Receptor for advanced glycation end products (RAGE)-mediated neurite outgrowth and activation of NF-kappaB require the cytoplasmic domain of the receptor but different downstream signaling pathways.
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Receptor for advanced glycation end products (RAGE) mediates neurite outgrowth in vitro on amphoterin-coated substrates. Ligation of RAGE by two other ligands, advanced glycation end products or amyloid beta-peptide, is suggested to play a role in cell injury mechanisms involving cellular oxidant stress and activation of the transcription factor NF-kappaB. However, the RAGE signaling pathways in neurite outgrowth and cell injury are largely unknown. Here we show that transfection of RAGE to neuroblastoma cells induces extension of filopodia and neurites on amphoterin-coated substrates. Furthermore, ligation of RAGE in transfected cells enhances NF-kappaB-dependent transcription. Both the RAGE-mediated neurite outgrowth and activation of NF-kappaB are blocked by deletion of the cytoplasmic domain of RAGE. Moreover, dominant negative Rac and Cdc42 but not dominant negative Ras inhibit the extension of neurites induced by RAGE-amphoterin interaction. In contrast, the activation of NF-kappaB is inhibited by dominant negative Ras but not Rac or Cdc42. These data suggest that distinct signaling pathways are used by RAGE to induce neurite outgrowth and regulate gene expression through NF-kappaB. (+info)
Use of tumor-specific gene expression for the differential diagnosis of neuroblastoma from other pediatric small round-cell malignancies.
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The differential diagnosis of neuroblastoma from other small round-cell tumors of childhood, although clinically of great importance, is sometimes difficult due to the almost indistinguishable appearance of such tumors by conventional microscopy. Because neuroblastomas are characterized by the synthesis of catecholamines, we investigated the possibility that expression of genes involved in this pathway could serve as a molecular marker for this disease. A reverse transcriptase polymerase chain reaction assay was used to analyze expression of tyrosine hydroxylase and dopa decarboxylase in 84 pediatric malignancies including 55 neuroblastomas, 6 Ewing's sarcomas/primitive neuroectodermal tumors, 7 lymphomas, 6 leukemias, 2 rhabdomyosarcomas, 6 osteosarcomas, and 2 phaeochromocytomas. Of the 55 neuroblastoma samples analyzed, 54 expressed clearly detectable levels of both genes. The one sample that did not express either of the genes was rediagnosed both clinically and by molecular genetic analysis as a Ewing's sarcoma. Of the 29 non-neuroblastoma tumor samples examined, the only tumor samples that expressed clearly detectable levels of both tyrosine hydroxylase and dopa decarboxylase were phaeochromocytomas. Like neuroblastomas, these tumors are characterized by high levels of catecholamines. These findings suggest that expression of genes involved in catecholamine biosynthesis may be useful for differentiating neuroblastoma from other small round-cell tumors of childhood. (+info)
Hyperphosphorylated tau in SY5Y cells: similarities and dissimilarities to abnormally hyperphosphorylated tau from Alzheimer disease brain.
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Unlike normal tau, abnormally hyperphosphorylated tau (AD P-tau) from Alzheimer disease (AD) does not promote but instead inhibits microtubule assembly and disrupts already formed microtubules. Tau in the human neuroblastoma cell line SH-SY5Y is hyperphosphorylated at several of the same sites as AD P-tau, and accumulates in the cell body without any association to the cellular microtubule network. The aim of the present study was to elucidate why the SY5Y tau does not affect the viability of the cells. We found that, like AD P-tau, SY5Y tau because of hyperphosphorylation does not bind to microtubules and inhibits the tau-promoted assembly of microtubules. However, the tau/HMW MAP ratio is about 10 times less in SY5Y cells than in AD brain. These findings suggest that the hyperphosphorylated tau from SY5Y cells has similar biological characteristics as AD P-tau from AD brain, but is not lethal to the SY5Y cells because of its low tau/HMW MAP ratio. (+info)
Sphingolipid depletion increases formation of the scrapie prion protein in neuroblastoma cells infected with prions.
