Biologically active phorbol esters specifically alter affinity of epidermal growth factor membrane receptors

TPA (12-O-tetradecanoyl-phorbol-13-acetate) reversibly inhibits the binding of (125)I-labelled epidermal growth factor (EGF) to treated mouse and human cells, but does not affect the binding of various other ligands to their membrane receptors. It alters the affinity of the receptors for EGF without changing the total number of available receptors per cell. Those phorbol esters which stimulate cell growth in culture and have tumour-promoting activity in vivo alter the EGF-receptor affinity, while the biologically inactive derivatives fail to change the affinity of EGF for its receptors.     

Desensitisation of cultured glial cells to epidermal growth factor by receptor down-regulation.

Epidermal growth factor (EGF), which can be purified from the mouse submaxillary gland or from pregnant human urine, is a potent multiplication-stimulating factor for several types of cultured cells, including human fibroblasts and glial cells. The molecule binds with high affinity and saturation kinetics to a cell-surface receptor, is subsequently internalised and finally degraded. The binding event is accompanied by a reduction in the number of EGF receptors. This phenomenon--'receptor down-regulation'--has been demonstrated with several hormones and may be a general principle for the modulation of binding groups on the outer cell surface. Further, it has been proposed that receptor loss acts to regulate the cellular response to the binding ligand. The present study provides direct experimental support for this hypothesis. It demonstrates that down-regulation of EGF receptors on glial cells causes desensitisation of the mitogenic response of these cells to subsequent stimulation with EGF.     

Diminished in vitro tyrosine kinase activity of the EGF receptor of senescent human fibroblasts

Fibroblastic cultures derived from normal human tissues undergo a finite number of population doublings when serially subcultivated in vitro (see refs 1, 2 for reviews). Epidermal growth factor (EGF) serves as a mitogen for early doubling level cultures of the human fetal lung-derived cell strain, WI-38, under serum-free conditions. The ability of cells from late doubling level cultures to respond mitogenically to EGF is lost, however, despite undiminished binding of EGF throughout the replicative lifespan. The ultimate effects of EGF, that is DNA synthesis and mitosis (see ref. 4 for review), occur after a sequence of events initiated by binding of ligand to specific cellular receptors. The receptor for EGF has been characterized as a 145,000-165,000 (145 K-165 K) molecular weight doublet, and, like the receptors for platelet-derived growth factor and insulin, and the transforming proteins of certain of the RNA tumour viruses, is a tyrosine-specific protein kinase with autophosphorylating activity. Moreover, several of the cellular target molecules of tyrosine phosphorylation have been found to be substrates for two or more of these kinases. The hypothesis that tyrosine phosphorylation underlies a common mechanism of growth control prompted us to ask whether the loss of responsiveness to EGF by late doubling level WI-38 cells is accompanied by altered expression of the EGF receptor, and specifically whether changes occur in the ability of receptors from populations of cells of various in vitro ages to catalyse tyrosine autophosphorylation. We show here that autophosphorylating activity is absent from the EGF receptor of cells which have lost their mitogenic responsiveness to EGF.     

Transformation by murine and feline sarcoma viruses specifically blocks binding of epidermal growth factor to cells.

Normal cells in culture have membrane receptors for epidermal growth factor (EGF); EGF stimulates cells to divide by binding to these receptors. Cells transformed by murine and feline sarcoma viruses rapidly lose the ability to bind EGF, whereas cells transformed by the DNA tumour viruses, polyoma and SV40, or infected with non-transforming RNA tumour viruses have normal levels of functional EGF receptors. The results suggest that a product of the sarcoma virus genome specifically changes cell EGF receptors; the sarcoma gene product may, then, be functionally related to EGF.     

