Key words: neuroblastoma; N-myc; oncogene; amplification; chromosome; gene regulation; pediatric cancers; childhood tumors; prognosis; transcription factors; promoter signaling; apoptosis; proliferation; growth factors

Introduction
Proper spatio-temporal expression of the N-myc gene is important as a lack of N-myc expression causes severe defects of embryonic development while overexpression of N-myc is associated with the development of several types of childhood tumors. Despite its clinical relevance, the regulation of N-myc expression has received little attention in the past and, accordingly, surprisingly little is known as yet. This review aims at providing a comprehensive overview of what we have learned about the transcriptional regulation of N-myc since its cloning 18 years ago. Since N-myc overexpression is a major determinant of the clinical course of neuroblastomas, it will be a focus of this review.

N-myc and tumorigenesis
The transcription factors encoded by the myc genes form part of a complex regulatory network implicated in the control of diverse aspects of cellular physiology including cell proliferation and apoptosis [1]. The experimental evidence for a contribution of the myc genes to tumorigenesis, and the functional consequences of myc overexpression have been the focus of several recent reviews [2,3].

The N-myc gene is amplified in about 25 % of neuroblastomas [for a review see: 4]. This amplification is well established as a marker for poor prognosis [5-7]. Consistent with a critical role of N-myc in the development of neuroblastomas are transgenic mice that overexpress N-myc in neuroectodermal cells and develop neuroblastoma [8]. N-myc is also amplified in several additional pediatric tumors of mostly, but not exclusively, neuroectodermal origin, including rhabdomyosarcoma, medulloblastoma, retinoblastoma, astrocytoma, glioblastoma, and small cell lung carcinoma [9-11]. In addition, N-myc is overexpressed in many Wilms´ tumors that lack a functional WT1 [12].

Although the vast majority of functional studies have focused on the c-Myc protein, N-Myc functions in a very similar manner in a number of cell culture assays. Indeed, when the coding region of the mouse c-myc gene was replaced with the N-myc coding sequence by homologous recombination, the resulting homozygous knock-in mice, in which the c-myc promoter drives the synthesis of N-myc mRNA instead of c-myc mRNA, were viable and appeared normal suggesting that N-Myc can functionally replace c-Myc [13].

There are three features that, taken together, make N-myc an attractive target for tumor therapy. Firstly, there is a clear association between N-myc amplification on the one hand and tumor aggressiveness and poor prognosis on the other. Secondly, two mouse models of tumorigenesis with experimentally controlled, reversible c-myc expression suggest that a tumor requires continuous myc expression and that down-regulation of myc expression results in tumor regression [14,15]. Thirdly, due to its restricted expression pattern after birth, side effects of even systemic down-regulation of N-myc expression can be expected to be moderate.

The spatio-temporal expression profile of N-myc
In contrast to c-myc which is expressed in virtually all proliferating cells of an organism, the expression of N-myc is more restricted. During murine development N-myc mRNA can be detected as early as day 7.5 in the primitive streak [16]. A peak of expression is reached between days 9.5 and 11.5, followed by a sharp decrease after day 12.5 [17,18]. During this time N-myc mRNA is present in many tissues including heart, limb buds, and neural tube [18]. N-myc mRNA has also been detected during organogenesis in tissues such as hair follicles, lung, liver, and stomach [19,20]. Data from frog and chicken by and large suggest a similar expression profile as compared to the mouse [21,22]. At least in Xenopus laevis, N-myc is initially expressed as a maternal RNA [21].

Within a tissue N-myc expression is not homogeneous. For example, in the heart, expression is restricted to the myocardium; in the liver it occurs mainly in the peripheral layer; in the neural crest it is initially expressed homogeneously, but after colonization of ganglion areas becomes restricted to those cells undergoing neuronal differentiation; in the human fetal kidney it is observed exclusively in the epithelially differentiating mesenchyme; in the somites it is stronger in the posterior than in the anterior half; and in the brain it is observed in the neural precursor cells but becomes more restricted after lineage commitment [23-28]. N-myc expression is dynamic not only in space but also in time. For example, N-myc mRNA levels are increased 20-fold within 3 hours after partial hepatectomy and then decline rapidly [29]. Other transient peaks of N-myc expression have been observed after IL-1 stimulation of pre-B-cells and during HMBA triggered differentiation of human embryonal carcinoma cells [30,31]. At birth N-myc is still expressed in the brain, kidney, intestine, lung, and heart but then becomes down-regulated within several days or weeks depending on the tissue [17,32,33]. In adults, N-myc expression has mainly been detected at early stages of B-cell development. However, weak expression may be maintained in the adult brain, testis and heart [17, but see:34].

Consistent with the embryonic expression pattern, three independently generated strains of N-myc null mice all were embryonic lethal at around day 11.5, showing abnormalities in the heart and nervous system, and additional defects in other tissues were reported for one of the null-alleles but not others [35-37]. Moreover, a hypomorphic allele of N-myc resulted in defects in lung branching morphogenesis and kidney development late in embryogenesis [24,38,39]. These findings suggest that lack of N-myc in any tissue where it is normally expressed results in developmental defects. An exception to this seems to be lymphocyte development, which proceeds normally in N-myc deficient embryos despite the expression of N-myc at certain stages of B-cells development [40].

Taken together, these results reveal a complex expression pattern of N-myc which should be reflected in a correspondingly complex promoter with multiple tissue-specific, stage-specific, and signal-dependent regulatory elements.

The promoters of c-myc and N-myc
As the different expression patterns would lead one to expect, the regulatory regions of c-myc and N-myc are divergent. However, there is a short region of about 40 base pairs with high homology between c-myc and N-myc located proximal to the second promoter of c-myc, termed P2, and the multiple transcription start sites of N-myc, respectively. This region contains a binding site for E2F transcription factors in both c-myc and N-myc (see below). Overlapping with the E2F-site of the c-myc promoter is a TGFb inhibitory element, TIE, which mediates TGFb dependent down-regulation of c-myc via Smad proteins [41,42]. The N-myc promoter also contains a putative TIE adjacent to the E2F-site (Figure 1). In addition, this region of c-myc and N-myc contains a GC-rich element termed ME1a1 in the c-myc promoter and CT-box in N-myc (Figure 1). The ME1a1 site in the c-myc promoter was initially identified by in-vitro-footprinting and later confirmed as a physiologically relevant regulatory element by in-vivo-footprinting [43-45]. Recent nucleosome mapping experiments indicate that the ME1a1 site is required for maintaining an open chromatin structure at the c-myc P2 promoter [46]. The CT-box is located next to a site that is strongly hypersensitive towards modification by DMS in vivo, specifically in N-myc expressing neuroblastoma cell lines [47]. A ME1a1 binding site oligonucleotide competes with the CT-box for the binding of proteins present in nuclear extracts of neuroblastoma cells suggesting that both elements may be recognized by the same transcription factors [47]. The ME1a1 site is bound by several proteins including the zinc finger protein MAZ and the homeodomain protein CUT [48-50]. in vitro, both ME1a1 and CT-box bind Sp1. The positions of these potential Sp1 binding sites relative to the E2F binding sites are reminiscent of Sp1 binding sites in other E2F-responsive genes such as dihydrofolate reductase and cyclin D1. Several recent studies have pointed to a cooperation between the E2F-sites and Sp1-sites both in the activation and repression of these promoters [51-56].

Despite their different expression profiles there are similarities between c-myc and N-myc regarding their transcriptional regulation. Both genes are activated by IL-7 signaling in pre-B-cells and can be repressed by TGFb [57-60]. In addition, both genes are targets of NFkB [30,61]. Finally, there is evidence that c-myc as well as N-myc can be repressed by the WT1 tumor suppressor protein [62,63]. In summary, there is some evidence that there may exist regulatory events that c-myc and N-myc have in common despite their different expression profiles.

