N-myc Gene Expression Research Articles

Expression of protein kinase C-alpha (PKC-α) and MYCN mRNAs in human neuroblastoma cells and modulation during morphological differentiation induced by retinoic acid

It is known that PKC is differently expressed in brain and the peripheral nervous system and is involved in cellular differentiation. We have analyzed 9 human neuronal-derived crest-cell lines for PKC-α mRNA. Seven out of nine expressed 9.0 kb and 4.0 kb PKC-α mRNAs but three had high level of 9.0 kb transcription. The different expression of the two messenger RNAs may result from alternative splicing and a different degree of cell maturation. The same cell lines were studied for MYCN gene expression. A possible relation between the two genes is discussed. One cell line expressing high levels of both PKC-α mRNA was treated with 10 −1 M retinoic acid (RA). The expression of both messenger RNAs was suppressed when the cells achieved a morphological differentiation and showed neurite-like processes. A decrease of PKC-α gene expression was associated to down regulation of MYCN mRNA. These preliminary results suggest that PKC suppression of PKC-α mRNA is associated with reversion of the malignant phenotype.

Association of expression between N-myc gene and major histocompatibility complex class I gene in surgically resected human neuroblastoma

Amplification of the N-myc gene in neuroblastoma correlates with advanced stage and poor prognosis. Association of the expression between N-myc and major histocompatibility complex (MHC) class I genes in 33 neuroblastomas obtained from Japanese children was investigated. Amplification of the N-myc gene was observed in two of five cases in Stage III, six of 11 cases in Stage IV, and one of five cases in Stage IV-S. In each case, the expression of N-myc gene was significantly increased. The expression was also increased in cases without amplification of the N-myc gene, the origin being from the suprarenal region. Expression of the MHC class I gene was significantly decreased in five of these nine with a high level of N-myc expression with amplification. These results suggest that the down-modulation of the MHC class I expression may be associated with the high level of expression and amplification of N-myc gene in the advanced stage of neuroblastoma.

DNA integration sites and hepatocellular carcinoma

This study is part of an ongoing analysis of woodchuck hepatitis virus integration sites in the host genome of hepatocellular carcinomas. The study of woodchuck hepatitis virus-DNA integration sites may shed light on the oncogenic mechanisms involved in cellular transformation and tumor formation. Viral integration enhancing cellular proto-oncogene expression is one such mechanism and has been well documented for oncogenic retroviruses such as mouse mammary tumor virus and interleukin-1. By cloning a woodchuck hepatitis virus integration site from a woodchuck hepatocellular carcinoma the authors were able to identify a new member of the myc gene family, N-myc-2. Examination of 30 additional woodchuck hepatomas revealed viral integration commonly occurred near N-myc loci with an additional five woodchuck hepatitis virus integrants near the N-myc-2 gene and one viral integrant near N-myc-1. Three of these N-myc-2 viral integrations were further evaluated and found to be localized within 200 bp of the translation stop codon. This 3′ noncoding region has recently been identified as a common site of murine leukemia virus integration in virally induced T-cell lymphomas and results in increased expression of the N-myc gene. Similar mechanisms can be proposed for hepatocellular carcinoma formation. Woodchuck hepatitis virus integration near cell-growth related protooncogenes, such as N-myc, can juxtapose viral enhancer elements and growth-regulatory genes. Virally induced overexpression of proto-oncogene messenger RNA could result from enhanced transcription or increased messenger RNA stability. To search for such effects the authors analyzed N-myc-2 RNA levels in 30 woodchuck hepatitis virus-related hepatomas. Increased levels of N-myc-2 RNA were found in 18 of 30 tumors, whereas nontumorous portions of the same livers had no detectable N-myc-2 RNA. Taken together these findings suggest that woodchuck hepatitis virus integration can result in altered N-myc-2 gene expression in a significant proportion of woodchuck hepatocellular carcinomas. The deregulation of N-myc gene expression could result in cellular transformation and ultimately tumor formation. Such examples of hepadnavirus-specific oncogenic mechanisms lend credence to theories of hepatitis B virus-induced tumorigenesis and provide models to design molecular investigations of human hepatocellular carcinoma formation.

Mechanism of endogenous myc gene down-regulation in E mu-N-myc tumors.

