Patterns of oncogene activation in human neuroblastoma cells

The authors report 7 cases of intestinal carcinoids. They examine the clinical aspects and describe and discuss both surgical and medical treatment strategies. They also critically evaluate the value of monitoring some oncological markers and their prognostic significance. Each patient underwent an in-depth evaluation of tumour evolution (CAT, ultrasonography, NMR, angiography) and urinary 5HIAA and platelet 5HT were monitored. Surgery took the form of ileal or ileocolic resection, gastric resection, exeresis of the tumour using a transanal route, ligature of the right branch of the hepatic artery afferent to the metastasised lobe of the liver. Five patients were treated using chemotherapy and three, also suffering from carcinoid syndrome, with octreotide. On the basis of their personal experience the authors underline the limited value of the study of 5HT and 5HIAA tumour markers in the diagnosis of small carcinoid tumours. This is compensated by the outstanding role of these markers in the diagnosis of the hepatic and/or lymphoglandular diffusion of the tumour. These markers were not influenced by octreotide treatment in cases in which longastatin was successfully used to combat carcinoid syndrome. Their behaviour allowed useful information to be acquired regarding the tumour evolution following surgery.     

Gamma radiation-induced differentiation on human neuroblastoma cells in culture

We have studied the effect of gamma radiation on differentiation in neuroblastoma cell lines AF8 and SJ-N-KP. Growth inhibition and morphological and biochemical differentiation have been examined following radiation exposure to 1-10 Gy. Gamma radiation induced marked growth inhibition and morphological differentiation in a dose-and time-dependent manner in both cell lines, and induced biochemical differentiation in AF8 cells.     

Effects and metabolism of steroid hormones in human neuroblastoma cells

The development of the central nervous system is influenced by sex steroids and by their metabolites. However, little information on the possible effects of steroid hormones on neuroblastoma cells is available. Human neuroblastoma cell lines have been used as a model of human neuroblasts in vitro to study the metabolism of steroid hormones; in addition, the effects of steroids and steroid antagonists on neuroblastoma cell growth have also been investigated. The results obtained show that SH-SY5Y human neuroblastoma cells may actively metabolize testosterone and progesterone to their respective 5 alpha-reduced metabolites and that differentiation of neuroblastoma cells is paralleled by a significant increase in expression of the type-1 5 alpha-reductase and of the formation of steroid metabolites. All these data are suggestive of a potential role of steroid 5 alpha-reduced metabolites in the biology of neuroblastoma cells. Studies performed to analyze the role of steroid hormones on neuroblastoma cell proliferation show that progesterone at low doses may induce minor stimulation, and at higher doses, a toxic effect on the neuroblastoma cell line SK-N-SH is seen. Moreover, the antiprogestin 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl-1)-17-(prop-1-ynyl)estra-4,9-dien+ ++-3-one (RU486) decreases the proliferation of these cells in a dose-dependent manner. The effect of RU486 is not antagonized by either progesterone or dexamethasone, a result that seems to exclude the action of RU486 via classic intracellular steroid hormone receptors.     

Cholinergic-induced production of reactive oxygen species in human neuroblastoma cells

Stimulation of human SH-SY5Y neuroblastoma cells by a muscarinic receptor agonist, carbachol (CCh; 1 mM), elevated levels of free intracellular calcium and subsequently increased the production of reactive oxygen species (ROS). Quinuclidinylbenzilate (QNB) binding increased at 1 h after CCh, but returned back to the control level at 3 h. Production of ROS increased, however, during the 3 h time period. CCh also increased the translocation of protein kinase C (PKC) to the membrane. ROS production was completely blocked by atropine and a PKC inhibitor, Ro 31-8220. These results show that increased ROS production was a result of muscarinic receptor stimulation, and that PKC had an active role in this cellular stimulation. ROS production upon cellular stimulation by CCh was completely inhibited also by superoxide dismutase, and partially by catalase, indicating that the formation of superoxide anion dominated in cholinergic-induced generation of ROS in human neuroblastoma cells. These results also show that muscarinic stimulation causes sustained ROS production in human neuroblastoma cells. The slow increase in ROS production by CCh suggest a stepwise cascade of events leading to oxidative stress with a triggering role of cholinergic muscarinic receptors in this process.     