Retinoids specifically enhance the number of epidermal growth factor receptors

Retinoids elicit many biological and biochemical responses from cells in vitro. One widely used criterion for the responsiveness of cells to retinoids is inhibition of growth; retinoids reduce the saturation density and/or growth rate of many normal and tumorigenic cell lines. Propagation of eukaryotic cells has been demonstrated to be dependent on the presence of macromolecular growth factors such as epidermal growth factor (EGF), which can stimulate proliferation of epithelial and fibroblastic cell lines. We now describe the effect of retinoids on the binding of EGF to its receptor. Retinoic acid enhances binding of 125I-labelled EGF to various fibroblastic and epidermal cell lines. It has no marked effect on the affinity of this growth factor for its receptor, but increases the number of EGF receptor sites. Retinoic acid has little effect on the binding of concanavalin A (Con A) and insulin, indicating the specific nature of the action of retinoids on cell-surface glycoproteins. Treatment of cells with the phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) and retinoic acid shows poor antagonism between these compounds on EGF binding. It has been previously shown that retinoids induce or stimulate differentiation of embryonal carcinoma cells. EGF binding can be used as a marker to monitor differentiation of these cells.     

Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin

Epidermal growth factor (EGF), through interaction with specific cell surface receptors, generates a pleiotropic response that, by a poorly defined mechanism, can induce proliferation of target cells. Subversion of the EGF mitogenic signal through expression of a truncated receptor may be involved in transformation by the avian erythroblastosis virus (AEV) oncogene v-erb-B, suggesting that similar EGF receptor defects may be found in human neoplasias. Overexpression of EGF receptors has been reported on the epidermoid carcinoma cell line A431, in various primary brain tumours and in squamous carcinomas. In A431 cells the receptor gene is amplified. Here we show that 4 of 10 primary brain tumours of glial origin which express levels of EGF receptors that are higher than normal also have amplified EGF receptor genes. Amplified receptor genes were not detected in the other brain tumours examined. Further analysis of EGF receptor defects may show that such altered expression and amplification is a particular feature of certain human tumours.     

表皮生长因子对猕猴胚胎角膜上皮组织增殖效应的研究

离体培养试验表明，小鼠颌下腺表此生长因子对离体培养猕猴胚胎眼角膜上皮组织具有显著促进细胞增殖和分化的效应。培养４ｄ的实验组角膜上皮的厚度，上皮细胞增殖层烽以及上皮组织中基底层细胞核与表层细胞核两者的比率均较正常猴胚角膜增长３倍。与此同时培养４ｄ的实验组角膜上皮厚度和上皮细胞的层数，都达到成年猴正常角膜上皮的结构水平。     

一些神经营养因子对神经干细胞的增殖和分化的影响

胚胎和成年哺乳动物脑内存在能分化为神经元和神经胶质细胞的细胞干细胞,从成年脑和胚脑分离的神经干细胞能在体外分裂并进一步分化成神经元和胶质细胞,许多生长因子,如成纤维细胞生长因子和表皮生长因子等都参与了这一分裂、分化过程。对神经干细胞的增殖及分化产生一定的影响,但在不同的情况下,它们对增殖及分化的作用不同。     

A chimaeric receptor allows insulin to stimulate tyrosine kinase activity of epidermal growth factor receptor

The cell surface receptors for insulin and epidermal growth factor (EGF) appear to share a common evolutionary origin, as suggested by structural similarity of cysteine-rich regions in their extracellular domains and a highly conserved tyrosine-specific protein kinase domain. Only minor similarity is found outside this catalytic domain, as expected for receptors that have different ligand specificities and generate different biological signals. The EGF receptor is a single polypeptide chain but the insulin receptor consists of distinct alpha and beta subunits that function as an alpha 2 beta 2 heterotetrameric receptor complex. Provoked by this major structural difference in two receptors that carry out parallel functions, we have designed a chimaeric receptor molecule comprising the extracellular portion of the insulin receptor joined to the transmembrane and intracellular domains of the EGF receptor to investigate whether one ligand will activate the tyrosine kinase domain of the receptor for the other ligand. We show here that the EGF receptor kinase domain of the chimaeric protein, expressed transiently in simian cells, is activated by insulin binding. This strongly suggests that insulin and EGF receptors employ closely related or identical mechanisms for signal transduction across the plasma membrane.     

