Induction of morphological and functional differentiation of human neuroblastoma cells by miR-124

Neuroblastoma is the most common extracranial solid tumour in children, and differentiation is considered its most appropriate therapy. In this work, we studied effects of miR-124 overexpression on differentiation in M17 cell line as a model of neuroblastoma cancer. Influence of miR-124 overexpression on differentiation in M17 cells was studied. M17 cells were infected with lentivirus that contained miR-124 precursor sequence and followed for 2 weeks to differentiate. Ectopic expression of miR-124 in M17 cells changed the shape of spherical undifferentiated cells to cells with extended neurites that formed neuronal networks. Overexpression of MiR-124 respectively increased the expression level of markers of β-Tubulin III, MAP2, SYN, NF-M and Nestin by 16-, 5-, 4-, 2.3- and 2-folds at the messenger RNA level. MiR-124 overexpression also increased the protein levels of β-Tubulin III and MAP2. Moreover, exogenous expression of miR-124 significantly increased the intracellular calcium in differentiated M17 cells. Since miR-124 is naturally expressed in neuronal cells and is downregulated in neuroblastoma cancer cells, differentiation with this type of microRNA can be a novel treatment for neuroblastoma cancer.     

Apoptosis Induced by Differentiation or Serum Deprivation in an Immortalized Central Nervous System Neuronal Cell Line

Abstract: To characterize the nature of programmed cell death (PCD) induced in neuronal cells during development, three regulators of apoptosis were investigated: one, the bcl-2-related genes, modulate cell survival, and the other two, the interleukin-1β converting enzyme (ICE)-related enzymes and the tumor suppressor protein p53, have been implicated as mediators of apoptosis. These regulators were studied in H19-7 cells, an SV40 T^ts-immortalized rat hippocampal neuronal cell line that can be differentiated with basic fibroblast growth factor at the nonpermissive temperature, resulting in a rapid attrition of cells by apoptosis. PCD occurred by two mechanisms in H19-7 cells: The first was initiated by removal of serum from undifferentiated cells, and the second was a consequence of neuronal differentiation. In differentiated H19-7 cells, the survival time was increased by both human bcl-2 and bcl-x_L, and this could be reversed by bcl-x_S.Addition of a peptide inhibitor of the ICE enzyme family to H19-7 cells resulted in a transient protection against differentiation-associated apoptosis, whereas no further protection was observed in the BCL-2- or BCL-X_L-expressing cells. Shifting the differentiated cells to 33°C to inactivate p53 did not significantly affect the apoptotic process, indicating that apoptosis induced by neuronal differentiation is not dependent on the continued presence of p53. By contrast, in undifferentiated cells, cell loss induced by transfer to serum-free media occurred more rapidly on inactivation of large T, consistent with p53 involvement. This medium-induced decrease in cell survival could not be rescued by the ICE inhibitor but was partially rescued by BCL-2 or BCL-X_L. Furthermore, studies involving expression of BCL-2 and BCL-X_L alone or together revealed differences in the survival dependent on the cellular environment. These results suggest that apoptosis of neuronal cellsoccurs by at least two processes: one in undifferentiated cells initiated by removal of serum and one linked to differentiation. The data implicate the ICE enzyme family but not p53 in apoptosis induced by differentiation and demonstrate that either BCL-2 or BCL-X_L can prolong the survival of differentiated neuronal cells.     

The cyclin-dependent kinase inhibitor p21 (WAF1) is required for survival of differentiating neuroblastoma cells.

We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.     

Expression of Calmodulin-Dependent Phosphodiesterase, Calmodulin-Dependent Protein Phosphatase, and Other Calmodulin-Binding Proteins in Human SMS-KCNR Neuroblastoma Cells

Calmodulin (CaM)-dependent enzymes, such as CaM-dependent phosphodiesterase (CaM-PDE), CaM-dependent protein phosphatase (CN), and CaM-dependent protein kinase II (CaM kinase II), are found in high concentrations in differentiated mammalian neurons. In order to determine whether neuroblastoma cells express these CaM-dependent enzymes as a consequence of cellular differentiation, a series of experiments was performed on human SMS-KCNR neuroblastoma cells; these cells morphologically differentiate in response to retinoic acid and phorbol esters [12-O-tetradecanoylphorbol 13-acetate (TPA)]. Using biotinylated CaM overlay procedures, immunoblotting, and protein phosphorylation assays, we found that SMS-KCNR cells expressed CN and CaM-PDE, but did not appear to have other neuronal CaM-binding proteins. Exposure to retinoic acid, TPA, or conditioned media from human HTB-14 glioma cells did not markedly alter the expression of CaM-binding proteins; 21-day treatment with retinoic acid, however, did induce expression of novel CaM-binding proteins of 74 and 76 kilodaltons. Using affinity-purified polyclonal antibodies, CaM-PDE immunoreactivity was detected as a 75-kilodalton peptide in undifferentiated cells, but as a 61-kilodalton peptide in differentiated cells. CaM kinase II activity and subunit autophosphorylation was not evident in either undifferentiated or neurite-bearing cells; however, CaM-dependent phosphatase activity was seen. Immunoblot analysis with affinity-purified antibodies against CN indicated that this enzyme was present in SMS-KCNR cells regardless of their state of differentiation. Although SMS-KCNR cells did not show a complete pattern of neuronal CaM-binding proteins, particularly because CaM kinase II activity was lacking, they may be useful models for examination of CaM-PDE and CN expression. It is possible that CaM-dependent enzymes can be used as sensitive markers for terminal neuronal differentiation.     

