Transformation Of Chicken Embryo Fibroblasts Research Articles

Cloning and characterisation of the mouse fra-2 gene.

Transcription factor AP-1 is comprised of multiple protein complexes that include members of a family of genes related to the proto-oncogene c-fos. In this report, we have extended the analysis of one member of this family, fos-related antigen-2 (fra-2), by isolating and characterising genomic and cDNA clones encoding the mouse fra-2 homolog. The overall gene structure (number and positions of introns) was similar to that of both the chicken fra-2 gene and other members of the fos family, and the relative positions of putative enhancers in the 5' regulatory region were well conserved between the mouse and chicken fra-2 genes. High levels of fra-2 mRNA were detected in ovary, stomach, small and large intestine, brain, lung and heart. The mouse Fra-2 protein showed 94% and 87.5% conservation with human and chicken Fra-2, respectively, and mouse Fra-2, like the chicken homolog, induced transformation of chicken embryo fibroblasts. The characterisation of the mouse fra-2 gene provides a basis for analysis of Fra-2 function in the whole animal.

Overexpression of Sox3 is Associated with Diminished Prognosis in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is a lethal malignancy lacking valid prognostic biomarkers. As a member of the High Mobility Group domain-containing DNA-binding proteins, Sox3 has been reported to induce oncogenic transformation of chicken embryo fibroblasts. However, the expression and prognostic value of Sox3 in ESCC remain unclear. A total of 30 pairs of ESCC with a corresponding non-neoplastic esophageal epithelium (NE) specimen were investigated for Sox3 expression using RT-PCR and western blot analysis. Tissue microarrays containing 118 ESCC and 30 NE samples were detected for Sox3 expression using immunohistochemical staining. The relationship of Sox3 staining with various clinicopathological characteristics and survival of patients was statistically analyzed. Sox3 expression in ESCC was 3.1- and 2.7-fold higher than in NE at mRNA (P < 0.001) and protein level (P < 0.001), respectively. Positive staining of Sox3 was observed in 77.1 % of the ESCC and 16.7 % of the NE samples (P < 0.001). High expression of Sox3 was significantly correlated with the regional lymph nodes metastasis (RLNM) (P = 0.022) and advanced TNM stage (P = 0.011). Moreover, high expression of Sox3 was significantly associated with poor overall survival (P < 0.001) and recurrence-free survival (P < 0.001) in ESCC patients. Both Sox3 expression (P < 0.001) and RLNM (P = 0.002) were independent prognostic factors for patients with ESCC. Sox3 might play a positive role in tumor development and could serve as an independent predictor of poor prognosis for ESCC.

Peroxisome Proliferator-activated Receptor γ (PPARγ) Mediates a Ski Oncogene-induced Shift from Glycolysis to Oxidative Energy Metabolism

Overexpression of the Ski oncogene induces oncogenic transformation of chicken embryo fibroblasts (CEFs). However, unlike most other oncogene-transformed cells, Ski-transformed CEFs (Ski-CEFs) do not display the classical Warburg effect. On the contrary, Ski transformation reduced lactate production and glucose utilization in CEFs. Compared with CEFs, Ski-CEFs exhibited enhanced TCA cycle activity, fatty acid catabolism through β-oxidation, glutamate oxidation, oxygen consumption, as well as increased numbers and mass of mitochondria. Interestingly, expression of PPARγ, a key transcription factor that regulates adipogenesis and lipid metabolism, was dramatically elevated at both the mRNA and protein levels in Ski-CEFs. Accordingly, PPARγ target genes that are involved in lipid uptake, transport, and oxidation were also markedly up-regulated by Ski. Knocking down PPARγ in Ski-CEFs by RNA interference reversed the elevated expression of these PPARγ target genes, as well as the shift to oxidative metabolism and the increased mitochondrial biogenesis. Moreover, we found that Ski co-immunoprecipitates with PPARγ and co-activates PPARγ-driven transcription.