Signaling to the N-myc promoter
The c-myc promoter is the target of multiple mitogenic and antimitogenic signals underscoring the central role of the c-myc protein in the control of proliferation. The mitogenic signals that activate c-myc expression include v-Abl via the Ras/Raf1-signaling cascade and PDGF via Src/Vav2/Rac [64-67]. Among the anti-mitogenic signals that repress c-myc expression are TGFβ, APC, glucocorticoids, and IFNg [60,68-71]. Signals that induce differentiation also down-regulate c-myc expression [eg: 72]. In some instances the promoter element that mediates the inducing or repressing effect has been mapped to the E2F-site of the c-myc promoter [65,70,73-75].

Not much is known about the upstream signals regulating the activity of the N-myc promoter. The available information concerns N-myc regulation in three very different cellular settings: human neuroblastoma cell lines, mouse embryonic lung organ cultures, and primary and established pre-B-cells. In pre-B-cells N-myc as well as c-myc expression is induced by IL-7 [57]. The transcription factor Pax-5 is essential for this induction [76]. A recent DNA microarray analysis of genes whose expression after stimulation of pre-B-cells with IL-1 or LPS depends on NEMO/ IkB signaling revealed N-myc as a target gene of NF k B [30]. In fact, N-myc was by far the most dramatically activated gene among the 11 800 genes analysed, activation was transient as it was detected only 2 hours but no longer 12 hours after stimulation, and activation was independent of protein synthesis [30].

As for neuroblastomas, the Insulin-like growth factor is the only signal that has been shown to induce N-myc expression [77]. This induction of N-myc was prevented by a neutralizing antibody against the IGF-I-receptor as well as the MEK1 inhibitor PD98059. More is known about signals that down-regulate N-myc expression. Upon treatment of neuroblastoma cells with the morphogen retinoic acid, down-regulation of N-myc expression is an early event preceding both cell cycle arrest and neurite outgrowth [78-80]. Furthermore, the reconstitution of a functional TrkA signaling pathway in a neuroblastoma cell line resulted in down-regulation of N-myc in the presence of NGF [81]. Both, NGF and retinoic acid, induce growth arrest and differentiation of neuroblastoma cells which are blocked by persistent N-myc expression [82,83]. Therefore, down-regulation of N-myc is a prerequisite for differentiation, and the failure to down-regulate N-myc may contribute to tumorigenesis by blocking differentiation. In line with this, in-situ hybridization showed that N-myc expression is heterogeneous in a tumor with strong N-myc expression predominantly in undifferentiated neuroblasts [5,84]. Another signal that down-regulates N-myc expression in neuroblastoma cells is iron chelation via deferoxamine mesylate [85]. Finally, in embryonic lung organ cultures N-myc expression is down-regulated by TGFb in a pRb dependent manner [58,59]. Although the B-cell work demonstrates an impressive signal responsiveness of the N-myc gene, we know very little about how the N-myc promoter is linked to cellular signaling networks.

Positively acting regulatory elements in the N-myc promoter
Transcription of the human N-myc gene is initiated from more than a dozen start sites spread out over a region of 130 bp in neuroblastoma cell lines both with and without amplification of the gene [86]. When the mouse gene is analysed in human neuroblastoma cells several start sites within 10 bp from each other are used [87]. The human as well as the murine N-myc promoter contain a sequence that looks like a TATA-box [86,87]. In the human gene a cluster of start sites that is preferentially employed for transcriptional initiation is located 35 base pairs downstream of this putative TATA-box [86]. In addition, several of the start sites in the human gene are a perfect or almost perfect match to the initiator element consensus sequence YYA+1NT/AYY. Which of these potential core promoter elements are required for N-myc expression has not been tested.

Two studies describe the use of transgenic mice to delineate the N-myc promoter [32,88]. In one case, a 16 kb region encompassing the entire human N-myc gene including 4.5 kb of upstream sequence and 6 kb of downstream sequence produced N-myc expression in the brain of both embryos at day 17 of development and adults [88]. In the other study a human N-myc transgene containing 3.5 kb of upstream sequence and 3 kb of downstream sequence was shown to recapitulate the expression pattern of the endogenous N-myc gene in newborns [32]. Neither one of these studies analysed the expression of the transgenes before day 17 of embryonic development or in pre-B-cells. Therefore, it remains unknown at present whether the transgene constructs are sufficient to completely reproduce the expression pattern of the endogenous gene or whether additional sequences further upstream or downstream are required.

Studies aimed at mapping regulatory elements with the help of transient reporter assays identified several regions of potential importance (Figure 2). A 200 base pair long region immediately upstream of the multiple transcription start sites was shown to govern basal transcription [88]. Negative autoregulation of N-myc expression, which has been found to be disrupted in neuroblastoma with N-myc amplification, maps in this region close to the transcription start sites [89]. Furthermore, one of two DNase I-hypersensitive sites identified in the murine N-myc promoter in pre-B-cells maps to this region [90]. DMS-in-vivo-footprinting of the proximal N-myc promoter visualized several features exclusively in neuroblastoma cells with strong N-myc expression that were absent in both neuroblastoma and non-neuroblastoma cells that lack N-myc expression [47]. Among these was a site that was strongly hypersensitive towards modification by DMS and may correspond to the DNase I-hypersensitive site present in pre-B-cells. Taken together, these data suggest that the proximal promoter is involved in different aspects of N-myc regulation including basal transcription, autoregulation, and tissue specificity.

This region is to 94% conserved in the N-myc genes of human and mouse and contains a large number of potential transcription factor binding sites (Figure 2). There are two canonical binding sites for Sp1 and related factors. A non-canonical Sp1 binding site, the CT-box, at least in vitro also binds Sp1 as demonstrated by supershift assay (Lutz, unpublished results). However, mutation of individual Sp1 binding sites had no effect on promoter activity in transient assays [47]. In addition to the Sp1 binding sites there are two overlapping, oppositely oriented binding sites for E2F transcription factors. An additional putative E2F-site is located adjacent to the others overlapping with the TGFb inhibitory element; in transient co-transfection assays with E2F expression plasmids it is functional in a N-myc promoter construct in which the other two E2F binding sites have been destroyed (Lutz, unpublished results). A potential octamer binding site is situated between the E2F- and the Sp1-binding sites. Furthermore, there is an almost perfect match to a Gfi-1 binding site [91]. In addition, multiple WT1 binding sites, which bind WT1 in vitro, are scattered in this area, and in transient assays overexpression of wt1 repressed a N-myc dependent reporter construct [63]. The close spacing of multiple regulatory elements is a common theme in the proximal promoters of many genes and serves the goal of synergistic, concerted action of several cooperating transcription factors [92,93].

Beside the proximal promoter only one other region, located less then 1 kb upstream, has been shown to stimulate promoter activity in transient reporter assays [88]. This regulatory region may correspond to a DNase I-hypersensitive site located 650 bp upstream of the murine N-myc start sites in pre-B-cells. The only transcription factor known to bind in this region is HBP1 which, however, represses the N-myc promoter in transient assays and is therefore unlikely to be the factor responsible for activation from this remote promoter position [94].

