The role of nerve growth factor in vitro in cell resistance to 6-hydroxydopamine toxicity.

Cell resistance to the catecholaminergic neurotoxin 6-hydroxydopamine was studied in various cell lines: human neuroblastoma lines SK-N-MC. SK-N-SH, and SK-N-SH-SY5Y; and non-neuroblastoma lines CHO-K1, S-180, C-6, and L-M, the latter three of which synthesize nerve growth factor. Cells were treated one day after seeding for 1 h with 6OHDA. Cytotoxicity of the drug was quantified as the percent live cells, determined by the trypan blue exclusion test, 24 h after treatment. At 100 μg/ml, 6OHDA lethal toxicity was confined mainly to neuroblastoma cells. However, drug specificity was dependent not only on cell type, but also on cell density and presence of NGF. Thus, the non-neuroblastoma cell strain S-180-A, which produces less NGF than its parent line S-180, lost resistance to toxicity at low cell density, and even at high density was less resistant than SK-N-MC neuroblastoma cells. Moreover, when mouse β-NGF (500 BU/ml) was administered to human neuroblastoma clones SY5Y and IN one day after seeding for 24 h before drug treatment, the cell survival rate increased significantly, although only SY5Y cells were protected by a lower concentration (1 BU/ml) of exogenous NGF. Finally, cell line S-180 became susceptible to 6OHDA killing when incubated one hour with high titer anti-mouse β-NGF immediately prior to drug treatment, whereas cell line C-6 did not. NGF was therefore proposed to have an important, though not determinative, role in cell resistance to 6OHDA toxicity.     

Phenotypic differentiation of aphidicolin-selected human neuroblastoma cultures after long-term exposure to nerve growth factor

Human neuroblastoma SH-SY5Y (SY5Y) cultures, exposed to murine 7 S nerve growth factor (NGF) for 5 weeks and selected with aphidicolin (Aph) for 1 week, acquire several properties indicative of mature peripheral nerve cells. The mitotic activity of treated cultures decreases prior to Aph selection and ultimately reaches a level approximately 3% that of untreated cultures by Week 4 of treatment. The measured plasma membrane resting potential of the cells increases from -5 mV for untreated cells to -(45-56) mV for NGF/Aph-treated cells. Intracellular stores of monoamines are increased as determined by histochemical staining, and levels of neuron-specific enolase antigen increase as a result of NGF/Aph treatment. The resulting outgrowth of neurites is extensive and large bundles of processes commonly exceed 300 micron in length. NGF/Aph-treated cells acquire a dependence upon NGF for survival; however, with continued administration of NGF, the cultures appear to be capable of surviving indefinitely. Retinoic acid will also promote certain aspects of a differentiated phenotype under similar culture conditions. As judged by these criteria, cells of the SY5Y human neuroblastoma cell line have the potential for phenotypic and irreversible differentiation in vitro and can survive for prolonged periods under these culture conditions.     

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc mRNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC8) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA and DiC8 it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.     

Differences in early and late responses between neurotrophin-stimulated trkA- and trkC-transfected SH-SY5Y neuroblastoma cells.

Despite their sympathetic neuroblast origin, highly malignant neuroblastoma tumors and derived cell lines have no or low expression of the neurotrophin receptor genes, trkA and trkC. Expression of exogenous trkA in neuroblastoma cells restores their ability to differentiate in response to nerve growth factor (NGF). Here we show that stable expression of trkC in SH-SY5Y neuroblastoma cells resulted in morphological and biochemical differentiation upon treatment with neurotrophin-3 (NT-3). To some extent, trkA- and trkC-transfected SH-SY5Y (SH-SY5Y/trkA and SH-SY5Y/trkC) cells resembled one another in terms of early signaling events and neuronal marker gene expression, but important differences were observed. Although induced Erk 1/2 and Akt/PKB phosphorylation was stronger in NT-3-stimulated SH-Y5Y/trkC cells, activation of the immediate-early genes tested was more prominent in NGF-treated SH-SY5Y/ trkA cells. In particular, c-fos was not induced in the SH-SY5Y/trkC cells. There were also phenotypic differences. The concentrations of norepinephrine, the major sympathetic neurotransmitter, and growth cone-located synaptophysin, a neurosecretory granule protein, were increased in NGF-treated SH-SY5Y/trkA but not in NT-3-treated SH-SY5Y/trkC cells. Our data suggest that NT-3/p145trkC and NGF/p140trkA signaling differ in some aspects in neuroblasoma cells, and that this may explain the phenotypic differences seen in the long-term neurotrophin-treated cells.     

