Neuronal Differentiation Of Neuroblastoma Cells Research Articles

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.

ATG5 expression induced by MDMA (ecstasy), interferes with neuronal differentiation of neuroblastoma cells.

The amphetamine derivative 3, 4-methylenedioxymethamphetamine (MDMA) has become a popular recreational drug, and has also been shown to cause serotonergic neurotoxicity. This report shows that MDMA impairs brain development in a whole mouse embryo culture. The results of quantitative real-time PCR analysis showed that autophagy-related protein 5 (Atg5) expression is elevated in mouse embryo and neuroblastoma cells after MDMA treatment. This elevated Atg5 expression interferes with the neuronal differentiation of neuroblastoma cells such as SH-SY5Y and PC12 cells. Thus, our results suggest that the use of MDMA during pregnancy may impair neuronal development via an induction of Atg5 expression.

An IRBIT homologue lacks binding activity to inositol 1,4,5‐trisphosphate receptor due to the unique N‐terminal appendage

IRBIT is an inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-binding protein that inhibits the activation of IP(3)R by competing with IP(3) for the common binding site on IP(3)R. In this study, we characterize an IRBIT homologue, termed Long-IRBIT. Long-IRBIT is highly homologous to IRBIT ( approximately 88%) and heteromerizes with IRBIT. In spite of complete conservation of critical amino acids required for the interaction with IP(3)R and comparable phosphorylations on critical four Ser residues for IP(3)R-binding, Long-IRBIT retains little ability to interact with IP(3)R. Deletion mutagenesis analysis revealed that this low affinity to IP(3)R is attributable to an inhibitory effect of the Long-IRBIT specific N-terminal appendage (LISN domain). Immunohistochemical analysis revealed the distinct distribution of Long-IRBIT and IRBIT in mouse cerebellar cortex, that is, Long-IRBIT is mainly expressed in interneurons such as basket cells, while IRBIT is mainly expressed in glial cells. Furthermore, Long-IRBIT, but not IRBIT, underwent phosphorylation during neuronal differentiation in neuroblastoma cells and this phosphorylation was dependent on the LISN domain. These results suggest that Long-IRBIT has a different function from IRBIT.

Long‐Term Ethanol Exposure Impairs Neuronal Differentiation of Human Neuroblastoma Cells Involving Neurotrophin‐Mediated Intracellular Signaling and in Particular Protein Kinase C

Revealing the molecular changes in chronic ethanol-impaired neuronal differentiation may be of great importance for understanding ethanol-related pathology in embryonic development but also in the adult brain. In this study, both acute and long-term effects of ethanol on neuronal differentiation of human neuroblastoma cells were investigated. We focused on several aspects of brain-derived neurotrophic factor (BDNF) signaling because BDNF activates the extracellular signal-regulated kinase (ERK) cascade, promoting neuronal differentiation including neurite outgrowth. The effects of ethanol exposure on morphological differentiation, cellular density, neuronal marker proteins, basal ERK activity, and ERK responsiveness to BDNF were measured over 2 to 4 weeks. qRT-PCR and Western blotting were performed to investigate the expression of neurotrophin receptor tyrosin kinase B (TrkB), members of the ERK-cascade, protein kinase C (PKC) isoforms and Raf-Kinase-Inhibitor-Protein (RKIP). Chronic ethanol interfered with the development of a neuronal network consisting of cell clusters and neuritic bundles. Furthermore, neuronal and synaptic markers were reduced, indicating impaired neuronal differentiation. BDNF-mediated activation of the ERK cascade was found to be continuously impaired by ethanol. This could not be explained by expressional changes monitored for TrkB, Raf-1, MEK, and ERK. However, BDNF also activates PKC signaling which involves RKIP, which finally leads to ERK activation as well. Therefore, we hypothesized that ethanol impairs this branch of BDNF signaling. Indeed, both PKC and RKIP were significantly down-regulated. Chronic ethanol exposure impaired neuronal differentiation of neuroblastoma cells and BDNF signaling, particularly the PKC-dependent branch. RKIP, acting as a signaling switch at the merge of the PKC cascade and the Raf/MEK/ERK cascade, was associated with neuronal differentiation and significantly reduced in ethanol treatment. Moreover, PKC expression itself was even more strongly reduced. In contrast, members of the Raf-1/MEK/ERK cascade were less affected and the observed changes were not associated with impaired differentiation. Thus, reduced RKIP and PKC levels and subsequently reduced positive feedback on ERK activation provide an explanation for the striking effects of long-term ethanol exposure on BDNF signal transduction and neuronal differentiation, respectively.