Transgenic mouse lines carrying the N-myc oncogene deregulated by the immunoglobulin heavy-chain enhancer spontaneously develop B-lymphoid tumors (R. Dildrop, A. Ma, K. Zimmerman, E. Hsu, A. Tesfaye, R. DePinho, and F. W. Alt, EMBO J. 8:1121-1128, 1989; H. Rosenbaum, E. Webb, J. M. Adams, S. Cory, and A. W. Harris, EMBO J. 8:749-755). Permanent cell lines derived from these tumors (E mu-N-myc cell lines) express extremely high levels of the N-myc transgene but little or no detectable endogenous N-myc or c-myc. We have employed nuclear run-on assays to show that down-regulation of endogenous N- and c-myc expression occurs at the transcriptional level. To determine whether the lack of endogenous myc gene transcription is a direct effect of high-level N-myc transgene expression, we have generated Abelson murine leukemia virus (A-MuLV)-transformed cell lines from prelymphomatous E mu-N-myc mice (A-MuLV/E mu-N-myc cell lines). Although these A-MuLV/E mu-N-myc lines express very high levels of the N-myc transgene, they continue to transcribe the endogenous c-myc gene. These findings demonstrate that high-level N-myc gene expression alone does not necessarily lead to down-regulation of endogenous myc gene expression and suggest that events associated with transformation by N-myc may be critical to this process.

Mechanism of Endogenous myc Gene Down-Regulation in Eμ-N-myc Tumors

Transgenic mouse lines carrying the N-myc oncogene deregulated by the immunoglobulin heavy-chain enhancer spontaneously develop B-lymphoid tumors (R. Dildrop, A. Ma, K. Zimmerman, E. Hsu, A. Tesfaye, R. DePinho, and F. W. Alt, EMBO J. 8:1121-1128, 1989; H. Rosenbaum, E. Webb, J. M. Adams, S. Cory, and A. W. Harris, EMBO J. 8:749-755). Permanent cell lines derived from these tumors (E mu-N-myc cell lines) express extremely high levels of the N-myc transgene but little or no detectable endogenous N-myc or c-myc. We have employed nuclear run-on assays to show that down-regulation of endogenous N- and c-myc expression occurs at the transcriptional level. To determine whether the lack of endogenous myc gene transcription is a direct effect of high-level N-myc transgene expression, we have generated Abelson murine leukemia virus (A-MuLV)-transformed cell lines from prelymphomatous E mu-N-myc mice (A-MuLV/E mu-N-myc cell lines). Although these A-MuLV/E mu-N-myc lines express very high levels of the N-myc transgene, they continue to transcribe the endogenous c-myc gene. These findings demonstrate that high-level N-myc gene expression alone does not necessarily lead to down-regulation of endogenous myc gene expression and suggest that events associated with transformation by N-myc may be critical to this process.

Regulation of N-myc gene expression: use of an adenovirus vector to demonstrate posttranscriptional control.

We present evidence that differences in the levels of N-myc mRNA among different cell types are the result of posttranscriptional control. First, we noted that while steady-state mouse N-myc mRNA could be detected only in fetal mouse brain, it was transcribed at an equivalent rate in adult brain, liver, spleen, and placenta and in fetal brain. Similarly, the human N-myc gene was transcribed at an equivalent rate in HeLa cells, which do not accumulate this RNA in the cytoplasm, and cell lines G401 (a Wilms tumor-derived cell line) and SKNMc (established from a primitive neuroepithelioma), which do express N-myc RNA. As expected, the N-myc promoter functioned at equivalent rates, as demonstrated by the level of a reporter gene, when introduced into these cell types by using a recombinant adenovirus vector. The suggestion that posttranscriptional mechanisms control the level of this RNA was supported by the observation that sequences in the N-myc third exon specifically decreased the level of E1A mRNA when these sequences were placed downstream of the E1A promoter in a recombinant adenovirus. Finally, we further localized these sequences to a 600-bp fragment of the third exon by introducing various subclones of this sequence downstream of the E1A promoter in both viral and plasmid vectors.

