Down modulation of N-myc, heat-shock protein 70, and nucleolin during the differentiation of human neuroblastoma cells.

Cultured human neuroblastoma (GOTO) cells were induced to differentiate by dibutyryl cyclic AMP (Bt2cAMP) and/or retinoic acid (RA). A combination of Bt2cAMP (1 mM) and RA (1 microM) yielded the most significant networks of neurites after 3 to 4 days, this being associated with the reduction of N-myc mRNA levels. Next, we examined several cellular genes that were possibly linked with changes in N-myc gene expression under these conditions. Among the genes examined, both nucleolin and a major heat-shock protein (hsp70) mRNAs showed changes concomitant with those in N-myc mRNA levels when induced by Bt2cAMP and RA. Dibutyryl cAMP alone induced several short cellular processes and caused a marked decrease in N-myc mRNA within 2 days. RA alone induced a few long and straight neurites along the longitudinal axis of individual cells and a significant decrease in growth rate but showed neither network formation nor a decrease in N-myc gene expression. These results indicate differential effects of Bt2cAMP and RA on the regulatory mechanisms of both cell proliferation and differentiation and also indicate a possible association of expression of N-myc gene with those of hsp70 and nucleolin genes.     

Nerve growth factor-induced differentiation of human neuroblastoma and neuroepithelioma cell lines

A series of neuroepithelioma and neuroblastoma cell lines were screened for nerve growth factor (NGF)-induced differentiation. All three neuroepithelioma cell lines and all nine neuroblastoma cell lines with amplified N-myc oncogene did not show any apparent NGF-induced differentiation. However, neurite extension was observed for three of six neuroblastoma cell lines with single-copy N-myc oncogene. The three responsive lines had a neuronal phenotype (short processes) which was enhanced by the addition of NGF. The three nonresponsive cell lines were flat without any processes. The addition of NGF to the responsive cell lines resulted in an up-regulation of neurofilament mRNA expression. Peripherin and synapsin, two markers of terminal neuronal differentiation, were not induced. There was little effect of NGF on the rate of cell growth or colony formation on soft agar. Binding of NGF to eight of the cell lines was analyzed by the method of Scatchard. Two responsive neuroblastoma cell lines and one nonresponsive neuroepithelioma cell line expressed both low- and high-affinity binding sites. Two nonresponsive neuroblastoma cell lines expressed only a small number of high-affinity binding sites, and two other nonresponsive neuroblastoma cell lines did not detectably bind NGF. Hence, NGF-induced differentiation is confined to a particular class of neural-related tumors, and, even for these cell lines, differentiation is incomplete.     

Combined RAR alpha- and RXR-specific ligands overcome N-myc-associated retinoid resistance in neuroblastoma cells

Retinoids induce human neuroblastoma cells to undergo growth inhibition and neuritic differentiation in vitro, through interactions with nuclear retinoid receptor proteins. In this study, we found that three different neuroblastoma cell lines exhibited wide variation in their responsiveness to the growth inhibitory effects of the retinoic acid receptor (RAR) agonist, all-trans-retinoic acid (aRA). Resistance to the growth inhibitory effect of aRA correlated with the presence of N-myc gene amplification and not aRA-induced RAR beta levels. Over-expression of N-myc in a neuroblastoma cell line with no endogenous N-myc expression caused a marked reduction in retinoid-induced growth inhibition. Combination of receptor-specific retinoid agonists for RXR and RAR alpha significantly enhanced the sensitivity of N-myc-amplified neuroblastoma cells to the growth inhibitory effects of aRA. Our results indicate that combination receptor-specific retinoid therapy can overcome N-myc-mediated retinoid resistance and may be a more effective chemo-preventive strategy in the disease.     

Expression of a drug resistance gene in human neuroblastoma cell lines: modulation by retinoic acid-induced differentiation.

Expression of a multidrug resistance gene (mdr1) and its protein product, P-glycoprotein (Pgp), has been correlated with the onset of multidrug resistance in vitro in human cell lines selected for resistance to chemotherapeutic agents derived from natural products. Expression of this gene has also been observed in normal tissues and human tumors, including neuroblastoma. We therefore examined total RNA prepared from human neuroblastoma cell lines before and after differentiation with retinoic acid or sodium butyrate. An increase in the level of mdr1 mRNA was observed after retinoic acid treatment of four neuroblastoma cell lines, including the SK-N-SH cell line. Western blot (immunoblot) analysis demonstrated concomitant increases in Pgp. However, studies of 3H-vinblastine uptake failed to show a concomitant Pgp-mediated decrease in cytotoxic drug accumulation. To provide evidence that Pgp was localized on the cell surface, an immunotoxin conjugate directed against Pgp was added to cells before and after treatment with retinoic acid. Incorporation of [3H]leucine was decreased by the immunotoxin in the retinoic acid-treated cells compared with the undifferentiated cells. These results demonstrate that whereas expression of the mdr1 gene can be modulated by differentiating agents, increased levels of expression are not necessarily associated with increased cytotoxic drug accumulation.     