Secretogranin II: a key AP-1-regulated protein that mediates neuronal differentiation and protection from nitric oxide-induced apoptosis of neuroblastoma cells

Identification of AP-1 target genes in apoptosis and differentiation has proved elusive. Secretogranin II (SgII) is a protein widely distributed in nervous and endocrine tissues, and abundant in neuroendocrine granules. We addressed whether SgII is regulated by AP-1, and if SgII is involved in neuronal differentiation or the cellular response to nitrosative stress. Nitric oxide (NO) upregulated sgII mRNA dependent on a cyclic AMP response element (CRE) in the sgII promoter, and NO stimulated SgII protein secretion in neuroblastoma cells. Upregulation of sgII mRNA, sgII CRE-driven gene expression and SgII protein synthesis/export were attenuated in cells transformed with dominant-negative c-Jun (TAM67), which became sensitized to NO-induced apoptosis and failed to undergo nerve growth factor-dependent neuronal differentiation. Stable transformation of TAM67 cells with sgII restored neuronal differentiation and resistance to NO. RNAi knockdown of sgII in cells expressing functional c-Jun abolished neuronal differentiation and rendered the cells sensitive to NO-induced apoptosis. Therefore, SgII represents a key AP-1-regulated protein that counteracts NO toxicity and mediates neuronal differentiation of neuroblastoma cells.

Enhanced autophagic cell death in expanded polyhistidine variants of HOXA1 reduces PBX1‐coupled transcriptional activity and inhibits neuronal differentiation

HOXA1 is a member of the homeobox gene family and is involved in early brain development. In our previous study, we identified novel variants of polyhistidine repeat tract in HOXA1 gene and showed that ectopic expression of expanded variants led to enhanced intranuclear aggregation and accelerated cell death in a time-dependent manner. Here, we further investigate the implications of polyhistidine variants on HOXA1 function. Aside from intranuclear aggregation, we observed cytosolic aggregates during the early stages of expression. Rapamycin, an autophagy inducer, resulted in decreased protein aggregation and cell death. Here, we also show an interaction between variants of HOXA1 and one of the HOX protein known cofactors, PBX1. Expanded HOXA1 variants exhibited reduced PBX1-coupled transcriptional activity through a regulatory enhancer of HOXB1. Moreover, we demonstrate that both deleted and expanded variants inhibited neurite outgrowth in retinoic acid-induced neuronal differentiation in neuroblastoma cells. These results provide further evidence that expanded polyhistidine repeats in HOXA1 enhance aggregation and cell death, resulting in impaired neuronal differentiation and cooperative binding with PBX1.

Unnatural Amino Acid-Substituted (Hydroxyethyl)urea Peptidomimetics Inhibit γ-Secretase and Promote the Neuronal Differentiation of Neuroblastoma Cells

Gamma-secretase, exhibiting characteristics of aspartyl protease, mediates the intramembranous proteolysis of beta-amyloid precursor protein (APP) and Notch, and it is considered to be a prime pharmacological target in the development of therapeutics for Alzheimer's disease (AD). To identify compounds that block gamma-secretase-mediated proteolysis, we used a highly sensitive cell-based reporter gene assay for gamma-secretase in which Gal4/VP16-tagged C99-APP was expressed as the immediate substrate of gamma-secretase, and Gal4/VP16-tagged APP intracellular domain released by the gamma-secretase cleavage then activated the expression of the Gal4-driven luciferase reporter gene. Using this reporter assay, we demonstrated that the newly synthesized (hydroxyethyl)urea peptidomimetics, which contain unnatural amino acid moieties at positions P1' and/or P3', can effectively inhibit gamma-secretase activity and significantly reduce Abeta production. The gamma-secretase-dependent S3 cleavage of Notch was also consistently blocked by these (hydroxyethyl)ureas as evidenced by the decreased generation of the Notch intracellular domain, a prerequisite for the activation of Notch signaling. The inhibition of Notch signaling by active Jia compounds efficiently promotes the neuronal differentiation of neuroblastoma cells, intervening in tumorigenesis and the malignancy of neuroblastomas. Our results suggest that (hydroxyethyl)urea peptidomimetics containing unnatural amino acid substitutions could represent a novel class of gamma-secretase inhibitors with enhanced stability, providing the basis for the further development of effective therapeutics for AD and neuroblastomas.

