N-myc expression switched off and class I human leukocyte antigen expression switched on after somatic cell fusion of neuroblastoma cells.

Neuroblastomas often show amplification and high expression of the N-myc oncogene. N-myc expression could be explained as a consequence of gene amplification, but an alternative possibility is that expression primarily results from the inactivation or loss of some factor that normally represses the N-myc gene. To test this idea, we fused N-myc-overexpressing neuroblastoma cell lines with lines that do not express N-myc. In the resulting hybrids, N-myc expression turned out to be switched off, although amplified N-myc copies were still present. This suggests that N-myc overexpression in neuroblastomas results, at least in part, from the inactivation of a suppressor gene that is present in normal cells. In rat neuroblastomas, it has been found that N-myc can switch off class I major histocompatibility complex (MHC) expression. Therefore, we analyzed in our hybrid cells whether suppression of N-myc results in reexpression of human class I MHC genes. Because this was found to be the case, the picture emerges of a hierarchic pathway that connects a putative tumor-suppressor gene with the expression of N-myc and consequently of class I MHC, thus affecting the potential immunogenic properties of neuroblastomas.     

The N-Myc oncoprotein is associated in vivo with the phosphoprotein Max(p20/22) in human neuroblastoma cells.

Proteins encoded by the proto-oncogenes c-myc, L-myc, and N-myc contain at their carboxy-terminus a tripartite segment comprising a basic DNA binding region (BR), a helix-loop-helix (HLH) and a leucine zipper motif (Zip), that are believed to be involved in DNA binding and protein-protein interaction. The N-Myc oncoprotein is overexpressed in certain human tumors that share neuroectodermal features due to amplification of the N-myc gene. Using a monoclonal antibody directed against an N-terminal epitope of the N-Myc protein in immunoprecipitations performed with extracts of neuroblastoma cells, two nuclear phosphoprotein, p20/22, forming a hetero-oligomeric complex with N-Myc are identified. Both proteins are phosphorylated by casein kinase II in vitro. By partial proteolytic maps we show that p20 and p22 are structurally related to each other and that p20 is identical with Max, a recently described in vitro binding partner of myc proteins. Time course experiments show the presence of the complex in cellular extracts immunoprecipitated within a 5 min interval after the preparation of the cell extract. While the expression of N-myc is restricted, expression of both Max(p20/22) and the murine homolog Myn(p20/22) was observed in cells of diverse human and murine embryonal lineages as detected by heterologous complex formation. By introduction of expression vectors containing the wild type N-myc gene or N-myc genes with in frame deletions or point mutations into recipient cells and subsequent immunoprecipitation of the resulting N-Myc proteins we show that the HLH-Zip region is essential to the formation of the N-Myc-p20/22 complex.     

N-myc down regulates neural cell adhesion molecule expression in rat neuroblastoma.

In human neuroblastoma, amplification of the N-myc oncogene is correlated with increased metastatic ability. We recently showed that transfection of the rat neuroblastoma cell line B104 with an N-myc expression vector resulted in an increase in metastatic ability and a significant reduction in the expression of major histocompatibility complex class I antigens. We examined whether N-myc causes additional phenotypic changes in these cells. We showed that expression of N-myc leads to a dramatic reduction in the levels of neural cell adhesion molecule (NCAM) polypeptides and mRNAs. Spontaneous revertants of the high N-myc phenotype were found to have regained significant levels of NCAM expression, indicating that the continued expression of N-myc is required to maintain the low NCAM phenotype. NCAM was not reduced in B104 cells transfected with the neomycin resistance vector alone, and other neuronal markers were not specifically reduced in N-myc-transfected B104 cells. As NCAM functions in cell-cell adhesion, decreased NCAM expression could contribute significantly to the increased metastatic potential of N-myc-amplified neuroblastomas.     

N-myc amplification causes down-modulation of MHC class I antigen expression in neuroblastoma

Amplification of the N-myc gene is correlated with increased metastatic ability of human neuroblastomas. We show here that overexpression of the N-myc gene in a rat neuroblastoma cell line following gene transfer causes down-modulation of class I histocompatibility antigen expression and increases in the in vivo growth rate and metastatic ability of these cells. N-myc-mediated down-modulation of MHC class I antigen expression could be reversed by treatment with interferon without affecting the steady state level of N-myc mRNA. No effect on MHC class I antigen expression was found when the N-myc gene was expressed in rat fibroblasts, indicating that some of the effects caused by N-myc gene amplification are cell-type-specific.     

