Neuronal Differentiation Of Neuroblastoma Cells Research Articles

KDM2B regulates choline kinase expression and neuronal differentiation of neuroblastoma cells.

The process of neuronal differentiation is associated with neurite elongation and membrane biogenesis, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. During neuroblast differentiation, the transcription of two genes involved in PtdCho biosynthesis are stimulated: Chka gene for choline kinase (CK) alpha isoform and Pcyt1a gene for CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. Here we show that CKα is essential for neuronal differentiation. In addition, we demonstrated that KDM2B regulates CKα expression and, as a consequence, neuronal differentiation. This factor is up-regulated in the course of the neuroblasts proliferative and undifferentiated state and down-regulated during differentiation induced by retinoic acid (RA). During proliferation, KDM2B binds to the Box2 located in the Chka promoter repressing its transcription. Interestingly, KDM2B knockdown enhances the levels of CKα expression in neuroblast cells and induces neuronal differentiation even in the absence of RA. These results suggest that KDM2B is required for the appropriate regulation of CKα during neuronal differentiation and to the maintaining of the undifferentiated stage of neuroblast cells.

Radiation-Induced Calreticulin Expression Leads to MYCN Suppression and Neuronal Differentiation in Neuroblastoma Cells

Radiation-Induced Calreticulin Expression Leads to MYCN Suppression and Neuronal Differentiation in Neuroblastoma Cells

Synthetic cannabinoids enhance neuronal differentiation in neuroblastoma cells at in vivo relevant concentrations

Synthetic cannabinoids enhance neuronal differentiation in neuroblastoma cells at in vivo relevant concentrations

CHL1 gene acts as a tumor suppressor in human neuroblastoma.

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system that accounts for 15% of pediatric cancer deaths. A distal portion of human chromosome 3p is often deleted in neuroblastoma, this region may contain one or more putative tumor suppressor genes. A 2.54 Mb region at 3p26.3 encompassing the smallest region of deletion pinpointed CHL1 gene, the locus for neuronal cell adhesion molecule close homolog of L1. We found that low CHL1 expression predicted poor outcome in neuroblastoma patients. Here we have used two inducible cell models to analyze the impact of CHL1 on neuroblastoma biology. Over-expression of CHL1 induced neurite-like outgrowth and markers of neuronal differentiation in neuroblastoma cells, halted tumor progression, inhibited anchorage-independent colony formation, and suppressed the growth of human tumor xenografts. Conversely, knock-down of CHL1 induced neurite retraction and activation of Rho GTPases, enhanced cell proliferation and migration, triggered colony formation and anchorage-independent growth, accelerated growth in orthotopic xenografts mouse model. Our findings demonstrate unambiguously that CHL1 acts as a regulator of proliferation and differentiation of neuroblastoma cells through inhibition of the MAPKs and Akt pathways. CHL1 is a novel candidate tumor suppressor in neuroblastoma, and its associated pathways may represent a promising target for future therapeutic interventions.

VEGF expression correlates with neuronal differentiation and predicts a favorable prognosis in patients with neuroblastoma

Neuroblastoma (NB) is a childhood cancer with a low survival rate and great metastatic potential. Vascular endothelial growth factor (VEGF), an angiogenesis factor, has been found to be involved in CRT-related neuronal differentiation of NB cells. In this study, we further confirmed the role VEGF in NB through mouse xenograft model and clinical analysis from NB patients. In xenograft experiments, CRT overexpression effectively inhibited the tumor growth. In addition, the mRNA and protein levels of VEGF and differentiation marker GAP-43 were upregulated by induced CRT expression. However, no significant correlation between the expression level of VEGF and microvessel density was observed in human NB tumors, suggesting a novel mechanism of VEGF participating in NB tumorigenesis through an angiogenesis-independent pathway. In NB patients’ samples, mRNA expression levels of CRT and VEGF were positively correlated. Furthermore, positive VEGF expression by immunostaining of NB tumors was found to correlate well with histological grade of differentiation and predicted a favorable prognosis. In conclusion, our findings suggest that VEGF is a favorable prognostic factor of NB and might affect NB tumor behavior through CRT-driven neuronal differentiation rather than angiogenesis that might shed light on a novel therapeutic strategy to improve the outcome of NB.