IL-1对β-淀粉样前体蛋白基因在人神经母细胞瘤株中表达的调节作用

β-淀粉样前体蛋白及白介素-1β与老年性痴呆病的发生发展有密切关系．用SH-SY5Y细胞株研究了rh-IL-1β对细胞中APPmRNA表达的影响．Northern杂交结果显示,IL-1β可诱导SH-SY5Y细胞中APPmRNA的表达增加,且有时间剂量反应关系．通过转录延伸实验证实,IL-1β对APP基因表达的诱导作用主要发生在转录水平．     

Functional receptors for nerve growth factor on Ewing's sarcoma and Wilm's tumor cells

Quantification of changes in levels of c-fos RNA was used as an indicator of the presence of functional responses to nerve growth factor in several human non-neuronal cell lines which have previously been shown to express high levels of NGF receptors. Four Ewing's sarcomas, one Wilm's tumor, and one melanoma were examined. Of these cell lines, the Ewing's sarcoma IARC-EW1 showed greatly increased levels (10-20-fold) of c-fos RNA after 1 hour of exposure to NGF. Except for the melanoma line, the other tumor lines exhibited small, but reproducible, elevation of c-fos RNA expression. In IARC-EW1 cells, this induction was analyzed for kinetics, dose-response, and suppression by selective inhibitors of NGF action. The results indicate that these cells bear high-affinity receptors for NGF, which utilize signal pathways similar to NGF receptors on PC12 cells. Thus, we report new types of cells with functional responses to NGF and indicate that these may constitute a new model which will usefully complement those presently used for studying the mechanism of action of NGF.     

Up-regulation of the integrin alpha 1/beta 1 in human neuroblastoma cells differentiated by retinoic acid: correlation with increased neurite outgrowth response to laminin.

Retinoic acid (RA) is known to induce differentiation of neuroblastoma cells in vitro. Here we show that treatment of two human neuroblastoma cell lines, SY5Y and IMR32, with RA resulted in a fivefold increase of the integrin alpha 1/beta 1 expression. The effect was selective because expression of the alpha 3/beta 1 integrin, also present in these cells, was not increased. The up-regulation of the alpha 1/beta 1 differentiated SY5Y cells correlated with increased neurite response to laminin. In fact, RA-treated SY5Y cells elongated neurites on laminin-coated substratum more efficiently compared with untreated cells or cells treated with nerve growth factor, insulin, or phorbol 12-myristate 13-acetate. These three agents induced partial morphological differentiation but did not increase alpha 1 integrin expression. Neurite extension in RA-treated cells was more efficient on laminin than on fibronectin or collagen type I and was inhibited with beta 1 integrin antibodies on all three substrates. Affinity chromatography experiments showed that alpha 1/beta 1 is the major laminin receptor in both untreated and RA-treated SY5Y cells. These data show that RA, a naturally occurring morphogen implicated in embryonic development, can selectively regulate the expression of integrin complexes in neuronal cells and suggest an important role of the alpha 1/beta 1 laminin receptor in the morphological differentiation of nerve cells.     