Ethanol exposure increases expression of c-jun and junD in human neuroblastoma cells

The aim of this study was to investigate the effect of ethanol exposure on the expression of fos and jun genes. Exposure of human neuroblastoma SH-SY5Y cells to ethanol for 2-4 days caused a dose-dependent increase in c-jun and junD mRNA levels, whereas mRNAs for c-fos, fosB and junB were not detectable in control or ethanol-treated cells. Four days of ethanol exposure also enhanced the AP-1 binding activity. Experiments with actinomycin D demonstrated that ethanol did not influence the degradation of c-jun and junD mRNAs. These results demonstrate that long-term exposure to ethanol increases c-jun and junD expression. This effect may be one of the mechanisms through which ethanol influences the gene regulatory system in neuronal cells.     

Inverse expression pattern of REST and synapsin I in human neuroblastoma cells

The zinc finger protein REST is a repressor of neuronal genes in nonneuronal tissues. We have analyzed the expression of REST, together with the expression of a REST target gene, encoding synapsin I, in human neuroblastoma cells. It was found that REST and synapsin I are coexpressed in neuroblastoma cell lines, although the expression of REST was inversely proportional to the levels of synapsin I mRNA. Thus, increased expression of synapsin I was directly correlated with decreased expression of REST. These expression data are in excellent correlation with synapsin I promoter activity measured in neuroblastoma cells showing that an increase in the REST concentration switched off synapsin I promoter activity. We conclude that the concentration of REST determines the expression level of neuronal genes such as the synapsin I gene.     

Cytotoxic effects of MPTP on SH-SY5Y human neuroblastoma cells

Morphological and metabolic endpoints were used to evaluate MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) toxicity to SH-SY5Y human neuroblastoma cells. After 8 hours of exposure, MPTP was found to affect cell viability only at a very high concentration (3 x 10(-3) M), but its metabolite MPP+ could decrease viability at 10(-4) M. MPTP, via its metabolite MPP+, inhibited NADH dehydrogenase activity when concentrations exceeded 10(-4) M (for MPP+ 10(-5)M). The Ki were 2.4 x 10(-3) M and 3 x 10(-4)M for MPTP and MPP+, respectively. MPTP at concentrations greater than 10(-4) M altered cell morphology as early as one hour after exposure. These changes included formation of cell surface blebs and attenuated neurites. After 8 hours at 10(-3) M and 24 hrs at 10(-4) M, MPTP caused ultrastructural changes of mitochondria with increased electron-density of the matrix and disorganization of cristae, as well as abnormal aggregation of filamentous material of the cytoskeleton. Because these changes of structure and function took place at concentrations lower than those needed to affect cell viability, they may play a role in MPTP neurotoxicity in SH-SY5Y cell culture.     

Induction of apoptosis in human neuroblastoma cells by abrogation of integrin-mediated cell adhesion

The survival, proliferation and differentiation of neuroblastoma (NB) cells are largely dependent on adhesion to extracellular matrix (ECM) proteins. Integrin occupancy seems to play a primary role. To elucidate the role of integrin heterodimers during neuronal cell death, we have analysed the changes in integrin expression in 2 human NB cell lines which represent different stages of neuronal maturation. Retinoic acid (RA) had different effects on the 2 NB cell lines: on LAN-5 cells it acted as a differentiation-promoting agent, while it had an anti-proliferative effect on GI-LI-N cells, driving them to apoptosis. Indeed, this occurrence was evidenced by the visualization of a "DNA ladder" on gel electrophoresis, by propidium iodide staining, and by DNA flow cytofluorimetric analysis. RA treatment rapidly and drastically decreased integrin expression and cell adhesion on GI-LI-N cells. These findings were also obtained by treating both NB cell lines with the apoptotic agent fenretinide. Furthermore, treatment of NB cells with anti-sense oligonucleotides to beta 1 integrin chain specifically induced chromatin condensation and nucleosomal DNA laddering. Moreover, blocking cell-matrix interactions by means of perturbing antibody against beta 1 subunit resulted in the induction of typical features of apoptotic cells. In conclusion, these findings indicate that abrogation of cell adhesion through down-modulation of integrin receptors plays a crucial role in the induction of neuroblastoma programmed cell death.     