Negatively acting regulatory elements in the N-myc promoter
Many observations demonstrate the importance of negative controls in the regulation of N-myc. In particular, when N-myc amplified, overexpressing neuroblastoma cells were fused to non-expressing cells, N-myc expression was switched off [95]. A major negatively acting regulatory region was mapped to the 3´end of the first exon and the first intron [ but see: 32,88,96]. This region seems to harbor several distinct repressive elements: one resides in the central portion of the non-coding exon 1 where transcriptional attenuation occurs [97]. The second, termed tissue specific element (TSE), has been mapped to a 116 base pair sequence located in intron 1 and contributes to tissue specificity by repressing N-myc expression in non-expressing cells [97]. The TSE, however, is not a silencer since it does not work from an upstream position. It may instead act at a posttranscriptional level, although there is only indirect evidence for this claim [97]. A region overlapping with the TSE was previously suggested to function as a silencer and a sequence with homology to the chicken lysozyme gene silencer has been proposed as the silencer element [96]. However, this hypothesis has not been substantiated, and the sequence is not conserved in the murine gene. Consistent with the existence of negatively acting elements in exon1/intron1 are data showing that N-myc expression constructs that lack this region have a higher oncogenic potency in the rat embryo fibroblast transformation assay than the corresponding full-length constructs including exon 1 and intron 1 [98]. The enhanced oncogenic potency correlated with an increased amount of N-myc mRNA in these cells relative to cells carrying the full-length construct which experienced a block to transcriptional elongation [98]. This transcriptional attenuation required a stem-loop structure followed by a stretch of thymidines in the first exon of the mouse gene [99]. The stretch of thymidines is not conserved in the human N-myc. Recently, sites of transcriptional pausing have been mapped to the non-coding exon 1 and intron 1 of N-myc [100]. Transcriptional attenuation of N-myc also seems to play a major role in N-myc down-regulation during embryogenesis, and a release from an attenuation block is responsible for part of the increase in N-myc mRNA in pre-B-cells in response to IL-7 [57,101]. In neuroblastomas, attenuation of N-myc has also been observed and mapped to the central piece of exon 1 [97]. However, in small cell lung cancer N-myc, in contrast to c-myc and L-myc, is not regulated by attenuation [102]. Together, these data establish a critical role for sequences downstream of the transcription start site for negative regulation of N-myc. However, the factors involved and the mechanism of repression remain to be worked out.

Transcription factors implicated in N-myc regulation
In pre-B-cells, Pax-5 is an important activator of N-myc [76]. N-myc mRNA was dramatically reduced in pre-B-cells from Pax-5 deficient mice, and induction of a Pax-5-estrogen receptor fusion protein by β-estradiol in a pre-B-cell line increased N-myc steady-state mRNA levels even in the presence of cycloheximide [76]. The binding site through which Pax-5 regulates N-myc expression has not been identified as yet, and no data concerning the potential contribution of Pax-5 to the expression of N-myc in pediatric tumors are available. A recent DNA microarray analysis implicated NFkB as a second transcription factor regulating N-myc in pre-B-cells [30].

Several lines of evidence suggest a role of E2F complexes in the regulation of N-myc. Firstly, it was found that the adenoviral protein E1A stimulates N-myc expression in an E2F-site dependent manner in LMTK- cells [103]. A similar activation of N-myc was subsequently observed in the neuroectodermal tumors of transgenic mice that express E1A and E1B [104]. Indeed, co-transfected E2F proteins activate a N-myc -promoter driven reporter construct in an E2F-site dependent manner in neuroblastoma cells (Strieder and Lutz, unpublished results). Secondly, when human embryonal carcinoma cells were induced to differentiate, N-myc expression was transiently down-regulated 12-24 hours after the addition of HMBA, and this down-regulation correlated with the loss of protein binding to the E2F-site in the N-myc promoter as demonstrated by EMSA [31]. Thirdly, DMS-in-vivo-footprinting revealed that the two overlapping E2F-sites are bound by proteins exclusively in human neuroblastoma cells with strong N-myc expression but not in cells that do not express N-myc [47]. In line with this, chromatin-immunoprecipitation assays revealed binding of E2F proteins to the N-myc E2F-sites specifically in cells with N-myc overexpression (Strieder and Lutz, unpublished results). However, a look at the literature reveals a complex functional interplay between myc and E2F proteins with E2F not always being upstream of Myc. During granulopoiesis, c-myc is indeed downstream of E2F [73]. However, in U2OS cells Myc and E2F appear to regulate largely independent genetic programs [105]. In contrast, during mitogenic stimulation and G0-S-phase progression of mouse embryo fibroblasts c-myc expression occurs before the activation of the proliferation stimulating E2F-family members E2F1, E2F2 and E2F3, and indeed Myc appears to function upstream of E2Fs in the control of the cell cycle [106,107]. In support of this, none of two recent DNA-microarray analyses of U2OS cells and mouse embryo fibroblasts recorded c-myc or N-myc as E2F target genes [108,109]. Instead, the Myc proteins appear to be able to activate several of the E2F genes [110-112]. Thus, E2F can regulate myc expression and, conversely, Myc can regulate the expression of E2F genes. Therefore, N-Myc and E2F proteins may form a positive feedback-loop in neuroblastomas with N-myc amplification that erroneously maintains N-myc expression (Figure 3). This feedback loop may be assisted by a second, indirect feedback loop. Among the genes activated by Myc-proteins are Id2 and cyclin D2 [113-115]. The products of both genes inactivate pRb by sequestration and phosphorylation, respectively, thereby liberating E2F which then may activate N-myc to close the loop.

Database searches with the consensus binding sites of particular transcription factors revealed some additional, putative regulators of N-myc expression. The orphan nuclear receptors RORa and RVR bind to a sequence in the first intron of both human and mouse N-myc, which is located approximately 400 base pairs upstream of, and is therefore distinct from, the tissue specific element [116]. In transient assays in COS-1 cells the two receptors showed opposite effects on a N-myc promoter-controlled reporter gene. In a N-myc /ras embryo fibroblast cotransformation assay the disruption of the receptor binding site in the N-myc construct increased the number of foci arguing for a silencer function of this element at least in fibroblasts. Another transcription factor implicated in N-myc regulation based on database searching is the HMG box transcriptional repressor and pRb-binding protein HBP1 [94]. The rat N-myc gene contains a perfect match to the putative HBP1 binding site at position –480, and the human promoter contains a similar site at position –850. The binding site in the rat gene is bound by HBP1 in vitro. No information is available on binding of HBP1 to the site in the human gene. HBP1 repressed the rat N-myc promoter in transient assays, and it was speculated that the HBP1 and E2F binding sites allow for the integration of antagonistic regulatory signals at the N-myc promoter via pRb. There is no direct evidence as yet to support a regulation of the endogenous N-myc by either RORa, RVR, or HBP1.

Chromatin structure, DNA methylation, co-regulator requirements and epigenetic control
Given that our picture of the proteins controling N-myc expression is patchy, it is not surprising that very little is known about changes in chromatin structure accompanying N-myc activation and the transcriptional co-factors involved. What is known is that actively transcribed N-myc in both murine and human cells is associated with changes in chromatin structure which make specific residues in the proximal promoter more accessible to DNase I and DMS, respectively [47,90]. E1A activates N-myc expression, but it is unknown which of the biochemical activities of E1A is required for this [103]. Since in embryonic lung organ cultures N-myc expression is down-regulated by TGFb in a pRb dependent manner, dissociation of pRb from repressing E2F complexes may be involved [58,59].

A recent DNA microarray analysis of DNA methyltransferase 1 deficient mouse embryonic fibroblasts suggested a link between DNA methylation and N-myc expression [117]. In this screen a large number of genes were up-regulated in the absence of functional DNA methyltransferase. A smaller number of genes was, however, down-regulated, and the most dramatically down-regulated gene turned out to be N-myc. Although this may be an indirect effect caused by the up-regulation of a repressor, there is some evidence that DNA methylation may directly control N-myc expression as in neuroblastomas the paternal N-myc allele is preferentially amplified [118]. More than 10 years ago, Zimmerman et al. noted that the same N-myc constructs that failed to reproduce the expression pattern of the endogenous N-myc in transfection assays faithfully reproduced it in transgenic mice; based on this they suggested that epigenetic events in the germ line or during early development may be important for the proper expression of N-myc [32].

