Differentiation Of Human Neuroblastoma Research Articles

Inhibition of WNT signaling reduces differentiation and induces sensitivity to doxorubicin in human malignant neuroblastoma SH-SY5Y cells.

Neuroblastoma is one of the most common cancers in infancy, arising from the neuroblasts during embryonic development. This cancer is difficult to treat and resistance to chemotherapy is often found; therefore, clinical trials of novel therapeutic approaches, such as targeted-cancer signaling, could be an alternative for a better treatment. WNT signaling plays significant roles in the survival, proliferation, and differentiation of human neuroblastoma. In this report, WNT signaling of a malignant human neuroblastoma cell line, SH-SY5Y cells, was inhibited by XAV939, a specific inhibitor of the Tankyrase enzyme. XAV939 treatment led to the reduction of β-catenin within the cells, confirming its inhibitory effect of WNT. The inhibition of WNT signaling by XAV939 did not affect cell morphology, survival, and proliferation; however, the differentiation and sensitivity to anticancer drugs of human neuroblastoma cells were altered. The treatment of XAV939 resulted in the downregulation of mature neuronal markers, including β-tubulin III, PHOX2A, and PHOX2B, whereas neural progenitor markers (PAX6, TFAP2α, and SLUG) were upregulated. In addition, the combination of XAV939 significantly enhanced the sensitivity of SH-SY5Y and IMR-32 cells to doxorubicin in both 2D and 3D culture systems. Microarray gene expression profiling suggested numbers of candidate target genes of WNT inhibition by XAV939, in particular, p21, p53, ubiquitin C, ZBED8, MDM2, CASP3, and FZD1, and this explained the enhanced sensitivity of SH-SY5Y cells to doxorubicin. Altogether, these results proposed that the altered differentiation of human malignant neuroblastoma cells by inhibiting WNT signaling sensitized the cells to anticancer drugs. This approach could thus serve as an effective treatment option for aggressive brain malignancy.

Tryptanthrin induces growth inhibition and neuronal differentiation in the human neuroblastoma LA-N-1 cells

Neuroblastoma is one of the most common extracranial solid cancers found in young children. The prognosis of neuroblastoma patients in advanced stages having N-myc amplification remains poor despite intensive multimodal therapy. Agents that trigger neuroblastoma cells to undergo cellular differentiation and thereby stop proliferation have attracted considerable interest as an alternative therapy. Tryptanthrin (12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline) is a weakly basic alkaloid isolated from the dried roots of medicinal indigo plants known as Banlangen. It has been shown to possess various biological activities, such as anti-microbial, anti-inflammatory and anti-tumor activities. However, its effects and mechanism(s) of action on human neuroblastoma cells remain poorly understood. Therefore, the objective of this study is to investigate the effects of tryptanthrin on the growth and differentiation of human neuroblastoma LA-N-1 cells with N-myc amplification. Our results show that tryptanthrin inhibited the growth of the human neuroblastoma cells in a dose- and time-dependent manner. Mechanistic studies indicated that tryptanthrin induced cell cycle arrest of the human neuroblastoma LA-N-1 cells at the G0/G1 phase. Tryptanthrin also induced neuronal differentiation of LA-N-1 cells, as assessed by morphological criteria, enhancement of acetylcholine esterase activity and up-regulation of various differentiation markers. Moreover, tryptanthrin treatment led to the significant reduction of N-myc expression in LA-N-1 cells while siRNA directed against N-myc induced morphological differentiation of LA-N-1 cells. These results, when taken together, suggest that tryptanthrin suppressed the growth and induced neuronal differentiation in the human neuroblastoma LA-N-1 cells and might be exploited as a potential therapeutic candidate for the treatment of high-risk neuroblastomas with N-myc-amplification.

