In vitro modulation and relationship between N-myc and HLA class I RNA steady-state levels in human neuroblastoma cells.

Neuroblastoma cell lines and tumors are characterized by low HLA class I expression. The majority of neuroblastoma cell lines and a high percentage of disseminated tumors display amplification of the nuclear protooncogene N-myc. An inverse correlation between HLA class I expression and N-myc amplification and overexpression has been recently described in neuroblastomas (NBs). In this study we have shown that cytokines (recombinant gamma-interferon, recombinant alpha-tumor necrosis factor), differentiation agents (dibutyryl cyclic AMP, phorbol myristate acetate) and growth factors (nerve growth factor, epithelial growth factor) were able to influence the growth rate and surface expression of HLA class I molecules as well as of a tumor-associated antigen on 2 representative NB cell lines. Induced decreased growth rate in NB cells was not always related to decreased N-myc expression. Analysis at the mRNA level revealed that both N-myc and HLA class I RNA steady-state levels could be modulated by several substances, including recombinant gamma-interferon, phorbol myristate acetate, dibutyryl cyclic AMP, and epithelial growth factor and were not necessarily linked. An inverse correlation between N-myc and HLA mRNA levels was observed only after exposure of NB cells to recombinant alpha-tumor necrosis factor. We conclude that N-myc and HLA class I RNA steady-state levels can be modulated independently and suggest that they are not necessarily inversely regulated.     

Increased N-myc expression following progressive growth of human neuroblastoma

Many human neuroblastoma tumors and cells in culture contain amplified N-myc DNA and this is associated with tumor stage. We have analyzed pairs of neuroblastoma cell cultures derived from two patients at the time of diagnosis and after tumor progression following the initiation of therapy. Cell cultures derived after progression have increased expression of N-myc RNA. In one pair this increase is associated with increased N-myc DNA amplification; in the other, amplification decreases and activation of N-myc is most likely the result of a regulatory change. Analysis of the pattern of DNA amplification in these cell cultures demonstrates additional changes that might be associated with tumor progression.     

Neuroblastoma with DNA amplification and rearrangement in the N-myc gene region

We detected a rearrangement in the N-myc gene region in a neuroblastoma from a 9-month-old girl. In this case, the N-myc gene was amplified 50-fold in the primary tumor, the lymph node metastasis, and the hepatic metastasis. The rearrangement was detected only in the primary tumor and the lymph node metastasis, whereas N-myc RNA and protein expression were detected only in the primary tumor and the hepatic metastasis. Both the rearranged N-myc gene and the normal N-myc gene were amplified 25-fold, and the rearrangement occurred 723 nucleotides downstream from the 3' end of exon 3. The cell line (NH-6) derived from the primary tumor showed N-myc gene amplification without this rearrangement. These results suggest the following: (a) the primary tumor had a least two clones; (b) the rearrangement interrupted N-myc gene expression; and (c) oncogenesis preceded N-myc gene amplification.     

Autoregulation of the N-myc gene is operative in neuroblastoma and involves histone deacetylase 2

BACKGROUND: Autoregulation of the myc gene family is a negative feedback mechanism known to occur at high levels of Myc expression. Loss of this mechanism and associated Myc overexpression has been observed in human tumors, thereby contributing to tumorigenesis. The childhood tumor neuroblastoma is characterized by N-myc amplification in aggressive and highly proliferative tumors that occur in a subset of patients. The precise molecular mechanism of autoregulation is unknown, and previous observations indicated that N-myc autoregulation was intact only in single-copy neuroblastoma cell lines. METHODS: Transient reporter assays and trichostatin A (TSA) experiments were performed to evaluate several candidate genes, including Mxi1, c-myc promoter binding protein 1 (MBP-1), Miz, and histone deacetylase 2 (HDAC2), for their involvement in N-myc autoregulation. Mxi1 and HDAC2 were examined further for their expression levels and effects on endogenous N-myc levels. Finally, their recruitments to the N-myc promoter were investigated by chromatin immunoprecipitation (ChIP). RESULTS: The autoregulatory circuit was operative, even in amplified cell lines. Mxi1 consistently showed a modest effect in down-regulating N-myc in transient reporter assays. Overexpression of the c-myc, Mxi1, and mHDAC2 genes resulted in a threefold to fourfold decrease in endogenous N-myc levels. Mxi1 and HDAC2 were up-regulated by N-Myc in an myc-inducible cell line and in N-myc-expressing cell lines. In addition, down-regulation of the N-myc promoter was relieved in the presence of TSA. Increased association of HDAC2 with the autoregulatory region within the N-myc promoter by ChIP was observed upon down-regulation of endogenous N-myc. CONCLUSIONS: The autoregulatory circuit was intact in both amplified and single-copy neuroblastoma cell lines. Furthermore, myc gene autoregulation occurred through histone deacetylation.     