微波辐射诱导人神经母细胞瘤SH-SY5Y细胞凋亡的可能机制

目的:观察微波辐射对人神经母细胞瘤SH-SY5Y细胞凋亡的影响,并探讨其机制.方法:采用10、30和50 mW·cm-2辐射强度的微波辐射SH-SY5Y细胞5 min,以假辐射组(0 mW·cm-2)为对照,光镜下观察细胞形态变化;荧光显微镜下观察DAPI染色细胞核的形态;CTAB法提取细胞基因组DNA,电泳观察DNA ladder;台盼蓝拒染法检测细胞存活率;AnnexinV-FITC和PI双染色,流式细胞仪检测细胞凋亡率;MTT法检测细胞相对活性;蛋白质印迹检测细胞凋亡相关蛋白的表达.结果:微波辐射后SH-SY5Y细胞形态即刻改变,胞核、胞质结构欠清,细胞皱缩甚至脱壁;细胞核内的染色质出现不规则凝集,碎裂成2～5个微核;细胞DNA出现明显的梯状条带;细胞存活率随微波辐射强度增大逐渐降低,10 mW·cm-2辐射组与假辐射组比较,细胞存活率差异无统计学意义(P>0.05),但30、50 mW·cm-2辐射组细胞存活率明显低于假辐射组(P<0.05);微波辐射后6 h的细胞凋亡率随辐射强度的增加逐渐升高,各辐射组细胞凋亡率均明显高于假辐射组(P<0.05);微波辐射后24和48 h,细胞相对活力随辐射强度的增加明显降低,各辐射组的细胞相对活力均明显低于假辐射组(P<0.05).微波辐射后,细胞凋亡相关蛋白Bcl-2、survivin表达水平逐渐下降,Bax、caspase-3 和caspase-7表达水平逐渐升高,caspase-8和-9表达水平无明显变化.结论:微波辐射可诱导SH-SY5Y细胞凋亡,其作用机制可能与survivin、Bcl-2蛋白表达水平下调、Bax蛋白表达水平上调及caspase-3和caspase-7的作用有关,而与caspase-8和caspase-9介导的caspase-3活化这一作用无密切关系.     

Carbachol but not bradykinin blocks the enkephalin-induced calcium transient in human neuroblastoma SK-N-SH cells

Early nursing reforms in the 19th century are usually associated with Nightingale, although later emphasis has been placed on similar movements in the Poor Law sector. Extension of nursing influence over decision-making in terms of nursing practice and education is charted, using examples from 19th century Minutes of hospital committees and more recent experience based mainly on the observations made by one of the writers, who had substantial input into steering the hospital through the stages prior to achieving National Health Service (NHS) Trust status. The significance of nurse executive power following the 1990s NHS reforms is highlighted and means of extending the use of this authority are explored.     

Swelling-induced arachidonic acid release via the 85-kDa cPLA2 in human neuroblastoma cells