Plasma membrane redox activity correlates with N-myc expression in neuroblastoma cells.

In different neuroblastoma cell lines and transfected clones, an increasing plasma membrane redox activity correlates with amplification and enhanced expression of the N-myc oncogene. Furthermore, plasma membrane redox activity is partially inhibited by retinoic acid in neuroblastoma cells with multiple copies of the N-myc oncogene but not in neuroblastoma cells with only one copy of this gene.     

Sub-micromolar concentrations of retinoic acid induce morphological and functional neuronal phenotypes in SK-N-SH neuroblastoma cells

Neuroblastoma cells are neural crest derivatives that can differentiate into neuron-like cells in response to exogenous agents, and are known to be particularly sensitive to retinoic acid. The spectrum of neuroblastoma responses, ranging from proliferation, migration, differentiation, or apoptosis, is difficult to predict due to the heterogeneity of these tumors and to the broad effective range of retinoic acid. Our study focused on the effects of nanomolar concentrations of retinoic acid on neuroblastoma differentiation in two cell lines cells: SK-N-SH (HTB-11) and IMR-32. Each cell line was treated with retinoic acid from 1 to 100 nM for up to 6 d. Morphological changes were quantified; immunocytochemistry was used to observe changes in neuronal protein expression and localization, while live-cell calcium imaging utilizing pharmacological agents was conducted to identify neuron-like activity. Retinoic acid-treated HTB-11 but not IMR-32 cells developed specific neuronal phenotypes: acquisition of long neurite-like processes, expression of neurofilament-200, increased responsiveness to acetylcholine, and decreased responsiveness to nicotine and epinephrine. In addition, nanomolar levels of retinoic acid elicited increased nuclear trafficking of the CRABP2, which is traditionally associated with gene expression of cellular pathways related to neuronal differentiation. Collectively, these results show that nanomolar concentrations of retinoic acid are capable of inducing both structural and functional neuron-like features in HTB-11 cells using CRABP2, suggesting differentiation in neuroblastoma cells into neuronal phenotypes. These have important implications for both chemotherapeutic design and for the use of neuroblastomas as in vitro models for neuron differentiation.     

Expression and function of TRK-B and BDNF in human neuroblastomas.

There is considerable interest in the role of the TRK family of neuotrophin receptors in regulating growth and differentiation in normal and neoplastic nerve cells. A neuroblastoma is a common pediatric tumor derived from the neural crest, and the majority of favorable neuroblastomas express a high level of TRK-A mRNA. However, little is known about the expression or function of TRK-B in these tumors. TRK-B encodes a tyrosine kinase that binds to brain-derived neuotrophic factor (BDNF), as well as neurotrophin-3 (NT-3) and NT-4/5. We have studied the N-myc-amplified human neuroblastoma cell line, SMS-KCN, which expresses both TRK-B and BDNF. Exogenous BDNF induces tyrosine phosphorylation of TRK-B as well as phosphorylation of phospholipase C-gamma 1, the extracellular signal-regulated kinases 1 and 2, and phosphatidylinositol-3 kinase. BDNF also induces expression of the immediate-early genes c-FOS and NGFI-A but not NGFI-B or NGFI-C. In addition, BDNF appears to promote cell survival and neurite outgrowth. SMS-KCN cells also express TRK-A, which is phosphorylated in response to nerve growth factor. However, the downstream TRK-A signaling is apparently defective. Finally, we determined that in a series of 74 primary neuroblastomas, 36% express TRK-B mRNA, 68% express BDNF mRNA, and 31% express both. Truncated TRK-B appears to be preferentially expressed in more-differentiated tumors (ganglioneuromas and ganglioneuroblastomas), whereas full-length TRK-B is expressed almost exclusively in immature neuroblastomas with N-myc amplification. Our findings suggest that in TRK-B-expressing human neuroblastomas, BDNF promotes survival and induces neurite outgrowth in an autocrine or paracrine manner. The BDNF/TRK-B pathway may be particularly important for growth and differentiation of neuroblastomas with N-myc amplification.     

N-myc down regulates neural cell adhesion molecule expression in rat neuroblastoma.

In human neuroblastoma, amplification of the N-myc oncogene is correlated with increased metastatic ability. We recently showed that transfection of the rat neuroblastoma cell line B104 with an N-myc expression vector resulted in an increase in metastatic ability and a significant reduction in the expression of major histocompatibility complex class I antigens. We examined whether N-myc causes additional phenotypic changes in these cells. We showed that expression of N-myc leads to a dramatic reduction in the levels of neural cell adhesion molecule (NCAM) polypeptides and mRNAs. Spontaneous revertants of the high N-myc phenotype were found to have regained significant levels of NCAM expression, indicating that the continued expression of N-myc is required to maintain the low NCAM phenotype. NCAM was not reduced in B104 cells transfected with the neomycin resistance vector alone, and other neuronal markers were not specifically reduced in N-myc-transfected B104 cells. As NCAM functions in cell-cell adhesion, decreased NCAM expression could contribute significantly to the increased metastatic potential of N-myc-amplified neuroblastomas.     