Levels of N-myc regulation subsequent to transcription initiation
One way of controling N-myc expression beside the regulation of transcriptional initiation, transcriptional attenuation, has already been mentioned. There is, however, evidence for several additional levels of control. One is the generation of endogenous antisense transcripts from the N-myc locus. The N-myc locus may encode a second gene, N-cym, which is transcribed from the opposite strand, overlaps with exon 1 of N-myc, and may encode a protein of 109 amino acids [119]. This antisense transcript is produced during fetal development as well as in neuroblastoma and small cell lung cancer cells [102,119]. The antisense RNA can form duplexes with the sense N-myc mRNA within the cell [120]. Presently it is unclear what this observation means in the light of several cellular machineries dedicated to the elimination of double stranded RNAs such as RNA interference [121]. Since N-myc antisense RNA is abundant in neuroblastoma cell lines with N-myc overexpression, it is either not involved in the negative control of N-myc or these neuroblastoma cells are defective in the antisense RNA dependent repression of N-myc [119,120].

Another level of control is the processing of the pre-mRNA and the stability of the mRNA. The fusion of part of the 3´-UTR of N-myc to a heterologous mRNA reduces the level of the resulting chimaeric mRNA [122]. This destabilization of the mRNA may involve members of the ELAV-like RNA-binding proteins, p40, HuD, and Hel-N1, all of which can bind to elements in the 3´UTR of the N-myc mRNA [123-125]. Whereas HuD expression is prognostically favorable in neuroblastomas, p40 expression correlates with enhanced N-myc expression [124,126]. Recently, an internal ribosome entry sequence, which appears to enhance translation initiation in neuronal cells, was identified in the N-myc gene [127]. In addition, posttranslational regulatory mechanisms such as redistribution of the N-myc protein to the cytoplasm and an increased half life of the N-myc protein have been described [28,128,129]. A physiological relevance of these post-transcriptional control mechanisms for the regulation of N-Myc protein levels or function has not been established as yet.

The relationship between amplification and overexpression
Gene amplification is a means to increase mRNA levels without the need to increase the efficiency of transcriptional initiation of the individual gene copy. In neuroblastomas the N-myc gene is amplified between less than 10-fold to more than 300-fold [6]. A similar extent of amplification is seen in retinoblastoma and small cell lung cancer [11,130]. Amplification is usually present at the time of diagnosis and there is no evidence for progressive amplification in the course of the disease or during metastatis [9,131]. Amplification does not lead to qualitative changes in gene structure or sequence but results in a quantitative change in mRNA levels with increases of up to 80-fold [5,132]. Amplification is only one of several means of a tumor cell to increase expression of a particular gene. For example, in breast cancer both cyclin D1 and ErbB2 are amplified only in a subpopulation of the tumors with increased mRNA levels of the two genes [133,134]. In addition, in the case of ErbB2 only part of the increased expression is due to the amplification of the gene because the amplified gene copies show a higher transcriptional activity than the single copy gene [133]. A similar analysis has not been performed for the N-myc gene. It is clear, however, that although neuroblastomas without N-myc amplification show a heterogeneity in the level of N-myc expression, they never produce as much N-myc as tumors with amplification do. In fact, several studies failed to find a correlation between the expression of N-myc in non-amplified tumors and an unfavourable outcome, suggesting that a threshold level of N-myc expression has to be exceeded for an unfavourable outcome to occur [135,136]. In contrast, a more recent report claimed that N-Myc protein levels do predict outcome in non-amplified tumors [137]. A point that should be stressed is that amplified N-myc can respond to certain differentiation triggering signals such as pharmacological concentrations of retinoic acid or reconstitution of a functional NGF signaling pathway with down-regulation of transcriptional activity just as the non-amplified gene does [78,81,138]. This indicates that tumor cells rather than activating N-myc during tumor progression may instead fail to down-regulate it and thereby maintain its expression in an untimely fashion. If so, amplification of the gene rather than being the primary cause of overexpression would be a secondary event subsequent to a loss of transcriptional control. Hence, the reconstitution of a functional signaling pathway that has become inactivated in the course of tumorigenesis may be a promising way of interfering with N-myc expression in the tumor.

Outlook
Clearly, more work is required before we will be able to describe in detail the events that control N-myc expression during development and disease. Which factors in addition to those already identified contribute to the regulation of N-myc? The sophisticated expression pattern during development in particular indicates that many more regulatory events take place at the N-myc gene than have been described as yet. Which co-factors are involved? What are the signals that control N-myc expression? How important are epigenetic events? The answers to these questions will without doubt hold some surprises. Most importantly, however, we will be rewarded with ideas of how to interfere with N-myc expression in tumor cells.