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Sphingolipid-rich rafts play an essential role in the posttranslational (Borchelt, D. R., Scott, M., Taraboulos, A., Stahl, N., and Prusiner, S. B. (1990) J. Cell Biol. 110, 743-752)) formation of the scrapie prion protein PrP(Sc) from its normal conformer PrP(C) (Taraboulos, A., Scott, M., Semenov, A., Avrahami, D., Laszlo, L., Prusiner, S. B., and Avraham, D. (1995) J. Cell Biol. 129, 121-132). We investigated the importance of sphingolipids in the metabolism of the PrP isoforms in scrapie-infected ScN2a cells. The ceramide synthase inhibitor fumonisin B(1) (FB(1)) reduced both sphingomyelin (SM) and ganglioside GM1 in cells by up to 50%, whereas PrP(Sc) increased by 3-4-fold. Whereas FB(1) profoundly altered the cell lipid composition, the raft residents PrP(C), PrP(Sc), caveolin 1, and GM1 remained insoluble in Triton X-100. Metabolic radiolabeling demonstrated that PrP(C) production was either unchanged or slightly reduced in FB(1)-treated cells, whereas PrP(Sc) formation was augmented by 3-4-fold. To identify the sphingolipid species the decrease of which correlates with increased PrP(Sc), we used two other reagents. When cells were incubated with sphingomyelinase for 3 days, SM levels decreased, GM1 was unaltered, and PrP(Sc) increased by 3-4-fold. In contrast, the glycosphingolipid inhibitor PDMP reduced PrP(Sc) while increasing SM. Thus, PrP(Sc) seems to correlate inversely with SM levels. The effects of SM depletion contrasted with those previously obtained with the cholesterol inhibitor lovastatin, which reduced PrP(Sc) and removed it from detergent-insoluble complexes. (+info)
Glycosphingolipid-enriched signaling domain in mouse neuroblastoma Neuro2a cells. Mechanism of ganglioside-dependent neuritogenesis.
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Differentiation and neuritogenesis of mouse neuroblastoma Neuro2a cells are induced by exogenous ganglioside but are not induced by nerve growth factor because its receptor is absent in these cells. In view of the emerging concept of the "glycosphingolipid-enriched domain" (GEM), we studied the mechanism of the ganglioside effect, focusing on the structure and function of such a domain. GEM in Neuro2a cells, separated as a low density membrane fraction, contains essentially all glycosphingolipids and sphingomyelin, together with five signal transducer molecules (c-Src, Lyn, Csk, Rho A, Ha-Ras). (3)H-Labeled Il(3)NeuAc-LacCer (GM3), Gb4Cer (globoside), and Il(3)NeuAc-Gg4Cer (GM1) added exogenously to cells were incorporated and concentrated in the low density GEM fraction. In contrast, more than 50% of glycerophospholipids and 30% of cholesterol were found in the high density fraction. (3)H-Labeled phosphatidylcholine added exogenously to cells was incorporated exclusively in the high density fraction. c-Src, the predominant signal transducer in the microdomain, was coimmunoprecipitated with anti-GM3 antibody DH2 or with anti-Csk; reciprocally, Csk was coimmunoprecipitated with anti-c-Src, indicating a close association of GM3, c-Src, and Csk. Brief stimulation of an isolated GEM fraction by the exogenous addition of GM3, but not lactosylceramide, caused enhanced c-Src phosphorylation with a concomitant decrease of Csk level in GEM. A decreased Csk/c-Src ratio in GEM may cause activation of c-Src because Csk is a negative regulator of c-Src. The effect of exogenous GM3 on c-Src activity was also observed in intact Neuro2a cells. Activation of c-Src was followed by rapid and prolonged (60 min) enhancement of mitogen-activated protein kinase activity leading to neuritogenesis. Thus, the ganglioside induction of neuritogenesis in Neuro2a cells is mediated by GEM structure and function. (+info)
Tumor-targeted IL-2 amplifies T cell-mediated immune response induced by gene therapy with single-chain IL-12.
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Induction, maintenance, and amplification of tumor-protective immunity after cytokine gene therapy is essential for the clinical success of immunotherapeutic approaches. We investigated whether this could be achieved by single-chain IL-12 (scIL-12) gene therapy followed by tumor-targeted IL-2 using a fusion protein containing a tumor-specific recombinant anti-ganglioside GD(2) antibody and IL-2 (ch14.18-IL-2) in a poorly immunogenic murine neuroblastoma model. Herein, we demonstrate the absence of liver and bone marrow metastases after a lethal challenge with NXS2 wild-type cells only in mice (five of six animals) vaccinated with scIL-12-producing NXS2 cells and given a booster injection of low-dose ch14.18-IL-2 fusion protein. This tumor-protective immunity was effective 3 months after initial vaccination, in contrast to control animals treated with a nonspecific fusion protein or an equivalent mixture of antibody and IL-2. Only vaccinated mice receiving the tumor-specific ch14.18-IL-2 fusion protein revealed a reactivation of CD8(+) T cells and subsequent MHC class I-restricted tumor target cell lysis in vitro. The sequential increase in the usage of TCR chains Vbeta11 and -13 in mouse CD8(+) T cells after vaccination and amplification with ch14.18-IL-2 suggests that the initial polyclonal CD8(+) T cell response is effectively boosted by targeted IL-2. In conclusion, we demonstrate that a successful boost of a partially protective memory T cell immune response that is induced by scIL-12 gene therapy could be generated by tumor-specific targeting of IL-2 with a ch14.18-IL-2 fusion protein. This approach could increase success rates of clinical cancer vaccine trials. (+info)
Metalloproteinase-mediated release of the ectodomain of L1 adhesion molecule.
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The L1 adhesion molecule is an approx. 200-220 kDa type I membrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. L1 can bind in a homotypic fashion and was shown to support integrin-mediated binding via RGDs in the 6th Ig-like domain. In addition to its cell-surface expression, L1 can occur in the extracellular matrix (ECM). Here we demonstrate that L1 is constitutively released from the cell surface by membrane-proximal cleavage. L1 shed from B16F10 melanoma cells remains intact and can serve as substrate for integrin-mediated cell adhesion and migration. The release of L1 occurs in mouse and human cells and is blocked by the metalloproteinase inhibitor TAPI (Immunex compound 3). This compound has been shown previously to block release of L-selectin and TNF-alpha which is mediated by the membrane-bound metalloproteinase TNF-alpha converting enzyme (TACE). Using CHO cells that are low in TACE expression and do not release L-selectin we demonstrate that L1 release is distinct from L-selectin shedding. We propose that cell-surface release may be necessary for the conversion of L1 from a membrane into an ECM protein. (+info)
Expression of B-myb in neuroblastoma tumors is a poor prognostic factor independent from MYCN amplification.
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The transcription factors of the Myb family are expressed in several tissues and play an important role in cell proliferation, differentiation, and survival In this study, the expression of A-myb, B-myb, and c-myb was investigated in a group of 64 neuroblastomas at different dinical stages by a sensitive reverse transcription-PCR tchnique and correlated with patients' survival. All of the myb genes were frequently expressed in neuroblastoma tumors. Interestingly, the expression of B-myb, which was detected in 33 cases, was associated with an increased risk of death (P = 0.027 in a univariate analysis), whereas there was no correlation with A-myb and c-myb expression. In addition, in a multivariate Cox regression analysis that included myb gene expression, MYCN status, age at diagnosis, and tumor staging, MYCN amplification and B-myb expression were independently associated to an increased risk (P < 0.01 and P = 0.015, respectively). In overall survival curves obtained by stratifying the neuroblastoma cases on the basis of MYCN status and B-myb expression, the group of patients without MYCN amplification and positive for B-myb expression had worse survival probability than that without MYCN amplification and nonexpressing B-myb (P < 0.01). In summary, these findings provide the first demonstration that B-myb expression can be a useful prognostic marker in human neuroblastoma. Moreover, B-myb expression has a prognostic value complementary to MYCN amplification and can identify a group of high-risk patients that would not be predicted on the basis of the MYCN status only. (+info)