Up-regulation of Jun/AP-1 during differentiation of N1E-115 neuroblastoma cells.

Neuroblastoma cell lines isolated from neuroblastoma tumors can be induced to differentiate into neuronal cell types by treatment with chemical agents, such as dimethyl sulfoxide and retinoic acid. The molecular mechanisms underlying this differentiation process, however, are completely obscure. In this paper, we show that neuronal differentiation of mouse N1E-115 neuroblastoma cells by dimethyl sulfoxide is accompanied by a prolonged rise in c-jun, junB, and junD expression and AP-1 activity. Multiple sequence elements in the Jun promoters are involved in this process. Furthermore, we show that c-jun and junD, but not junB, are expressed at high levels in the neuronal cell types obtained after dimethyl sulfoxide treatment. These results suggest an important role for c-jun and junD in neuronal differentiation of N1E-115 cells.     

Suppression of tumorigenicity in neuroblastoma cells by upregulation of human vasoactive intestinal peptide receptor type 1

We hypothesize that vasoactive intestinal peptide (VIP) promotes neural crest differentiation through VIP receptor type I (VPAC1). In order to test this hypothesis, SKNSH neuroblastoma cells were stably transfected with VPAC1 and receptor expression was verified by real-time RT-PCR. Overexpression of VPAC1 in SKNSH cells resulted in upregulation of endogenous retinoic acid receptor expression for both RARalpha and RXRalpha with no change in expression of RARbeta. Transfected cells demonstrated high affinity binding of VIP (K(D)=0.2 nM) and VIP-mediated stimulation of adenylate cyclase and a shift in cell cycle kinetics to a near triploid DNA index in G1. SKNSH/VPAC1 cells treated with VIP were observed to express a more differentiated phenotype compared to wild type cells as characterized by an increase in tissue transglutaminase II and a decrease in bcl-2 immunostaining. VIP-induced differentiation effects were potentiated by retinoic acid. This differentiation resulted in decreased proliferative potential in a xenograft model. Whereas, wild type SKNSH cells induced tumor growth in 100% of nude mice within 13 days post-injection, SKNSH transfected with VPAC1 demonstrated no tumor formation in xenografts followed for 6 months. Taken together, these data support the hypothesis that VIP modulation of neural crest differentiation is mediated via VPAC1 and that high expression of VPAC1 induces differentiation in and decreases tumorigenicity of neuroblastoma cells.     

Retinoic acid-induced blr1 expression promotes ERK2 activation and cell differentiation in HL-60 cells

Retinoids are known to induce the differentiation and cell cycle arrest of human myeloid leukemia cells in vitro. Differential display was used to identify putative early regulatory genes that are differentially expressed in HL-60 human promyelocytic leukemia cells treated with retinoic acid. One of the cDNAs cloned encodes sequences identifying Burkitt's lymphoma receptor 1 (BLR1), a recently described chemokine receptor. Northern blot analysis demonstrates that blr1 mRNA expression increases within 9 h of retinoic acid treatment, well before functional differentiation or G(1)/G(0) growth arrest at 48 h or onset of morphological changes, suggesting a possible regulatory function. The expression of blr1 mRNA is transient, peaking at 72 h when cells are differentiated. blr1 mRNA also is induced by other differentiation-inducing agents, 1alpha,25-dihydroxyvitamin D(3) and DMSO. Induction of blr1 mRNA by retinoic acid is not blocked by the protein synthesis inhibitor cycloheximide. In HL-60 cells stably transfected with blr1 cDNA, ectopic expression of blr1 causes an increase in ERK2 MAPK activation and promotes retinoic acid-induced G(1)/G(0) growth arrest and cell differentiation. The early expression of blr1 mRNA during differentiation, its ability to increase ERK2 activation, and its enhancement of retinoic acid-induced differentiation suggest that blr1 expression may be involved in retinoic acid-induced HL-60 differentiation.     

Rapid and transient decrease of N-myc expression in retinoic acid-induced differentiation of OTF9 teratocarcinoma stem cells.

The level of expression of N-myc in mouse teratocarcinoma stem cells is very high. Previous studies have shown that N-myc expression significantly decreases when the stem cells are subjected to long-term induction for differentiation by retinoic acid (RA). We found that in a stem cell line, OTF9, a steep yet transient decrease of N-myc expression takes place much earlier, immediately after induction by RA. To examine whether this decrease is responsible for differentiation, we constructed a gene, miwNmyc, to express N-myc cDNA constitutively and transformed OTF9 cells with this gene construct. Transformants under the constitutive expression of miwNmyc differentiated normally, as judged by morphological changes and by modulation of c-myc, Hox1.1, and laminin B1 expression. Therefore, transient decrease of N-myc expression may be the consequence of RA-induced differentiation, even though it occurs very early in the process. Alternatively, in addition to N-myc decrease, there may be redundant mechanisms which lead to OTF9 differentiation after induction by RA, so that suppression of N-myc decrease is bypassed by at least one other mechanism.