[1] C. Grandori, S.M. Cowley, L.P. James,R.N. Eisenman, The Myc/Max/Mad network and the transcriptional control of cell behavior, Annu Rev Cell Dev Biol 16 (2000) 653-699.
[2] B. Amati, S.R. Frank, D. Donjerkovic,S. Taubert, Function of the c-Myc oncoprotein in chromatin remodeling and transcription, Biochim Biophys Acta 1471 (2001) M135-145.
[3] R.N. Eisenman, Deconstructing myc, Genes Dev 15 (2001) 2023-2030.
[4] L. Savelyeva,M. Schwab, Amplification of oncogenes revisited: from expression profiling to clinical application, Cancer Lett 167 (2001) 115-123.
[5] M. Schwab, J. Ellison, M. Busch, W. Rosenau, H.E. Varmus,J.M. Bishop, Enhanced expression of the human gene N-myc consequent to amplification of DNA may contribute to malignant progression of neuroblastoma, Proc Natl Acad Sci U S A 81 (1984) 4940-4944.
[6] R.C. Seeger, G.M. Brodeur, H. Sather, A. Dalton, S.E. Siegel, K.Y. Wong,D. Hammond, Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas, N Engl J Med 313 (1985) 1111-1116.
[7] G.M. Brodeur, R.C. Seeger, M. Schwab, H.E. Varmus,J.M. Bishop, Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage, Science 224 (1984) 1121-1124.
[8] W.A. Weiss, K. Aldape, G. Mohapatra, B.G. Feuerstein,J.M. Bishop, Targeted expression of MYCN causes neuroblastoma in transgenic mice, Embo J 16 (1997) 2985-2995.
[9] A.J. Wong, J.M. Ruppert, J. Eggleston, S.R. Hamilton, S.B. Baylin,B. Vogelstein, Gene amplification of c-myc and N-myc in small cell carcinoma of the lung, Science 233 (1986) 461-464.
[10] J.A. Garson, P.G. McIntyre,J.T. Kemshead, N-myc amplification in malignant astrocytoma, Lancet 2 (1985) 718-719.
[11] W.H. Lee, A.L. Murphree,W.F. Benedict, Expression and amplification of the N-myc gene in primary retinoblastoma, Nature 309 (1984) 458-460.
[12] P.D. Nisen, K.A. Zimmerman, S.V. Cotter, F. Gilbert,F.W. Alt, Enhanced expression of the N-myc gene in Wilms' tumors, Cancer Res 46 (1986) 6217-6222.
[13] B.A. Malynn, I.M. de Alboran, R.C. O'Hagan, R. Bronson, L. Davidson, R.A. DePinho,F.W. Alt, N-myc can functionally replace c-myc in murine development, cellular growth, and differentiation, Genes Dev 14 (2000) 1390-1399.
[14] S. Pelengaris, T. Littlewood, M. Khan, G. Elia,G. Evan, Reversible activation of c-Myc in skin: induction of a complex neoplastic phenotype by a single oncogenic lesion, Mol Cell 3 (1999) 565-577.
[15] D.W. Felsher,J.M. Bishop, Reversible tumorigenesis by MYC in hematopoietic lineages, Mol Cell 4 (1999) 199-207.
[16] K.M. Downs, G.R. Martin,J.M. Bishop, Contrasting patterns of myc and N-myc expression during gastrulation of the mouse embryo, Genes Dev 3 (1989) 860-869.
[17] K.A. Zimmerman, G.D. Yancopoulos, R.G. Collum, R.K. Smith, N.E. Kohl, K.A. Denis, M.M. Nau, O.N. Witte, D. Toran-Allerand, C.E. Gee,et al., Differential expression of myc family genes during murine development, Nature 319 (1986) 780-783.
[18] A. Jakobovits, M. Schwab, J.M. Bishop,G.R. Martin, Expression of N-myc in teratocarcinoma stem cells and mouse embryos, Nature 318 (1985) 188-191.
[19] G. Mugrauer, F.W. Alt,P. Ekblom, N-myc proto-oncogene expression during organogenesis in the developing mouse as revealed by in situ hybridization, J Cell Biol 107 (1988) 1325-1335.
[20] U. Hirning, P. Schmid, W.A. Schulz, G. Rettenberger,H. Hameister, A comparative analysis of N-myc and c-myc expression and cellular proliferation in mouse organogenesis, Mech Dev 33 (1991) 119-125.
[21] P.D. Vize, A. Vaughan,P. Krieg, Expression of the N-myc proto-oncogene during the early development of Xenopus laevis, Development 110 (1990) 885-896.
[22] S. Sawai, K. Kato, Y. Wakamatsu,H. Kondoh, Organization and expression of the chicken N-myc gene, Mol Cell Biol 10 (1990) 2017-2026.
[23] R.A. Conlon, A.G. Reaume,J. Rossant, Notch1 is required for the coordinate segmentation of somites, Development 121 (1995) 1533-1545.
[24] C.B. Moens, B.R. Stanton, L.F. Parada,J. Rossant, Defects in heart and lung development in compound heterozygotes for two different targeted mutations at the N-myc locus, Development 119 (1993) 485-499.
[25] S. Giroux,J. Charron, Defective development of the embryonic liver in N-myc-deficient mice, Dev Biol 195 (1998) 16-28.
[26] O. Bernard, J. Drago,H. Sheng, L-myc and N-myc influence lineage determination in the central nervous system, Neuron 9 (1992) 1217-1224.
[27] H. Hirvonen, M. Sandberg, H. Kalimo, V. Hukkanen, E. Vuorio, T.T. Salmi,K. Alitalo, The N-myc proto-oncogene and IGF-II growth factor mRNAs are expressed by distinct cells in human fetal kidney and brain, J Cell Biol 108 (1989) 1093-1104.
[28] Y. Wakamatsu, Y. Watanabe, H. Nakamura,H. Kondoh, Regulation of the neural crest cell fate by N-myc: promotion of ventral migration and neuronal differentiation, Development 124 (1997) 1953-1962.
[29] M. Corral, B. Paris, C. Guguen-Guillouzo, D. Corcos, J. Kruh,N. Defer, Increased expression of the N-myc gene during normal and neoplastic rat liver growth, Exp Cell Res 174 (1988) 107-115.
[30] J. Li, G.W. Peet, D. Balzarano, X. Li, P. Massa, R.W. Barton,K.B. Marcu, Novel NEMO/IkappaB kinase and NF-kappa B target genes at the pre-B to immature B cell transition, J Biol Chem 276 (2001) 18579-18590.
[31] E. Hara, S. Okamoto, S. Nakada, Y. Taya, S. Sekiya,K. Oda, Protein phosphorylation required for the formation of E2F complexes regulates N-myc transcription during differentiation of human embryonal carcinoma cells, Oncogene 8 (1993) 1023-1032.
[32] K. Zimmerman, E. Legouy, V. Stewart, R. Depinho,F.W. Alt, Differential regulation of the N-myc gene in transfected cells and transgenic mice, Mol Cell Biol 10 (1990) 2096-2103.
[33] I. Semsei, S.Y. Ma,R.G. Cutler, Tissue and age specific expression of the myc proto-oncogene family throughout the life span of the C57BL/6J mouse strain, Oncogene 4 (1989) 465-471.
[34] J. Squire, A.D. Goddard, M. Canton, A. Becker, R.A. Phillips,B.L. Gallie, Tumour induction by the retinoblastoma mutation is independent of N-myc expression, Nature 322 (1986) 555-557.
[35] J. Charron, B.A. Malynn, P. Fisher, V. Stewart, L. Jeannotte, S.P. Goff, E.J. Robertson,F.W. Alt, Embryonic lethality in mice homozygous for a targeted disruption of the N-myc gene, Genes Dev 6 (1992) 2248-2257.
[36] S. Sawai, A. Shimono, K. Hanaoka,H. Kondoh, Embryonic lethality resulting from disruption of both N-myc alleles in mouse zygotes, New Biol 3 (1991) 861-869.
[37] B.R. Stanton, A.S. Perkins, L. Tessarollo, D.A. Sassoon,L.F. Parada, Loss of N-myc function results in embryonic lethality and failure of the epithelial component of the embryo to develop, Genes Dev 6 (1992) 2235-2247.
[38] C.B. Moens, A.B. Auerbach, R.A. Conlon, A.L. Joyner,J. Rossant, A targeted mutation reveals a role for N-myc in branching morphogenesis in the embryonic mouse lung, Genes Dev 6 (1992) 691-704.