Amplification and expression of different myc-family genes in a tumor specimen and 3 cell lines derived from one small-cell lung cancer patient during longitudinal follow-up

In a small-cell lung carcinoma (SCLC) tumor specimen as well as in 3 cell lines derived from SCLC biopsies obtained from the same patient at successive times during the clinical course, either the N-myc gene or the c-myc gene appeared to be amplified and expressed. The initial tumor specimen, a lymph-node metastasis, was amplified for N-myc, as was the cell line GLC-14 derived from this metastasis. The cell lines GLC-16 and GLC-19, derived from the recurrent primary tumor biopsies after a complete remission, were amplified for c-myc. This finding implies independent amplification events and supports the idea that the amplification of myc genes is probably a secondary event correlated with tumor progression. Although all 3 cell lines could be classified as classic SCLC cell lines according to their histological characteristics, GLC-16 and GLC-19 clearly possess, in their c-myc amplification and derivation from therapy-resistant tumor cells, features of variant SCLC lines. This may question the significance of the classic/variant classification.     

Coamplification of the L-myc and N-myc oncogenes in a neuroblastoma cell line

The L-myc, N-myc and c-myc genes are members of the myc oncogene family. In particular, L-myc is novel, and amplification of L-myc is still unknown except in small cell lung carcinoma. We examined L-myc amplification in 30 human neuroblastomas using Southern blot hybridization, and found that the L-myc gene was amplified approximately 5-fold in GOTO, a human neuroblastoma cell line. The N-myc gene was also amplified approximately 60-fold and furthermore, over-expression of L-myc and N-myc genes was observed in this cell line. In this report, we describe the coamplification of the myc gene family in the GOTO neuroblastoma cell line.     

Sequence of a germ-line N-myc gene and amplification as a mechanism of activation

Amplification of the N-myc gene has been reported in a number of tumour types including neuroblastoma, retinoblastoma and small cell lung carcinoma. Two amplified human N-myc genes have been sequenced. To determine whether any sequence changes accompany gene amplification and hence to define more precisely the mechanism of activation of the N-myc gene, we have sequenced a germ-line N-myc gene. This sequence differs from the amplified gene sequences by only a single base substitution, which may represent a polymorphic site. This comparison indicates that neither changes in gene structure nor nucleotide sequence accompany gene amplification. Analyses of four small cell lung carcinoma cell lines with amplification of the N-myc gene using restriction endonucleases and Northern blotting reveal no structural aberrations of the majority of the gene copies, substantiating the concept that amplification of the N-myc gene results in high levels of structurally normal N-myc mRNA.     

Genistein modulates neuroblastoma cell proliferation and differentiation through induction of apoptosis and regulation of tyrosine kinase activity and N-myc expression

Genistein is a specific inhibitor of protein tyrosine kinase (PTK) and is considered as a therapeutic candidate for various cancers. In this paper we investigate the effects of genistein on cell proliferation and differentiation in neuroblastoma (NB) cell lines and its possible mechanism of action. Genistein substantially inhibited the growth of five (N2A, JC, SKNSH, MSN and Lan5) of the six tumor cell lines examined in a dose-dependent manner with an IC50 value of approximately 5 microg/ml. The exception was GC cells. N2A cells were treated with genistein for 6 days and exhibited morphological features of differentiation, as evidenced by the development of dendritic extensions. Terminal deoxynucleotidyl transferase (TDT) histochemical staining showed a significant elevation in darkly stained nuclei in genistein-treated N2A cells compared with controls, indicating the occurrence of apoptosis. Fluorescent quantitation of DNA fragments confirmed apoptosis in genistein-treated N2A cells. To further elucidate the possible mechanisms by which genistein modulates NB cell growth and differentiation we investigated the effect of genistein on the activities of PTK and mitogen-activated protein (MAP) kinase and N- myc proto-oncogene expression in N2A cells. The results showed that genistein down-regulated intrinsic PTK activity by approximately 33% and inhibited insulin-like growth factor (IGF)-stimulated PTK activity by 75%. The effect of genistein on the intrinsic activity of MAP kinase was insignificant. In addition, genistein significantly reduced N-myc expression in a dose-dependent fashion. Our study suggests that genistein arrests cell growth and induces NB cell differentiation by mediating apoptosis and modulating PTK activity and N-myc proto- oncogene expression.      