Inhibitory effects of fucoxanthin, a natural carotenoid, on N- myc expression and cell cycle progression in human malignant tumor cells

Fucoxanthin, a natural carotenoid prepared from brown algae, inhibited the growth of GOTO cells, a human neuroblastoma cell line. Fucoxanthin at 10 μg/ml reduced the growth rate of GOTO cells to 38% of the control at day 3 after drug treatment. Flowcytometric analysis revealed that fucoxanthin caused the arrest in the G 0–G 1 phase of cell cycle. Expression of N-myc gene was proved to be decreased by fucoxanthin as early as 4 h after treatment at 10 μg/ml and that may be important for the mechanism of anti-proliferative action of the carotenoid.

Increased expression of c- jun gene during spontaneous hepatocarcinogenesis in LEC rats

We have studied the expressions of nine proto-oncogenes (c-myc, N-myc, c-fos, C-jun, p53, H-ras, N-ras, c-raf, hst) and two other genes (PCNA, GST-P) during the spontaneous development of hepatocellular carcinomas (HCCs) in LEC rats. Expression of c-myc, H-ras, N-ras, C-raf, p53 and PCNA genes was detected, but this did not significantly change during the development of HCCs in LEC rats. Expression of N-myc and hst genes was not detectable. Expression of c-fos gene was detected in one HCC case out of four. Significantly increased expression of c-jun gene was observed in the liver tissues of LEC rats aged 8 months. This high expression was decreased with the development of HCCs. On the other hand, the expression of GST-P gene increased in parallel with the clinical course of the development of HCCs in LEC rats. The pattern of c-jun mRNA augmentation was different from that of GST-P mRNA. These observations suggest that c-jun gene may play a role in the spontaneous development of HCCs in LEC rats.

Frequent activation of N-myc genes by hepadnavirus insertion in woodchuck liver tumours

The recent finding of c-myc activation by insertion of woodchuck hepatitis virus DNA in two independent hepatocellular carcinoma has given support to the hypothesis that integration of hepatitis B viruses into the host genome, observed in most human and woodchuck liver tumours, might contribute to oncogenesis. We report here high frequency of woodchuck hepatitis virus DNA integrations in two newly identified N-myc genes: N-myc1, the homologue of known mammalian N-myc genes, and N-myc2, an intronless 'complementary DNA gene' or 'retroposon' that has retained extensive coding and transforming homology with N-myc. N-myc2 is totally silent in normal liver, but is overexpressed without genetic rearrangements in most liver tumours. Moreover, viral integrations occur within either N-myc1 or N-myc2 in about 20% of the tumours, giving rise to chimaeric messenger RNAs in which the 3' untranslated region of N-myc was replaced by woodchuck hepatitis virus sequences encompassing the viral enhancer. Insertion sites were clustered in a short sequence of the third exon that coincides with a retroviral integration hotspot within the murine N-myc gene, recently described in T-cell lymphomas induced by murine leukaemia virus. Thus, comparable mechanisms, leading to deregulated expression of N-myc genes, may operate in the development of tumours induced either by hepatitis virus or by nonacute retroviruses in rodents. Activation of myc genes by insertion of hepadnavirus DNA now emerges as a common event in the genesis of woodchuck hepatocellular carcinoma.

Organization and expression of the chicken N-myc gene.

We cloned the chicken N-myc gene and analyzed its structure and expression. We found that it consisted of three exons with coding regions in exons 2 and 3. Comparison to mammalian N-myc genomic sequence indicated that nucleotide sequences of the 5'-flanking region, noncoding exon 1, and introns were not conserved, but coding and 3' noncoding sequences showed significant homology to mammalian N-myc. Alignment of deduced amino acid sequences of chicken and mammalian N-myc proteins revealed nine conserved domains interrupted by different lengths of nonhomologous sequences. Two of the domains were specific to N-myc proteins, and the other seven were common to c-myc proteins. Northern blot (immunoblot) and in situ hybridization analyses of 3.5-day-old chicken embryos revealed that high-level expression of the N-myc gene was confirmed to certain tissues, e.g., the central nervous system, neural crest derivatives, and mesenchyme of limb buds. In the beak and limb primordia, N-myc expression in the mesenchyme was higher toward the distal end, suggesting possible involvement in positional assignment of the tissue within the rudimentary structures.