BM88 Is a Dual Function Molecule Inducing Cell Cycle Exit and Neuronal Differentiation of Neuroblastoma Cells via Cyclin D1 Down-regulation and Retinoblastoma Protein Hypophosphorylation

Control of cell cycle progression/exit and differentiation of neuronal precursors is of paramount importance during brain development. BM88 is a neuronal protein associated with terminal neuron-generating divisions in vivo and is implicated in mechanisms underlying neuronal differentiation. Here we have used mouse neuroblastoma Neuro 2a cells as an in vitro model of neuronal differentiation to dissect the functional properties of BM88 by implementing gain- and loss-of-function approaches. We demonstrate that stably transfected cells overexpressing BM88 acquire a neuronal phenotype in the absence of external stimuli, as judged by enhanced expression of neuronal markers and neurite outgrowth-inducing signaling molecules. In addition, cell cycle measurements involving cell growth assays, BrdUrd incorporation, and fluorescence-activated cell sorting analysis revealed that the BM88-transfected cells have a prolonged G(1) phase, most probably corresponding to cell cycle exit at the G(0) restriction point, as compared with controls. BM88 overexpression also results in increased levels of the cell cycle regulatory protein p53, and accumulation of the hypophosphorylated form of the retinoblastoma protein leading to cell cycle arrest, with concomitant decreased levels and, in many cells, cytoplasmic localization of cyclin D1. Conversely, BM88 gene silencing using RNA interference experiments resulted in acceleration of cell proliferation accompanied by impairment of retinoic acid-induced neuronal differentiation of Neuro 2a cells. Taken together, our results suggest that BM88 plays an essential role in regulating cell cycle exit and differentiation of Neuro 2a cells toward a neuronal phenotype and further support its involvement in the proliferation/differentiation transition of neural stem/progenitor cells during embryonic development.

Molecular mechanisms involved in the adenosine A1 and A2A receptor‐induced neuronal differentiation in neuroblastoma cells and striatal primary cultures

Adenosine A1 receptors (A1Rs) and adenosine A(2A) receptors (A(2A)Rs) are the major mediators of the neuromodulatory actions of adenosine in the brain. In the striatum A1Rs and A(2A)Rs are mainly co-localized in the GABAergic striatopallidal neurons. In this paper we show that agonist-induced stimulation of A1Rs and A(2A)Rs induces neurite outgrowth processes in the human neuroblastoma cell line SH-SY5Y and also in primary cultures of striatal neuronal precursor cells. The kinetics of adenosine-mediated neuritogenesis was faster than that triggered by retinoic acid. The triggering of the expression of TrkB neurotrophin receptor and the increase of cell number in the G1 phase by the activation of adenosine receptors suggest that adenosine may participate in early steps of neuronal differentiation. Furthermore, protein kinase C (PKC) and extracellular regulated kinase-1/2 (ERK-1/2) are involved in the A1R- and A(2A)R-mediated effects. Inhibition of protein kinase A (PKA) activity results in a total inhibition of neurite outgrowth induced by A(2A)R agonists but not by A1R agonists. PKA activation is therefore necessary for A(2A)R-mediated neuritogenesis. Co-stimulation does not lead to synergistic effects thus indicating that the neuritogenic effects of adenosine are mediated by either A1 or A(2A) receptors depending upon the concentration of the nucleoside. These results are relevant to understand the mechanisms by which adenosine receptors modulate neuronal differentiation and open new perspectives for considering the use of adenosine agonists as therapeutic agents in diseases requiring neuronal repair.

Polysialic acid directs tumor cell growth by controlling heterophilic neural cell adhesion molecule interactions.

Polysialic acid (PSA), a carbohydrate polymer attached to the neural cell adhesion molecule (NCAM), promotes neural plasticity and tumor malignancy, but its mode of action is controversial. Here we establish that PSA controls tumor cell growth and differentiation by interfering with NCAM signaling at cell-cell contacts. Interactions between cells with different PSA and NCAM expression profiles were initiated by enzymatic removal of PSA and by ectopic expression of NCAM or PSA-NCAM. Removal of PSA from the cell surface led to reduced proliferation and activated extracellular signal-regulated kinase (ERK), inducing enhanced survival and neuronal differentiation of neuroblastoma cells. Blocking with an NCAM-specific peptide prevented these effects. Combinatorial transinteraction studies with cells and membranes with different PSA and NCAM phenotypes revealed that heterophilic NCAM binding mimics the cellular responses to PSA removal. In conclusion, our data demonstrate that PSA masks heterophilic NCAM signals, having a direct impact on tumor cell growth. This provides a mechanism for how PSA may promote the genesis and progression of highly aggressive PSA-NCAM-positive tumors.