Fighting Neuroblastomas with NBAT1.

Fighting Neuroblastomas with NBAT1.

Genome-wide analysis of HOXC9-induced neuronal differentiation of neuroblastoma cells.

Induction of differentiation is a therapeutic strategy in neuroblastoma, a common pediatric cancer of the sympathetic nervous system. The homeobox protein HOXC9 is a key regulator of neuroblastoma differentiation. To gain a molecular understanding of the function of HOXC9 in promoting differentiation of neuroblastoma cells, we conducted a genome-wide analysis of the HOXC9-induced differentiation program by microarray gene expression profiling and chromatin immunoprecipitation in combination with massively parallel sequencing (ChIP-seq). Here we describe in detail the experimental system, methods, and quality control for the generation of the microarray and ChIP-seq data associated with our recent publication [1].

GLS2 is transcriptionally regulated by p73 and contributes to neuronal differentiation

The amino acid Glutamine is converted into Glutamate by a deamidation reaction catalyzed by the enzyme Glutaminase (GLS). Two isoforms of this enzyme have been described, and the GLS2 isoform is regulated by the tumor suppressor gene p53. Here, we show that the p53 family member TAp73 also drives the expression of GLS2. Specifically, we demonstrate that TAp73 regulates GLS2 during retinoic acid-induced terminal neuronal differentiation of neuroblastoma cells, and overexpression or inhibition of GLS2 modulates neuronal differentiation and intracellular levels of ATP. Moreover, inhibition of GLS activity, by removing Glutamine from the growth medium, impairs in vitro differentiation of cortical neurons. Finally, expression of GLS2 increases during mouse cerebellar development. Although, p73 is dispensable for the in vivo expression of GLS2, TAp73 loss affects GABA and Glutamate levels in cortical neurons. Together, these findings suggest a role for GLS2 acting, at least in part, downstream of p73 in neuronal differentiation and highlight a possible role of p73 in regulating neurotransmitter synthesis.

Type III TGF-β receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma

Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the pediatric tumor neuroblastoma remain unclear. Comparison of mRNA from benign neuroblastic tumors and neuroblastomas revealed that expression of the type III TGF-β receptor (TGFBR3) decreases with advancing stage of neuroblastoma and this loss correlates with a poorer prognosis. Patients with MYCN oncogene amplification and low TGFBR3 expression were more likely to have an adverse outcome. In vitro, TβRIII expression was epigenetically suppressed by MYCN-mediated recruitment of histone deacetylases to regions of the TGFBR3 promoter. TβRIII bound FGF2 and exogenous FGFR1, which promoted neuronal differentiation of neuroblastoma cells. TβRIII and FGF2 cooperated to induce expression of the transcription factor inhibitor of DNA binding 1 via Erk MAPK. TβRIII-mediated neuronal differentiation suppressed cell proliferation in vitro as well as tumor growth and metastasis in vivo. These studies characterize a coreceptor function for TβRIII in FGF2-mediated neuronal differentiation, while identifying potential therapeutic targets and clinical biomarkers for neuroblastoma.

The transcription factor activating protein 2 beta (TFAP2B) mediates neuronal differentiation in neuroblastoma cells

We here aimed to investigate the role of the transcription factor activating protein 2 beta (TFAP2B) in neuroblastoma pathogenesis. TFAP2 is a family of transcription factors that play a crucial role in embryonic differentiation and development. Oligonucleotide-microarray analysis of 649 neuroblastoma specimens demonstrated that TFAP2B downregulation is associated with unfavourable prognostic markers such as disseminated stage 4, age > 18 months, MYCN amplification, unfavourable gene expression-based classification and adverse patient outcome.

Abstract 5041: The type III TGF-beta receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma.

Abstract Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the pediatric tumor neuroblastoma remain unclear. Here we report that expression of type III TGF-beta receptor (TβRIII) mRNA and protein decreases with advancing stage of neuroblastoma and positively correlates with prognosis. TβRIII expression is epigenetically suppressed by MYCN oncogene amplification and TβRIII expression can be used as a prognostic marker in neuroblastoma patients with MYCN amplification. TβRIII expression in neuroblastoma cells promotes neuronal differentiation and enhances the differentiating effects of FGF2 treatment. Mechanistically, glycosaminoglycan modifications on TβRIII bind FGF2 and FGFR1 to promote neuronal differentiation via Erk MAPK and the transcription factor ID1. TβRIII-mediated differentiation suppresses tumor cell proliferation in vitro and in vivo. These studies characterize a novel co-receptor function for TβRIII in FGF2-mediated neuronal differentiation of neuroblastoma cells, while identifying potential therapeutic targets and clinical biomarkers for advanced-stage disease. More generally, our results suggest that the targeting of growth factor receptors and downstream signaling pathways may prove useful in promoting neuronal differentiation to suppress neuroblastoma tumor growth. Citation Format: Erik H. Knelson, Angela L. Gaviglio, Alok K. Tewari, Michael B. Armstrong, Andrew B. Nixon, Mark D. Starr, Karthikeyan Mythreye, Gerard C. Blobe. The type III TGF-beta receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5041. doi:10.1158/1538-7445.AM2013-5041

Functional Dissection of HOXD Cluster Genes in Regulation of Neuroblastoma Cell Proliferation and Differentiation

Retinoic acid (RA) can induce growth arrest and neuronal differentiation of neuroblastoma cells and has been used in clinic for treatment of neuroblastoma. It has been reported that RA induces the expression of several HOXD genes in human neuroblastoma cell lines, but their roles in RA action are largely unknown. The HOXD cluster contains nine genes (HOXD1, HOXD3, HOXD4, and HOXD8-13) that are positioned sequentially from 3′ to 5′, with HOXD1 at the 3′ end and HOXD13 the 5′ end. Here we show that all HOXD genes are induced by RA in the human neuroblastoma BE(2)-C cells, with the genes located at the 3′ end being activated generally earlier than those positioned more 5′ within the cluster. Individual induction of HOXD8, HOXD9, HOXD10 or HOXD12 is sufficient to induce both growth arrest and neuronal differentiation, which is associated with downregulation of cell cycle-promoting genes and upregulation of neuronal differentiation genes. However, induction of other HOXD genes either has no effect (HOXD1) or has partial effects (HOXD3, HOXD4, HOXD11 and HOXD13) on BE(2)-C cell proliferation or differentiation. We further show that knockdown of HOXD8 expression, but not that of HOXD9 expression, significantly inhibits the differentiation-inducing activity of RA. HOXD8 directly activates the transcription of HOXC9, a key effector of RA action in neuroblastoma cells. These findings highlight the distinct functions of HOXD genes in RA induction of neuroblastoma cell differentiation.

Nuclear GRP75 Binds Retinoic Acid Receptors to Promote Neuronal Differentiation of Neuroblastoma

Retinoic acid (RA) has been approved for the differentiation therapy of neuroblastoma (NB). Previous work revealed a correlation between glucose-regulated protein 75 (GRP75) and the RA-elicited neuronal differentiation of NB cells. The present study further demonstrated that GRP75 translocates into the nucleus and physically interacts with retinoid receptors (RARα and RXRα) to augment RA-elicited neuronal differentiation. GRP75 was required for RARα/RXRα-mediated transcriptional regulation and was shown to reduce the proteasome-mediated degradation of RARα/RXRαin a RA-dependent manner. More intriguingly, the level of GRP75/RARα/RXRα tripartite complexes was tightly associated with the RA-induced suppression of tumor growth in animals and the histological grade of differentiation in human NB tumors. The formation of GRP75/RARα/RXRα complexes was intimately correlated with a normal MYCN copy number of NB tumors, possibly implicating a favorable prognosis of NB tumors. The present findings reveal a novel function of nucleus-localized GRP75 in actively promoting neuronal differentiation, delineating the mode of action for the differentiation therapy of NB by RA.

HOXC9 Links Cell-Cycle Exit and Neuronal Differentiation and Is a Prognostic Marker in Neuroblastoma

Differentiation status in neuroblastoma strongly affects clinical outcomes and inducing differentiation is a treatment strategy in this disease. However, the molecular mechanisms that control neuroblastoma differentiation are not well understood. Here, we show that high-level HOXC9 expression is associated with neuroblastoma differentiation and is prognostic for better survival in neuroblastoma patients. HOXC9 induces growth arrest and neuronal differentiation in neuroblastoma cells by directly targeting both cell-cycle-promoting and neuronal differentiation genes. HOXC9 expression is upregulated by retinoic acid (RA), and knockdown of HOXC9 expression confers resistance to RA-induced growth arrest and differentiation. Moreover, HOXC9 expression is epigenetically silenced in RA-resistant neuroblastoma cells, and forced HOXC9 expression is sufficient to inhibit their proliferation and tumorigenecity. These findings identified HOXC9 as a key regulator of neuroblastoma differentiation and suggested a therapeutic strategy for RA-resistant neuroblastomas through epigenetic activation of HOXC9 expression.

Abstract 2025: REST and SCFβ-TRCP expression determine the response of neuroblastoma cells to retinoic acid

Abstract BACKGROUND: Retinoic acid (RA) is a mainstay of neuroblastoma treatment as it promotes tumor differentiation. However, the mechanism underlying RA-mediated neuronal differentiation is not well understood. The purpose of this study is to better define the molecular targets through which RA promotes differentiation in neuroblastoma cells. The RE-1 Silencing Transcription Factor (REST) is a repressor of neuronal genes. It is expressed in embryonic stem cells and neural progenitors, and is downregulated during neurogenesis. REST is overexpressed in many neuroblastoma tumor samples and cell lines, and its expression is associated with an undifferentiated phenotype. Since REST is critical for neuronal differentiation, we hypothesized that RA promotes differentiation in neuroblastoma cell lines by downregulating REST expression. METHODS AND RESULTS: Quantitative reverse transcription polymerase chain reaction, immunofluorescence assay and western blotting revealed REST expression to be elevated in a number of human neuroblastoma tumors and cell lines. Treatment with RA promoted neuronal differentiation in some but not all cell lines. In RA-responsive cells, induction of neuronal differentiation was associated with a decline in REST protein levels and upregulation of REST-target gene expression. However, REST protein levels were elevated upon RA treatment in resistant cells, REST transcript was upregulated in both RA-sensitive and resistant cells as expected. These results suggested that RA downregulated REST through post-transcriptional mechanisms, which was confirmed by measurement of REST protein levels in the presence of cycloheximide. Treatment with MG-132 blocked RA-mediated degradation of REST, indicating the involvement of the proteasome. The E3-ligase, SCFβ-TRCP, is known to be involved in REST ubiquitination and degradation. Low levels SCFβ-TRCP were associated with elevated REST protein in a number of human neuroblastoma tumors and cell lines. We observed that SCFβ-TRCP transcript and protein were elevated upon RA-treatment in responsive cell lines but not in RA-resistant cells with high REST levels. Ectopic expression of SCFβ-TRCP promoted REST ubiquitination and degradation in RA-resistant cells and formed a complex with ubiquitinated REST as determined by immunoprecipitation and co-immunoprecipitation experiments. Since these events were associated with induction of neuronal differentiation, our findings suggest that a failure to degrade REST blocks neuronal differentiation upon RA treatment. CONCLUSION: Our study indicates that deranged proteasomal machinery may contribute to maintenance of REST expression and the blockade of neuronal differentiation in neuroblastoma cells. Agents with potential to upregulate SCFβ-TRCP expression may have therapeutic value in patients that fail to respond to RA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2025. doi:10.1158/1538-7445.AM2011-2025

CAMTA1, a 1p36 Tumor Suppressor Candidate, Inhibits Growth and Activates Differentiation Programs in Neuroblastoma Cells

A distal portion of human chromosome 1p is often deleted in neuroblastomas and other cancers and it is generally assumed that this region harbors one or more tumor suppressor genes. In neuroblastoma, a 261 kb region at 1p36.3 that encompasses the smallest region of consistent deletion pinpoints the locus for calmodulin binding transcription activator 1 (CAMTA1). Low CAMTA1 expression is an independent predictor of poor outcome in multivariate survival analysis, but its potential functionality in neuroblastoma has not been explored. In this study, we used inducible cell models to analyze the impact of CAMTA1 on neuroblastoma biology. In neuroblastoma cells that expressed little endogenous CAMTA1, its ectopic expression slowed cell proliferation, increasing the relative proportion of cells in G(1)/G(0) phases of the cell cycle, inhibited anchorage-independent colony formation, and suppressed the growth of tumor xenografts. CAMTA1 also induced neurite-like processes and markers of neuronal differentiation in neuroblastoma cells. Further, retinoic acid and other differentiation- inducing stimuli upregulated CAMTA1 expression in neuroblastoma cells. Transciptome analysis revealed 683 genes regulated on CAMTA1 induction and gene ontology analysis identified genes consistent with CAMTA1-induced phenotypes, with a significant enrichment for genes involved in neuronal function and differentiation. Our findings define properties of CAMTA1 in growth suppression and neuronal differentiation that support its assignment as a 1p36 tumor suppressor gene in neuroblastoma.

Nuclear localized GRP75 binds to retinoic acid receptors (RAR/RXR) and promotes retinoic acid‐induced neuronal differentiation of neuroblastoma

Neuroblastoma (NB) is the most common and deadly extracranial solid tumor in children. Retinoic acid (RA) has been used in clinic to treat NB by inducing neuronal differentiation of tumor cells. We previously found that glucose‐regulated protein 75 (GRP75) is correlated with the RA‐elicited neuronal differentiation of NB cells. In the present study, we further demonstrated that GRP75 can be translocated into nucleus and interact with retinoic acid receptor (RAR)α and retinoid X receptor (RXR)α to augment RA‐elicited neuronal differentiation. Down‐regulation of GRP75 diminishes the recruitment of RARα/RXRα to the retinoic acid response element (RARE) in the regulatory region of RA target gene promoters, resulting in a significant decrease in RA‐triggered gene expression. GRP75 also plays an essential role in stabilizing RARα/RXRα complexes in the presence of RA by reducing the proteasome‐mediated degradation of RARα and RXRα. Most intriguingly, the interaction between GRP75 and RARα/RXRα receptors was tightly associated with favorable outcome in NB patients and in a xenograft NB mouse model. Together, our present findings delineate a mechanism underlying the GRP75‐dependent regulation of RARα/RXRα‐mediated gene expression in the RA‐induced neuronal differentiation of NB, providing the basis for the development of novel therapeutics for NB.Grant Funding Source: Academia Sinica

Notch1 Expression Predicts an Unfavorable Prognosis and Serves as a Therapeutic Target of Patients with Neuroblastoma

Notch signaling has been implicated to play a critical role in the tumorigenesis of neuroblastoma (NB) and can modulate calreticulin (CRT) expression that strongly correlates with tumor differentiation and favorable prognosis of NB. We thus sought to determine how Notch regulates CRT expression and affects NB tumor behavior. The Notch-dependent regulation of CRT expression in cultured NB cells was analyzed by confocal microscopy and Western blotting. Notch1 protein expression in 85 NB tumors was examined by immunohistochemistry and correlated with the clinicopathologic/biological characters of NB patients. The progression of NB tumors in response to attenuated Notch signaling was examined by using a xenograft mouse model. We showed that CRT is essential for the neuronal differentiation of NB cells elicited by inhibition of Notch signaling. This effect was mediated by a c-Jun-NH(2)-kinase-dependent pathway. Furthermore, NB tumors with elevated Notch1 protein expression were strongly correlated with advanced tumor stages, MYCN amplification, an undifferentiated histology, as well as a low CRT expression level. Most importantly, the opposing effect between Notch1 and CRT could reciprocally affect the survival of NB patients. The administration of a gamma-secretase inhibitor into a xenograft mouse model of NB significantly suppressed the tumor progression. Our findings provide the first evidence that a c-Jun-NH(2)-kinase-CRT-dependent pathway is essential for the neuronal differentiation elicited by Notch signaling blockade and that Notch1 and CRT can synergistically predict the clinical outcomes of NB patients. The present data suggest that Notch signaling could be a therapeutic target for NB.

Abstract 5254: NLRR family proteins play distinct roles in deciding cell fate and affect the clinical outcome in neuroblastomas

Abstract We have previously generated neuroblastoma (NB)-prone oligo-capping cDNA libraries from primary neuroblastomas and identified novel important genes. Among those, we selected human neuronal leucine-rich repeat protein (NLRR) family genes for understanding of their functional roles in biology and clinical behavior of NB. In our previous observations, the expression level of NLRR1 mRNA is significantly higher in unfavorable NBs than favorable ones and its high expression is associated with poor clinical outcome, whereas that of NLRR3 is significantly high in favorable NBs in association with good prognosis. These findings suggested that NLRR1 and NLRR3 may have a different and opposite role in regulating cell growth and differentiation in NB. On the other hand, NLRR2 is similarly expressed in both subsets of NB. However, their real function in NB still remains elusive. Our present studies showed that cell proliferation was increased by overexpression of NLRR1 with enhanced EGF and IGF signals. NLRR1 proteins localized in cholesterol-rich lipid raft microdomain of cellular membrane and its expression altered the membrane distribution of EGFR and IGF-1R. Treatment of methyl-β-cyclodextrin to deplete membrane cholesterol and disrupt lipid rafts diminished the effect of NLRR1 on EGFR, suggesting that NLRR1 accelerates growth signaling pathway through lipid raft microdomains. Transcriptional study revealed that stimulation of EGF and IGF signaling enhanced the transcriptional activity of MYCN gene and MYCN in turn transactivates NLRR1 transcription, suggesting that NLRR1 and MYCN, both of which are poor prognostic factors in NBs, form a positive feedback loop via EGFR and IGF-1R. On the contrary, we observed opposite results in the study on NLRR3. The transcription was negatively regulated by MYCN with Max and Miz-1. Interestingly, enforced expression of NLRR3 induced neuronal differentiation and siRNA-mediated knockdown of NLRR3 reduced retinoic acid-induced neuronal differentiation in NB cells. Quantitative PCR also showed the inverse relationship between NLRR3 and MYCN in primary NB tissues. The combination of low NLRR3 and low Miz-1 expression or that of low NLRR3 and high MYCN expression was significantly associated with a poor prognosis. The current study on NLRR2 so far indicated its involvement in endoplasmic reticulum-stress response. Collectively, our results suggest that the transcriptional regulation varies between NLRR family genes and they play different roles in cell fate decision and contribute to the clinical outcome of NBs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5254.

Effects of bone morphogenetic protein 2 on Id expression and neuroblastoma cell differentiation

Bone morphogenetic proteins (BMPs) are secretory signal molecules that have a variety of regulatory functions during embryonic morphogenesis. BMP2 has been shown to induce differentiation in many cell types, mediated through the activation of its target genes: the inhibitors of differentiation (Id1-3) and key transcription factors. In this study, we investigated the effects of BMP2 on mouse neuroblastoma (Neuro2a) cell differentiation and regulation of the expression of Id1-3 and neural-specific transcription factors. Our results showed that BMP2 stimulation upregulated Id1-3 expression at the early stage of application by involvement of the Smad signaling pathway. BMP2 caused phosphorylation of Smad1/5/8 followed by upregulation of Id1-3. Co-incubation with Noggin, a BMP antagonist, or Smad1 siRNA transfection significantly inhibited phosphorylation of Smad1/5/8 and upregulation of Id protein. Furthermore, our results showed that BMP2-induced differentiation of Neuro2a cells into neurons by downregulating the expression of Id1-3 proteins and upregulating the expression of neural-specific transcriptional factors Dlx2, Brn3a, and NeuroD6. The results suggested that the transient upregulation of Id1-3 expression during the early phase of BMP stimulation may play a role in lineage specification and promote differentiation of neuroblastoma cells towards a neuronal phenotype. Subsequently, a coordinated increase in expression of proneural transcription factors and a decrease in Id1-3 expression may culminate in the transition from proliferation to neurogenesis and the terminal neuronal differentiation of neuroblastoma cells.