Amplified N-myc in human neuroblastoma cells is often arranged as clustered tandem repeats of differently recombined DNA.

Human neuroblastoma cells often carry amplified DNA encompassing the gene N-myc. Amplified N-myc has been found localized in "double minutes" in direct tumor cell preparations. In contrast, later passages carried amplified N-myc almost exclusively within a single homogeneously staining chromosomal region located at a chromosomal site different from the normal location of N-myc. We used pulsed field gel electrophoresis to define the structural arrangement of the amplified DNA. Long-range mapping was facilitated by the presence of several sites for rare cutting restriction endonucleases in the 5' region of N-myc. Amplified DNAs of different neuroblastoma cell lines were heterogeneous in size and had undergone recombination at various distances from N-myc. N-myc occupied a central position within the amplified DNA, and in no case was the coding region affected by recombination. Among neuroblastoma cells, varying proportions of amplified DNA (in some instances close to 100%) consisted of multiple tandem arrays of DNA segments ranging in size from 100 to 700 kilobase pairs. Tumor cells with low degrees of amplification revealed regions of amplified DNA in excess of 1,500 kilobase pairs without apparent rearrangement. Our observations, in concert with the cytogenetic findings, suggest a model of gene amplification which involves unscheduled DNA replication, recombination, and formation of extrachromosomal DNA followed by integration into a chromosome and subsequent in situ multiplication. The central position which N-myc occupies within the amplified sequences and the lack of recombination within the coding region of N-mc indicate that N-myc rather than other genetic information provides the selective advantage for retention of the amplified DNA.     

Isolation and structural analysis of a 1.2-megabase N-myc amplicon from a human neuroblastoma.

Oncogene amplification is observed frequently in human cancers, but little is known about the mechanism of gene amplification or the structure of amplified DNA in tumor cells. We have studied the N-myc amplified domain from a representative neuroblastoma cell line, SMS-KAN, and compared the map of the amplicon in this cell line with that seen in normal DNA. The SMS-KAN cell line DNA was cloned into yeast artificial chromosomes (YACs), and clones were identified by screening the YAC library with amplified DNA probes that were obtained previously (B. Zehnbauer, D. Small, G. M. Brodeur, R. Seeger, and B. Vogelstein, Mol. Cell. Biol. 8:522-530, 1988). In addition, YAC clones corresponding to the normal N-myc locus on chromosome 2 were obtained by screening two normal human YAC libraries with these probes, and the restriction maps of the two sets of overlapping YACs were compared. Our results suggest that the amplified domain in this cell line is a approximately 1.2-Mb circular molecule with a head-to-tail configuration, and the physical map of the normal N-myc locus generally is conserved in the amplicon. These results provide a physical map of the amplified domain of a neuroblastoma cell line that has de novo amplification of an oncogene. The head-to-tail organization, the general conservation of the normal physical map in the amplicon, and the extrachromosomal location of the amplified DNA are most consistent with the episome formation-plus-segregation mechanism of gene amplification in these tumors.     

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.     

Characterization of N-myc amplification units in human neuroblastoma cells.

A set of DNA clones comprising 48 independent HindIII fragments (215 kilobases of sequence) was derived from the N-myc amplification unit of the neuroblastoma cell line NGP. These clones were used to investigate N-myc amplification units in NGP cells and 12 primary neuroblastoma tumors. Three parameters were evaluated: (i) the number of rearrangements from germ line configuration that had occurred during the amplification process; (ii) the homogeneity of amplification units within individual tumors; and (iii) the conservation of amplified sequences among different tumors. The results indicated that remarkably few rearrangements had occurred during amplification, that the amplification units within any one tumor were quite homogeneous, and that although each tumor contained a unique pattern of amplified DNA fragments, there was considerable similarity between the amplification units of different tumors. In particular, the amplification units were strikingly similar over a contiguous domain of at least 140 kilobases surrounding the N-myc structural gene.     

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.     

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.     

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells.

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.     

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 oncogene overexpression down-regulates leukemia inhibitory factor in neuroblastoma.