MYCN silencing induces differentiation and apoptosis in human neuroblastoma cells

MYCN amplification strongly correlates with unfavorable outcomes in patients with neuroblastoma. The aim of this study was to investigate the role of MYCN in neuroblastoma cell differentiation and apoptosis. We used the technique of RNA interference to inhibit MYCN gene expression in neuroblastoma cells with variable expression of MYCN. Our results showed that inhibition of MYCN gene expression in MYCN amplified cells induced apoptosis and suppressed cell growth; neuronal differentiation also occurred after MYCN gene silencing. Moreover, N-myc downregulation was associated with decreased Bcl-xL protein levels and caspase-3 activation. These data show that small interfering RNA directed to MYCN, which plays a crucial role in neuroblastoma cell survival, may provide a potential novel therapeutic option for aggressive neuroblastomas.     

N-myc down-regulates activin A

N-myc oncogene amplification is frequent in human neuroblastoma and predicts poor prognosis, but the molecular consequences have remained obscure. We report here that enhanced N-myc expression correlates with low or undetectable expression of activin A, but not other closely related members of the transforming growth factor-beta superfamily. N-myc interacts with the activin A promoter, eventually inducing down-regulation of activin A mRNA and protein. This study demonstrates for the first time N-myc-induced down-regulation of a gene implicated in signal transduction. Down-regulation of activin A could deprive neuroblastomas from a signal with growth-inhibitory activities toward the tumor and its stroma and thereby permit neuroblastoma progression.     

VIP receptor 1 (VPAC1) promoter targets the expression of a reporter gene to cerebellum and adrenal medulla in transgenic mice

Vasoactive intestinal peptide (VIP) is a neurotransmitter with neurotropic effects. VIP functions through two distinct G-protein-coupled receptor subtypes (VPAC1 and VPAC2). We have demonstrated expression of VPAC1 in pediatric nervous system tumors, including medulloblastoma arising in the cerebellum and neuroblastoma arising in the adrenal medulla. More recently, we have reported the differentiation of neuroblastoma cells by upregulation of VIP type 1 receptor suggesting a role for VPAC1 in neuronal development.To understand the molecular mechanisms regulating VPAC1 expression in both cerebellum and adrenal medulla, we have cloned the human VPAC1 gene and sequenced 2.6-kb of the 5'-flanking sequence. Expression of the luciferase reporter gene under the control of this 2.6-kb human VPAC1 promoter was induced 35-fold in a human medulloblastoma cell line (DAOY) and 36-fold in a human neuroblastoma cell line (SKNSH). Analysis of 5'-unidirectional deletion derivatives of the 2.6-kb fragment demonstrated that a 241-bp sequence immediately upstream of the VPAC1 coding region retains high activity, suggesting that it contains the core promoter region. Quantitative RT-PCR analysis demonstrated that VPAC1 is expressed in mouse cerebellar and adrenal tissues. The VPAC1 promoter also directed expression of a reporter gene in cerebellum and adrenal medulla in transgenic mice. Along with our previous findings, these results suggest that VPAC1 may play a functional role in development of both cerebellum and adrenal medulla.     

Combined RAR alpha- and RXR-specific ligands overcome N-myc-associated retinoid resistance in neuroblastoma cells

Retinoids induce human neuroblastoma cells to undergo growth inhibition and neuritic differentiation in vitro, through interactions with nuclear retinoid receptor proteins. In this study, we found that three different neuroblastoma cell lines exhibited wide variation in their responsiveness to the growth inhibitory effects of the retinoic acid receptor (RAR) agonist, all-trans-retinoic acid (aRA). Resistance to the growth inhibitory effect of aRA correlated with the presence of N-myc gene amplification and not aRA-induced RAR beta levels. Over-expression of N-myc in a neuroblastoma cell line with no endogenous N-myc expression caused a marked reduction in retinoid-induced growth inhibition. Combination of receptor-specific retinoid agonists for RXR and RAR alpha significantly enhanced the sensitivity of N-myc-amplified neuroblastoma cells to the growth inhibitory effects of aRA. Our results indicate that combination receptor-specific retinoid therapy can overcome N-myc-mediated retinoid resistance and may be a more effective chemo-preventive strategy in the disease.     

