表皮生长因子和前列腺素E1对小鼠精原细胞增殖的促进作用

利用生殖细胞-体细胞无血清共培养模型研究了表皮生长因(EGF)和前列腺素E1(PGE1)对小鼠A型精原细胞增殖的影响.A型精原细胞在ITS培养液(添加胰岛素、转铁蛋白和亚硒酸钠的DMEM)中培养24 h后进行c-kit、EGF、表皮生长因子受体(EGFR)、环氧化酶-1(COX-1)及环氧化酶-2(COX-2)的免疫细胞化学检测,72 h后测定其形成集落教的情况.结果显示,A型精原细胞呈c-kit阳性,EGF、EGFR、COX-1及COX-2主要表达于精原细胞.EGF(10-7～10-6mol/L)或PGE,(10-8.10一mol/I_.)均可显著促进精原细胞集落的形成.此外,前列腺素(PG)受体拮抗剂SCl9220(10-6～10-5mol/L)可抑制PGE1对精原细胞的促增殖作用,COX-1抑制剂SC560(10-7～10-5mol/L)和COX-2抑制剂NS398(10-7～10-5mol/L)能抑制EGF促进精原细胞增殖的作用.因此,EGF可通过促进局部PG的产生而刺激精原细胞的增殖.     

不同浓度HGF、EGF优化大鼠胎肝干细胞体外培养的研究

目的:研究不同浓度肝细胞生长因子(Hepatocyte growth factor,HGF)和表皮生长因子(epidermal growth factor,EGF)对大鼠胎肝干细胞体外增殖的影响,探索二者间有无协同作用.方法:取孕14天胎龄F344大鼠胚胎的胎肝,经三步分离法分离纯化后,配置不同浓度HGF、EGF及HGF和EGF联合组培养基,将胎肝干细胞分组培养.光镜下观察细胞增殖状况,MTT法观察不同浓度和不同时间HGF、EGF及HGF和EGF联合对大鼠胎肝干细胞增殖的影响,并进行统计学分析.结果:HGF 10～80 ng/mL各浓度组,EGF 10～80 ng/mL各浓度组增殖效应均大于对照组.当HGF为20ng/mL,增殖效应明显大于对照组(P＜0.01),当HGF浓度继续增高时,增殖效应无明显增高.将20 ng/mL HGF组与不同浓度EGF组合后分组培养细胞,发现20 ng/ml HGF和10 ng/mLEGF联合组增殖效应明显升高,继续升高联合组中EGF浓度,增殖效应无明显提高.结论:HGF和EGF具有明显改善大鼠胎肝干细胞体外无血清培养条件的作用,二者对大鼠胎肝干细胞体外培养具有协同促进作用.其中20 ng/mL HGF和10 ng/mL EGF联合培养促进大鼠胎肝干细胞增殖效果最明显.     

卵泡刺激素和雌激素对培养的鸡胚卵巢生殖细胞增殖的影响

在动物体内多种激素共同调控着卵巢生殖细胞的生长、发育和成熟,其中促性腺激素起着至关重要的作用。本实验建立了鸡胚(18日胚龄)卵巢构建生殖细胞和体细胞的共培养模型,并研究卵泡刺激素(FSH)、17B雌二醇(E2)以及二者联合处理对生殖细胞增殖的影响。结果发现FSH(0．25-1．0IU／mL)、E2(10-1000ng／mL)处理48h后均可显著促进生殖细胞的增殖,FSH与E2共同处理作用更加显著且高于其单独处理组,表明FSH和E2可协同刺激鸡胚卵巢生殖细胞的增殖,FSH可能是通过促进雌激素或其受体的合成而发挥作用。     

