Child ; Epigenesis, Genetic ; Humans ; Infant ; Neuroblastoma/genetics*

We used retroviral-mediated gene transfer of the human interleukin (IL)-2 gene into murine neuroblastoma cells to investigate whether locally-secreted IL-2 is able to influence the generation of anti-tumor immune responses. Supernatant obtained from cultures of approximately 1 x 10(6) IL-2 gene-transduced, G-418 selected neuro-2a cells was assayed for human IL-2 production by ELISA kit. First, to estimate whether the local secretion of IL-2 from the genetically-modified tumor cells would affect their tumorigenicity in vivo, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice and tumor growth was measured weekly. And to estimate whether IL-2 transfected neuroblastoma cells protect mice from tumor development after wild-type tumor cell challenge, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice. Seven days after IL-2 gene-transfected neuroblastoma cell injection, unmodified neuro-2a cells were s.c. injected into the contralateral site of A/J mice and tumor growth was measured weekly. Finally, to estimate IL-2 effect on pre-established large tumor burdens, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice with established tumor and its growth was measured weekly. The IL-2 gene-transduced neuro-2a clones secreted 120.25-177.3 IU of IL-2 per ml per 10(6) cells during 24 hr. None of the mice injected with IL-2-secreting neuro-2a cells developed tumors within 6 weeks, while all of the mice injected with wild-type neuro-2a cells developed tumors. Immunization of mice with IL-2 gene-transfected, irradiated neuro-2a cells protected these animals against a subsequent challenge with wild-type tumor cells. Finally, the size of large neuroblastomas decreased after IL-2-secreting neuro-2a cell injection into mice. Local secretion of IL-2 gene-transduced tumor cells abrogates their tumorigenicity and induces protective immunity and may inhibit the growth of neuroblastoma.
Animal ; Antibody Formation ; Gene Transfer Techniques* ; Human ; Immunization/methods ; Interleukin-2/therapeutic use ; Interleukin-2/genetics* ; Mice ; Neoplasm Transplantation ; Neuroblastoma/therapy ; Neuroblastoma/prevention & control ; Neuroblastoma/pathology ; Neuroblastoma/genetics* ; Retroviridae/genetics* ; Tumor Cells, Cultured

Arrhythmias, Cardiac ; Genetic Diseases, X-Linked/genetics* ; Gigantism/genetics* ; Heart Defects, Congenital/genetics* ; Humans ; Intellectual Disability/genetics* ; Male ; Neuroblastoma

OBJECTIVETo investigate mutations of anaplastic lymphoma kinase (ALK) in Chinese children with neuroblastoma (NB).

METHODSGenomic DNA was extracted from 22 cases of paraffin-embedding NB tumor tissues. Gene mutations in the exons 20-26 which were mutational hotspots of ALK were analyzed by PCR-DNA direct sequencing.

RESULTSA novel synonymous mutation C3586T (Leu1196Leu) and a known synonymous mutation C3375A (Gly1125Gly) were found and located at exon 23 and exon 21 of ALK respectively. There were 10 cases (46%) of known synonymous mutation C3375A in 22 cases of NB. The C3375A allelic frequency was 27%. No statistically significant correlation was found between mutation C3375A and clinical parameters of NB such as age, sex, metastasis and tumor differentiation. Mutation was not found in the other 5 exons.

CONCLUSIONSA novel ALK gene synonymous mutation C3586T was identified using PCR-DNA sequencing. A known mutation C3375A in ALK was successfully identified in children, and its incidence is not influenced by the clinical features of childhood NB.

Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation ; Neuroblastoma ; genetics ; Polymerase Chain Reaction ; Receptor Protein-Tyrosine Kinases ; genetics

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

This study was purposed to analyze the relation of N-myc gene copy number with clinical staging, pathological types and tumor biological factors in children with neuroblastoma (NB), and to investigate the influence of chemotherapy on N-myc gene expression and explore the relationship of N-myc gene copies with prognosis of NB children. The newly diagnosed children with NB from 1 March 2007 to 31 January 2011 were enrolled in this study. The treatment was carried out by BCH-NB-2007 based on Hongkong NB-07 protocol, and the patients were follow up to 31 January 2012. The N-myc gene in NB children was detected by FISH. According to number of N-myc gene copies, the NB children were divided into 3 groups. A group (N-myc gene negative) had less than 2 copies, B group (N-myc gene gains) had 3 to 9 copies, and C group (N-myc amplification) had more than 10 copies. The results showed that the N-myc gene expression in 58 cases of NB was observed. There were 36 males and 22 females. NB children aged from 6.5 to 138 months (median age 47.5 months), all patients were followed up for 11 - 57 months with an average of 31.5 months. INSS stages I-IV were 1, 5, 8 and 44 cases, respectively. Twenty-five cases had primary post mediastinal tumor, thirty-three cases had retroperitoneal and pelvic tumor, three of which also companied with post mediastinal tumor. Thirty-five cases had bone metastasis (60.3%), thirty-two cases had bone marrow metastasis (55%). Of the 54 patients with fully known biologic features, seventeen cases had ganglioneuroblastoma, thirty-seven cases had neuroblastoma (15 displayed differentiated, 7 poorly differentiated or undifferentiated, 15 with pathological changes after chemotherapy), four cases had bone marrow metastasis only detected by bone marrow biopsy. Eleven cases had N-myc gene negative, forty-three had N-myc gains, four had N-myc amplification. The average copy number of N-myc gene copies in 58 cases was 5.96 ± 7.81 in which 28 children were non chemotherapy cases, their average copy number was 4.00 ± 1.88, thirty cases out of 58 cases received preoperation chemotherapy (chemotherapy group), and their average copy number was 7.80 ± 10.46, the difference is significant (P = 0.064). The clinic stage, the location of primary tumor, pathological classification, urine VMA and serum neurogenic specific enolase had no effects on the N-myc gene expression, but the serum LDH level had influence (P < 0.01). Single factor Kaplan-Meier analysis showed that the number of N-myc gene copies in NB patients were closely related with the poor prognosis. The more copies of N-myc gene, the more poor prognosis, the difference is statistically significant (P < 0.05). It is concluded that the number of N-myc gene copies correlates with the rapid growth of NB and its poor prognosis, detecting the N-myc amplification can help to estimate the prognosis and decide the program of treatment. Serum LDH, which correlated with the rapid growth of NB, had effect on the N-myc gene expression and is closely related with the poor prognosis of NB.
Child ; Child, Preschool ; Female ; Gene Amplification ; Genes, myc ; Humans ; Infant ; Male ; Neoplasm Staging ; Neuroblastoma ; diagnosis ; genetics ; pathology ; Prognosis