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.

Two Novel Genes, Human neugrin and Mouse m-neugrin, Are Upregulated with Neuronal Differentiation in Neuroblastoma Cells

We herein report two new genes, human neugrin and mouse homologue m-neugrin, found by screening the cDNA library for the human spinal cord. The neugrin mRNA encodes 219 amino acids and its deduced amino acid sequence contains an NLS-like domain. No previously known motif is found in it. m-neugrin mRNA encodes 233 amino acids. Neugrin and m-Neugrin are 70% homologous in amino acid sequence. Northern analysis revealed that neugrin was strongly expressed in the heart, brain, and skeletal muscle, and m-neugrin in the liver, kidney, and brain. A transfection study indicated that these proteins are localized in the nucleus. Although the expression of neugrin was found to be ubiquitous in the nervous system, in situ hybridization showed that both neugrin and m-neugrin were expressed mainly in the neurons rather than the glial cells. Their expression was highly upregulated with the neurite outgrowth associated with neuronal differentiation in neuroblastoma cell lines. These results indicate that neugrin and m-neugrin are mainly expressed in neurons in the nervous system, and play an important role in the process of neuronal differentiation.

Induction of Neuronal Differentiation by p73 in a Neuroblastoma Cell Line

The p53-related p73 and p63 genes encode proteins that share considerable structural and functional homology with p53. Despite similarities, their deletion in mice has different outcomes, implying that the three genes may play distinct roles in vivo. Here we show that endogenous p73 levels increase in neuroblastoma cells induced to differentiate by retinoic acid and that exogenously expressed p73, but not p53, is sufficient to induce both morphological (neurite outgrowth) and biochemical (expression of neurofilaments and neural cell adhesion molecule (N-CAM); down-regulation of N-MYC and up-regulation of pRB) markers of neuronal differentiation. This activity is shared, to different extents, by all p73 isoforms, whereas the transcriptionally inactive mutants of p73 isoforms are ineffective. Conversely, blockage of endogenous p73 isoforms with a dominant negative p73 results in the abrogation of retinoid-induced N-CAM promoter-driven transcription. Our results indicate that the p73 isoforms activate a pathway that is not shared by p53 and that is required for neuroblastoma cell differentiation in vitro.

Induced neuroblastoma cell differentiation, associated with transient HES-1 activity and reducedHASH-1 expression, is inhibited by Notch1

Neuroblastoma is a childhood tumor that originates from the sympathetic nervous system. The tumor cells have embryonic features, presumably as a consequence of an impaired capacity to respond to signals and transcriptional control mechanisms operating during normal differentiation. Two basic helix-loop-helix transcription factors, human achaete-scute homologue-1 (HASH-1) and hairy/enhancer of split homologue-1 (HES-1), are crucial for proper development of some neuronal cells. Here, their potential roles during sympathetic differentiation of human neuroblastoma cells have been investigated. In all tested protocols for induction of differentiation of SH-SY5Y and SK-N-BE(2) neuroblastoma cells, HASH-1 expression was rapidly decreased with a concomitant, often transient, increase in HES-1 expression. In gel mobility shift assays, using extracts from neuroblastoma cells, HES-1 bound to an oligonucleotide corresponding to a sequence in the HASH-1 promoter including the so-called N-box, suggesting that the transiently increased HES-1 activity in differentiating neuroblastoma cells is involved in down-regulation of HASH-1. Constitutive expression of the intracellular domain of Notch1, which activates the HES-1 promoter in SH-SY5Y cells, inhibited spontaneous and induced morphological differentiation of these neuroblastoma cells. Our data show that functional sympathetic neuronal differentiation of neuroblastoma cells is associated with transient activation of HES-1 and down-regulation of HASH-1 expression.

Significance of gangliosides in neuronal differentiation of neuroblastoma cells and neurite growth in tissue culture.