Epidermal growth factor receptor occupancy inhibits vaccinia virus infection

Vaccinia virus encodes VGF, an early protein of relative molecular mass 19,000 (19K) which, from amino-acid residues 45 to 85, is homologous in 19 residues to epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha). The conserved sequence includes a region of high homology (6 out of 10 amino acids) from residues 71 to 80, corresponding to the third disulphide loop of both EGF and TGF-alpha. This region has recently been shown to contain a binding region of TGF-alpha for the EGF receptor, and this raises the question of whether vaccinia virus utilizes the EGF receptor in order to bind to and infect cells. We now show that occupancy of the EGF receptor inhibits vaccinia virus infection. Inhibition is observed in a dose-dependent fashion by pre-treatment with either EGF or synthetic decapeptide antagonists of EGF's mitogenic activity which correspond to the sequence of the third disulphide loop of VGF or TGF-alpha. The relative ability of the peptides to inhibit vaccinia virus infection parallels their binding affinity to the EGF receptor.     

大鼠胎脑神经干细胞的分离和培养

胚龄14．5～16．5d(E14．5～16．5)的大鼠胎脑组织获得大鼠胎脑神经干细胞(rat fetal neural stem cells，rFNSCs)，培养于含有碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)和表皮生长因子(epidermal growth factor，EGF)的无血清培养液DMEM／F12中，用^3[H]胸苷掺入试验检测EGF和bFGF对大鼠胚胎神经干细胞分裂和增殖的影响．BrdU结合反应和nestin免疫组化检测显示培养细胞在早期时代约90％以上具有分裂增殖能力并显示nestln阳性，而且这些细胞在培养过程中可以分化神经元、星形胶质细胞和少突胶质细胞，证明分离培养的是神经干细胞，可用于移植、定向分化和基因转移的研究．     

Transforming growth factorβ1 and epidermal growth factor decrease the expression of 17β-hydroxysteroid dehydrogenase type 2 in endometrial carcinoma cells

Estradiol (E2) is the major molecular form of estrogens. Its biological effects are determined by estrogen receptors and intracellular E2 concentration in target cells. Regulation of intracellular E2 concentration involves the action of 17β(-hydroxysteroid dehydrogenase (17HSD) type 2, the enzyme inactivating E2 to estrone. It has been demonstrated that 17HSD type 2 is expressed in normal endometrial epithelia and emdometrial carcinoma cells (RL 95-2). However, the regulatory mechanism of 17HSD type 2 expression in emdometrial cancer cells remains unknown. In the present study, the effects of transforming growth factor-(1 (TGF-β1) and epidermal growth factor (EGF) on 17HSD type 2 expression in RL 95-2 cells have been investigated using enzyme activity assay and Northern blot analysis. After stimulation with TGF-β1 or EGF, the in vivo oxidative 17HSD activity in RL 95-2 cells was significantly decreased. It appeared that the inhibitory effect of TGF-β1 and EGF on the enzyme activity of 17HSD type 2 is dose- and time-dependent. Northern blot analysis further revealed that treatment of cells for 48 h with 10 ng/mL TGF-1β And 50 ng/mL EGF reduced the expression 17HSD type 2 mRNA to 30% and 20% of the control level, respectively. The data demonstrate that 17HSD type 2 expression in endometrial carcinoma cells is down-regulated by certain growth factors.     

野花生豆(Crotalaria mucronata)凝集素的细胞表面受体的研究

研究了异硫氰基荧光素和~(125)I标记的野花生豆凝集素,对A型细胞和牛精细胞表面的凝集素受体的定位、分布和数量。结果表明这些细胞表面的凝集素受体比较复杂,难以用一般动力学方法加以说明。D-葡萄糖、D-甘露糖、D-半乳糖和N-乙酰半乳糖胺等糖类和Con A、PHA、BSA对标记凝集素与A型细胞结合的影响各不相同。通过饱和曲线计算出A型细胞和牛精细胞表面的凝集素受体数目,分别为1.29×10~7和3.09×10~6个/细胞.     