Amplification of N-myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N-myc oncogene might regulate angiogenesis in neuroblastoma. Here, we report that experimental N-Myc overexpression results in down-regulation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation. Reporter assays using the LIF promoter and a series of N-Myc mutants clearly demonstrated that down-regulation of the LIF promoter was independent of Myc/Max interaction and required a contiguous N-terminal N-Myc domain. STAT3, a downstream signal transducer, was essential for LIF activity as infection with adenoviruses expressing a phosphorylation-deficient STAT3 mutant rendered endothelial cells insensitive to the antiproliferative action of LIF. LIF did not influence neuroblastoma cell proliferation suggesting that, at least in the context of neuroblastoma, LIF is involved in paracrine rather than autocrine interactions. Our data shed light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastoma.     

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.     

Characterization of DNA rearrangements of N-myc gene amplification in three neuroblastoma cell lines by pulsed-field gel electrophoresis

We characterized N-myc gene amplification in three human neuroblastoma cell lines (IMR-32, TGW, GOTO). Rearrangements in long-range regions surrounding amplified N-myc genes were examined by pulsed-field gel electrophoresis. Since rare-cutting enzymes completely digested DNA at the middle of the N-myc gene, we were able to construct a physical map upstream and downstream of the germline N-myc gene, and to obtain information on restriction sites surrounding amplified N-myc genes. This method enables us to envisage the organization of amplified units over a long range. Digestion patterns differed considerably among the germline and the three cell lines, but were simple in each case. We estimated that the minimal distance between neighboring N-myc genes is at least several hundred kilobases. Our data suggest that amplification units contain several DNA fragments derived from ditterent loci, but that they are homogeneous.     

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.     

Hypoxia promotes apoptosis of human neuroblastoma cell lines with enhanced N-myc expression

We have examined the effect of hypoxia and nutrient depletion on the growth of human neuroblastoma cells with normal or enhanced expression of the N-myc oncogene. The combination of both conditions reduced the growth of neuroblastoma cells with normal N-myc expression. However, this effect was much more pronounced in neuroblastoma cells with enhanced N-myc expression and eventually resulted in apoptosis, presumably by the up-regulation of CD95. Our data suggest that therapeutic induction of tumor hypoxia and nutrient depletion (for example, by anti-angiogenesis) could help to improve the outcome of patients with neuroblastomas carrying the prognostically unfavourable N-myc amplification.     

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.     

Amplification of the N-myc oncogene in an adenocarcinoma of the lung

c-myc oncogene is the most extensively studied member of the myc gene family, which now consists of three characterized members, namely the c-myc, N-myc, and L-myc genes. Deregulation owing to amplification and/or rearrangements of the c-myc gene have been described in a variety of human malignancies. Several neuroblastomas have amplifications of the N-myc genes. The c-myc, N-myc, or L-myc oncogenes are also found amplified in different cell lines from small cell carcinomas of the lung. In this study, we have examined the c-myc, N-myc, and c-erbB oncogenes in 34 clinical and autopsy tumor specimens representing various histopathological types of human lung cancer, including nine small cell lung cancers. A 30-fold amplification of the N-myc gene was found in a tumor histopathologically and histochemically verified as a typical adenocarcinoma. No amplifications of the c-myc or c-erbB oncogenes were seen in any of the tumors. In the DNA of one small cell carcinoma, an extra c-myc and N-myc cross-hybridizing restriction fragment was observed, possibly owing to an amplification of a yet uncharacterized myc-related gene.     

Transrepression of the N-myc expression by c-myc protein

In the human neuroblastoma cell line IMR32 the N-myc gene happens to be amplified and actively expressed, whereas no stable c-myc RNA can be detected in the same cells. In this report, we show that in IMR32 cells the expression of the N-myc gene is repressed by introduction of a c-myc expression vector (c-myc cDNA conjugated with an SR promoter). Moreover, dose response experiments showed that the amount of endogenous c-myc protein present in HeLa cells (which express c-myc but not N-myc) is enough to repress the expression of N-myc in IMR32 cells.