Organization and Expression of the Chicken N-myc Gene

We cloned the chicken N-myc gene and analyzed its structure and expression. We found that it consisted of three exons with coding regions in exons 2 and 3. Comparison to mammalian N-myc genomic sequence indicated that nucleotide sequences of the 5'-flanking region, noncoding exon 1, and introns were not conserved, but coding and 3' noncoding sequences showed significant homology to mammalian N-myc. Alignment of deduced amino acid sequences of chicken and mammalian N-myc proteins revealed nine conserved domains interrupted by different lengths of nonhomologous sequences. Two of the domains were specific to N-myc proteins, and the other seven were common to c-myc proteins. Northern blot (immunoblot) and in situ hybridization analyses of 3.5-day-old chicken embryos revealed that high-level expression of the N-myc gene was confirmed to certain tissues, e.g., the central nervous system, neural crest derivatives, and mesenchyme of limb buds. In the beak and limb primordia, N-myc expression in the mesenchyme was higher toward the distal end, suggesting possible involvement in positional assignment of the tissue within the rudimentary structures.

Effect of human interferons on morphological differentiation and suppression of N-myc gene expression in human neuroblastoma cells.

The activity of human interferons (HuIFNs) to induce morphological changes and the suppression of N‐myc gene expression on human neuroblastoma cells (GOTO and KP‐N‐RT) was evaluated. Morphological differentiation, characterized as the extension and bifurcation of neurites, the formation of multinucleated giant cells and the formation of neurite networks, was induced by treatment with recombinant HuIFN‐γ (rHuIFN‐γ and also with natural HuIFN‐γ on human neuroblastoma cells (GOTO and KP‐N‐RT). But recombinant HuIFN‐αA and recombinant HuIFN‐βdid not induce any changes. The rHuIFN‐β and rHuIFN‐γ inhibited the growth of GOTO and KP‐N‐RT cells more strongly than the rHuIFN‐αA did. The expression of N‐myc gene was suppressed in GOTO cells treated with rHuIFN‐γ. The suppressive effect of rHuIFN‐γ was dependent on the duration of the treatment. However, rHuIFN‐αA and rHuIFN‐β did not suppress N‐myc gene expression. Moreover, both morphological differentiation and the supprcssive effect on N‐myc gene expression by rHuIFN‐γ were inhibited in the presence of cycloheximide. These results suggest that the morphological changes and N‐myc gene expression in neuroblastoma cells are closely related. Furthermore, this decreased N‐myc gene expression during the morphological differentiation may be related to the proteins induced by HuIFN‐γ.

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.

IgH enhancer-mediated deregulation of N-myc gene expression in transgenic mice: generation of lymphoid neoplasias that lack c-myc expression.

We have generated transgenic mouse lines that carry one of three different constructs in which the murine N-myc gene is expressed under the control of the immunoglobulin heavy chain transcriptional enhancer element (E mu-N-myc genes). High-level expression of the E mu-N-myc transgenes occurred in lymphoid tissues; correspondingly, many of these E mu-N-myc lines reproducibly developed pre-B- and B-lymphoid malignancies. The E mu-N-myc transgene also appeared to participate in the generation of a T cell malignancy that developed in one E mu-N-myc mouse. These tumors and cell lines adapted from them expressed exceptionally high levels of the E mu-N-myc transgene; the levels were comparable to those observed in human neuroblastomas with highly amplified N-myc genes. In contrast, all of the E mu-N-myc cell lines had exceptionally low or undetectable levels of the c-myc RNA sequences, consistent with the possibility that high-level N-myc expression can participate in the negative 'cross-regulation' of c-myc gene expression. Our findings demonstrate that deregulated expression of the N-myc gene has potent oncogenic potential within the B-lymphoid lineage despite the fact that the N-myc gene has never been implicated in naturally occurring B-lymphoid malignancies. Our results also are discussed in the context of differential myc gene activity in normal and transformed cells.

Differential expression of N-myc and c-src proto-oncogenes during neuronal and schwannian differentiation of human neuroblastoma cells.

Neuroblastoma (NB) cells can be induced to differentiate bidirectionally into either neuronal or schwannian cells by different inducers. However, the underlying mechanisms are poorly understood. We examined the expression of N-myc and c-src genes in 3 human NB-cell lines during either neuronal or schwannian differentiation in vitro, since proto-oncogenes are considered to play a pivotal role in regulating cell proliferation and differentiation. Decreased N-myc expression and increased c-src expression were observed during neuronal differentiation by retinoic acid, polyprenoic acid (E5166) and dibutyryl cyclic AMP, whereas the expression of N-myc and c-src genes was considerably reduced during schwannian differentiation by bromodeoxyuridine, demonstrating that the expression of N-myc and c-src genes was regulated independently in the bipolar differentiation processes of NB cells. Our results suggest that enhanced N-myc expression might be closely linked to the undifferentiated phenotypes of NB cells, that c-src expression might be related to the neuronal differentiation of NB cells, and that N-myc and c-src genes might be regulated independently in the determination of the bipolar differentiation of NB cells.