Human neuroblastoma tumor cell lines correspond to the arrested differentiation of chromaffin adrenal medullary neuroblasts

We have examined the hypothesis that nonhematopoietic malignancies may contain cells corresponding to those which occur during the differentiation of tissue precursors. Neuroblastoma, an embryonal tumor of the adrenal medulla, was studied because of its well described ability to differentiate both in vivo and in vitro. We examined the expression of four genes during development of the human adrenal medulla: tyrosine hydroxylase, chromagranin A, pG2, and beta-2-microglobulin. The sequential expression of these genes by adrenal neuroblasts marks successive stages during maturation of the chromaffin lineage. We also observed a population of neuroblasts during adrenal medullary development that did not express any of these four genes, suggestive of adrenal medullary cells differentiating along nonchromaffin lineage(s). We then evaluated 27 neuroblastoma cell lines for the expression of these genes and found that 24 expressed chromaffin markers, with 19 of these mimicking the pattern of gene expression found during development. Three cell lines did not express tyrosine hydroxylase, chromogranin A, or pG2, consistent with either a very undifferentiated neural crest cell or maturation along a nonchromaffin lineage. These data indicate that neuroblastoma tumor cells correspond to adrenal neuroblasts arrested during morphogenesis of the adrenal medulla and raise the possibility that malignant transformation of cells at different stages of tissue maturation may contribute to the diversity that characterizes tumors of solid tissues.     

Plasma membrane redox activity correlates with N-myc expression in neuroblastoma cells.

In different neuroblastoma cell lines and transfected clones, an increasing plasma membrane redox activity correlates with amplification and enhanced expression of the N-myc oncogene. Furthermore, plasma membrane redox activity is partially inhibited by retinoic acid in neuroblastoma cells with multiple copies of the N-myc oncogene but not in neuroblastoma cells with only one copy of this gene.     

Regulation of Id1 and its association with basic helix-loop-helix proteins during nerve growth factor-induced differentiation of PC12 cells.

Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of helix-loop-helix (HLH) proteins during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor. Gel mobility shift assays using PC12 cell nuclear extracts demonstrated that active basic HLH complexes exist throughout differentiation. Addition of exogeneous Id1 protein, a negative regulator of basic HLH proteins, disrupted specific complexes formed by PC12 cell nuclear extracts on a CANNTG consensus oligonucleotide. To identify possible novel basic HLH proteins in these complexes, a glutathione S-transferase-Id1 fusion protein was used to screen a PC12 cell cDNA expression library. A single clone representing the rat E2-2 gene was identified. Sequential immunoprecipitations with antibodies to each HLH protein revealed an association between Id1 and E2-2 that could be detected in both untreated and nerve growth factor-treated PC12 cell lysates. These experiments define a new HLH interaction between Id1 and E2-2 in neuronal cells and suggest that neuronal differentiation may be regulated by HLH proteins in a distinctive manner.     

Characterization of N-myc amplification in a human neuroblastoma cell line by clones isolated following the phenol emulsion reassociation technique and by hexagonal field gel electrophoresis.

The N-myc amplification of human neuroblastomas was characterized by the amplified DNA cloned from the cell line MC-NB-1 using the phenol emulsion reassociation technique (PERT). A number of PERT clones exhibiting amplification in this cell line were tested for amplification in other neuroblastoma cell lines. In almost all cell lines examined, only a few clones were co-amplified with N-myc and most of the others were exclusively amplified in a subset of the cell lines. The total aggregate size of the Hind III fragment identified by the PERT clones was approximately 350 kb. Most of the PERT clones were mapped to human chromosome (chr) 2p23-2pter, where the N-myc gene is located. Four types of amplicons, the 100, 420, 480 and 520 kb fragments, shown to be Not I fragments, were identified by hexagonal field gel electrophoresis. Three fragments are ordered in a head-to-tail array, and the remaining fragment is either ordered in a tail-to-head array or something else. Despite the extremely unusual construction of the amplified sequences in this cell line as compared with others, there was a low degree of sequence heterogeneity among the amplicons within this cell line. These observations lead to the idea that the complex rearrangements that give rise to the heterogeneous organization of the amplified sequences among the different cell lines precede the amplification of these sequences.     

Retinoic acid treatment of human neuroblastoma cells is associated with decreased N-myc expression

Cells from human neuroectodermal tumors (retinoblastoma and neuroblastoma) and from neuroblastoma cell lines express a gene, N-myc, which is frequently amplified in these tumors. We report here that N-myc mRNA content is markedly decreased in cells of a neuroblastoma cell line (LA-N-5) following differentiation induced with retinoic acid. Exposure of the cells to retinoic acid induced morphologic changes consistent with neuronal differentiation, and led to a 75% decrease in expression of N-myc mRNA. These results suggest that N-myc expression is intimately related to an undifferentiated phenotype in neuroblastoma cells, and support other studies which relate N-myc expression to the malignant phenotype in neuroblastoma tumors.