Pleiotropic Action of AP-1 in v-Src-Transformed Cells

The activation of AP-1 is a hallmark of cell transformation by tyrosine kinases. In this study, we characterize the role of AP-1 proteins in the transformation of chicken embryo fibroblasts (CEF) by v-Src. In normal CEF, the expression of a dominant negative mutant of c-Jun (TAM67) induced senescence. In contrast, three distinct phenotypes were observed when TAM67 was expressed in v-Src-transformed CEF. While senescent cells were also present, the inhibition of AP-1 caused apoptosis in a fraction of the v-Src-transformed cells. In addition, cells containing lipid-rich vesicles accumulated, suggesting that a subpopulation of the v-Src-transformed cells underwent differentiation in response to the inhibition of AP-1. JunD and Fra-2 were the main components of this factor, while c-Jun accounted for a minor fraction of AP-1 in v-Src-transformed CEF. The downregulation of c-Jun expression by short hairpin RNA (shRNA) induced senescence in normal and v-Src-transformed cells. In contrast, a high incidence of apoptosis was caused by the downregulation of JunD, suggesting that it is required for the survival of v-Src-transformed CEF. Levels of the p53 tumor suppressor were elevated under conditions of JunD inhibition. Repression of p53 by shRNA enhanced the survival and anchorage-independent proliferation of v-Src-transformed CEF with JunD/AP-1 inhibition. The inhibition of Fra-2 had no visible phenotype in normal CEF but caused the appearance of lipid-rich vesicles in v-Src-transformed CEF. Therefore, AP-1 facilitated transformation by acting as a survival factor, by inhibiting premature entry into senescence, and by blocking the differentiation of v-Src-transformed CEF.

Akt Increases Sarcoplasmic Reticulum Ca2+ Cycling by Direct Phosphorylation of Phospholamban at Thr17

Cardiomyocytes adapt to physical stress by increasing their size while maintaining cell function. The serine/threonine kinase Akt plays a critical role in this process of adaptation. We previously reported that transgenic overexpression of an active form of Akt (Akt-E40K) in mice results in increased cardiac contractility and cell size, as well as improved sarcoplasmic reticulum (SR) Ca(2+) handling. Because it is not fully elucidated, we decided to study the molecular mechanism by which Akt-E40K overexpression improves SR Ca(2+) handling. To this end, SR Ca(2+) uptake and the phosphorylation status of phospholamban (PLN) were evaluated in heart extracts from wild-type and Akt-E40K mice and mice harboring inducible and cardiac specific knock-out of phosphatidylinositol-dependent kinase-1, the upstream activator of Akt. Moreover, the effect of Akt was assessed in vitro by overexpressing a mutant Akt targeted preferentially to the SR, and by biochemical assays to evaluate potential interaction with PLN. We found that when activated, Akt interacts with and phosphorylates PLN at Thr(17), the Ca(2+)-calmodulin-dependent kinase IIdelta site, whereas silencing Akt signaling, through the knock-out of phosphatidylinositol-dependent kinase-1, resulted in reduced phosphorylation of PLN at Thr(17). Furthermore, overexpression of SR-targeted Akt in cardiomyocytes improved Ca(2+) handling without affecting cell size. Thus, we describe here a new mechanism whereby the preferential translocation of Akt to the SR is responsible for enhancement of contractility without stimulation of hypertrophy.

Ski and SnoN, potent negative regulators of TGF-β signaling

Ski and the closely related SnoN were discovered as oncogenes by their ability to transform chicken embryo fibroblasts upon overexpression. While elevated expressions of Ski and SnoN have also been reported in many human cancer cells and tissues, consistent with their pro-oncogenic activity, emerging evidence also suggests a potential anti-oncogenic activity for both. In addition, Ski and SnoN have been implicated in regulation of cell differentiation, especially in the muscle and neuronal lineages. Multiple cellular partners of Ski and SnoN have been identified in an effort to understand the molecular mechanisms underlying the complex roles of Ski and SnoN. In this review, we summarize recent findings on the biological functions of Ski and SnoN, their mechanisms of action and how their levels of expression are regulated.

Disruption of the MYC transcriptional function by a small-molecule antagonist of MYC/MAX dimerization

Inhibition of MYC/MAX dimerization by a small-molecule antagonist (IIA6B17) has been shown to interfere with MYC-induced transformation of chick embryo fibroblasts, suggesting that the functional inhibitors of the MYC family of oncoproteins have potential as therapeutic agents. In the present study, a functional MYC reporter gene assay has been developed, using a luciferase gene construct under the control of the ornithine decarboxylase (ODC) gene promoter. This luciferase gene construct has been stably transfected into the MYCN amplified neuroblastoma cell line (NGP) and MYCC-overexpressed neuroepithelioma cell line (NB100). After exposure of the cell lines to IIA6B17 for 24 h, a significant reduction of luciferase activity was only observed in the NB100 cells, with IC50 values of approximately 28+/-9 microM, indicating that IIA6B17 has cell line-specific activity which may be selective for individual members of the MYC family.

Oncogenic signaling of class I PI3K isoforms

The catalytic subunits of class I PI3Ks comprise four isoforms: p110alpha, p110beta, p110delta and p110gamma. Cancer-specific gain-of-function mutations in p110alpha have been identified in various malignancies. Cancer-specific mutations in the non-alpha isoforms of class I PI3K have not yet been identified, however overexpression of either wild-type p110beta, p110gamma or p110delta is sufficient to induce cellular transformation in chicken embryo fibroblasts. The mechanism whereby these non-alpha isoforms of class I mediate oncogenic signals is unknown. Here we show that potently transforming class I isoforms signal via Akt/mTOR, inhibit GSK3beta and cause degradation of FoxO1. A functional Erk pathway is required for p110gamma and p110beta transformation but not for transformation by p110delta or the H1047R mutant of p110alpha. Transformation and signaling by p110gamma and p110beta are sensitive to loss of interaction with Ras, which acts as a membrane anchor. Mutations in the C2 domain of p110delta reduce transformation, most likely by interfering with membrane association. Several small molecule inhibitors potently and specifically inhibit the oncogenic signaling and transformation of each of the class I PI3K, and, when used in combination with MEK inhibitors, can additively reduce the transformation induced by p110beta and p110gamma.

Rare cancer-specific mutations in PIK3CA show gain of function

Fifteen rare cancer-derived mutants of PIK3CA, the gene coding for the catalytic subunit p110alpha of phosphatidylinositol 3-kinase (PI3K), were examined for their biological and biochemical properties. Fourteen of these mutants show a gain of function: they induce rapamycin-sensitive oncogenic transformation of chicken embryo fibroblasts, constitutively activate Akt and TOR-mediated signaling, and show enhanced lipid kinase activity. Mapping of these mutants on a partial structural model of p110alpha suggests three groups of mutants, defined by their location in distinct functional domains of the protein. We hypothesize that each of these three groups induces a gain of PI3K function by a different molecular mechanism. Mutants in the C2 domain increase the positive surface charge of this domain and therefore may enhance the recruitment of p110alpha to cellular membranes. Mutants in the helical domain map to a contiguous surface of the protein and may affect the interaction with other protein(s). Mutants in the kinase domain are located near the hinge of the activation loop. They may alter the position and mobility of the activation loop. Arbitrarily introduced mutations that have no detectable phenotype map either to the interior of the protein or are positioned on a surface region that lies opposite to the exposed surfaces containing gain-of-function mutants. Engineered mutants that exchange acidic or neutral residues for basic residues on the critical surfaces show a gain of function.

Nuclear addressing provides a clue for the transforming activity of amino‐truncated CCN3 proteins

CCN3 is a founding member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators. These secreted proteins are key regulators in embryonic development, and are associated with severe pathologies including fibrotic diseases and cancers. CCN3 was discovered as a MAV integration site in an avian nephroblastoma. Previous work established that the amino-truncated protein expressed in this tumor was inducing morphological transformation of chicken embryo fibroblasts, whereas the full-length secreted CCN3 protein was inhibiting cell growth. Amino-truncated variants were identified in cancer cell lines. Since the lack of signal peptide was expected to alter the fate of the truncated proteins, we hypothesized that modifications of CCN3 subcellular addressing could be responsible for the oncogenic activities of CCN3. The CCN proteins are composed of four structural modules (IGFBP, TSP1, VWC, and CT). We report that amino-truncated variants of CCN3 are addressed to the nucleus and that the carboxyterminal (CT) module of CCN3 is responsible for the nuclear addressing. Furthermore, our data identify nuclear CCN3 variants as potential transcriptional regulators. In this context, the CT module confers on nuclear CCN3 proteins a negative regulatory effect on transcription. We propose that the nuclear localization of amino-truncated CCN3 proteins be correlated to oncogenicity.

The suppression of SH3BGRL is important for v-Rel-mediated transformation

The v-rel oncogene is the most efficient transforming member of the Rel/NF-kappaB family of transcription factors. v-Rel induces avian and mammalian lymphoid cell tumors and transforms chicken embryo fibroblasts in culture by the aberrant regulation of genes under the control of Rel/NF-kappaB proteins. Here we report that the expression of SH3BGRL, a member of the SH3BGR (SH3 domain-binding glutamic acid-rich) family of proteins, is downregulated in v-Rel-expressing fibroblasts, lymphoid cells, and splenic tumor cells. Chromatin immunoprecipitation experiments demonstrated that v-Rel binds to the sh3bgrl promoter in transformed cells. Coexpression of SH3BGRL with v-Rel in primary splenic lymphocytes reduced the number of colonies formed by 76%. Mutations in the predicted SH3-binding domain of SH3BGRL abolished the suppressive effect on v-Rel transformation and resulted in colony numbers comparable to those formed by v-Rel alone. However, mutations in the predicted EVH1-binding domain of SH3BGRL only had a modest effect on suppression of v-Rel transformation. This study provides the first example of a gene that is downregulated in v-Rel-expressing cells that also plays a role in v-Rel transformation.

Inhibition of Protein Synthesis by Y Box-Binding Protein 1 Blocks Oncogenic Cell Transformation

The multifunctional Y box-binding protein 1 (YB-1) is transcriptionally repressed by the oncogenic phosphoinositide 3-kinase (PI3K) pathway (with P3K as an oncogenic homolog of the catalytic subunit) and, when reexpressed with the retroviral vector RCAS, interferes with P3K- and Akt-induced transformation of chicken embryo fibroblasts. Retrovirally expressed YB-1 binds to the cap of mRNAs and inhibits cap-dependent and cap-independent translation. To determine the requirements for the inhibitory role of YB-1 in P3K-induced transformation, we conducted a mutational analysis, measuring YB-1-induced interference with transformation, subcellular localization, cap binding, mRNA binding, homodimerization, and inhibition of translation. The results show that (i) interference with transformation requires RNA binding and a C-terminal domain that is distinct from the cytoplasmic retention domain, (ii) interference with transformation is tightly correlated with inhibition of translation, and (iii) masking of mRNAs by YB-1 is not sufficient to block transformation or to inhibit translation. We identified a noncanonical nuclear localization signal (NLS) in the C-terminal half of YB-1. A mutant lacking the NLS retains its ability to interfere with transformation, indicating that a nuclear function is not required. These results suggest that YB-1 interferes with P3K-induced transformation by a specific inhibition of translation through its RNA-binding domain and a region in the C-terminal domain. Potential functions of the C-terminal region are discussed.

V-Jun downregulates the alpha 2 (I) collagen target gene indirectly through Sp1/3

Transformation of chick embryo fibroblasts (CEFs) by the v-Jun oncoprotein correlates with a downregulation of the alpha 2 (I) collagen gene. To investigate whether this gene constitutes a direct target of v-Jun, an analysis of a large proximal fragment of the promoter, extending from position -1080 to +109, was performed. Transient transfections with -1080/+109 and deleted derivatives revealed that a short proximal fragment, -433/+11, is the target for repression by v-Jun. Extensive analysis, conducted in CEFs and in Sp1/3-deficient Drosophila SL2 cells, further showed that (i) high constitutive activity of -433/+11 requires a direct binding of the ubiquitous Sp1 and/or Sp3 transcription factors acting on two distinct motifs, that is, a proximal TCC-rich region and an upstream GC box, and that (ii) repression by v-Jun does not require any direct binding of the oncoprotein to the DNA, but an indirect binding within a v-Jun-Sp1/3-DNA chromatin-associated complex. This situation is reminiscent of a situation previously reported with the tata-less, SPARC (secreted protein, acidic, and rich in cysteine) target promoter that regulates the expression of another extracellular matrix component in the same model of cell transformation. Taken together, these data reinforce the view that, at least in CEFs, v-Jun downregulates a family of direct target genes by binding to the DNA indirectly through Sp1/3.

Proteasomal degradation of the FoxO1 transcriptional regulator in cells transformed by the P3k and Akt oncoproteins.

The P3k oncoprotein [homolog of the catalytic subunit p110alpha of class 1A phosphoinositide 3-kinase (PI3K)] and its downstream effector Akt induce oncogenic transformation in cultures of chicken embryo fibroblasts (CEF). The winged helix transcription factor FoxO1 is a growth-attenuating and proapoptotic protein and serves as a substrate of Akt. Here we show that FoxO1 expression is constitutively suppressed in CEF transformed by P3k or Akt. The FoxO1 protein level is high in serum-starved normal CEF, but platelet-derived growth factor treatment induces rapid phosphorylation and disappearance of FoxO1. PI3K inhibitors or the proteasome inhibitor lactacystin interfere with this process. These data suggest that phosphorylation-dependent degradation of FoxO1 by means of proteasomes plays a role in oncogenic transformation by P3k and Akt. A dominant negative mutant of FoxO1 containing the repressor domain of the Drosophila Engrailed protein induces partial oncogenic transformation of CEF and interferes with FoxO1-dependent transcriptional activation. The FoxG1 oncoprotein also inhibits transcriptional activation by FoxO1. Inhibition of FoxO1, albeit by different mechanisms, appears to be a common denominator of the PI3K and FoxG1 oncogenic pathways.

Expression of c- maf and mafB genes in the skin during rat embryonic development

The maf oncogene (v- maf) was initially identified in an avian oncogenic retrovirus, AS42, which induces musculoaponeurotic fibrosarcoma in vivo and transforms chicken embryo fibroblasts in vitro. Genes of the maf family have important roles in embryonic development and cellular differentiation. Both genes are expressed in a wide variety of tissues including spleen, kidney, lens and liver. The present study was performed to analyze expression of c- maf-1 and mafB genes in skin of embryonic stages from 15 days onwards using in situ hybridization. Expression of c- maf mRNA was first detected on embryonic day (ED) 16 in the nuclei of cells in the basal layer in developing epidermis. On ED 19, high expression was detected in the nucleus of basal keratinocytes and developing hair germs. On postnatal day (PD) 3, expression of c- maf had disappeared in epidermis and hair follicles. MafB showed similar expression patterns as c- maf. Our findings indicate that c- maf and mafB are involved in embryonic development of epidermis and hair follicles.

Microarray analysis identifies Autotaxin, a tumour cell motility and angiogenic factor with lysophospholipase D activity, as a specific target of cell transformation by v-Jun.

We have used chicken cDNA microarrays to investigate gene-expression changes induced during transformation of chick embryo fibroblasts (CEF) by the viral Jun oncoprotein encoded by ASV17. This analysis reveals that v-Jun induces increases and decreases of varying magnitude in the expression of genes involved in diverse cellular functions, most of which have not been detected in previous screens for putative v-Jun targets. In all, 27 individual genes were identified, whose expression is increased threefold or more in v-Jun-transformed cells, including genes involved in energy generation, protein synthesis, and gene transcription. Interestingly, this group includes the hypoxia-inducible factor-1 alpha (Hif-1alpha) transcription factor and the glycolytic enzyme enolase, suggesting that adaptation to hypoxia could play a role in tumorigenesis by v-Jun. We also identified 32 genes whose expression is decreased threefold or more, including chaperones, components of the cytoskeleton, and, unexpectedly, DNA replication factors. The gene whose expression is upregulated most dramatically (approximately 100-fold) encodes Autotaxin (ATX), a secreted tumor motility-promoting factor with lysophospholipase D activity. Strikingly, v-Jun-transformed CEF secrete catalytically active ATX and chemotactic activity, which can be detected in conditioned medium. ATX is not detectably expressed in normal CEF or CEF transformed by the v-Src or v-Myc oncoproteins, indicating that induction of this putative autocrine/paracrine factor is a specific consequence of cell transformation by v-Jun. ATX has been implicated in both angiogenesis and invasion, and could therefore play an important role in tumorigenesis by v-Jun in vivo.

The Transforming Activity of Ski and SnoN Is Dependent on Their Ability to Repress the Activity of Smad Proteins

The regulation of cell growth and differentiation by transforming growth factor-beta (TGF-beta) is mediated by the Smad proteins. In the nucleus, the Smad proteins are negatively regulated by two closely related nuclear proto-oncoproteins, Ski and SnoN. When overexpressed, Ski and SnoN induce oncogenic transformation of chicken embryo fibroblasts. However, the mechanism of transformation by Ski and SnoN has not been defined. We have previously reported that Ski and SnoN interact directly with Smad2, Smad3, and Smad4 and repress their ability to activate TGF-beta target genes through multiple mechanisms. Because Smad proteins are tumor suppressors, we hypothesized that the ability of Ski and SnoN to inactivate Smad function may be responsible for their transforming activity. Here, we show that the receptor regulated Smad proteins (Smad2 and Smad3) and common mediator Smad (Smad4) bind to different regions in Ski and SnoN. Mutation of both regions, but not each region alone, markedly impaired the ability of Ski and SnoN to repress TGF-beta-induced transcriptional activation and cell cycle arrest. Moreover, when expressed in chicken embryo fibroblasts, mutant Ski or SnoN defective in binding to the Smad proteins failed to induce oncogenic transformation. These results suggest that the ability of Ski and SnoN to repress the growth inhibitory function of the Smad proteins is required for their transforming activity. This may account for the resistance to TGF-beta-induced growth arrest in some human cancer cell lines that express high levels of Ski or SnoN.

V-Jun downregulates the SPARC target gene by binding to the proximal promoter indirectly through Sp1/3.

Transformation of chick embryo fibroblasts by the v-Jun oncoprotein correlates with a downregulation of the extracellular matrix protein SPARC and repression of the corresponding mRNA. Repression of SPARC contributes to the oncogenic process by facilitating tumor development in vivo. A proximal promoter fragment, designated -124/+16, is responsible for high constitutive activity of the SPARC gene and is the target of repression by v-Jun. In this paper, using electrophoretic mobility shift and pull-down assays in vitro, and transient transfections and chromatin immunoprecipitation assays in Sp1/3-deficient Drosophila SL2 cells and in chick embryo fibroblasts, we show that (i) Sp1 and/or Sp3 is required for constitutive activation of SPARC transcription, by binding directly to the GGA-rich -92/-57 fragment; and (ii) v-Jun does not bind -124/+16 directly, but binds to the GGA-rich fragment indirectly, most likely through a physical interaction with Sp1/3. Moreover, a transactivation-proficient v-Jun derivative, designated v-Jun/cebp/glz, which cannot bind Jun DNA motifs anymore and cannot heterodimerize, is still capable of downregulating SPARC efficiently. Taken together, these data strongly suggest that v-Jun downregulates SPARC through the formation of a DNA-Sp1/3-v-Jun, chromatin-associated complex.

Partial oncogenic transformation of chicken embryo fibroblasts by Jun dimerization protein 2, a negative regulator of TRE- and CRE-dependent transcription.

Jun dimerization protein 2 (JDP2) was identified as a bZIP protein that forms dimers with Jun proteins. JDP2 represses transcriptional activation of reporter constructs containing 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive elements (TRE) or cyclic AMP responsive elements (CRE). JDP2, overexpressed by the avian retroviral vector RCAS, induces partial oncogenic transformation of chicken embryo fibroblasts. JDP2-expressing cells form multilayered foci in monolayer cultures but do not show anchorage-independent growth. Both the carboxyl and the amino terminus of JDP2 are required for the transforming activity. Chimeric constructs of JDP2 carrying the leucine zipper domain of Fos, GCN4 or EB1 fail to transform CEF. The leucine zipper of Fos mediates only heterodimerization; it cannot homodimerize. In contrast, the leucine zippers of GCN4 and of EB1 exclusively homodimerize and do not form dimers with other bZip proteins. The results with the JDP2 chimeras suggest that the JDP2 homodimer and the JDP2/Jun heterodimer (or other bZip heterodimers formed with the Fos leucine zipper) are nontransforming, leaving as possible transforming combination the JDP2/Fos heterodimer. The unexpected transforming activity of a negative regulator of TRE- and CRE-dependent transcription raises an important question concerning the mechanisms of transformation by the related bZIP proteins Jun and Fos that address the same target sequences.

The MYCN oncoprotein as a drug development target

The transcription factor and proto-oncogene MYCN is reviewed as a potential specific target for cancer therapy. Amplification of MYCN is frequently found in a number of advanced-stage tumours, including neuroblastoma (25%), small cell lung cancers (7%), alveolar rhabdomyosarcoma and retinoblastoma. It is associated with rapid tumour progression and poor outcome in human neuroblastoma. MYCN is a member of the myc family of proto-oncogenes which encode nuclear proteins that form heterodimers with MAX protein through their conserved HLHZip domains. The MYC/MAX complexes transactivate a number of MYC-target genes in a sequence-specific manner. MYC-MAX interaction is essential for MYC-induced cell cycle progression, cellular transformation, and transcriptional activation. A causal link between the transformed phenotype and MYCN has been established by a range of in vitro and in vivo studies, including a transgenic model of neuroblastoma in which MYCN overexpression is targeted to neuronal tissue by the use of a tyrosine hydroxylase promoter. Downregulation of MYCN expression either by antisense treatment targeted against MYCN mRNA or by retinoids has been shown to decrease proliferation and/or induce neuronal differentiation of neuroblastoma cells. Inhibition of MYC-MAX dimerisation by small-molecule antagonists has recently been shown to interfere with MYC-induced transformation of chick embryo fibroblasts, indicating that functional inhibitors of the MYC family of oncoproteins have potential as therapeutic agents. Finally, we describe the development and validation of a functional MYCN reporter gene assay using neuroblastoma cells (NGP) which have been stably transfected with a luciferase gene construct under control of the ornithine decarboxylase gene promoter. This assay has been used for a pilot screen of 2800 compounds from the Cancer Research-UK collection, identifying five compounds showing a consistent significant reduction of MYCN-dependent luciferase activity (>50%) in repeated screens. This cell-based, MYCN reporter gene assay will be scaled up for high throughput screens of compound libraries and will aid in the future development of specific therapeutic strategies in neuroblastoma and other tumours in which MYCN amplification has been implicated.