Arachidonic acid or its metabolites have been implicated in the regulatory volume decrease (RVD) response after hypotonic cell swelling in some mammalian cells. The present study investigated the role of arachidonic acid (AA) during RVD in the human neuroblastoma cell line CHP-100. During the first nine minutes of hypo-osmotic exposure the rate of 3H-arachidonic acid (3H-AA) release increased to 250 +/- 19% (mean +/- SE, n = 22) as compared with cells under iso-osmotic conditions. This release was significantly inhibited after preincubation with AACOCF3, an inhibitor of the 85-kDa cytosolic phospholipase A2 (cPLA2). This indicates that a PLA2, most likely the 85-kDa cPLA2 is activated during cell swelling. In contrast, preincubation with U73122, an inhibitor of phospholipase C, did not affect the swelling-induced release of 3H-AA. Swelling-activated efflux of 36Cl and 3H-taurine were inhibited after preincubation with AACOCF3. Thus the swelling-induced activation of cPLA2 may be essential for stimulation of both 36Cl and 3H-taurine efflux during RVD. As the above observation could result from a direct effect of AA or its metabolite leukotriene D4 (LTD4), the effects of these agents were investigated on swelling-induced 36Cl and 3H-taurine effluxes. In the presence of high concentrations of extracellular AA, the swelling-induced efflux of 36Cl and 3H-taurine were inhibited significantly. In contrast, addition of exogenous LTD4 had no significant effect on the swelling-activated 36Cl efflux. Furthermore, exogenous AA increased cytosolic calcium levels as measured in single cells loaded with the calcium sensitive dye Fura-2. On the basis of these results we propose that cell swelling activates phospholipase A2 and that this activation via an increased production of AA or some AA metabolite(s) other than LTD4 is essential for RVD.     

The presence of amyloid beta-protein in the detergent-insoluble membrane compartment of human neuroblastoma cells

To investigate the intracellular compartmentalization of amyloid beta-protein (Abeta), human neuroblastoma SH-SY5Y cells were fractionated and the Abeta content in each fraction was quantitated by the well-characterized two-site enzyme-linked immunosorbent assay (ELISA). Subcellular fractionation of the cell revealed two distinct pools of Abeta within the cells: a Triton-soluble and a Triton-insoluble pools with the latter being larger than the former. Because Triton insolubility points to caveolae-like domains, we prepared detergent-insoluble, low-density membrane domains from SH-SY5Y cells using two different protocols. The low-density membrane fraction prepared by either protocol was found to contain a substantial proportion of intracellular Abeta40 and Abeta42. These results indicate that the distinct membrane domains are involved in the generation and/or trafficking of Abeta.     

Serum deprivation and protein synthesis inhibition induce two different apoptotic processes in N18 neuroblastoma cells

N18 are murine neuroblastoma cells that underwent cell death upon serum deprivation or inhibition of protein synthesis by means of cycloheximide (CHX). In both cases, an ultrastructural morphology and an internucleosomal pattern of DNA fragmentation typical of apoptosis were found. However, electron microscopy revealed abundant lipid vesicles in the cytoplasm of CHX-treated cells that were not found in their serum-deprived counterparts. In addition, when both types of apoptotic cells were compared by means of flow cytometry and chromatin staining with propidium iodide, the former showed consistently less fluorescence than the latter. Therefore, in N18 cells, both apoptotic processes seemed to differ at a structural level. At a functional level, we found that apoptosis was blocked by the protease inhibitor TLCK in CHX-treated but not in serum-deprived cells. On the other hand, we generated N18 clones that overexpressed Bcl-2 protein. After a period of 48 h we found that identical levels of Bcl-2 protein were able to block apoptosis in serum-deprived but not in CHX-treated cells. In conclusion, two different biochemical pathways leading to apoptosis seem to coexist in N18 neuroblastoma cells.     

Control of glycogen synthesis in cultured human muscle cells

The regulation of glycogen synthesis and associated enzymes was studied in human myoblasts and myotubes maintained in culture. Both epidermal growth factor (EGF) and insulin stimulated glycogen synthesis approximately 2-fold, this stimulation being accompanied by a rapid and stable activation of the controlling enzyme glycogen synthase (GS). EGF also caused inhibition of glycogen synthase kinase 3 (GSK-3) and activation of the alpha isoform of protein kinase B (PKB) with the time-course and magnitude of its effects being similar to those induced by insulin. An inhibitor of the mitogen-activated protein (MAP) kinase pathway did not prevent stimulation of GS by EGF, suggesting that this pathway is not essential for the effect. A partial decrease in the fold activation of GS was, however, observed when p70(S6k) activation was blocked with rapamycin, suggesting a contribution of this pathway to the control of GS by either hormone. Wortmannin, a selective inhibitor of phosphatidylinositol 3'-kinase (PI-3 kinase) completely blocked the effects of both EGF and insulin in these cells. These results demonstrate that EGF, like insulin, activates glycogen synthesis in muscle, acting principally via the PKB/GSK-3 pathway but with a contribution from a rapamycin-sensitive component that lies downstream of PI-3 kinase.     