Addition of DL-threo-1-phenyl-2-decanolylamine-3-morpholino-1-propanol HCl (PDMP; 7-24 microM) or Fumonisin B1 (FB1; 30-50 microM) to SH-SY5Ytrk-A human neuroblastoma cells results within 4 days in a 40% decrease of the ganglioside content and in a reduction of nerve-growth-factor (NGF)-induced outgrowth of neuritic processes. NGF-induced enhancement of GAP-43 expression was not affected. However, unlike controls, immunostained GAP-43 appeared concentrated in defined areas of cell perikarya and mostly absent from cell processes. Presence of 20-microM exogenous GM1 for 4 days in NGF and PDMP containing cell cultures led to an increase of cell-associated GM1(15-fold), GM2 (10-fold), GM3 (15 fold), GD1a (4-fold), GD2, GD1b, and GT1b (all 3-fold), and partially reversed the PDMP (and FB1) effects on neurite growth and GAP-43 distribution. In a newly developed neuronal tissue culture system, PDMP and FB1 led to a comparable dose-dependent inhibition of neurite outgrowth from embryonic chicken spinal cord explants, which had been embedded into a fibrin matrix. In this system, addition of GM1 led to a further inhibition of neurite growth, probably due to an interaction with growth-promoting components present in the surrounding fibrin matrix.

Long-term exposure to retinoic acid induces the expression of IRK1 channels in HERG channel-endowed neuroblastoma cells.

The modulation of inward K+ conductances was studied during neuronal differentiation of human SH-SY5Y neuroblastoma cells. Under standard culture conditions, these cells express the herg gene, and the HERG current is the main inward K+ current regulating their Vrest. After 10-20 days exposure to Retinoic Acid (RA), SH-SY5Y cells showed, in addition to HERG currents, a novel current characterized by inward rectification, dependence on the extracellular K+ concentration, and blockade by Cs+ and Ba2+, the main features of the IRK1 current. The appearance of this current is accompanied by a strong hyperpolarisation of Vrest. RT-PCR experiments confirmed that a transcript of the IRK1 (Kir 2.1) gene actually appears in SH-SY5Y cells treated for 10-20 days with RA. On the whole, data here presented demonstrate that RA-induced neuronal differentiation of neuroblastoma cells is accompanied by the switch from a HERG-driven to a IRK1-driven control of Vrest, similarly to what happens in normal differentiating neurons; however, in tumor cells, this switch does not imply the abolition of HERG channel expression.

Decrease in neuron-restrictive silencer factor (NRSF) mRNA levels during differentiation of cultured neuroblastoma cells

Non-neuronal cells and undifferentiated neuronal progenitors express the neuron-restrictive silencer factor (NRSF), a silencer protein which represses neuronal gene transcription in these cell types. Neuroblastoma, a childhood tumor of neuroectodermal origin, shares some biological properties with neuronal progenitor cells and can acquire neuronal phenotypes in response to a variety of agents, including cyclic AMP and staurosporine. We report here that NRSF mRNA content was markedly decreased in a human neuroblastoma cell line following differentiation induced by staurosporine plus cyclic AMP, with a concomitant increase in mRNA levels of synapsin I, whose expression is restricted to neuronal cell types. Our novel finding suggests that NRSF expression is related to an undifferentiated state and regarded as a biochemical marker of neuronal differentiation in neuroblastoma cells.

Induction of TrkB by retinoic acid mediates biologic responsiveness to BDNF and differentiation of human neuroblastoma cells

Retinoic acid (RA) induces the neuronal differentiation of many human neuroblastoma cell lines. In this study, we show that RA treatment of neuroblastoma cells induces the expression of TrkB, the receptor for the neurotrophins BDNF, NT-3, and NT-4/5. BDNF addition to RA-treated SH-SY5Y neuroblastoma cells stimulated the tyrosine phosphorylation of TrkB and neuronal differentiation. RA treatment of KCNR neuroblastoma cells, which constitutively express BDNF mRNA, resulted in the expression of TrkB and differentiation in the absence of added BDNF. Finally, in 15N neuroblastoma cells, which express BDNF mRNA but do not differentiate in response to RA, RA induced only a truncated form of TrkB. 15N cells transfected with full-length TrkB differentiated in the absence of RA. These results indicate that RA induces the neuronal differentiation of neuroblastoma cells by modulating the expression of neurotrophin receptors.

Nerve Growth Factor (NGF) Induces Neuronal Differentiation in Neuroblastoma Cells Transfected with the NGF Receptor cDNA

Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment.

Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA.

Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment.