Apoptosis ; Calcineurin ; metabolism ; Chymotrypsin ; metabolism ; Humans ; Lysosomes ; enzymology ; Mitochondria ; metabolism ; pathology ; Mitochondrial Dynamics ; Neuroblastoma ; metabolism ; pathology

OBJECTIVETo observe the cellular uptake of beta amyloid protein (Abeta) by cultured human neuroblastoma (SH-SY5Y) cells and the location of Abeta in the subcellular structures.

METHODSThe time course of cellular uptake of Abeta1-42-fluo in the SH-SY5Y cells was observed directly under laser scanning confocal microscope (LSCM). Image analysis was conducted to compare the differences of cellular Abeta uptake after treatment of the cells with different concentrations of extracellular Abeta for 24 h. Multiple immunofluorescence staining was employed to identify the location of Abeta in the subcellular structures.

RESULTSSH-SY5Y cells showed Abeta internalization after incubation with Abeta1-42-fluo (200 nmol/L) for 1 h, and the quantity of Abeta uptake was time-dependent. A higher concentration of extracellular Abeta1-42-fluo resulted in increased Abeta uptake, which differed significantly between the 3 groups with treatment at different concentrations (P<0.01 or 0.05). Immunofluorescence staining revealed a co-localization of part of the Abeta and Lamp-1 (a lysosome marker) in the cytosome.

CONCLUSIONSH-SY5Y cells can clear Abeta through a time- and dose-dependent cellular uptake mechanism. Part of the Abeta uptaken in the cytoplasm is located in the lysosome .

Alzheimer Disease ; metabolism ; Amyloid beta-Peptides ; metabolism ; Cell Line, Tumor ; Humans ; Neuroblastoma ; metabolism ; pathology ; Peptide Fragments ; metabolism

OBJECTIVETo evaluate the human neuroblastoma SH-SY5Y cell line as an in vitro model of dopaminergic (DAergic) neurons for Parkinson's disease (PD) research and to determine the effect of differentiation on this cell model.

DATA SOURCESThe data of this review were selected from the original reports and reviews related to SH-SY5Y cells published in Chinese and foreign journals (Pubmed 1973 to 2009).

STUDY SELECTIONAfter searching the literature, 60 articles were selected to address this review.

RESULTSThe SH-SY5Y cell line has become a popular cell model for PD research because this cell line posses many characteristics of DAergic neurons. For example, these cells express tyrosine hydroxylase and dopamine-beta-hydroxylase, as well as the dopamine transporter. Moreover, this cell line can be differentiated into a functionally mature neuronal phenotype in the presence of various agents. Upon differentiation, SH-SY5Y cells stop proliferating and a constant cell number is subsequently maintained. However, different differentiating agents induce different neuronal phenotypes and biochemical changes. For example, retinoic acid induces differentiation toward a cholinergic neuronal phenotype and increases the susceptibility of SH-SY5Y cells to neurotoxins and neuroprotective agents, whereas treatment with retinoic acid followed by phorbol ester 12-O-tetradecanoylphorbol-13-acetate results in a DAergic neuronal phenotype and decreases the susceptibility of cells to neurotoxins and neuroprotective agents. Some differentiating agents also alter kinetics of 1-methyl-4-phenyl-pyridinium (MPP(+)) uptake, making SH-SY5Y cells more similar to primary mesencephalic neurons.

CONCLUSIONSDifferentiated and undifferentiated SH-SY5Y cells have been widely used as a cell model of DAergic neurons for PD research. Some differentiating agents afford SH-SY5Y cells with more potential for studying neurotoxicity and neuroprotection and are thus more relevant to experimental PD research.

Cell Differentiation ; physiology ; Cell Line, Tumor ; Dopamine ; metabolism ; Humans ; Neuroblastoma ; metabolism ; pathology ; Neurons ; metabolism ; pathology ; Parkinson Disease ; metabolism ; pathology