应用原代软骨细胞与SW1353软骨肉瘤细胞系建立体外骨关节炎模型的效果评价

目的通过比较白细胞介素-1β(interleukin-1β,IL-1β)处理对原代软骨细胞与SW1353软骨肉瘤细胞系增殖活力、炎症因子与炎症通路表达水平变化的影响,为骨关节炎体外研究用细胞提供多重选择。方法免疫细胞化学法与甲苯胺蓝染色分别检测细胞中Ⅱ型胶原与蛋白多糖,鉴定所培养的原代细胞是否为软骨细胞。CCK-8法检测IL-1β(10ng/ml)处理24h、48h、72h对原代软骨细胞增殖活力的影响,IL-1β(1、10、20、40ng/ml)处理24h对SW1353软骨肉瘤细胞系增殖活力的影响。IL-1β(10ng/ml)分别处理原代软骨细胞与SW1353软骨肉瘤细胞系细胞24h后,ELISA法检测细胞培养上清中白细胞介素6(interleukin-6,IL-6)与基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)的表达水平。Real-time PCR法检测核因子-κB(nuclear factor-κB,NF-κB)mRNA表达水平。结果所培养的原代细胞为原代软骨细胞。IL-1β(10ng/ml)处理可显著抑制原代软骨细胞增殖活力,但IL-1β(1、10、20、40 ng/ml)处理对SW1353软骨肉瘤细胞系增殖活力无明显影响。IL-1β(10ng/ml)处理可使IL-6、MMP-13表达水平及NF-κB mRNA的表达量均显著增加。结论IL-1β作用下原代软骨细胞与SW1353软骨肉瘤细胞系均可表现出骨关节炎样炎症反应,二者均可用于骨关节炎的体外实验研究。     

NGF 对大鼠胚胎中脑神经细胞体外增殖和分化的影响

目的:探讨神经生长因子(nerve growth factor,NGF)对大鼠胚胎中脑神经细胞体外增殖和分化的影响。方法:在体外分离培养大鼠胚胎中脑神经细胞的培养液中加入不同浓度(10、50、100、200ng/ml)的NGF,培养不同时间,以不加神经营养因子的细胞为对照组,通过MTT法检测细胞活性,神经元特异性烯醇化酶免疫细胞荧光技术鉴定神经细胞,光镜下形态学观察各组大鼠中脑神经细胞体外增殖和分化情况。结果:胚胎中脑神经细胞胞体增大、突起延长且有丰富的神经纤维连结成网络状,细胞集落数增加,显示出剂量-效应关系。结论:一定剂量的NGF能促进大鼠中脑神经细胞分化和增殖,增强其活性。     

单细胞因子支持人胚胎干细胞生长的培养体系

[目的]为2株中国人胚胎干细胞系建立培养液中仅添加1种细胞因子的培养体系。[方法]两株人胚胎干细胞h ES-846XX和h ES-18 46XY分别使用含有成纤维细胞生长因子(b FGF)160ng/ml、转化生长因子(TGFβ1)20ng/ml和noggin 200ng/ml的培养液进行无饲养层培养,观察细胞的生长情况并对所得细胞进行鉴定。[结果]培养液中添加b FGF 160ng/ml可以有效地支持人胚胎干细胞的长期增殖,传代8次的细胞仍然保持胚胎干细胞的特性。添加TGFβ1或noggin的培养基无法维持人胚胎干细胞的长期增殖,3代以后细胞完全分化。[结论]证明培养液单独添加高浓度b FGF 160ng/ml支持2株中国人胚胎干细胞长期保持未分化状态增殖,实际未分化率为68.2±1.08%。     

表皮生长因子对精原干细胞的作用研究

应用不连续Percoll梯度液和选择性贴壁法分离纯化精原干细胞:c-kit细胞免疫组化鉴定细胞类型;MTT法研究EGF对精原干细胞增殖的效应;加入MAPK-ERK信号通路特异性抑制剂PD98059,探讨EGF对精原干细胞增殖作用的可能机制.证明:1)c-kit细胞免疫组化结果显示分离得到细胞为精原千细胞;2)MTT结果显示各实验组比对照组细胞数量均有显著增多(p＜0.01),且20 ng/mL剂量组的增殖作用最明显;3)与对照组相比,加入PD98095组的活细胞数有显著下降(p＜0.01).结论:EGF能够促进精原干细胞的增殖,并且可以通过MAPK-ERK信号通路起作用.     

人参总皂甙对人GM-CSF和GM-CSFR表达的调控

为探讨人参调控粒细胞发生的生物学机制,采用造血祖细胞和骨髓基质细胞体外培养、造血生长因子生物学活性检测、免疫细胞化学、核酸分子原位杂交、免疫沉淀和蛋白印迹等现代生物学技术,研究人参总皂甙(total saponins of Panax ginaeng,TSPG)对人粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)和粒-巨噬细胞集落刺激因子受体α(GM-CSFRα)表达的影响。结果：(1)经TSPG(50μg／m1)诱导制备的骨髓基质细胞、胸腺细胞、脾细胞、血管内皮细胞和单核细胞条件培养液可显著提高粒单系造血祖细胞(CFU-GM)的集落产率;(2)经TSPG(50μg/ml)诱导后,上述细胞的GM-CSF蛋白(诱导24h)和mRNA(诱导12h)表达显著提高;(3)经TSPG(50μg/ml)诱导24h骨髓造血细胞的GM-CSFRα蛋白表达增强;(4)经TSPG(50μg/ml)刺激后2min,GM-CSFRα和Shc发生酪氨酸磷酸化,5min时达高峰,随后去磷酸化。上述结果表明,TSPG可能通过直接和／或间接途径促进淋巴细胞与骨髓基质细胞合成与分泌GM-CSF,诱导骨髓造血细胞表达GM-CSFRα,并刺激GM-CSFRα和Shc的酪氨酸可逆磷酸化,从而通过调控GM-CSF的信号转导过程,促进CFU-GM的增殖。     

SD大鼠骨髓间充质干细胞钙离子波动的探究

本实验旨在通过化学药物促进或抑制骨髓间充质干细胞(BMMSCs)增殖以研究胞内Ca2+浓度在此过程中的变化规律.同步化于G1期的MSCs分别用10%胎牛血清(FBS)、15 ng/mL表皮生长因子(EGF)做短期(1 h)或持续(32 h)刺激,或用10 μg/mL丝裂霉素C(Mi C)短期(1 h)刺激和刺激2.5 h后除去,检测胞内Ca2+浓度变化.结果 表明在增殖相关的化学信号瞬时刺激下,MSCs胞内Ca2+信号瞬时增高,然后回复到一个稳态水平:促增殖时维持高稳态水平,抑增殖则低;持续的抑制导致胞内Ca2+波动弱.     

阿霉素肾病大鼠表皮生长因子及其受体的表达

目的研究阿霉素肾病大鼠肾组织中表皮生长因子(EGF)及其受体EGFR的表达分布以及表达量与尿蛋白之间关系。方法选择第5天、14天、28天作为动态观察的时点,同期设立正常对照。采用荧光定量RT-PCR、免疫组织化学及计算机图像定量分析EGF mRNA以及EGF、EGFR蛋白在肾组织的表达,同时测定24 h尿蛋白定量。WT1和EGFR双重免疫组化确定EGFR在肾小球内确切细胞定位。结果阿霉素注射后第5天,EGFmRNA即较正常增高,28 d明显增高并高于5 d和14 d。正常对照组EGF阳性细胞主要分布于远曲小管和髓袢,阿霉素组EGF还在集合管和近曲小管上表达;EGF阳性表达范围和强度随尿蛋白增加而增加;EGFmRNA表达量以及EGF在肾小管中的表达强度与24 h尿蛋白量呈正相关。肾小管上皮细胞广泛表达EGFR,阿霉素组EGFR在小管表达均高于正常,但组间各时点差异无显著性;随尿蛋白增加EGFR在肾小球内表达逐渐增多。EGFR在肾小球和肾小管中的表达强度均与24 h尿蛋白量呈正相关。WT1和EGFR双重免疫组化显示阿霉素肾病组EGFR可在足突细胞上表达,正常组则无。结论阿霉素肾病大鼠的肾小球脏层上皮有EGFR的表达。EGF/EGFR可能参与了阿霉素肾病的发病过程以及蛋白尿的形成。     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones

The mitogenic activity of several growth factors on androgen responsive LNCaP human prostate tumor cells was studied. A two-fold stimulation of cell proliferation was observed after a culture period of 6 days in 1 ng EGF/ml, 10 ng TGF-alpha/ml or 20 ng basic FGF/ml. TGF-beta (0.02 ng/ml), which did not affect cell proliferation when added alone to the culture medium, inhibited the EGF- and TGF-alpha-induced growth. The synthetic androgen R1881 (0.1 nM) stimulated cell proliferation three-fold and increased the number of EGF receptors from 11500 to 28500 sites/cell. One of the mechanisms involved in androgen action on these cells is therefore an increased EGF receptor expression and increased sensitivity to EGF. TGF-beta did not directly affect androgen-responsive growth but inhibited the synergistic effect of EGF. A considerable expression of TGF alpha (precursors) could be demonstrated on the cells by immunohistochemical staining. However the staining intensity was not affected by androgens. These results make it less likely that androgen-responsive growth is mediated by regulation of secretion of an EGF- or TGF alpha-like activity, which in turn acts in an autocrine manner to stimulate growth. Estrogens, progestagens and antiandrogens do not inhibit androgen responsive growth of LNCaP cells but have striking growth stimulatory effects, increase EGF receptor level and increase acid phosphatase secretion. LNCaP cells contain a modified androgen receptor system with respect to both steroid specificity and antiandrogen sensitivity. It has recently been shown that the stimulatory effects are due to a mutated amino acid in the steroid binding domain of the androgen receptor.     

Inhibition of human neuroblastoma cell proliferation and EGF receptor phosphorylation by gangliosides GM1, GM3, GD1A and GT1B

The inhibitory action of gangliosides GT1B, GD1A, GM3 and GM1 on cell proliferation and epidermal growth factor receptor (EGFR) phosphorylation was determined in the N-myc amplified human neuroblastoma cell line NBL-W. The IC50 of each ganglioside was estimated from concentration-response regressions generated by incubating NBL-W cells with incremental concentrations (5-1000 microm) of GT1B, GD1A, GM3 or GM1 for 4 days. Cell proliferation was quantitatively determined by a colourimetric assay using tetrazolium dye and spectrophotometric analysis, and EGFR phosphorylation by densitometry of Western blots. All gangliosides assayed, with the exception of GM1, inhibited NBL-W cell proliferation in a concentration-dependent manner. The IC50s for gangliosides GT1B [molecular weight (MW) 2129], GM3 (MW 1236), and GD1A (MW 1838) were (mean +/- SEM) 117 +/- 26, 255 +/- 29, and 425 +/- 44 m, respectively. In contrast, the IC50 for GM1 (MW 1547) could not be determined. Incubation of NBL-W cells with epidermal growth factor (EGF) concentrations ranging from 0.1 to 1000 ng/ml progressively increased cell proliferation rate, but it plateaued at concentrations above 10 ng/ml. EGFR tyrosine phosphorylation, however, was incrementally stimulated by EGF concentrations from 1 to 100 ng/ml. The suppression of EGF-induced EGFR phosphorylation differed for each ganglioside, and their respective inhibitory potencies were as follows: EGFR phosphorylation [area under curve (+ EGF)/area under curve (- EGF)]: control (no ganglioside added) = 8.2; GM1 = 8.3; GD1A = 6.7; GM3 = 4.87, and GT1B = 4.09. The lower the ratio, the greater the inhibitory activity of the ganglioside. Gangliosides GD1A and GT1B, which have terminal N-acetyl neuraminic acid moieties, as well as one and two N-acetyl neuraminic acid residues linked to the internal galactose, respectively, both inhibited cell proliferation and EGFR phosphorylation. However, GD1A was a more potent suppressor of cell proliferation and GT1B most effective against EGFR phosphorylation. GM3, which only has a terminal N-acetyl neuraminic acid, inhibited cell proliferation and EGFR phosphorylation almost equivalently. These data suggest that gangliosides differ in their potency as inhibitors of NBL-W neuroblastoma cell proliferation and EGFR tyrosine phosphorylation, and that perturbations in the differential expression of membrane glycosphingolipids may play a role in modulating neuroblastoma growth.     