兔ES样细胞系的建立及其特性分析

报道从２３７枚家兔胚胎中建成７个可连续传代的ＥＳ样细胞系。建系条件为，使用小鼠原始胚胎成纤维细胞（ＭＥ）作饲养层，以含１０％胎年血清和１０％兔血清的ＤＭＥＭ／Ｆ１２为培养基，添加白血病抑制因子（ＬＩＦ）或上皮生长因子（ＥＧＦ），胚龄为９０，９６ｈ。该细胞系的细胞。在许多方面类似于小鼠ＥＳ细胞，具干细胞的形态特征，呈集落型生长，可连续传代并保持其形态特征，具有一定的自发分化和诱导分化的能力，悬浮培养     

Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells

The complete 1,210-amino acid sequence of the human epidermal growth factor (EGF) receptor precursor, deduced from cDNA clones derived from placental and A431 carcinoma cells, reveals close similarity between the entire predicted v-erb-B mRNA oncogene product and the receptor transmembrane and cytoplasmic domains. A single transmembrane region of 23 amino acids separates the extracellular EGF binding and cytoplasmic domains. The receptor gene is amplified and apparently rearranged in A431 cells, generating a truncated 2.8-kilobase mRNA which encodes only the extracellular EGF binding domain.     

不同培养基对山羊毛囊体外培养的影响研究

为得到1种简捷而适宜的山羊毛囊体外培养方法,采用3因子3水平的拉丁方正交试验设计(L9(34))方法,对比研究了无血清的Williams E培养基、无血清的DMEM培养基和添加10％FCS(胎牛血清)的DMEM培养基,及其在3种培养基中分别添加0、2和10ng／mL 3种不同质量浓度的血管内皮细胞生长因子(VEGF)和表皮细胞生长因子(EGF)时对毛囊体外培养的影响。将成年青山羊皮肤上的单根毛囊完整地分离出来,并同时平行培养在9种培养基中,借助于倒置显微镜和目镜测微尺随时不断地观察毛囊生长的形态变化和测量毛根的长度。结果发现:山羊游离毛囊在9种培养基中均能很好地生长;但以无血清的培养基和在Williams E培养基的效果最好;分别和同时添加2％～10％的VEGF和EGF时可加快毛囊的生长;添加10％FCS和不添加细胞生长因子时毛囊也能生长,但生长速度较慢。     

Selective inhibition of the anchorage-independent growth of myc-transfected fibroblasts by retinoic acid

Fischer rat 3T3 (FR3T3) fibroblasts transfected with a cellular myc gene can be induced to grow and form colonies in soft agar by treatment either with epidermal growth factor (EGF) alone or with the combination of platelet-derived growth factor (PDGF) and type-beta transforming growth factor (TGF-beta). We now show that induction of anchorage-independent growth by each of these sets of growth factors involves different cellular pathways which can be distinguished by their sensitivity to retinoic acid. Colony formation induced by the combined action of PDGF and TGF-beta is 100-fold more sensitive to inhibition by retinoic acid than is colony formation induced by treatment of the myc-transfected cells with EGF. Moreover, retinoic acid (10(-8) M) is inhibitory for colony growth whenever TGF-beta is present, regardless of whether the effects of TGF-beta are stimulatory, as occurs in the presence of PDGF, or inhibitory, as found in the presence of EGF.     

Transient expression of IL-2 receptor precedes the differentiation of immature thymocytes

The growth of mature T lymphocytes after activation by antigen is regulated by the binding and endocytosis of interleukin-2 (IL-2). In the thymus, approximately 50% of adult thymocytes that carry neither the CD4 nor the CD8 antigen and day 14-15 fetal CD4-8- thymocytes express receptors for IL-2(IL-2R). The CD4-8- (double-negative) subpopulation of thymocytes contains the precursors of cells that can differentiate along an unknown pathway into thymocytes bearing either CD8 or CD4, with the characteristics of mature T lymphocytes. The basis for IL-2R expression by double-negative thymocytes is unclear as they appear to lack a functional T-cell receptor/CD3 complex through which activation of peripheral T cells is mediated. The argument for a role for IL-2 in thymocyte differentiation has also been complicated by conflicting reports on the inability or capability of double-negative thymocytes to respond to IL-2 in vitro. At present, both the nature of the stimuli within the thymic micro-environment which induce IL-2R expression and its relevance to thymocyte differentiation are not known. We show here that the IL-2R-bearing subset has a greater potential to differentiate into phenotypically mature T lymphocytes than do IL-2R-negative thymocytes. In addition, progeny of IL-2R-negative donor cells transiently express IL-2R in the thymuses of adoptive hosts before generating CD8 and/or CD4-positive thymocytes. These results identify the IL-2R-positive cells as a more differentiated double-negative thymocyte subset on the pathway to mature T lymphocytes.