Localization of neuropeptides in efferent terminals of the eye in the marine snail,Bulla gouldiana

Summary Retinoic acid (RA), a naturally occurring metabolite of vitamin A, increased the number of receptors for nerve growth factor (NGF) in cultured human neuroblastoma cells (LA-N-1), as indicated by an immunofluorescence assay of cell surface receptors and by specific binding of ¹²⁵I-NGF to solubilized receptors. Analysis of ¹²⁵I-NGF binding showed that RA increased the number of both high affinity and low affinity receptors for NGF without affecting the equilibrium dissociation constants. Neurite outgrowth similar to that produced by NGF occurred following RA-treatment in LA-N-1 cells, in the SY5Y subclone of SK-N-SH human neuroblastoma cells and in explanted chick dorsal root ganglia (DRG). Whether morphological changes following RA treatment are directly related to the increase in NGF receptors is unknown. Data presented here are consistent with literature reports that RA modifies cell surface glycoproteins, including those that act as cell surface receptors for epidermal growth factor and insulin.Abbreviations DRG dorsal root ganglia - NGF nerve growth factor - RA retinoic acid     

八氯腺苷诱导SH-SY5Y和SK-N-SH细胞G2/M期阻滞的分子机制不同

为探索八氯腺苷的抗肿瘤作用机制,以神经母细胞瘤SH-SY5Y和SK-N-SH细胞为对象,采用四唑盐比色实验（MTT法）证明,八氯腺苷具有明显的抑制肿瘤细胞增殖的作用,这种抑制作用呈剂量-时间依赖性.流式细胞分析显示,10 μmol/L八氯腺苷作用48 h后可导致靶细胞生长停滞于G ^₂/M期;SH-SY5Y细胞发生明显细胞凋亡,但SK-N-SH细胞却未见凋亡.Hoechst 33342染色显示,SK-N-SH细胞发生了核分裂异常.蛋白质免疫印迹分析证明,10 μmol/L 八氯腺苷处理SH SY5Y 48～72 h后,G^₂检验点调节蛋白ATM、Chk1、Cdc25C和Cdc2磷酸化形式明显上调,同时伴有caspase-3的激活,提示SH-SY5Y细胞发生了G^₂检验点通路和细胞凋亡途径的激活.与SH-SY5Y细胞不同,在SK-N-SH细胞中,八氯腺苷处理24～96 h时,磷酸化ATM、磷酸化Chk1/Chk2、磷酸化Cdc25C以及磷酸化Cdc2的水平呈现逐渐降低的趋势.结果提示,SK-N-SH细胞在八氯腺苷处理后发生了G^₂检验点失败.蛋白质免疫印迹分析还显示,八氯腺苷可诱导p53在SH-SY5Y细胞的表达,但却不能影响SK—N-SH细胞的p53组成性表达水平.p21在SK-N-SH的组成性表达随八氯腺苷处理时间延长而逐渐减少,但在处理前后的SH-SY5Y细胞均未检测到p21蛋白的表达.上述实验结果提示,八氯腺苷抑制两种细胞增殖的机制不同：在SH-SY5Y细胞,八氯腺苷可激活ATM-Chk-Cdc25C-Cdc2/cyclin途径和凋亡通路,使细胞发生G_²/M期阻滞和细胞凋亡;在SK-N-SH细胞,八氯腺苷诱导G^₂检验点失败,导致细胞阻滞在有丝分裂期,并发生有丝分裂异常.2种不同的细胞命运可能还与p53和p21表达不同有关.     

Multiple defects of the nerve growth factor receptor in human neuroblastomas

Neuroblastoma is a tumor of postganglionic sympathetic origin, and nerve growth factor (NGF) is normally required for the survival and differentiation of sympathetic neuroblasts. Since the biological activity of NGF is mediated by the NGF receptor (NGFR), we hypothesized that defects in the NGF/NGFR pathway may play a role in maintenance of the undifferentiated state of neuroblastomas. To test this hypothesis, we examined the structure of the NGFR at the DNA, RNA, and protein levels in a panel of 10 neuroblastoma cell lines. In addition, we examined the function of the NGFR in these lines by analysis of NGF binding kinetics, as well as by the ability of NGF to induce c-fos expression and neurite outgrowth. Southern blot analysis showed that all 10 cell lines possess apparently normal NGFR genes. Northern blot and ligand binding/immunoprecipitation assays revealed four receptor-positive cell lines (NGP, NLF, SK-N-SH, and LA-N-6), with NGFR mRNA and protein of expected sizes (3.8 kilobases and Mr approximately 75,000, respectively). NGF binding assays and Scatchard analyses were performed on the four NGFR-positive lines. The NGP line possesses only low-affinity receptor (Kd approximately 3.5 x 10(-9)), whereas the other three lines express both low- and high-affinity forms (Kd approximately 10(-9) and Kd approximately 10(-11), respectively). However, none of the 10 lines exhibited a response to NGF treatment as assayed by c-fos mRNA induction and neurite extension.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular-Regulated Kinases and Phosphatidylinositol 3-Kinase Are Involved in Brain-Derived Neurotrophic Factor-Mediated Survival and neuritogenesis of the Neuroblastoma Cell Line SH-SY5Y