[39] C.M. Bates, S. Kharzai, T. Erwin, J. Rossant,L.F. Parada, Role of N-myc in the developing mouse kidney, Dev Biol 222 (2000) 317-325.
[40] B.A. Malynn, J. Demengeot, V. Stewart, J. Charron,F.W. Alt, Generation of normal lymphocytes derived from N-myc-deficient embryonic stem cells, Int Immunol 7 (1995) 1637-1647.
[41] K. Yagi, M. Furuhashi, H. Aoki, D. Goto, H. Kuwano, K. Sugamura, K. Miyazono,M. Kato, c-myc is a downstream target of Smad pathway, J Biol Chem 277 (2002) 854-861.
[42] C.R. Chen, Y. Kang,J. Massague, Defective repression of c-myc in breast cancer cells: A loss at the core of the transforming growth factor beta growth arrest program, Proc Natl Acad Sci U S A 98 (2001) 992-999.
[43] C. Asselin, A. Nepveu,K.B. Marcu, Molecular requirements for transcriptional initiation of the murine c- myc gene, Oncogene 4 (1989) 549-558.
[44] A. Plet, N. Tourkine, N. Mechti, P. Jeanteur,J.M. Blanchard, In vivo footprints between the murine c-myc P1 and P2 promoters, Oncogene 7 (1992) 1847-1851.
[45] K.H. Moberg, T.J. Logan, W.A. Tyndall,D.J. Hall, Three distinct elements within the murine c-myc promoter are required for transcription, Oncogene 7 (1992) 411-421.
[46] T. Albert, J. Wells, J.O. Funk, A. Pullner, E.E. Raschke, G. Stelzer, M. Meisterernst, P.J. Farnham,D. Eick, The chromatin structure of the dual c-myc promoter P1/P2 is regulated by separate elements, J Biol Chem 276 (2001) 20482-20490.
[47] W. Lutz,M. Schwab, In vivo regulation of single copy and amplified N-myc in human neuroblastoma cells, Oncogene 15 (1997) 303-315.
[48] J.J. Pyrc, K.H. Moberg,D.J. Hall, Isolation of a novel cDNA encoding a zinc-finger protein that binds to two sites within the c-myc promoter, Biochemistry 31 (1992) 4102-4110.
[49] S.A. Bossone, C. Asselin, A.J. Patel,K.B. Marcu, MAZ, a zinc finger protein, binds to c-MYC and C2 gene sequences regulating transcriptional initiation and termination, Proc Natl Acad Sci U S A 89 (1992) 7452-7456.
[50] D. Dufort,A. Nepveu, The human cut homeodomain protein represses transcription from the c- myc promoter, Mol Cell Biol 14 (1994) 4251-4257.
[51] G. Watanabe, C. Albanese, R.J. Lee, A. Reutens, G. Vairo, B. Henglein,R.G. Pestell, Inhibition of cyclin D1 kinase activity is associated with E2F-mediated inhibition of cyclin D1 promoter activity through E2F and Sp1, Mol Cell Biol 18 (1998) 3212-3222.
[52] Y.C. Chang, S. Illenye,N.H. Heintz, Cooperation of E2F-p130 and Sp1-pRb complexes in repression of the Chinese hamster dhfr gene, Mol Cell Biol 21 (2001) 1121-1131.
[53] J. Karlseder, H. Rotheneder,E. Wintersberger, Interaction of Sp1 with the growth- and cell cycle-regulated transcription factor E2F, Mol Cell Biol 16 (1996) 1659-1667.
[54] A. Doetzlhofer, H. Rotheneder, G. Lagger, M. Koranda, V. Kurtev, G. Brosch, E. Wintersberger,C. Seiser, Histone deacetylase 1 can repress transcription by binding to Sp1, Mol Cell Biol 19 (1999) 5504-5511.
[55] R.J. Lee, C. Albanese, M. Fu, M. D'Amico, B. Lin, G. Watanabe, G.K. Haines, 3rd, P.M. Siegel, M.C. Hung, Y. Yarden, J.M. Horowitz, W.J. Muller,R.G. Pestell, Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway, Mol Cell Biol 20 (2000) 672-683.
[56] S.Y. Lin, A.R. Black, D. Kostic, S. Pajovic, C.N. Hoover,J.C. Azizkhan, Cell cycle-regulated association of E2F1 and Sp1 is related to their functional interaction, Mol Cell Biol 16 (1996) 1668-1675.
[57] M.A. Morrow, G. Lee, S. Gillis, G.D. Yancopoulos,F.W. Alt, Interleukin-7 induces N-myc and c-myc expression in normal precursor B lymphocytes, Genes Dev 6 (1992) 61-70.
[58] R. Serra, R.W. Pelton,H.L. Moses, TGF beta 1 inhibits branching morphogenesis and N-myc expression in lung bud organ cultures, Development 120 (1994) 2153-2161.
[59] R. Serra,H.L. Moses, pRb is necessary for inhibition of N-myc expression by TGF-beta 1 in embryonic lung organ cultures, Development 121 (1995) 3057-3066.
[60] J.A. Pietenpol, R.W. Stein, E. Moran, P. Yaciuk, R. Schlegel, R.M. Lyons, M.R. Pittelkow, K. Munger, P.M. Howley,H.L. Moses, TGF-beta 1 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains, Cell 61 (1990) 777-785.
[61] M.P. Duyao, D.J. Kessler, D.B. Spicer, C. Bartholomew, J.L. Cleveland, M. Siekevitz,G.E. Sonenshein, Transactivation of the c-myc promoter by human T cell leukemia virus type 1 tax is mediated by NF kappa B, J Biol Chem 267 (1992) 16288-16291.
[62] S.M. Hewitt, S. Hamada, T.J. McDonnell, F.J. Rauscher, 3rd,G.F. Saunders, Regulation of the proto-oncogenes bcl-2 and c-myc by the Wilms' tumor suppressor gene WT1, Cancer Res 55 (1995) 5386-5389.
[63] X. Zhang, G. Xing,G.F. Saunders, Proto-oncogene N-myc promoter is down regulated by the Wilms' tumor suppressor gene WT1, Anticancer Res 19 (1999) 1641-1648.
[64] J.L. Cleveland, M. Dean, N. Rosenberg, J.Y. Wang,U.R. Rapp, Tyrosine kinase oncogenes abrogate interleukin-3 dependence of murine myeloid cells through signaling pathways involving c-myc: conditional regulation of c-myc transcription by temperature-sensitive v-abl, Mol Cell Biol 9 (1989) 5685-5695.
[65] X. Zou, S. Rudchenko, K. Wong,K. Calame, Induction of c-myc transcription by the v-Abl tyrosine kinase requires Ras, Raf1, and cyclin-dependent kinases, Genes Dev 11 (1997) 654-662.
[66] M. Chiariello, M.J. Marinissen,J.S. Gutkind, Regulation of c-myc expression by PDGF through Rho GTPases, Nat Cell Biol 3 (2001) 580-586.
[67] M.V. Barone,S.A. Courtneidge, Myc but not Fos rescue of PDGF signalling block caused by kinase- inactive Src, Nature 378 (1995) 509-512.
[68] R.J. Coffey, Jr., C.C. Bascom, N.J. Sipes, R. Graves-Deal, B.E. Weissman,H.L. Moses, Selective inhibition of growth-related gene expression in murine keratinocytes by transforming growth factor beta, Mol Cell Biol 8 (1988) 3088-3093.
[69] T.C. He, A.B. Sparks, C. Rago, H. Hermeking, L. Zawel, L.T. da Costa, P.J. Morin, B. Vogelstein,K.W. Kinzler, Identification of c-MYC as a target of the APC pathway, Science 281 (1998) 1509-1512.
[70] K. Rhee, T. Ma,E.A. Thompson, The macromolecular state of the transcription factor E2F and glucocorticoid regulation of c-myc transcription, J Biol Chem 269 (1994) 17035-17042.
[71] C.V. Ramana, N. Grammatikakis, M. Chernov, H. Nguyen, K.C. Goh, B.R. Williams,G.R. Stark, Regulation of c-myc expression by IFN-gamma through Stat1-dependent and -independent pathways, Embo J 19 (2000) 263-272.
[72] B. Hoffman-Liebermann,D.A. Liebermann, Suppression of c-myc and c-myb is tightly linked to terminal differentiation induced by IL6 or LIF and not growth inhibition in myeloid leukemia cells, Oncogene 6 (1991) 903-909.