Anti-neuroblastoma activity of Helminthosporium carbonum (HC)-toxin is superior to that of other differentiating compounds in vitro

Treatment of high-risk neuroblastoma (NB) is difficult. Novel therapeutics improving survival rates are urgently required. We have previously shown that the histone deacetylase inhibitor (HDACI) Helminthosporium carbonum (HC)-toxin induces differentiation of neuroblastoma (NB) cells. Here, we show that HC-toxin inhibits the growth of both established NB cell lines and primary cultures with and without amplified MYCN stronger than retinoids (RAs) and other HDACIs (MS-275, n-butyric acid, suberoylanilide hydroxamic acid, trichostatin A, valproic acid). Nanomolar dosages suppress E2F-1, N-myc, Skp2, Mad2 and survivin proteins, found at high levels in high-risk NBs, more efficiently than both RAs and other HDACIs. The level of hypophosphorylated active retinoblastoma (RB) tumor suppressor protein is increased most effectively. HC-toxin's epoxy group is essential for inhibiting HDACs and promoting anti-NB activity. Without this functional group, those cellular effects are not observed. In conclusion, the anti-NB activity of HC-toxin is superior to that of RAs and that of all other HDACIs tested.     

Loss of one HuD allele on chromosome #1p selects for amplification of the N-myc proto-oncogene in human neuroblastoma cells

In human neuroblastoma tumors, amplification of the N-myc proto-oncogene and loss of all or part of the short arm of chromosome #1 are both associated with a poor prognosis. Accruing evidence indicates that it is the absence of one allele of the HuD (ELAVL4) gene, encoding the neuronal-specific RNA-binding protein HuD and localized to 1p34, that is linked to amplification. In 12 human neuroblastoma cell lines, N-myc amplification correlates with loss of one HuD allele and decreased HuD expression. Transfection experiments demonstrate that modulating HuD expression affects N-myc gene copy number as well as expression. Introduction of a sense HuD construct into two N-myc amplified cell lines considerably increases N-myc expression whereas gene copy number decreases. Conversely, expression of antisense HuD in N-myc nonamplified SH-SY5Y cells reduces HuD and N-myc mRNA levels even as cells show amplification of the N-myc gene. Thus, N-myc gene copy number is modulated by alteration of HuD expression. We propose that haploinsufficiency of HuD due to chromosome #1p deletion in neuroblastoma selects for cells that amplify N-myc genes. Application of these findings could lead to more effective therapies in the treatment of those patients with the worst prognosis.     

Heterogeneity of lung cancer cells with respect to the amplification and rearrangement of myc family oncogenes

Seventy lung tumors from 53 patients were analysed for alterations of myc family oncogenes, c-myc, N-myc and L-myc, to evaluate when activation of these genes occurs during tumor development. The 53 cases were 17 small cell carcinomas (SCCs), 18 adenocarcinomas, 12 squamous cell carcinomas (SqCs), 4 large cell carcinomas and 2 adenosquamous carcinomas. Either N-myc or L-myc was amplified in 4 of the 17 (one N-myc and 3 L-myc) SCCs (24%), while c-myc was amplified in 3 of the 12 SqCs (25%). In one SCC, amplification of N-myc was found in the primary tumor, a pulmonary hilar lymph node metastasis and a pleural metastasis, but not in a liver metastasis or a para-aortic lymph node metastasis. In one SqC, c-myc was amplified in a pleural metastasis and a lymph node metastasis, but not in the primary tumor. In 2 cases of SCCs, amplification or rearrangement of c-myc was detected only in the cell lines, but not in the original tumors taken from the same individuals. These results indicate that tumor cells were heterogeneous for amplification and rearrangement of myc family oncogenes, and suggest that activation of these oncogenes in SCCs and SqCs occurs not at the time of malignant transformation but during tumor progression.     

Expression of a MYCN-interacting isoform of the tumor suppressor BIN1 is reduced in neuroblastomas with unfavorable biological features.

PURPOSE: Amplification of the MYCN proto-oncogene is strongly correlated with poor outcome in neuroblastoma (NB), although deregulated MYCN is a potent inducer of apoptosis. BIN1 (2q14) encodes multiple isoforms of a Myc-interacting adaptor protein that has features of a tumor suppressor, including the ability to inhibit Myc-mediated cell transformation and to promote apoptosis. We hypothesized that BIN1 may function as a suppressor gene in NB, because Bin1 is highly expressed in neural tissues and binds the Myc Box motifs that are conserved in MycN. EXPERMENTAL DESIGN: Expression of MYCN, total BIN1, and BIN1 isoforms were determined in 56 primary NBs using the real-time PCR. Expression was correlated with biological and genetic features. To determine the functional significance of BIN1 expression we ectopically expressed BIN1 isoforms in NB cell lines with and without MYCN amplification, and assessed clonogenic growth. RESULTS: Four predominant BIN1 isoforms resulting from alternative splicing of exon 12A (a neural tissue-specific exon) and exon 13 (a Myc-binding domain encoding exon) were variably expressed in the 56 primary NBs. Expression of BIN1 was lower in: NBs with MYCN amplification (n = 10) compared with those without, P < 0.03; in International Neuroblastoma Risk Group high-risk NB (n = 19) compared with low- or intermediate-risk NB, P < 0.01; and in metastatic NB (n = 21) compared with localized NB, P < 0.06. BIN1 inactivation by deletion or genomic rearrangement was identified infrequently. Forced expression of BIN1 isoforms containing the Myc-binding domain (with or without exon 12A) inhibited colony formation in NB cell lines with MYCN amplification (P < 0.01) but not in those without. Forced expression of BIN1 isoforms with a MBD deletion did not inhibit colony formation in any cell line assessed. CONCLUSIONS: These data support that reduced BIN1 expression contributes to the malignant phenotype of childhood NB. As we reported previously, BIN1 may function to circumvent MycN-mediated apoptosis in NBs with MYCN amplification.     

N-myc gene expression and oncoprotein characterisation in medulloblastoma

Although medulloblastoma and neuroblastoma share many common biological, histological and immunological features, the frequency of N-myc amplification differs markedly between the two tumours. In this study, Southern blot analysis revealed that the N-myc gene was not amplified in any of the nine medulloblastoma samples analysed. In contrast, over-expression of the gene was found in six of 11 samples as determined by immunocytochemistry and/or Western blot analysis, using an antiserum raised against a synthetic peptide representing a sequence unique to the N-myc gene product. The specificity of this reagent was demonstrated by studies on a variety of cell lines expressing N-myc and/or c-myc oncoproteins. Of the 12 medulloblastoma samples collected over a two-year period and analysed in the course of this project, a trend towards longer disease-free survival was noted in the patients having low levels of the N-myc protein in their tumour.     

Amplification of N-myc and ornithine decarboxylase genes in human neuroblastoma and hydroxyurea-resistant hamster cell lines

The genes for the M2 subunit of ribonucleotide reductase (RRM2), ornithine decarboxylase (ODC1), and 55,000-Daltons protein (P5), are amplified in hydroxyurea-resistant hamster and human cell lines. These genomic sequences have been mapped to hamster chromosome 7 and to human chromosome 2p24-25 near the cytogenetic location of the N-myc gene. We now report that genomic sequences homologous to N-myc are amplified in hydroxyurea-resistant hamster lung cell line, 600H, and the N-myc gene segregates with hamster chromosome 7 in mouse-hamster somatic cell hybrids. The conserved linkage group consisting of the RRM2, ODC1, P5, and N-myc in the hamster and human genomes prompted our investigation of human neuroblastomas. We report here that genomic DNA from 1 of 6 primary neuroblastoma tumors containing amplified N-myc also contains amplified sequences homologous to a hamster ODC cDNA.