The localization of hnRNP A2/B1 in nuclear matrix and the aberrant expression during the RA‐induced differentiation of human neuroblastoma SK‐N‐SH cells

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is involved in the synthesis of RNA. Its expression is up-regulated in many tumor cell lines. In this study, we investigated the distribution of hnRNP A2/B1 in the nuclear matrix, including its co-localization with expression products of related genes. Results from 2-DE PAGE and MS showed that hnRNP A2/B1 is involved with components of nuclear matrix proteins of SK-N-SH cells, and that its expression level is down-regulated after retinoic acid (RA) treatment. Protein immunoblotting results further confirm the existence of hnRNP A2/B1 in the nuclear matrix, as well as its down-regulation after RA treatment. Immunofluorescence microscopy observation showed that hnRNP A2/B1 localized in nuclear matrix of SK-N-SH cells and its distribution regions were altered after RA treatment. Laser scanning confocal microscopy observation showed that hnRNP A2/B1 co-localized with c-Myc, c-Fos, P53, and Rb in SK-N-SH cells. The co-localized region was altered as a result of RA treatment. Our data proved that hnRNP A2/B1 is a nuclear matrix protein and can be up-regulated in human neuroblastoma. The expression and distribution of hnRNP A2/B1 can affect the differentiation of SK-N-SH cells, as well as its co-localization with related oncogenes and tumor suppressor genes.

Localization of Prohibitin in the Nuclear Matrix and Alteration of Its Expression During Differentiation of Human Neuroblastoma SK-N-SH Cells Induced by Retinoic Acid

The nuclear matrix-intermediate filament system of human neuroblastoma SK-N-SH cells before and after retinoic acid (RA) treatment was selectively extracted and the distribution of prohibitin (PHB) in the nuclear matrix, as well as its colocalization with related genes, was observed. Results of two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS) identification, and protein immunoblotting all confirm that PHB was present in the components of SK-N-SH nuclear matrix proteins and was down-regulated after RA treatment. Immunofluorescence microscopy observations show that PHB was localized in the nuclear matrix and its distribution was altered due to RA treatment. Laser confocal microscopy results reveal that PHB colocalized with the expression products of c-myc, c-fos, p53, and Rb, but the colocalization region was altered after RA treatment. Our results prove that PHB is a nuclear matrix protein and is localized in nuclear matrix fibers. The distribution of PHB in SK-N-SH cells and its colocalization with related proto-oncogenes and tumor suppressor genes suggest that PHB plays pivotal roles in the differentiation of SK-N-SH cells and deserves further study.

Localization of nucleophosmin in nuclear matrix and changes in its expression during the differentiation of human neuroblastoma induced by retinoic acid

In this article, we selectively extracted the nuclear matrix and intermediate filament system of human neuroblastoma SK-N-SH cells pre- and post-treated with retinoic acid (RA). The distribution of nucleophosmin (NPM) in the nuclear matrix and its colocalization with several products of related genes were investigated. Results from two-dimensional gel electrophoresis and MALDI-TOF showed that NPM was a component of the nuclear matrix and its expression in SK-N-SH cells post-treated with RA was down-regulated. Immunofluorescent microscopy observations further showed that NPM was localized in the nuclear matrix of SK-N-SH cells, and its expression level and distribution were altered after treatment with RA. The colocalization of NPM with c-myc, c-fos, p53, and Rb in SK-N-SH cells was observed under a laser scanning confocal microscope, but the colocalization region was changed by RA. Our results prove that NPM is a nuclear matrix protein, which is localized in nuclear matrix fibers. The colocalization of NPM with its related genes and oncogenes affect the differentiation of SK-N-SH cells. The expression of NPM and its distribution in the process of cell differentiation deserve more intensive investigation.

MYCN-regulated microRNAs repress estrogen receptor-α ( ESR1 ) expression and neuronal differentiation in human neuroblastoma

MYCN, a proto-oncogene normally expressed in the migrating neural crest, is in its amplified state a key factor in the genesis of human neuroblastoma (NB). However, the mechanisms underlying MYCN-mediated NB progression are poorly understood. Here, we present a MYCN-induced miRNA signature in human NB involving the activation and transrepression of several miRNA genes from paralogous clusters. Several family members derived from the miR-17 approximately 92 cluster, including miR-18a and miR-19a, were among the up-regulated miRNAs. Expression analysis of these miRNAs in NB tumors confirmed increased levels in MYCN-amplified samples. Specifically, we show that miR-18a and miR-19a target and repress the expression of estrogen receptor-alpha (ESR1), a ligand-inducible transcription factor implicated in neuronal differentiation. Immunohistochemical staining demonstrated ESR1 expression in human fetal sympathetic ganglia, suggesting a role for ESR1 during sympathetic nervous system development. Concordantly, lentiviral restoration of ESR1 in NB cells resulted in growth arrest and neuronal differentiation. Moreover, lentiviral-mediated inhibition of miR-18a in NB cells led to severe growth retardation, outgrowth of varicosity-containing neurites, and induction of neuronal sympathetic differentiation markers. Bioinformatic analyses of microarray data from NB tumors revealed that high ESR1 expression correlates with increased event-free survival in NB patients and favorable disease outcome. Thus, MYCN amplification may disrupt estrogen signaling sensitivity in primitive sympathetic cells through deregulation of ESR1, thereby preventing the normal induction of neuroblast differentiation. Collectively, our findings demonstrate the molecular consequences of abnormal miRNA transcription in a MYCN-driven tumor and offer unique insights into the pathology underlying MYCN-amplified NB.

Differential expression of nuclear matrix proteins during the differentiation of human neuroblastoma SK‐N‐SH cells induced by retinoic acid

To investigate the alteration of nuclear matrix proteins (NMPs) during the differentiation of neuroblastoma SK-N-SH cells induced by retinoic acid (RA), differentiation markers were detected by immunocytochemistry and NMPs were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. Immunocytochemical observation demonstrated that the expression of neuronal markers was up-regulated in SK-N-SH cells following RA treatment. Meanwhile, 52 NMPs (41 of which were identified) changed significantly during SK-N-SH differentiation; four of these NMPs were further confirmed by immunoblotting. This study suggests that the differentiation of neuroblastoma cells was accompanied by the altered expression of neuronal markers and NMPs. The presence of some differentially expressed NMPs was related to the proliferation and differentiation of neuroblastomas. Our results may help to reveal the relationship between NMPs and neuroblastoma carcinogenesis and reversion, as well as elucidate the regulatory principals driving neural cell proliferation and differentiation.

Reduction of neuropathy target esterase does not affect neuronal differentiation, but moderate expression induces neuronal differentiation in human neuroblastoma (SK-N-SH) cell line

Neuropathy target esterase (NTE) is inhibited and aged by organophosphorus compounds that induce delayed neuropathy in human and some sensitive animals. NTE has been proposed to play a role in neurite outgrowth and process elongation during neurodifferentiation. However, to date, there is no direct evidence of the relevance of NTE in neurodifferentiation under physiological conditions. In this study, we have investigated a possible role for NTE in the all- trans retinoic acid-induced differentiation of neuroblastoma cells. The functional inactivation of NTE by RNA interference indicated that reduction of NTE does not affect process outgrowth or differentiation of the cells, although moderate expression of NTE by expression of the NTE esterase domain accelerates the elongation of neurite processes. Mipafox, a neurotoxic organophosphate, was shown to block process outgrowth and differentiation in cells that have lowered NTE activity due to RNA interference, suggesting that mipafox may interact with other molecules to exert its effect in this context.

P21Waf1/Cip1 is a common target induced by short-chain fatty acid HDAC inhibitors (valproic acid, tributyrin and sodium butyrate) in neuroblastoma cells

Histone acetyltransferase and histone deacetylase (HDAC) determine the acetylation status of histones, and thereby control the regulation of gene expression. HDAC inhibitors have been found to inhibit the growth of a variety of tumor cells in vitro and in vivo. We demonstrated previously that the short-chain fatty acid compound butyrate and its derivative tributyrin (both HDAC inhibitors) arrest cell growth and induce differentiation in human neuroblastoma (NB) cells. In the current study we investigated the effect of the HDAC inhibitor valproic acid (VPA) on proliferation and differentiation in human NB cells (SJ-N-KP, AF8). Treatment with VPA resulted in a strong inhibition of cell proliferation and induction of cell differentiation, as revealed by neurite outgrowth and increase of acetylcholinesterase specific activity. Moreover, we addressed the question of whether the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) are involved in the mechanism of action of members of the short-chain fatty acids class (VPA, sodium butyrate and tributyrin) of HDAC inhibitors, in human NB cells. We demonstrated that p21(Cip1) is a common target of induction of transcription and protein expression for all the three compounds, while only VPA induced a concomitant increase of p27(Kip1) gene expression. These results suggest that p21(Cip1) could be involved in the inhibition of proliferation and induction of differentiation in human NB cells induced by treatment with VPA or tributyrin or sodium butyrate. Moreover, p21(Cip1) could be applied in the molecular monitoring of drug action in the possible therapeutic application of these short-chain fatty acid members of HDAC inhibitors for human NB treatment.

Inhibition of neuropathy target esterase expressing by antisense RNA does not affect neural differentiation in human neuroblastoma (SK-N-SH) cell line

Neuropathy target esterase (NTE) is phosphorylated and aged by oraganophosphorus compounds (OP) that induce delayed neuropathy in human and some animals. NTE has been proposed to play a role in neurite outgrowth and process elongation during neural differentiation. However, to date, there is no direct evidence of the relevance of NTE in neural differentiation under physiological conditions. In this study we have investigated a possible role for NTE in the all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells by antisense RNA. A NTE antisense RNA construct was generated and then transfected into human neuroblastoma SK-N-SH cells. A positive cell clone that can stably express NTE antisense RNA was obtained by G418 selection and then identified by western blotting. NTE activity was depressed in the transfected cells with only about 50% activity of the enzyme in the control cells. ATRA-induced differentiation of the neuroblastoma cells with lowered NTE activity revealed that inhibition of NTE expression does not affect neural differentiation in SK-N-SH cells. The result suggested that organophosphates may inhibit neural differentiation by initially acting on other targets other than NTE.

Identification of Dynamic Proteome Changes Upon Ligand Activation of Trk-Receptors Using Two-dimensional Fluorescence Difference Gel Electrophoresis and Mass Spectrometry

The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. Expression levels of TrkA/TrkB are important prognostic factors in a variety of embryonal tumors including neuroblastoma, the most common solid tumor of childhood. Because TrkA/TrkB exhibit a high level of sequence similarity and use overlapping pathways for signal transduction, the existence of specific effector molecules crucial for receptor and cell-type-specific response is likely. To identify these effectors by analyzing biological effects of TrkA and TrkB activation in a defined model, we performed a proteome study using the human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA. The use of the recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (Amersham Biosciences, Piscataway, NJ) allowed us to monitor differences in protein expression between samples in one gel. Proteomic changes were monitored in a time course of 0, 0.5, 1, 6, and 24 h following receptor activation. Using MALDI mass spectrometry, we identified, respectively, 22 and 9 differentially expressed proteins upon the addition of neurotrophin in SY5Y-TrkB and SY5Y-TrkA cells. Functional assignment revealed that the majority of these proteins are involved in organization and maintenance of cellular structures.

Induction of cyclin-dependent kinase 5 and its activator p35 through the extracellular-signal-regulated kinase and protein kinase A pathways during retinoic-acid mediated neuronal differentiation in human neuroblastoma SK-N-BE(2)C cells.