Induction of differentiation in mouse neuroblastoma cells

In the presence of 1--2% dimethyl sulfoxide (DMSO), mouse neuroblastoma cells are induced to differentiate morphologically as well as electrically. In addition, treatment of neurolbastoma cells with 2% DMSO results in a marked increase in the veratridine-activated K+ or Rb+ efflux. At 4% DMSO, neurite outgrowth is completely repressed and electrical activity is poorly developed. However, at this concentration, the cells have a relatively high resting potential which suggested that membrane components determining passive and active permeability properties are not necessarily under coordinated control. Induction of differentiation by 2% DMSO is also accompanied by an increase in a heavier molecular form of acetylcholinesterase sedimenting at 10.5S. The effect of other agents on the growth and differentiation of neuroblastoma cells is also presented.     

Characterization of phospholipase D activation by muscarinic receptors in human neuroblastoma SH-SY5Y cells

The cholinergic regulation of phospholipase D activity was studied in SH-SY5Y human neuroblastoma cells with phosphatidylethanol formation as a specific marker for the enzyme activity. The muscarinic antagonists, hexahydrosiladifenidol and pirenzepine, inhibited carbachol-induced phosphatidylethanol formation in a concentration-dependent manner and the inhibitory constants indicated that muscarinic M1 receptors are responsible for the major part of the phospholipase D activation. The mechanism of receptor-mediated phospholipase D activation varies between different cell types and receptors. In SH-SY5Y cells, the carbachol-induced phospholipase D activity was inhibited by protein kinase C inhibitors. Since both phospholipases D and C are activated by muscarinic stimulation in SH-SY5Y cells, most of the phospholipase D activation is probably secondary to the protein kinase C activation that follows phospholipase C-mediated increase in diacylglycerols. Other kinases may be involved in the regulation since also a tyrosine kinase inhibitor decreased the phosphatidylethanol formation. Stimulation of G-protein(s) and increase in the intracellular Ca2+ concentration activated phospholipase D and may be additional mechanisms for the muscarinic regulation of phospholipase D in SH-SY5Y cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, increased the carbachol-induced formation of phosphatidic acid at the expense of 1,2-diacylglycerol. This indicates that phospholipase D contributes to the formation of 1,2-diacylglycerol after carbachol stimulation in SH-SY5Y cells.     

The neurotoxic effect of 24-hydroxycholesterol on SH-SY5Y human neuroblastoma cells

PURPOSE: The purpose of this study was to compare two commercially available accelerometers with indirect calorimetry in a group of older adults (x +/- SD; 70.6+/-3.7 yr; N = 86, 44 males and 42 females). METHODS: The accelerometers (Caltrac and Tritrac, Hemokinetics, Madison, WI) were worn while performing three submaximal, discontinuous (5 min exercise, 2 min recovery), progressive levels of treadmill walking and bench stepping. The treadmill exercise averaged 3.4 mph, at 0.4% grade, 3.0% grade, and 5.1% grade, while the stepping work rates (24 steps x min(-1)) were performed on 15.2-, 20.3-, and 25.4-cm steps. Estimated energy expenditure (EE) from the two accelerometers was compared with EE as measured by indirect calorimetry. RESULTS: The Caltrac significantly (P < 0.05) overestimated EE at the three treadmill work rates (10-52% difference) and underestimated EE at the three stepping work rates (-19% to -28% difference). When comparing the changes in EE between work rates one, two and three, the Caltrac was not sensitive to the changes (increase in EE) that occurred during graded treadmill walking but did detect some changes in the stepping exercise. The Tritrac significantly (P < 0.05) underestimated EE for the three work rates of both the treadmill and stepping exercise when compared with indirect calorimetry but did detect differences in EE among work rates during stepping exercise (P < 0.05). CONCLUSIONS: These data indicate that the magnitude of the differences between measured and estimated EE is affected by exercise mode and intensity and that caution is warranted when using the accelerometers in an attempt to quantify EE in older adults.     

Multihormonal regulation of progesterone synthesis in cultured human midluteal cells

The objectives of this investigation were to examine in vivo insulin like-growth factor-I (IGF-I) secretion by the human midcorpora lutea (mid-CL) and the effects of IGF-I, hCG, FSH, and human GH on progesterone (P) production by CL cells obtained from patients at laparotomy. We first examined whether the CL produces IGF-I by measuring IGF-I levels in the ovarian vein from the ovary bearing the CL. The IGF-I concentration in the ovarian vein bearing the CL (206 +/- 31 ng/mL) was significantly increased compared to the concentration in the contralateral ovarian vein (179.2 +/- 32 ng/mL; P < 0.05). Luteal cells isolated from mid-CL were cultured in serum-free medium 199 in the presence and absence of hCG, FSH, GH, and graded concentrations of IGF-I. At the end of the incubation period (24 h), P levels in the medium were measured by RIA. The treatment with IGF-I (0.1-10 ng/mL) showed a dose-dependent stimulatory action of IGF-I on P synthesis in the luteal cell system, being maximal between 5-10 ng/mL. The treatment with hCG (10 IU/mL), IGF-I (5 ng/mL), and GH (1000 ng/mL) increased basal P synthesis by 300%, 80%, and 30%, respectively (P < 0.001 and P < 0.05). FSH (100 ng/mL), either alone or in combination with IGF-I, failed to stimulate P synthesis. Treatment with IGF-I monoclonal antibody (1:5000) completely reduced P synthesis induced by 5 ng/mL IGF-I and slightly reduced basal P synthesis as well as GH-stimulated P synthesis by human midluteal cells. To further evaluate the specific role of IGF-I on luteal steroidogenesis, IGF-I receptor was identified by chemical cross-linking of [125I]IGF-I to mid-CL membranes. Experiments conducted in the absence and presence of unlabeled IGF-I (500 ng) revealed proteins with characteristics of the type I IGF receptor. These results are consistent with multihormonal regulation of P synthesis by the human mid-CL. hCG and IGF-I play a major role in the stimulation of P synthesis and, to a lesser extent, human GH. These in vivo and in vitro data suggest that the CL is a site of secretion, action, and reception of IGF-I during the midluteal phase.     

Induction of ceruloplasmin synthesis by IFN-gamma in human monocytic cells

Ceruloplasmin is a 132-kDa glycoprotein abundant in human plasma. It has multiple in vitro activities, including copper transport, lipid pro- and antioxidant activity, and oxidation of ferrous ion and aromatic amines; however, its physiologic role is uncertain. Although ceruloplasmin is synthesized primarily by the liver in adult humans, production by cells of monocytic origin has been reported. We here show that IFN-gamma is a potent inducer of ceruloplasmin synthesis by monocytic cells. Activation of human monoblastic leukemia U937 cells with IFN-gamma increased the production of ceruloplasmin by at least 20-fold. The identity of the protein was confirmed by plasmin fingerprinting. IFN-gamma also increased ceruloplasmin mRNA. Induction followed a 2- to 4-h lag and was partially blocked by cycloheximide, indicating a requirement for newly synthesized factors. Ceruloplasmin induction in monocytic cells was agonist specific, as IL-1, IL-4, IL-6, IFN-alpha, IFN-beta, TNF-alpha, and LPS were completely ineffective. The induction was also cell type specific, as IFN-gamma did not induce ceruloplasmin synthesis in endothelial or smooth muscle cells. In contrast, IFN-gamma was stimulatory in other monocytic cells, including THP-1 cells and human peripheral blood monocytes, and also in HepG2 cells. Ceruloplasmin secreted by IFN-gamma-stimulated U937 cells had ferroxidase activity and was, in fact, the only secreted protein with this activity. Monocytic cell-derived ceruloplasmin may contribute to defense responses via its ferroxidase activity, which may drive iron homeostasis in a direction unfavorable to invasive organisms.