain α-PKC but not β-PKC, while G361 cells expressed β-PKC but not α-PKC. The growth of these melanoma cells was suppressed by TPA treatment, and the growth of the G361 cells lacking α-PKC was inhibited more efficiently than the other melanoma cell lines which lacked β-PKC. It was further shown that β-PKC was not detected in freshly isolated human primary or metastatic melanoma tissues. These results suggest that the expression of α-PKC or β-PKC may be altered during the malignant transformation of normal melanocytes and that loss of α-PKC or β-PKC may be related to the inhibitory effect of TPA on the growth of melanoma cells. © 1996 Wiley-Liss, Inc. 相似文献
15.
16.
W P Faulk G M Galbraith 《Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society (Great Britain)》1979,204(1154):83-97
Transferrin and specific transferrin receptors are demonstrated on the microvillous surface of syncytiotrophoblast in human immature and term placentae by immuno histological techniques with the use of light and electron microscopy. That the distribution of transferrin is limited to the materno-foetal interface supports the hypothesis that binding of maternal transferrin to trophoblast receptors is involved in the process of iron transport to the foetus. Parallel studies with baboon placentae demonstrate the presence of trophoblast receptors which bind both baboon and human transferrin, thereby putting forward an experimental model which might be used to test the biological significance of placental transferrin receptors in primates. In addition, investigation of a large number of human cell lines shows that many transformed cells, but no normal cells (such as blood lymphocytes) or cells from primary culture (such as neonatal foreskin fibroblasts), possess the ability to bind transferrin to their membranes. These findings suggest that transferrin receptors may play important biological roles in addition to that of iron transport from mother to foetus. One such role could be the limitation of iron in intervillous spaces, thus depriving iron-requiring microorganisms of iron, hence serving as a non-specific factor of resistance for placentae. Another role for foetal transferrin receptors on trophoblasts could be to bind maternal transferrin at the materno-foetal interface, thus frustrating maternal immunosurveillance. This is similar to a mechahism used by schistosomes in the host-parasite relation where host proteins are bound by the parasite to escape immunological recognition. The presence of transferrin receptors on transformed cells suggests that this mechanism might also be employed by tumour cells. Finally, in view of previous studies which show that transferrin is required by stimulated lymphocytes to pass from the G1 to the S phase of cellular replication, it is proposed that trophoblast transferrin receptors could limit the amount of transferrin in intervillous spaces and thus impede the proliferation and possible cytotoxicity of maternal activated lymphocytes at the materno-foetal interface. 相似文献
17.
Cyclic AMP-dependent protein kinases from Balb 3T3 cells and other 3T3 derived lines 总被引:1,自引:0,他引:1
Cyclic AMP-dependent protein kinase and 3H-cAMP-binding activities were determined in normal Balb 3T3 cells and compared with the same preparations from SV40, chemical, and spontaneous transformants of 3T3 cells. The cytosolic protein kinase activities and protein kinase activity ratios were similar in all cell lines, although when the normal 3T3 cytosol was prepared by homogenization it contained less 3H-cAMP binding activity than the transformed 3T3 cytosols. The Triton X-100 treated particulate fractions from the normal and transformed 3T3 cells contained similar protein kinase and binding activities. The isozymic profile of cAMP-dependent protein kinases was examined by DEAE-chromatography. The 3T3 cells contained only type II isozyme in either cytosolic or membrane fractions. All transformants of the 3T3 cells contained both type I and type II isozymes. Other cell cultures, including chicken embryo fibroblasts, rat kidney cells, and human or calf endothelial cells contained type I and type II isozymes. Binding of the photoaffinity analogue of cAMP, 8-N3 cAMP, to the regulatory subunits of protein kinases in sonicates obtained from Balb 3T3 and SV 3T3 cells followed by separation on SDS polyacrylamide electrophoresis showed that the amount of RII subunit was approximately equal in the two cell lines. RI in Balb 3T3 cells was detectable but in a much lower quantity than in SV 3T3 cells. The cyclic AMP dependent-protein kinases from Balb 3T3 cells appears to be different from SV 3T3 cells by three criteria: 3H-cAMP binding in homogenates, DEAE chromatographic separation of isozymes, and 8-N3 cAMP binding. 相似文献
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19.
The insulin receptors in normal and transformed lines of mouse Balb/3T3 fibroblasts have been studied. In the normal fibroblasts, the binding of insulin was low in growing cells and increased 2–9 fold in confluent stationary cells. Insulin binding was increased whether growth arrest was due to contact inhibition of growth or serum starvation. When serum-starved cells were stimulated to grow by the addition of fresh serum, insulin binding declined. In cells transformed by simian virus 40, Kirsten, Moloney, and Harvey sarcoma viruses, methylcholanthrene, X rays, or spontaneously, the binding was low, in the same range as growing normal cells. In simian virus 40-transformed cells, insulin binding increased 4 fold as the cells reached higher densities in culture. No relationship to changes in cell size was found. The differences in binding were due to changes in the concentration of the receptors, without changes in their affinity for the hormone. 相似文献
20.
Immortalization by c-myc, H-ras, and Ela oncogenes induces differential cellular gene expression and growth factor responses. 总被引:16,自引:6,他引:10
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Early-passage rat kidney cells were immortalized or rescued from senescence with three different oncogenes: viral promoter-driven c-myc, H-ras (Val-12), and adenovirus type 5 E1a. The normal c-myc and H-ras (Gly-12) were unable to immortalize cells under similar conditions. Quantitation of RNA in the ras-immortalized lines demonstrated that the H-ras oncogene was expressed at a level equivalent to that of the normal H-ras gene in established human or rat cell lines. Cell lines immortalized by different oncogenes were found to have distinct growth responses to individual growth factors in a short-term assay. E1a-immortalized cells were largely independent of serum growth factors, whereas c-myc-immortalized cells responded to serum better than to epidermal growth factor and insulin. H-ras-immortalized cells responded significantly to insulin alone and gave a maximal response to epidermal growth factor and insulin. Several cellular genes associated with platelet-derived growth factor stimulation, including c-myc, were expressed at high levels in the H-ras-immortalized cells, and c-myc expression was deregulated, suggesting that the H-ras oncogene has provided a "competence" function. H-ras-immortalized cells could not be morphologically transformed by secondary transfection with a long terminal repeat-c-myc oncogene, but secondary transfection of the same cells with H-ras (Val-12) produced morphologically transformed colonies that had 20- to 40-fold higher levels of H-ras oncogene expression. Thus, transformation in this system is dependent on high levels of H-ras oncogene expression rather than on the presence of activated H-ras and c-myc oncogenes in the same cell. 相似文献