Cyclin-dependent kinase 5 (Cdk5), a neuronal Cdc2-like kinase, exhibits a variety of functions in neuronal differentiation and neurocytoskeleton dynamics, as well as neuronal degeneration. However, its role and induction mechanisms in retinoic acid (RA)-induced neuronal differentiation have not been well understood. In this study we newly found that RA treatment of SK-N-BE(2)C, human neuroblastoma cells, increased the expression of Cdk5 and its neuron specific activator p35 through the extracellular-signal-regulated kinase1/2 (ERK1/2) and cAMP-dependent protein kinase A (PKA) pathway. Inhibition of Cdk5 activity either by an inhibitor, roscovitine, or by transfection with a dominant negative form of Cdk5 caused a dramatic decrease in RA-induced differentiation, suggesting the requirement of Cdk5 kinase activity for the RA-induced neurite outgrowth. Furthermore, Cdk5 and p35 expression was decreased by ERK1/2 inhibition with PD98059 and increased by overexpression of a constitutive active mitogen-activated protein kinase kinase 1 (MEK1) mutant, suggesting the critical role of ERK1/2 in the induction of Cdk5 and p35. In addition, a transcription factor early growth response 1 (Egr-1) was induced by RA through the ERK1/2 pathway, suggesting its possible involvement in the p35 induction. RA treatment also induced c-fos mediated AP-1 binding, and cAMP-responsive element binding protein (CREB) mediated CRE binding via ERK1/2 and PKA pathway, respectively, in the Cdk5 promoter region, resulting in the induction of Cdk5. Our results suggest that ERK1/2 and PKA-induced regulation of Cdk5 activity possibly through Egr-1, c-fos, and CREB plays a critical role in the RA-induced neuronal differentiation.

Distinct mechanisms of cell cycle arrest control the decision between differentiation and senescence in human neuroblastoma cells

Retinoic acid (RA) induces cell cycle arrest and differentiation of human neuroblastoma (NB) cells. Typically, NB cells differentiate along the neuronal lineage, but quiescent, "flat" cell types frequently have been described after treatment with differentiating agents. Two indistinguishable subclones of the cell line SK-N-SH, SK-N-SH-N (SH-N) and SK-N-SH-F (SH-F), display dramatically different responses to RA. In SH-N, RA induces neuronal differentiation, but in SH-F it transforms the small neuroblastic cells into large, flattened, epithelium-like cells. Here we analyze the mechanistic basis for the different effects of RA in the two NB subclones. First, we show that the flattened RA-treated SH-F expresses markers of cells undergoing replicative senescence. Inhibition of DNA synthesis by RA is significantly more rapid in SH-F than in SH-N. SH-F, which expresses basal amounts of p16(INK4A), responds to RA with elevation of p18(INK4C), marked down-regulation of cyclin D1, and swift inhibition of cyclin D-dependent kinases (cdks). Conversely, after addition of RA, SH-N retains cell cycling due to high expression of cyclin D1, the absence of Ink4 inhibitors, and accumulation of p21(Cip1). These changes result in sustained cdk activity. Accordingly, overexpression of p21(Cip1) but not p16(INK4A) induces neuronal differentiation of untreated NB cells. We propose that rapid inhibition of cdks by RA in NB leads to early cell cycle arrest, prevents neuronal differentiation, and results in a senescence-like state.

Retinoblastoma‐related protein pRb2/p130 and its binding to the Bmyb promoter increase during human neuroblastoma differentiation

Neuroblastoma cells can undergo neural differentiation upon treatment with a variety of chemical inducers and growth factors. During this process, many cell cycle–related genes are downregulated while differentiation-specific genes are triggered. The retinoblastoma family proteins, pRb, p107, and pRb2/p130, are involved in transcriptional repression of proliferation genes, mainly through their interaction with the E2F transcription factors. We report that pRb2/p130 expression levels increased during differentiation of neuroblastoma cell line LAN-5. On the other hand, both pRb and p107 decreased and underwent progressive dephosphorylation at late differentiation times. The expression of B-myb and c-myb, two targets of the retinoblastoma family proteins, were downregulated in association with the increase of pRb2/p130, which was detected as the major component of the complex with E2F on the E2F site of the B-myb promoter in differentiated cells. Interestingly, E2F4, a preferential partner of p107 and pRb2/p130, was upregulated and underwent changes in cellular localization during differentiation. In conclusion, our data suggest a major role of pRb2/p130 in the regulation of B-myb promoter during neural differentiation despite the importance of cofactors in modulating the function of the retinoblastoma family proteins. J. Cell. Biochem. 67:297–303, 1997. © 1997 Wiley-Liss, Inc.

Retinoblastoma-related protein pRb2/p130 and its binding to theB-myb promoter increase during human neuroblastoma differentiation

Neuroblastoma cells can undergo neural differentiation upon treatment with a variety of chemical inducers and growth factors. During this process, many cell cycle-related genes are downregulated while differentiation-specific genes are triggered. The retinoblastoma family proteins, pRb, p107, and pRb2/p130, are involved in transcriptional repression of proliferation genes, mainly through their interaction with the E2F transcription factors. We report that pRb2/p130 expression levels increased during differentiation of neuroblastoma cell line LAN-5. On the other hand, both pRb and p107 decreased and underwent progressive dephosphorylation at late differentiation times. The expression of B-myb and c-myb, two targets of the retinoblastoma family proteins, were downregulated in association with the increase of pRb2/p130, which was detected as the major component of the complex with E2F on the E2F site of the B-myb promoter in differentiated cells. Interestingly, E2F4, a preferential partner of p107 and pRb2/p130, was upregulated and underwent changes in cellular localization during differentiation. In conclusion, our data suggest a major role of pRb2/p130 in the regulation of B-myb promoter during neural differentiation despite the importance of cofactors in modulating the function of the retinoblastoma family proteins.

The functionally antagonistic POU family transcription factors Brn-3a and Brn-3b show opposite changes in expression during the growth arrest and differentiation of human neuroblastoma cells.

The Brn-3a and Brn-3b POU family transcription factors have previously been shown to have functionally antagonistic effects, with Brn-3a activating specific gene promoters which are repressed by Brn-3b. We show here that proliferating cell lines of human neuroblastoma origin have a high ratio of Brn-3b to Brn-3a compared with other cell lines derived from related tumours. Moreover, the level of Brn-3a rises and that of Brn-3b falls when neuroblastoma cell lines are exposed to treatments which induce a cessation of proliferation. Such treatments also result in the activation of a test promoter which is normally stimulated by Brn-3a and repressed by Brn-3b. Our findings suggest that these antagonistic factors may play a key role in regulation of gene expression during human neuroblastoma differentiation and could thus represent a potential therapeutic target for treatments designed to induce such differentiation.

Antitumor activity of sodium valproate in cultures of human neuroblastoma cells.

Valproic acid (VPA) is a simple branched-chain fatty acid that has anticonvulsant activity and is widely used in the treatment of epilepsy. VPA was found to effect growth and differentiation of human neuroblastoma (NB) cells in vitro at concentrations that have been achieved in humans with no significant adverse effects. Treatment of UKF-NB-2 and UKF-NB-3 NB cell lines with VPA at concentrations ranging from 0.5 to 2 mM resulted in neuronal morphological differentiation characterized by extension of cellular processes without significant effects on cell viability. Ultrastructural features of VPA-treated cells were consistent with the neuronal type of differentiation. VPA treatment of NB cells was associated with decreased expression of N-myc oncoprotein and increased expression of neural cell adhesion molecule in their membrane. Treatment of NB cells with 0.5 mM VPA increased their sensitivity to lymphokine-activated killer lysis. The results indicate that VPA, at non-toxic pharmacological concentrations, arrests the growth, induces differentiation and increases immunogenicity of NB cells through non-toxic mechanisms.

Differential expression of ubiquitous and neuronal kinesin heavy chains during differentiation of human neuroblastoma and PC12 cells.

Kinesin is a microtubule-based motor protein involved in intracellular organelle transport. Neurons are characterized by the presence of at least two isoforms of conventional kinesin: ubiquitous kinesin, expressed in all cells and tissues, and neuronal kinesin, whose pattern of expression is confined to neuronal cells. In order to investigate whether the two kinesin motors, which are encoded by different genes, may play distinct biological roles in neurons, we studied their expression during neuronal differentiation. Human neuroblastoma SH-SY5Y and IMR32 cells and rat phaeochromocytoma PC12 cells were used as an in vitro system for neuronal differentiation and were induced to differentiate in the presence of retinoic acid, a combination of dibutyryl cAMP and 5-bromodeoxyuridine, and nerve growth factor respectively. The expression level of each kinesin isoform was evaluated by quantitative immunoblot before and after pharmacological treatment. We found that in all cell types the expression level of neuronal kinesin, but not of ubiquitous kinesin, is stimulated during differentiation. In particular, SH-SY5Y cells show a 4.5-fold, IMR32 cells a 3-fold and PC12 cells a 7-fold increase in the level of expression of neuronal kinesin. By Northern blot analysis we found that the selective increase in the expression of neuronal kinesin is paralleled by an increase in its mRNA, indicating that there is a transcriptional control of the expression of this kinesin isoform during differentiation of neuroblastoma and PC12 cells. Our results suggest that these cells represent an adequate model to study the function of conventional kinesin and its isoforms.

3,4-dihydroxyphenylalanine (dopa) metabolism and retinoic acid induced differentiation in human neuroblastoma.

In mature cells of the sympathetic nervous system and the adrenal gland, the activity of dihydroxyphenylalanine decarboxylase (DDC) is higher than that of tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (dopa) does not accumulate in the cells. On the other hand, it is known that in some neuroblastoma cells there is a relative deficiency of DDC, resulting in accumulation and secretion of dopa. Such a relative deficiency of DDC is a characteristic of neural cells at an early stage of neural crest development, suggesting the neuroblastoma are cells arrested in early neural crest development. If this were the case, it is possible that agents such as retinoic acid (RA) could induce neuroblastoma to differentiate into mature cells with respect to their metabolism of catecholamines. We have measured the effect of RA on the metabolism of dopa and expression of tyrosine hydroxylase and DDC in human neuroblastoma cell lines, CHP-126, CHP-134, IMR-32, NB-69, and LA-N-5. When the cell cultures were treated with RA, they showed wide variations in response as measured by morphological change, growth inhibition, enzyme activities and enzyme expressions. The RA treatment modulated the activities of tyrosine hydroxylase and DDC, but does not increase DDC relative to tyrosine hydroxylase. It is concluded that RA does not induce biochemical differentiation of the neuroblastoma into mature cells even when there are extensive morphological changes and suppression of growth rate.

Retinoic acid-induced differentiation of human neuroblastoma SH-SY5Y cells is associated with changes in the abundance of G proteins.

Western blot analysis, using subtype-specific anti-G protein antibodies, revealed the presence of the following G protein subunits in human neuroblastoma SH-SY5Y cells: Gs alpha, Gi alpha 1, Gi alpha 2, Go alpha, Gz alpha, and G beta. Differentiation of the cells by all-trans-retinoic acid (RA) treatment (10 mumol/L; 6 days) caused substantial alterations in the abundance of distinct G protein subunits. Concomitant with an enhanced expression of mu-opioid binding sites, the levels of the inhibitory G proteins Gi alpha 1 and Gi alpha 2 were found to be significantly increased. This coordinate up-regulation is accompanied by functional changes in mu-opioid receptor-stimulated low-Km GTPase, mu-receptor-mediated adenylate cyclase inhibition, and receptor-independent guanosine 5'-(beta gamma-imido)triphosphate [Gpp(NH)p; 10 nmol/L]-mediated attenuation of adenylate cyclase activity. In contrast, increased levels of inhibitory G proteins had no effect on muscarinic cholinergic receptor-mediated adenylate cyclase inhibition. With respect to stimulatory receptor systems, a reciprocal regulation was observed for prostaglandin E1 (PGE1) receptors and Gs alpha, the G protein subunit activating adenylate cyclase. RA treatment of SH-SY5Y cells increases both the number of PGE1 binding sites and PGE1-stimulated adenylate cyclase activity, but significantly reduced amounts of Gs alpha were found. This down-regulation is paralleled by a decrease in the stimulatory activity of Gs alpha as assessed in S49 cyc- reconstitution assays.(ABSTRACT TRUNCATED AT 250 WORDS)