Expression of myc family oncogenes in primary human testicular cancer

In order to understand the role of myc family genes (c-myc, N-myc and L-myc) in the development and progression of human testicular cancer, we have analyzed the expression of myc family genes in three different types of primary human testicular cancer (seminoma, embryonal carcinoma and teratocarcinoma) and normal testis using Northern blot analysis. Expression of N-myc gene, which is usually limited in the neoplasms derived from neuroectoderm, was detected in seven out of ten cases of seminomas and two out of two cases of embryonal carcinomas. Gene amplification was not observed in these cases. Expression of N-myc gene was not detected in teratocarcinomas and normal testes. Expression of c-myc gene was observed in seminomas, embryonal carcinomas, teratocarcinomas and normal testes, but specific expression of c-myc gene was not seen in these cancers and normal testes. Expression of L-myc gene was not detected in all cases examined in our studies. Since N-myc gene expression was observed only in undifferentiated testicular neoplasm, such as seminoma and embryonal carcinoma, its expression may be positively related to the development and progression of special types of human testicular cancer.

Novel combination of c-myc, N-myc and N-ras oncogene alteration in brain tumors

We have examined forty human brain tumors (neoplasias presenting an important incidence in Mexico), for cellular myc (c-myc), N-myc and N-ras proto-oncogene alterations. An elevated amplification and/or rearrangement of the oncogenes was detected in most samples (60% presenting alteration for c-myc, 54% for N-myc, 6% for N-ras and 60% for ras-related genes). The tumors were of different histological types and for some of them we detected either amplification and/or rearrangement of the oncogenes. We describe, for the first time, the alterations of two related genes (c-myc and N-myc) in the same tumor samples; in 64% of the analyzed samples, oncogene alterations were accompanied by enhanced expression of N-myc and ras-related genes. These results suggest an important role for c-myc, N-myc and N-ras oncogenes, in the development and progression of brain tumors.

Identification and characterization of the NMYC gene product in human neuroblastoma cells by monoclonal antibodies with defined specificities.

Increased N-myc (now designated NMYC in human gene nomenclature) gene expression has been detected at the transcriptional level in certain types of neoplasms. As yet, the N-myc gene product has not been identified. To detect and characterize the N-myc gene product, we have developed monoclonal antibodies against the putative N-myc gene product made in Escherichia coli as a fusion protein. The antibodies that recognize the N-myc-specific regions were selected on the basis of their reactivities to different portions of the fusion protein. These monoclonal antibodies detect a pair of closely migrating polypeptides of 60 and 63 kDa in nuclear fractions of human neuroblastoma cells. The relative levels of the polypeptides are roughly proportional to the level of N-myc transcripts present in a panel of neuroblastoma lines. These two polypeptides have a half-life of approximately equal to 35 min, and they are indistinguishable from each other by their epitopic profiles.

Expression and amplification of the N-myc gene in primary retinoblastoma.

Retinoblastoma, the most common intraocular tumour of childhood, probably arises from embryonal cells and occurs in hereditary and non-hereditary forms. Recent evidence suggests that this retinoblastoma (Rb) susceptibility gene located at chromosome 13q14 is actually recessive. Knudson has proposed that the tumour is caused by two mutational events. This idea was extended by Comings, who suggested that dominantly inherited tumours may result from loss or inactivation of both alleles of regulatory or suppressor genes that, when active, prevent the expression of a structural transforming gene(s) (possibly an oncogene) normally active only during embryogenesis. Despite circumstantial evidence for this hypothesis, no activated oncogene has been identified. We now report that (1) the N-myc gene is amplified 10-200-fold in two primary retinoblastomas and a retinoblastoma cell line Y79 and (2) expression of N-myc gene is highly elevated in most of the retinoblastomas examined. This finding suggests that N-myc gene may have a primary role in the tumorigenesis of retinoblastoma.