Retinoic acid (RA) induces the differentiation of many cell lines, including those derived from neuroblastoma. RA treatment of SH-SY5Y cells induces the appearance of functional Trk B and Trk C receptors. Acute stimulation of RA-predifferentiated SH-SY5Y cells with brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), or neurotrophin 4/5 (NT-4/5), but not nerve growth factor (NGF), induces Trk autophosphorylation, followed by phosphorylation of Akt and the extracellular signal-regulated kinases (ERKs) 1 and 2. In addition, BDNF, NT-3, or NT-4/5, but not NGF, promotes cell survival and neurite outgrowth in serum-free medium. The mitogen-activated protein kinase and ERK kinase (MEK) inhibitor PD98059 blocks BDNF-induced neurite outgrowth and growth-associated protein-43 expression but has no effects on cell survival. On the other hand, the phosphatidylinositol 3-kinase inhibitor LY249002 reverses the survival response elicited by BDNF, leading to a cell death with morphological features of apoptosis.     

Differences in gene expression profile among SH-SY5Y neuroblastoma subclones with different neurite outgrowth responses to nerve growth factor

Nerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF-induced neurite-positive (NIN+) subclones that showed high responsiveness to NGF-induced neurite outgrowth and three NGF-induced neurite-negative (NIN-) subclones that abolished NGF-induced neurite outgrowth from parental SH-SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506-mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF-induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF-induced increases in mRNAs for the immediate-early genes (IEGs) c-fos and NGF inducible (NGFI) genes NGFI-A, NGFI-B and NGFI-C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN- cell line, particularly in terms of axonal vesicle-related genes and growth cone guidance-related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN- cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH-SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.     

Inhibition of Cx43 mediates protective effects on hypoxic/reoxygenated human neuroblastoma cells

Olfactory ensheathing cells (OECs), a special population of glial cells, are able to synthesise several trophic factors exerting a neuroprotective action and promoting growth and functional recovery in both in vitro and in vivo models. In the present work, we investigated the neuroprotective effects of OEC‐conditioned medium (OEC‐CM) on two different human neuron‐like cell lines, SH‐SY5Y and SK‐N‐SH (neuroblastoma cell lines), under normoxic and hypoxic conditions. In addition, we also focused our attention on the role of connexins (Cxs) in the neuroprotective processes. Our results confirmed OEC‐CM mediated neuroprotection as shown by cell adherence, proliferation and cellular viability analyses. Reduced connexin 43 (Cx43) levels in OEC‐CM compared to unconditioned cells in hypoxic conditions prompted us to investigate the role of Cx43‐Gap junctions (GJs) and Cx43‐hemichannels (HCs) in hypoxic/reoxygenation injury using carbenoxolone (non‐selective GJ inhibitor), ioxynil octanoato (selective Cx43‐GJ inhibitor) and Gap19 (selective Cx43‐HC inhibitor). We found that Cx43‐GJ and Cx43‐HC inhibitors are able to protect SH‐SY5Y and allow to these cultures to overcome the injury. Our findings support the hypothesis that both OEC‐CM and the inhibition of Cx43‐GJs and Cx43‐HCs offer a neuroprotective effect by reducing Cx43‐mediated cell‐to‐cell and cell‐to‐extracellular environment communications.