[73] L.M. Johansen, A. Iwama, T.A. Lodie, K. Sasaki, D.W. Felsher, T.R. Golub,D.G. Tenen, c-Myc is a critical target for c/EBPalpha in granulopoiesis, Mol Cell Biol 21 (2001) 3789-3806.
[74] K.K. Wong, X. Zou, K.T. Merrell, A.J. Patel, K.B. Marcu, S. Chellappan,K. Calame, v-Abl activates c-myc transcription through the E2F site, Mol Cell Biol 15 (1995) 6535-6544.
[75] S. Ishida, K. Shudo, S. Takada,K. Koike, A direct role of transcription factor E2F in c-myc gene expression during granulocytic and macrophage-like differentiation of HL60 cells, Cell Growth Differ 6 (1995) 229-237.
[76] S.L. Nutt, A.M. Morrison, P. Dorfler, A. Rolink,M. Busslinger, Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments, Embo J 17 (1998) 2319-2333.
[77] A. Misawa, H. Hosoi, A. Arimoto, T. Shikata, S. Akioka, T. Matsumura, P.J. Houghton,T. Sawada, N-Myc induction stimulated by insulin-like growth factor I through mitogen-activated protein kinase signaling pathway in human neuroblastoma cells, Cancer Res 60 (2000) 64-69.
[78] C.J. Thiele, C.P. Reynolds,M.A. Israel, Decreased expression of N-myc precedes retinoic acid-induced morphological differentiation of human neuroblastoma, Nature 313 (1985) 404-406.
[79] G. Han, B. Chang, M.J. Connor,N. Sidell, Enhanced potency of 9-cis versus all-trans-retinoic acid to induce the differentiation of human neuroblastoma cells, Differentiation 59 (1995) 61-69.
[80] G. Giannini, M.I. Dawson, X. Zhang,C.J. Thiele, Activation of three distinct RXR/RAR heterodimers induces growth arrest and differentiation of neuroblastoma cells, J Biol Chem 272 (1997) 26693-26701.
[81] H. Matsushima,E. Bogenmann, Expression of trkA cDNA in neuroblastomas mediates differentiation in vitro and in vivo, Mol Cell Biol 13 (1993) 7447-7456.
[82] E. Bogenmann, M. Torres,H. Matsushima, Constitutive N-myc gene expression inhibits trkA mediated neuronal differentiation, Oncogene 10 (1995) 1915-1925.
[83] F.A. Peverali, D. Orioli, L. Tonon, P. Ciana, G. Bunone, M. Negri,G. Della-Valle, Retinoic acid-induced growth arrest and differentiation of neuroblastoma cells are counteracted by N-myc and enhanced by max overexpressions, Oncogene 12 (1996) 457-462.
[84] E.F. Grady-Leopardi, M. Schwab, A.R. Ablin,W. Rosenau, Detection of N-myc oncogene expression in human neuroblastoma by in situ hybridization and blot analysis: relationship to clinical outcome, Cancer Res 46 (1986) 3196-3199.
[85] L. Fan, J. Iyer, S. Zhu, K.K. Frick, R.K. Wada, A.E. Eskenazi, P.E. Berg, N. Ikegaki, R.H. Kennett,C.N. Frantz, Inhibition of N-myc expression and induction of apoptosis by iron chelation in human neuroblastoma cells, Cancer Res 61 (2001) 1073-1079.
[86] N.E. Kohl, E. Legouy, R.A. DePinho, P.D. Nisen, R.K. Smith, C.E. Gee,F.W. Alt, Human N-myc is closely related in organization and nucleotide sequence to c-myc, Nature 319 (1986) 73-77.
[87] R.A. DePinho, E. Legouy, L.B. Feldman, N.E. Kohl, G.D. Yancopoulos,F.W. Alt, Structure and expression of the murine N-myc gene, Proc Natl Acad Sci U S A 83 (1986) 1827-1831.
[88] S. Hiller, S. Breit, Z.Q. Wang, E.F. Wagner,M. Schwab, Localization of regulatory elements controlling human MYCN expression, Oncogene 6 (1991) 969-977.
[89] L.E. Sivak, K.F. Tai, R.S. Smith, P.A. Dillon, G.M. Brodeur,W.L. Carroll, Autoregulation of the human N-myc oncogene is disrupted in amplified but not single-copy neuroblastoma cell lines, Oncogene 15 (1997) 1937-1946.
[90] R.K. Smith, K. Zimmerman, G.D. Yancopoulos, A. Ma,F.W. Alt, Transcriptional down-regulation of N-myc expression during B-cell development, Mol Cell Biol 12 (1992) 1578-1584.
[91] P.A. Zweidler-Mckay, H.L. Grimes, M.M. Flubacher,P.N. Tsichlis, Gfi-1 encodes a nuclear zinc finger protein that binds DNA and functions as a transcriptional repressor, Mol Cell Biol 16 (1996) 4024-4034.
[92] D.G. Tenen, R. Hromas, J.D. Licht,D.E. Zhang, Transcription factors, normal myeloid development, and leukemia, Blood 90 (1997) 489-519.
[93] M. Merika, A.J. Williams, G. Chen, T. Collins,D. Thanos, Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription, Mol Cell 1 (1998) 277-287.
[94] S.G. Tevosian, H.H. Shih, K.G. Mendelson, K.A. Sheppard, K.E. Paulson,A.S. Yee, HBP1: a HMG box transcriptional repressor that is targeted by the retinoblastoma family, Genes Dev 11 (1997) 383-396.
[95] R. Versteeg, C. van der Minne, A. Plomp, A. Sijts, A. van Leeuwen,P. Schrier, N-myc expression switched off and class I human leukocyte antigen expression switched on after somatic cell fusion of neuroblastoma cells, Mol Cell Biol 10 (1990) 5416-5423.
[96] K.A. Woodruff, J.D. Rosenblatt, T.B. Moore, R.H. Medzoyan, D.S. Pai, J.L. Noland, J.M. Yamashiro,R.K. Wada, Cell type-specific activity of the N-myc promoter in human neuroblastoma cells is mediated by a downstream silencer, Oncogene 10 (1995) 1335-1341.
[97] L.E. Sivak, G. Pont-Kingdon, K. Le, G. Mayr, K.F. Tai, B.T. Stevens,W.L. Carroll, A novel intron element operates posttranscriptionally To regulate human N-myc expression, Mol Cell Biol 19 (1999) 155-163.
[98] L. Xu, R. Wallen, V. Patel,R.A. DePinho, Role of first exon/intron sequences in the regulation of myc family oncogenic potency, Oncogene 8 (1993) 2547-2553.
[99] L. Xu, Y. Meng, R. Wallen,R.A. DePinho, Loss of transcriptional attenuation in N-myc is associated with progression towards a more malignant phenotype, Oncogene 11 (1995) 1865-1872.
[100] R.G. Keene, A. Mueller, R. Landick,L. London, Transcriptional pause, arrest and termination sites for RNA polymerase II in mammalian N- and c-myc genes, Nucleic Acids Res 27 (1999) 3173-3182.
[101] L. Xu, S.D. Morgenbesser,R.A. DePinho, Complex transcriptional regulation of myc family gene expression in the developing mouse brain and liver, Mol Cell Biol 11 (1991) 6007-6015.
[102] G. Krystal, M. Birrer, J. Way, M. Nau, E. Sausville, C. Thompson, J. Minna,J. Battey, Multiple mechanisms for transcriptional regulation of the myc gene family in small-cell lung cancer, Mol Cell Biol 8 (1988) 3373-3381.
[103] S.W. Hiebert, M. Blake, J. Azizkhan,J.R. Nevins, Role of E2F transcription factor in E1A-mediated trans activation of cellular genes, J Virol 65 (1991) 3547-3552.
[104] A. Fukamizu, M. Sagara, F. Sugiyama, H. Horiguchi, H. Kamma, T. Hatae, T. Ogata, K. Yagami,K. Murakami, Neuroectodermal tumors expressing c-, L-, and N-myc in transgenic mice that carry the E1A/E1B gene of human adenovirus type 12, J Biol Chem 269 (1994) 31252-31258.
[105] E. Santoni-Rugiu, J. Falck, N. Mailand, J. Bartek,J. Lukas, Involvement of Myc activity in a G(1)/S-promoting mechanism parallel to the pRb/E2F pathway, Mol Cell Biol 20 (2000) 3497-3509.
[106] G. Leone, J. DeGregori, R. Sears, L. Jakoi,J.R. Nevins, Myc and Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F, Nature 387 (1997) 422-426.