To establish vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) as secretary biomarkers for cell growth on topographic substrates, we have evaluated the secretion and expression of these 2 factors by SH-SY5Y human neuroblastoma cells on poly-L-lactide (PLLA) micropillar arrayed topographic substrates. We fabricated topographic substrates with UV lithography, silicon etching and polydimethylsiloxane-based replica molding, and interfaced SH-SY5Y human neuroblastoma cells with both the topographic substrates and PLLA flat substrates. Cell morphology and spreading were examined with scanning electron microscopy. The secretion and mRNA expression of VEGF and IL-8 were evaluated with enzyme linked immunosorbent assay (ELISA) and real time qPCR, respectively, 24 hours after cell plating. We successfully achieved 4 topographic substrates with a nominal pillar diameter of 2 microm and 4 microm, and a nominal pillar spacing of 2 microm and 7 microm. We found that the secretion and mRNA expression of VEGF and/or IL-8 by SH-SY5Y cells on 2-2 microm (pillar diameter-spacing), 4-2 microm and 4-7 microm topographic substrates were upregulated in comparison to those by cells on PLLA flat substrate, 24 hours after cell plating. Furthermore, both cytokines were even more substantially upregulated on the 2-7 microm substrate than on the other 3 topographic substrates. Compared to those on PLLA flat substrate, cells on topographic substrates showed significant changes in morphology (spreading area, perimeter and roundness), and the increase in the secretion and mRNA expression of VEGF and IL-8 was accompanied with a decrease in cell spreading areas. These results provided evidence that pillar arrayed topography was an important microenvironmental factor in affecting VEGF and IL-8 expression or secretion, and VEGF and IL-8 might serve as important secretary biomarkers for growth on topographic substrates by SH-SY5Y cells.
Biomarkers ; Cell Line ; Cell Proliferation ; Cellular Microenvironment ; Humans ; Interleukin-8 ; genetics ; secretion ; Neuroblastoma ; secretion ; Polyesters ; chemistry ; RNA, Messenger ; genetics ; Vascular Endothelial Growth Factor A ; genetics ; secretion

OBJECTIVE: To explore the effects of nuclear protein-like transcription factor nuclear receptor subfamily 2 group E member 1 (NR2E1) on the growth, division, and proliferation of neuroblastoma cell line IMR32. METHODS: A NR2E1 shiRNA plasmid vector was constructed and transfected into neuroblastoma cell line IMR32 using lipofedamine™2000. Subsequent cell growth was measured by cell counting and the protein expression of somatic nuclear division was examined by immunofluorescent staining. RESULTS: At 48 h after the neuroblastoma cells IMR32 were transfected with NR2E1-shiRNA vector, the related nuclear division protein and the proliferation of the transfected cells IMR32 were remarkably depressed. CONCLUSION Cells division and proliferation of neuroblastoma cell line IMR32 is inhibited through transfection with the NR2E1-shiRNA plasmid vector.
Cell Division ; genetics ; physiology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Neuroblastoma ; pathology ; RNA, Small Interfering ; genetics ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Transfection

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 investigate whether short interfering RNAs(siRNAs) of beta-site APP cleaving enzyme (BACE) can inhibit the expression of BACE in mammalian cells.

METHODSThe gene of EGFP, U6 promoter and beta-secretase targeting siRNA were cloned by PCR, respectively. The PCR products were inserted into plasmid pLXSN. The interfering vector pLXSN/EGFP-U6-siBACE was transferred into SK-N-SH cells to express BACE. The inhibition effect of BACE siRNA on BACE expression was investigated by fluoroscopy and immunohistochemistry method.

RESULTThe interfering vector pLXSN/EGFP-U6-siBACE was constructed successfully. The BACE siRNA inhibited the expression of BACE in the SK-N-SH cells specifically and effectively, and the production of A beta was reduced.

CONCLUSIONBACE siRNA can inhibit the expression of BACE gene of mammalian cells.

Amyloid Precursor Protein Secretases ; genetics ; metabolism ; Animals ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Mice ; Microscopy, Fluorescence ; NIH 3T3 Cells ; Neuroblastoma ; genetics ; metabolism ; pathology ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Tumor Cells, Cultured