Accumulative evidence has shown the adverse effects of a geomagnetic field shielded condition, so called a hypomagnetic field (HMF), on the metabolic processes and oxidative stress in animals and cells. However, the underlying mechanism remains unclear. In this study, we evaluate the role of HMF on the regulation of cellular reactive oxygen species (ROS) in human neuroblastoma SH-SY5Y cells. We found that HMF exposure led to ROS decrease, and that restoring the decrease by additional HO rescued the HMF-enhanced cell proliferation. The measurements on ROS related indexes, including total anti-oxidant capacity, HO and superoxide anion levels, and superoxide dismutase (SOD) activity and expression, indicated that the HMF reduced HO production and inhibited the activity of CuZn-SOD. Moreover, the HMF accelerated the denaturation of CuZn-SOD as well as enhanced aggregation of CuZn-SOD protein, in vitro. Our findings indicate that CuZn-SOD is able to response to the HMF stress and suggest it a mediator of the HMF effect.
Cell Line, Tumor ; Humans ; Hydrogen Peroxide ; metabolism ; Magnetic Fields ; Neoplasm Proteins ; metabolism ; Neuroblastoma ; metabolism ; Stress, Physiological ; Superoxide Dismutase-1 ; metabolism

Parkinson's disease is a multifactorial disorder with several genes linked to the familial types of the disease. ATP13A2 is one of those genes and encode for a transmembrane protein localized in lysosomes and late endosomes. Previous studies suggested the roles of this protein in lysosomal functions and cellular ion homeostasis. Here, we set out to investigate the role of ATP13A2 in lysosomal function and in metabolism of alpha-synuclein, another PD-linked protein whose accumulation is implicated in the pathogenesis. We generated non-sense mutations in both copies of ATP13A2 gene in SH-SY5Y human neuroblastoma cells. We examined lysosomal function of ATP13A2-/- cells by measuring the accumulation of lysosomal substrate proteins, such as p62 and polyubiquitinated proteins, induction of acidic compartments, and degradation of ectopically introduced dextran. None of these measures were altered by ATP13A2 deficiency. The steady-state levels of alpha-synuclein in cells or secretion of this protein were unaltered either in ATP13A2-/- compared to the normal cells. Therefore, the proposed roles of ATP13A2 in lysosomal functions may not be generalized and may depend on the cellular context. The ATP13A2-/- cells generated in the current study may provide a useful control for studies on the roles of PD genes in lysosomal functions.
alpha-Synuclein* ; Dextrans ; Endosomes ; Homeostasis ; Humans ; Lysosomes ; Metabolism* ; Neuroblastoma ; Parkinson Disease ; Polyubiquitin

OBJECTIVETo correlate the abnormal expression of anapastic lymphoma kinase (ALK) protein with the genetic and epigenetic changes of ALK, and to analyze its clinical application in pediatric neuroblastoma.

METHODSThree neuroblastoma (NB) cell lines (two ALK positive: SH-SY5Y and SK-N-SH, one ALK negative: SK-N-AS) and 43 paraffin-embedded NB tissues were included in the study. In both cell lines and clinical cases, immunohistochemistry was used to detect ALK protein expression; PCR and Sanger sequencing were used to detect ALK point mutation; fluorescence in situ hybridization (FISH) was used to detect ALK abnormality and bisulfite sequencing PCR (BSP) was used to detect methylation of CpG island in the promoter area of ALK.

RESULTSThe cell lines SH-SY5Y and SK-N-SH were positive for ALK expression (cytoplasm), while the SK-N-AS was negative; among the 43 cases of NB, 26 (60.5%, 26/43) were positive for ALK protein (membrane and cytoplasm), and the rest were negative. Survival analysis showed ALK protein expression was related to survival time, with ALK positive cases having shorter survival time than ALK negative cases (P = 0.020). But ALK protein expression had no association with tumor differentiation (P = 0.503), tumor sites (P = 1.000) and age of patients (P = 0.063). FISH showed ALK amplification in two cases (4.6%, 2/43), ALK gain was found in 30 cases (69.7%, 30/43), and the remaining cases had normal ALK copy (25.6%, 11/43). The presence of extra copies (amplification and gain) of ALK was associated with ALK positive protein expression (P = 0.020), but there was no association with tumor differentiation (P = 1.000), tumor sites (P = 0.775) and age of patients (P = 0.328). No point mutation was found in all three cell lines. Of the 43 NB cases, only one case (2.3%, 1/43) showed point mutation in exon 23, and was a synonymous mutation [A1200A (G4552C)]. The case was ALK negative, but the patient died two months after diagnosis. BSP analysis showed that CpG island in ALK promoter region were all unmethylated in three cell lines and 6 NB cases (including 3 ALK positive, 3 ALK negative).