TGFalpha and EGFR in ovine preimplantation embryos and effects on development

The present study aimed to assess location and relative amounts of transforming growth factor alpha (TGFalpha) and its receptor (EGFR) in ovine oocytes and preimplantation embryos by using immunohistochemical technique that was graded on a relative scale of 0-3, with 0 representing absence of staining, and 3 exhibiting prominent staining, and to evaluate the effects of TGFalpha/EGF on in vitro development of preimplantation embryos by adding different concentrations of EGF and TGFalpha to culture medium. The results showed that EGFR was abundant in cell plasma membranes in immature and mature oocytes, cumulus cells of immature cumulus-oocyte complexes (COC), fertilized oocytes and at different stages of embryo development. However, the relative amounts in inner cell mass (ICM) (1+) was less than that in trophectoderm (TE) cells (2+) at the blastocysts stage. The staining pattern for TGFalpha was a similar to EGFR. However, the staining for TGFalpha slightly increased in the fertilized oocytes (1-2+) as compared to immature and mature oocytes (1+). TGFalpha was mainly detected in the cytoplasm close to the membrane in both ICM and trophectoderm (TE) cells. The developmental rate of 8-cell stage embryos cultured with 5 ng/ml TGFalpha was increased as compared to other treatments (P<0.05). There was no significant difference in the rate of development of blastocysts cultured with 5 ng/ml TGFalpha, 20 ng/ml EGF, 20 ng/ml EGF+5 ng/ml TGFalpha or the control treatment (P>0.05). In addition, there was no significant difference in the number of cells in blastocyst stage as compared with different treatments (P>0.05). However, TGFalpha alone enhanced cell survival rated (P<0.01) and reduced apoptosis. We concluded that TGFalpha can improve development of ovine preimplantation embryos at the 8-cell and blastocyst stages in vitro.     

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.     

A Switch Role of Src in the Biphasic EGF Signaling of ER-Negative Breast Cancer Cells

It is well established that epidermal growth factor (EGF) is a potent mitogen in cells expressing EGF receptor (EGFR). However, a body of evidence indicated that the effects of mitogenic EGF signaling exhibit a non-monotonic, or biphasic dose response curve; EGF at low concentrations elicits a mitogenic signaling pathway to stimulate cell proliferation while at high concentrations, EGF inhibits cell growth. However, the molecular mechanism underlying this paradoxical effect of EGF on cell proliferation remains largely unknown. Here, we investigated the molecular mechanisms underlying the biphasic EGF signaling in ER-negative breast cancer MDA-MB-231 and MDA-MB-436 cells, both of which express endogenous EGFR. We found that EGF at low concentrations induced the phosphorylation of the Src-Y416 residue, an event to activate Src, while at high concentrations allowed Src-Y527 phosphorylation that inactivates Src. EGF at 10 ng/ml also induced phosphorylation of the MAPK/ERK and activated cyclin D1 promoter activity through the Src/EGFR/STAT5 pathways but not at a higher concentration (500 ng/ml). Our results thus demonstrated that Src functions as a switch of EGF signaling depending on concentrations of EGF.     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

Loss of cytotoxic effect of epidermal growth factor (EGF) on EGF receptor overexpressing cells is associated with attenuation of EGF receptor tyrosine kinase activity

The biological activity of epidermal growth factor (EGF) is mediated through the intrinsic tyrosine kinase activity of the EGF receptor (EGFR). In numerous cell types, binding of EGF to the EGFR stimulates the tyrosine kinase activity of the receptor eventually leading to cell proliferation. In tumor-derived cell lines, which overexpress the EGFR, however, growth inhibition is often seen in response to EGF. The mechanism for growth inhibition is unclear. To study the relationship between growth inhibition and EGFR kinase activity, we have used a cell line (PC-10) derived from a human squamous cell carcinoma that overexpresses EGFR. When exposed to 25 ng/ml EGF at low cell densities (1,300 cells/cm²), PC-10 cells exhibit cell death. In contrast, if EGF is added to high density cultures, no EGF mediated cell death is seen. When PC-10 cells were maintained at confluency in the presence of 25 ng/ml EGF for a period of 1 month, they were subsequently found competent to proliferate at low density in the presence of EGF. We designate these cells APC-10. The APC-10 cells exhibited a unique response to EGF, and no concentration of EGF tested could produce cell death. By ¹²⁵I-EGF binding analysis and [³⁵S]methionine labeling of EGFR, it was found that the total number of EGFR on the cell surface of APC-10 was not decreased relative to PC-10. No difference between PC-10 and APC-10 was seen in EGF binding affinity to the EGFR. Significantly, EGF stimulated autophosphorylation of the EGFR of APC-10 was 8–10-fold lower than that of PC-10. This reduced kinase activity was also seen in vitro in membrane preparations for EGFR autophosphorylation as well as phosphorylation of an exogenously added substrate. No difference between PC-10 and APC-10 in the overall pattern of EGFR phosphorylation in the presence or absence of EGF was detectable. However, the serine and threonine phosphorylation of the EGFR of APC-10 cells was consistently 2–3-fold lower than that seen in PC-10 cells. These results suggest a novel mechanism for EGFR overexpressing cells to survive EGF exposure, one that involves an attenuation of the tyrosine kinase activity of the EGFR in the absence of a change in receptor levels or receptor affinity. © 1994 Wiley-Liss, Inc.     

Promotion of spermatogonial proliferation by neuregulin 1 in newt (Cynops pyrrhogaster) testis

We have previously shown that mammalian follicle-stimulating hormone (FSH) promotes the proliferation of spermatogonia and their differentiation into primary spermatocytes in organ culture of newt testis. In the current study, we performed microarray analysis to isolate local factors secreted from somatic cells upon FSH treatment and acting on the germ cells. We identified neuregulin 1 (NRG1) as a novel FSH-upregulated clone homologous to mouse NRG1 known to control cell proliferation, differentiation and survival in various tissues. We further isolated cDNAs encoding two different clones. Amino acid sequences of the two clones were 75% and 94% identical to Xenopus leavis immunoglobulin (Ig)-type and cysteine-rich domain (CRD)-type NRG1, respectively, which had distinct sequences in their N-terminal region but identical in their epidermal growth factor (EGF)-like domain. Semi-quantitative and quantitative PCR analyses indicated that both clones were highly expressed at spermatogonial stage than at spermatocyte stage. In vitro FSH treatment increased newt Ig-NRG1 (nIg-NRG1) mRNA expression markedly in somatic cells, whereas newt CRD-NRG1 (nCRD-NRG1) mRNA was only slightly increased by FSH. To elucidate the function of newt NRG1 (nNRG1) in spermatogenesis, recombinant EGF domain of nNRG1 (nNRG1-EGF) was added to organ and reaggregated cultures with or without somatic cells: it promoted spermatogonial proliferation in all cases. Treatment of the cultures with the antibody against nNRG1-EGF caused remarkable suppression of spermatogonial proliferation activated by FSH. These results indicated that nNRG1 plays a pivotal role in promoting spermatogonial proliferation by both direct effect on spermatogonia and indirect effect via somatic cells in newt testes.     

Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture

A medium consisting of a rich basal nutrient mixture supplemented with bovine insulin (10 micrograms/ml), human transferrin (10 micrograms/ml), human cold-insoluble globulin (5 micrograms/ml), and ethanolamine (0.5 mM) supported the growth of the A431 human epidermoid cell line in the absence of serum with a generation time equal to that of cells in serum-containing medium. Addition of epidermal growth factor (EGF) to this culture medium at concentration mitogenic for other cell types resulted in a marked inhibition of A431 cell growth. Inhibitory effects of EGF were observed at 1 ng/ml and near-maximal effects were observed at 10 ng/ml. The inhibitory effect of EGF could be reversed by the omission of EGF in subsequent medium changes and could be prevented by the addition of anti-EGF antibody to the culture medium. Inhibition of A431 cell growth by EGF also could be demonstrated in serum-containing medium.