[107] G. Leone, R. Sears, E. Huang, R. Rempel, F. Nuckolls, C. Park, P. Giangrande, L. Wu, H.I. Saavedra, S.J. Field, M.A. Thompson, H. Yang, Y. Fujiwara, M.E. Greenberg, S. Orkin, C. Smith et al., Myc requires distinct e2f activities to induce s phase and apoptosis, Mol Cell 8 (2001) 105-113.
[108] H. Muller, A.P. Bracken, R. Vernell, M.C. Moroni, F. Christians, E. Grassilli, E. Prosperini, E. Vigo, J.D. Oliner,K. Helin, E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis, Genes Dev 15 (2001) 267-285.
[109] S. Ishida, E. Huang, H. Zuzan, R. Spang, G. Leone, M. West,J.R. Nevins, Role for E2F in control of both DNA replication and mitotic functions as revealed from DNA microarray analysis, Mol Cell Biol 21 (2001) 4684-4699.
[110] M.R. Adams, R. Sears, F. Nuckolls, G. Leone,J.R. Nevins, Complex transcriptional regulatory mechanisms control expression of the E2F3 locus, Mol Cell Biol 20 (2000) 3633-3639.
[111] R. Sears, K. Ohtani,J.R. Nevins, Identification of positively and negatively acting elements regulating expression of the E2F2 gene in response to cell growth signals, Mol Cell Biol 17 (1997) 5227-5235.
[112] S.M. Mac, C.A. D'Cunha,P.J. Farnham, Direct recruitment of N-myc to target gene promoters, Mol Carcinog 29 (2000) 76-86.
[113] A. Lasorella, M. Noseda, M. Beyna, Y. Yokota,A. Iavarone, Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins, Nature 407 (2000) 592-598.
[114] C. Bouchard, K. Thieke, A. Maier, R. Saffrich, J. Hanley-Hyde, W. Ansorge, S. Reed, P. Sicinski, J. Bartek,M. Eilers, Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27, Embo J 18 (1999) 5321-5333.
[115] I. Perez-Roger, S.H. Kim, B. Griffiths, A. Sewing,H. Land, Cyclins D1 and D2 mediate myc-induced proliferation via sequestration of p27(Kip1) and p21(Cip1), Embo J 18 (1999) 5310-5320.
[116] I. Dussault,V. Giguere, Differential regulation of the N-myc proto-oncogene by ROR alpha and RVR, two orphan members of the superfamily of nuclear hormone receptors, Mol Cell Biol 17 (1997) 1860-1867.
[117] L. Jackson-Grusby, C. Beard, R. Possemato, M. Tudor, D. Fambrough, G. Csankovszki, J. Dausman, P. Lee, C. Wilson, E. Lander,R. Jaenisch, Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation, Nat Genet 27 (2001) 31-39.
[118] J.M. Cheng, J.L. Hiemstra, S.S. Schneider, A. Naumova, N.K. Cheung, S.L. Cohn, L. Diller, C. Sapienza,G.M. Brodeur, Preferential amplification of the paternal allele of the N-myc gene in human neuroblastomas, Nat Genet 4 (1993) 191-194.
[119] B.C. Armstrong,G.W. Krystal, Isolation and characterization of complementary DNA for N-cym, a gene encoded by the DNA strand opposite to N-myc, Cell Growth Differ 3 (1992) 385-390.
[120] G.W. Krystal, B.C. Armstrong,J.F. Battey, N-myc mRNA forms an RNA-RNA duplex with endogenous antisense transcripts, Mol Cell Biol 10 (1990) 4180-4191.
[121] S.M. Hammond, A.A. Caudy,G.J. Hannon, Post-transcriptional gene silencing by double-stranded RNA, Nat Rev Genet 2 (2001) 110-119.
[122] L.E. Babiss,J.M. Friedman, Regulation of N-myc gene expression: use of an adenovirus vector to demonstrate posttranscriptional control, Mol Cell Biol 10 (1990) 6700-6708.
[123] D. Chagnovich, B.E. Fayos,S.L. Cohn, Differential activity of ELAV-like RNA-binding proteins in human neuroblastoma, J Biol Chem 271 (1996) 33587-33591.
[124] D. Chagnovich,S.L. Cohn, Binding of a 40-kDa protein to the N-myc 3'-untranslated region correlates with enhanced N-myc expression in human neuroblastoma, J Biol Chem 271 (1996) 33580-33586.
[125] D.L. Lazarova, B.A. Spengler, J.L. Biedler,R.A. Ross, HuD, a neuronal-specific RNA-binding protein, is a putative regulator of N-myc pre-mRNA processing/stability in malignant human neuroblasts, Oncogene 18 (1999) 2703-2710.
[126] N.S. Ball,P.H. King, Neuron-specific hel-N1 and HuD as novel molecular markers of neuroblastoma: a correlation of HuD messenger RNA levels with favorable prognostic features, Clin Cancer Res 3 (1997) 1859-1865.
[127] C.L. Jopling,A.E. Willis, N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells, Oncogene 20 (2001) 2664-2670.
[128] Y. Wakamatsu, Y. Watanabe, A. Shimono,H. Kondoh, Transition of localization of the N-Myc protein from nucleus to cytoplasm in differentiating neurons, Neuron 10 (1993) 1-9.
[129] S.L. Cohn, H. Salwen, M.W. Quasney, N. Ikegaki, J.M. Cowan, C.V. Herst, R.H. Kennett, S.T. Rosen, J.A. DiGiuseppe,G.M. Brodeur, Prolonged N-myc protein half-life in a neuroblastoma cell line lacking N-myc amplification, Oncogene 5 (1990) 1821-1827.
[130] M.M. Nau, B.J. Brooks, Jr., D.N. Carney, A.F. Gazdar, J.F. Battey, E.A. Sausville,J.D. Minna, Human small-cell lung cancers show amplification and expression of the N-myc gene, Proc Natl Acad Sci U S A 83 (1986) 1092-1096.
[131] G.M. Brodeur, F.A. Hayes, A.A. Green, J.T. Casper, J. Wasson, S. Wallach,R.C. Seeger, Consistent N-myc copy number in simultaneous or consecutive neuroblastoma samples from sixty individual patients, Cancer Res 47 (1987) 4248-4253.
[132] J.M. Ibson,P.H. Rabbitts, Sequence of a germ-line N-myc gene and amplification as a mechanism of activation, Oncogene 2 (1988) 399-402.
[133] D.P. Hollywood,H.C. Hurst, A novel transcription factor, OB2-1, is required for overexpression of the proto-oncogene c-erbB-2 in mammary tumour lines, Embo J 12 (1993) 2369-2375.
[134] Y. Hosokawa,A. Arnold, Mechanism of cyclin D1 (CCND1, PRAD1) overexpression in human cancer cells: analysis of allele-specific expression, Genes Chromosomes Cancer 22 (1998) 66-71.
[135] G.M. Brodeur, Genetics of embryonal tumours of childhood: retinoblastoma, Wilms' tumour and neuroblastoma, Cancer Surv 25 (1995) 67-99.
[136] I. Slavc, R. Ellenbogen, W.H. Jung, G.F. Vawter, C. Kretschmar, H. Grier,B.R. Korf, myc gene amplification and expression in primary human neuroblastoma, Cancer Res 50 (1990) 1459-1463.
[137] H.S. Chan, B.L. Gallie, G. DeBoer, G. Haddad, N. Ikegaki, J. Dimitroulakos, H. Yeger,V. Ling, MYCN protein expression as a predictor of neuroblastoma prognosis, Clin Cancer Res 3 (1997) 1699-1706.
[138] S. Han, R.K. Wada,N. Sidell, Differentiation of human neuroblastoma by phenylacetate is mediated by peroxisome proliferator-activated receptor gamma, Cancer Res 61 (2001) 3998-4002.

Figure 1. Regulatory sequences shared by the promoters of c-myc and N-myc

Figure 2. Location of potential regulatory elements and hypersensitive sites in the N-myc gene. DNaseI-hypersensitive sites have been determined in pre-B-cells, DMS-in-vivo-footprinting data have been obtained from human neuroblastoma cells. CR2 is a sequence of 15 bp that is conserved in the N-myc genes of man, mouse and chicken.

Figure 3. Hypothetical feedback loop between N-Myc and E2F proteins that may maintain N-myc expression in neuroblastomas.

Back . . .