CONCLUSIONSALK protein is expressed in most NB, and the expression indicates poor outcome. ALK expression is associated with extra copies of ALK, but there is no association with the methylation status of CpG island of ALK; the presence of extra copies of ALK is the most common genetic aberration in NB. Point mutation of ALK is rare, and may predict poor prognosis in pediatric NB.

Adolescent ; Cell Line, Tumor ; Child ; Exons ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Neuroblastoma ; enzymology ; genetics ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism

OBJECTIVETo investigate the influence of fluorosis on nicotinic acetylcholine receptors (nAChRs) in protein and gene levels in SH-SY5Y cells and the mechanism of the receptor modification.

METHODSSH-SY5Y cells, a human neuroblastoma cell line, were incubated with different concentrations of fluoride or with antioxidant for 48 hours. The functions of cells were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method, and protein oxidation detected by carbonyl content; the alpha3 and alpha7 nAChR subunits in protein level were measured by Western blotting and in mRNA level by RT-polymerase chain reaction (RT-PCR).

RESULTSIn high-dose group as compared to the control, the decreased MTT (49%), increased protein oxidation (72%), and lower expression of alpha3 (51%) and alpha7 (47%) nAChR subunit proteins were obviously observed in SH-SY5Y cells. There were no changes in expression of nAChR subunit mRNAs between the cells treated with fluoride and those un-treated in controls. Prior treatment with antioxidant resulted in preventing the decrease of nAChR protein in cells exposed to the high doses of fluoride.

CONCLUSIONFluorosis should result in damage of cells and the declined expression of nAChRs in protein levels, but no influences on gene expression of the receptors in human neuroblastoma neurons. The decreased nAChR proteins might be involved in the mechanism of oxidative stress induced by fluorosis.

Cell Line, Tumor ; Fluoride Poisoning ; metabolism ; Fluorides ; toxicity ; Humans ; Neuroblastoma ; metabolism ; pathology ; Protein Processing, Post-Translational ; drug effects ; Proteins ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Nicotinic ; biosynthesis ; genetics

OBJECTIVETo study the effect of antizyme 1 (ZA1) gene transfection on the cell proliferation, cell cycle and apoptosis of human neuroblastoma SH-SY5Y cells in vitro.

METHODSThe recombinant eukaryotic expression vector pAZ1m was constructed by cloning mutant AZ1 gene into the vector pEGFP-N1, and subsequently transfected in SH-SY5Y cells. The transfected cell proliferation was examined using MTT assay, and the changes in cell cycle and apoptosis were assayed using flow cytometry analysis. RT-PCR was performed to measure cyclin D1 and caspase-3 mRNA expressions, Western blotting carried out to examine cyclin D1 protein expression, and the changes in caspase-3 activity were detected using a caspase-3 detection kit.

RESULTSAZ1 gene transfection significantly inhibited the proliferation of SH-SY5Y cells, causing cell cycle arrest at G0/G1 stage and down-regulated cyclin D1 and up-regulated caspase-3 expressions. Obviously increased caspase-3 activity was also observed in the transfected cells.

CONCLUSIONExogenous AZ1 gene transfection can inhibit the proliferation and cause cell cycle arrest of SH-SY5Y cells by down-regulating cyclin D1 expression. Up-regulated caspase-3 expression resulting from AZ1 gene transfection may induce apoptosis of the neuroblastoma cells.

Apoptosis ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Down-Regulation ; Genetic Vectors ; Humans ; Neuroblastoma ; metabolism ; Proteins ; genetics ; Transfection ; Up-Regulation

Adult ; Axilla ; Cell Transformation, Neoplastic ; pathology ; Diagnosis, Differential ; Epithelioid Cells ; metabolism ; pathology ; Fibrosarcoma ; metabolism ; pathology ; Humans ; Male ; Neurilemmoma ; metabolism ; pathology ; surgery ; Neuroblastoma ; metabolism ; pathology ; S100 Proteins ; metabolism ; Soft Tissue Neoplasms ; metabolism ; pathology ; surgery ; Vimentin ; metabolism

Adult ; Diagnosis, Differential ; Follow-Up Studies ; Humans ; Infant ; Keratins ; metabolism ; Male ; Mandibular Neoplasms ; metabolism ; pathology ; surgery ; Melanoma ; metabolism ; pathology ; Neuroblastoma ; metabolism ; pathology ; Neuroectodermal Tumor, Melanotic ; metabolism ; pathology ; surgery ; Rhabdomyosarcoma, Embryonal ; metabolism ; pathology ; Synaptophysin ; metabolism