Retinoic acid-induced differentiation of cultured human neuroblastoma cells: a comparison with phorbolester-induced differentiation

Cultured human SH-SY5Y neuroblastoma cells differentiated in the presence of retinoic acid (RA) or 12-0-tetradecanoyl-phorbol-13-acetate (TPA). In both cases, the cells acquired long cell processes and the cell growth was partially inhibited. Treatment with RA or TPA resulted in an increased neuron-specific enolase activity, relative to the total cellular enolase activity. At the optimal concentration, TPA induced a 200-fold increase in the concentration of noradrenalin, whereas in RA-treated cells the corresponding increase was only fourfold. Cells treated with a combination of RA and TPA were morphologically differentiated and growth inhibited and had a high relative activity of neuron-specific enolase. The increase in the concentration of noradrenalin induced by TPA was inhibited by RA in a concentration-dependent fashion. However, despite this result there seemed to be no general antagonistic effect of RA on the TPA-induced differentiation. The phenotypes of the cells treated by RA, TPA, or the combination of RA and TPA, did, on the other hand, differ from each other. Our results suggest that RA and TPA induce the SH-SY5Y cells to differentiate along different pathways.     

Retinoic acid-induced differentiation in a human neuroblastoma cell line is associated with an increase in nitric oxide synthesis

The human neuroblastoma cell line SK-N-BE, after incubation with 10 μM retinoic acid (RA) or 20 nM phorbol 12-myristate 13-acetate (PMA), underwent biochemical and morphological signs of differentiation within 10–14 days. In parallel, SK-N-BE cells produced significantly higher amounts of nitric oxide (NO) in comparison with controls, as assessed by the measurement of nitrite and nitrate in the culture supernatant and of NO synthase (NOS) activity in the cell lysates (measured as ability to convert [³H]arginine into [³H]citrulline and as NADPH diaphorase activity). Nitrite/nitrate production was abolished by adding the NO scavenger hemoglobin in the culture medium and was inhibited by aminoguanidine (AG, a selective inhibitor of the inducible NOS isoform) but not by the less selective inhibitor N^G-nitro-L -arginine methylester (NAME). Western blotting experiments with monoclonal antibodies against the ncNOS and iNOS isoforms suggest that RA-elicited NOS activation is not attributable to an increased expression of the protein. NAME and AG were not able to revert inhibition of proliferation induced by RA, and the NO donor sodium nitroprusside did not mimic the effect of RA and PMA. These data indicate that increased NO synthesis does not mediate RA- or PMA-induced differentiation but may be an additional marker of differentiation into sympathetic-like neuronal cells. J. Cell. Physiol. 174:99–106, 1998. © 1998 Wiley-Liss, Inc.     

Retinoic acid- and staurosporine-induced bidirectional differentiation of human neuroblastoma cell lines.

The differentiation pattern of two related human neuroblastoma cell lines, SK-N-SHF and SK-N-SHN, induced by retinoic acid and staurosporine was studied. Immunohistochemical and electron microscopic examination of the cells indicated that the SHF variant could undergo differentiation along a melanocytic route when treated with retinoic acid and to neuronal cells when treated with retionic acid and staurosporine together. Treatment of SHN cells with either or both these agents caused neuronal differentiation. The melanocytic pathway was characterized in part by the flattening of the cells, the appearance of melanocytic antigens and various forms of melanosomes, an increase in tyrosinase activity, and the absence of neuronal marker proteins. The neuronal route was typified by the development of long neuritic processes containing microtubules and numerous neurosecretory granules as well as by immunohistochemical reactions for neural cell adhesion molecule, synaptophysin, and neurofilament proteins. The significance of these results is discussed in terms of the differentiation responses of neuroblastoma cells to chemical agents as well as some of the factors involved in the regulation of phenotype expressions of these cells.     

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.     

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67^phox, one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67^phox membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation.     

Identification of a FXIIIA variant in human neuroblastoma cell lines

FXIII is a transglutaminase consisting of two catalytic (FXIIIA) and two non-catalytic subunits (FXIIIB) in plasma, where this enzyme is responsible for stabilizing fibrin clots. Although possible functions of intracellular FXIIIA have been proposed, these remain to be established. We show that a 40 kDa protein species of FXIIIA is present in the human neuroblastoma cell lines SH-SY5Y and LAN5. These data reveal the presence of a new uncharacterised variant of FXIIIA, possibly due to an alternative splicing, in nervous cells.     

Retinoic acid-induced differentiation of F9 embryonal carcinoma cells

The ability of retinoic acid (RA) to induce differentiation in embryonal carcinoma (EC) cells was examined by growing mouse F9 cells in a medium containing 1 μM RA. The altered properties of the cells became apparent after a lag period of approx. 24 h and were fully expressed after 5 days. The RA-induced phenotype was characterized by changes in cell morphology, slowing of the rate of cell multiplication, reduced DNA and protein synthesis, altered pattern of polypeptide synthesis and changes in cell surface components. The slowing of cell multiplication and general reduction in the rate of protein synthesis was paralleled by changes in the relative rates at which different polypeptides were synthesized. Two-dimensional gel electrophoretic analysis of [³⁵S]methioninelabelled cell proteins showed an altered relative synthesis of at least fifty polypeptides. The relative rate of synthesis of two components of the cytoskeleton identified as vimentin and tropomyosin were shown to increase.     

Retinoic acid-induced neural differentiation of embryonal carcinoma cells.

We have previously shown that the P19 line of embryonal carcinoma cells develops into neurons, astroglia, and fibroblasts after aggregation and exposure to retinoic acid. The neurons were initially identified by their morphology and by the presence of neurofilaments within their cytoplasm. We have more fully documented the neuronal nature of these cells by showing that their cell surfaces display tetanus toxin receptors, a neuronal cell marker, and that choline acetyl-transferase and acetyl cholinesterase activities appear coordinately in neuron-containing cultures. Several days before the appearance of neurons, there is a marked decrease in the amount of an embryonal carcinoma surface antigen, and at the same time there is a substantial decrease in the volumes of individual cells. Various retinoids were able to induce the development of neurons in cultures of aggregated P19 cells, but it did not appear that polyamine metabolism was involved in the effect. We have isolated a mutant clone which does not differentiate in the presence of any of the drugs which are normally effective in inducing differentiation of P19 cells. This mutant and others may help to elucidate the chain of events triggered by retinoic acid and other differentiation-inducing drugs.     

Sodium currents during differentiation in a human neuroblastoma cell line

The electrophysiological properties of a human neuroblastoma cell line, LA-N-5, were studied with the whole-cell configuration of the patch clamp technique before and after the induction of differentiation by retinoic acid, a vitamin A metabolite. Action potentials could be elicited from current clamped cells before the induction of differentiation, suggesting that some neuroblasts of the developing sympathetic nervous system are excitable. The action potential upstroke was carried by a sodium conductance, which was composed of two types of sodium currents, described by their sensitivity to tetrodotoxin (TTX) as TTX sensitive and TTX resistant. TTX-sensitive and TTX-resistant sodium currents were blocked by nanomolar and micromolar concentrations of TTX, respectively. The voltage sensitivity of activation and inactivation of TTX-resistant sodium current is shifted -10 to -30 mV relative to TTX-sensitive sodium current, suggesting that TTX-resistant sodium current could play a role in the initiation of action potentials. TTX-sensitive current comprised greater than 80% of the total sodium current in undifferentiated LA-N-5 cells. The surface density of total sodium current increased from 24.9 to 57.8 microA/microF after cells were induced to differentiate. The increase in total sodium current density was significant (P less than 0.05). The surface density of TTX-resistant sodium current did not change significantly during differentiation, from which we conclude that an increase in TTX-sensitive sodium current underlies the increase in total current.     

Retinoic acid negatively regulates p34cdc2 expression during human neuroblastoma differentiation.

p34cdc2 is a protein kinase that has an important role in controlling cell cycle progression and may regulate tumor suppressor gene activity. In this work, we show that the arrest of cell growth and induction of differentiation in a tumorigenic neuroblastoma cell line by retinoic acid (RA) is associated with a 75-fold decrease in the level of p34cdc2 protein. The RA induced decrease in p34cdc2 levels does not simply reflect the arrest of cell growth, because p34cdc2 levels are not reduced when neuroblastoma cells are growth arrested by nutrient deprivation. Furthermore, dephosphorylation of the tumor suppressor gene product RB, a substrate for the p34cdc2 kinase activity, is observed only when p34cdc2 levels are decreased in RA treated cells. These studies link regulation of cdc2 level, RB phosphorylation state, and induction of differentiation by RA and suggest that alterations in the cdc2 gene or in genes controlling its regulation contribute to tumorigenesis.     

Retinoic acid-induced modifications in the growth and cell surface components of a human carcinoma (HeLa) cell line

The addition of retinoic acid to cultures of HeLa-S3 cells caused a reduction in cell proliferation rate which became apparent after 72 h and was linearly dependent on retinoic acid concentration in the range 10⁻⁹–10⁻⁵ M. After 72 h of exposure to retinoic acid, the cells assumed a flattened appearance and no longer formed multilayers. These changes were reversed within 48 h after removal of retinoic acid from the medium. Structural analogs of retinoic acid with a free ---COOH group at C-15 were usually more potent in growth inhibition than compounds with an alcohol, aldehyde, ether or ester group. A cellular retinoic acid-binding protein was detected in cell homogenates, and the binding of [³H]retinoic acid to the binding protein was inhibited by most, but not all, analogs possessing a free terminal ---COOH group. For example, the 4-oxo analog of retinoic acid, while capable of inhibiting cellular proliferation, failed to bind to the retinoic acid-binding protein. Analysis of cell surface and cellular glycoproteins by lactoperoxidase-catalysed ¹²⁵I iodination and by metabolic labeling with [³H]glucosamine revealed that a 190000 D glycoprotein which was labeled by both methods and a 230000 D glycoprotein which was labeled only with [³H]glucosamine were labeled more intensely in retinoic acid-treated cells compared with untreated cells. The electrophoretic mobility of the 230000 D glycoprotein could be modified by treatment of intact cells with either neuraminidase or proteolytic enzymes, suggesting that this glycoprotein is also exposed on the cell surface. The cell surface alterations were detected much earlier than the onset of growth inhibition and appeared as early as 24 h after exposure to retinoic acid. The possible relationship between retinoic acid-induced changes in cell membrane structure, cell morphology, and cell proliferation is discussed.     

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.     

Retinoic acid-induced spina bifida: evidence for a pathogenetic mechanism

Treatment of C57Bl/6J mice with three successive doses of all-trans retinoic acid (28 mg kg-1 body weight) on 8 day, 6 h (8d,6h), 8d,12h, and 8d,18h of gestation resulted in a high incidence (79%, 31/39 fetuses) of spina bifida with myeloschisis (spina bifida aperta) in near term fetuses. Twelve hours following the last maternal dose (9d,6h), the caudal aspects of treated embryos, were abnormal, with eversion of the neural plate at the posterior neuropore, as compared to its normal concavity in comparably staged control specimens. This eversion persisted in affected embryos through the time that the posterior neuropore should normally close. The distribution of cell death in control and experimental embryos was determined using vital staining with Nile blue sulphate and with routine histological techniques. Twelve hours following the maternal dosing regimen, experimental embryos showed evidence of excessive cell death, predominantly in the mesenchyme associated with the primitive streak and in the endoderm of the tail gut, both of which are readily identifiable sites of physiological cell death at this stage of development. In addition, the presumptive trunk neural crest cells located in the dorsal midline, cranial to the posterior neuropore, exhibited a marked amount of cell death in the experimental embryos. We propose that the major factor in the generation of spina bifida in this model is excessive cell death in the tail gut and mesenchyme ventral to the neuroepithelium of the posterior neuropore.(ABSTRACT TRUNCATED AT 250 WORDS)     

An autocrine loop involving ret and glial cell-derived neurotrophic factor mediates retinoic acid-induced neuroblastoma cell differentiation

In several neuroblastoma cell lines, retinoic acid (RA)-induced differentiation is coupled to increased expression of functional neurotrophic factor receptors, including Trk family receptors and the glial cell-derived neurotrophic factor receptor, Ret. In several cases, increased expression is dependent on signaling through TrkB. Unlike TrkA and TrkB, Ret has never been implicated as a prognostic marker for neuroblastomas. SK-N-BE(2) cells do not express any of Trk family receptors; therefore, they are a choice system to study the specific role of Ret in RA-induced differentiation. Using a 2'-fluoro-RNA aptamer and a truncated Ret protein as specific inhibitors of Ret, we show that RA-induced differentiation is mediated by a positive autocrine loop that sustains Ret downstream signaling and depends on glial cell-derived neurotrophic factor expression and release. This report shows that in SK-N-BE(2) cells, stimulation of Ret is a major upstream mechanism needed to mediate RA-induced differentiation. These results provide important insights on the molecular mechanism of RA action, which might be relevant for the development of biologically based therapeutic strategies.     

Retinoic acid directs neuronal differentiation of human pluripotent stem cell lines in a non-cell-autonomous manner

Differentiation of human embryonic stem (ES) cells and embryonal carcinoma (EC) cells provides an in vitro model to study the process of neuronal differentiation. Retinoic acid (RA) is frequently used to promote neural differentiation of pluripotent cells under a wide variety of culture conditions. Through systematic comparison of differentiation conditions we demonstrate that RA induced neuronal differentiation of human ES and EC cells requires prolonged RA exposure and intercellular communication mediated by high cell density. These parameters are necessary for the up-regulation of neural gene expression (SOX2, PAX6 and NeuroD1) and the eventual appearance of neurons. Forced over-expression of neither SOX2 nor NEUROD1 was sufficient to overcome the density dependency of neuronal differentiation. Furthermore, inhibition of GSK3β activity blocked the ability of RA to direct cell differentiation along the neural lineage, suggesting a role for appropriately regulated WNT signalling. These data indicate that RA mediated neuronal differentiation of human EC and ES cell lines is not a cell autonomous program but comprises of a multi-staged program that requires intercellular input.     

Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro

The human embryonal carcinoma cell lines $\text{NT2}\text{D1}$ and $\text{NT2}\text{B9}$, clonally derived from Tera-2, differentiate extensively in vitro when exposed to retinoic acid. This differentiation is marked by the appearance of several morphologically distinct cell types and by changes in cell surface phenotype, particularly by the disappearance of stage-specific embryonic antigen-3 (SSEA-3), which is characteristically expressed by human EC cells. Among the differentiated cells are neurons, which form clusters interconnected by extended networks of axon bundles, and which express tetanus toxin receptors and neurofilament proteins. These observations constitute the first instance of extensive somatic differentiation of a clonal human EC cell line in vitro.     

Effect of cytidine analogs on cell growth and differentiation on a human neuroblastoma line

The cytidine analog 5-AZA-2'-deoxycytidine (5-AZA-CdR) has been demonstrated to induce cellular differentiation; on the other hand, induction of differentiation has been suggested as a possible form of therapy for leukemic cells. We have evaluated the possibility that the neuroblastoma malignant tumor growth could be controlled by treatment that promotes the differentiation of immature tumor cells. We have previously reported on differentiation of murine neuroblastoma cells (41A3) treated with 5-AZA-CdR. In this paper, we describe the effect of 5-AZA-CdR on human neuroblastoma cell line CHP-100. The drug-treated cells show some degree of differentiation, demonstrated by morphological and biochemical markers. A significant DNA hypomethylation and partial inhibition of DNA synthesis and cell proliferation is also observed. This effect is more stable than that caused by another cytidine analog, Cytosine-beta-D-Arabinofuranoside (ARA-C).