The neuronal differentiation process involves a series of antioxidant proteins

Summary. Involvement of individual antioxidant proteins (AOXP) and antioxidants in the differentiation process has been already reported. A systematic search strategy for detecting differentially regulated AOXP in neuronal differentiation, however, has not been published so far. The aim of this study was to provide an analytical tool identifying AOXP and to generate a differentiation-related AOXP expressional pattern. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments: We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical (MALDI-TOF-TOF) identification of proteins to generate a map of AOXP. 16 AOXP were unambiguously determined in both cell lines; catalase, thioredoxin domain-containing protein 4 and hypothetical glutaredoxin/glutathione S-transferase C terminus-containing protein were detectable in the undifferentiated cells only. Five AOXP were observed in both, undifferentiated and differentiated cells and thioredoxin, thioredoxin-like protein p19, thioredoxin reductase 1, superoxide dismutases (Mn and Cu-Zn), glutathione synthetase, glutathione S-transferase P1 and Mu1 were detected in differentiated cells exclusively. Herein a differential expressional pattern is presented that reveals so far unpublished antioxidant principles involved in neuronal differentiation by a protein chemical approach, unambiguously identifying AOXP. This finding not only shows concomitant determination of AOXP but also serves as an analytical tool and forms the basis for design of future studies addressing AOXP and differentiation per se.     

Caspase 8L, a novel inhibitory isoform of caspase 8, is associated with undifferentiated neuroblastoma

Caspase 8 is a key apoptotic factor in the receptor/ligand apoptosis-signaling cascade. Absent caspase 8 expression is shown to correlate with poor prognosis in neuroblastoma. Paradoxically, the caspase 8 gene can produce as plice variant and novel inhibitor of itself-caspase 8l. The presence of caspase 8 alone in tumors may not necessarily portend a good prognosis. We sought to determine whether caspase 8l is present in neuroblastoma and whether over-expression of this protein could inhibit caspase 8-dependent apoptosis. Six of 6 histologically undifferentiated and 2 of 5 differentiated neuroblastoma tumors expressed the caspase 8l isoform, whereas caspase 8l was absent in 3 of 3 ganglioneuromas. Seven human neuroblastoma cell lines were surveyed. Two of the 5 cell lines that expressed caspase 8 also expressed the caspase 8l isoform and both were of a less differentiated neuronal phenotype. Over-expression of caspase 8l in cell lines afforded protection against TRAIL, but not against etoposide induced apoptosis. Conversely, blockade of Caspase 8l in cells that express this splice variant made them more sensitive to apoptosis induced cell death. We demonstrate the caspase 8l isoform is present in neuroblastoma and appears to be associated with undifferentiated cell lines and tumors. Furthermore, it suppresses caspase 8-dependent apoptosis.     

Metabolism of two Go alpha isoforms in neuronal cells during differentiation

We have previously shown that undifferentiated N1E-115 neuroblastoma cells express only one isoform of Go alpha (pI = 5.8), whereas differentiated neuroblastoma cells expressed, in addition to this isoform, another Go alpha with a more acidic pI (5.55). Moreover, primary cultures of cerebellar granule cells, which are extremely well differentiated cells yielding a high density of synapses, expressed only a single Go alpha isoform with a pI of 5.55 (Brabet, P., Pantaloni, C., Rodriguez Martinez, J., Bockaert, J., and Homburger, V. (1990) J. Neurochem. 54, 1310-1320). In this report, using biosynthetic labeling with [35S]methionine and specific quantitative immunoprecipitation with a polyclonal antibody raised against the purified Go alpha protein, we have determined 1) the degradation rate of total Go alpha (sum of the two isoforms) in differentiated as well as in undifferentiated neuroblastoma cells and in cerebellar granule cells, 2) the degradation rates of each isoform in differentiated neuroblastoma cells. The t 1/2 for total Go alpha protein degradation was very different in the three neuronal cell populations and was 28 +/- 5 h (n = 5), 58 +/- 9 h (n = 5), and 154 +/- 22 h (n = 6) in undifferentiated, differentiated neuroblastoma, and granule cells, respectively. Using two-dimensional gel analysis of immunoprecipitates, we have also determined the individual t 1/2 for degradation of each Go alpha isoform in differentiated neuroblastoma cells, in which the two Go alpha isoforms were expressed. Results indicated that the two Go alpha isoforms exhibit similar t1/2 for degradation (49 +/- 5 h, n = 3). Thus, the t1/2 for degradation of the more basic Go alpha isoform is higher in differentiated neuroblastoma cells (49 +/- 5 h, n = 3) than in undifferentiated neuroblastoma cells (28 +/- 5 h, n = 5) which expressed only the more basic Go alpha isoform. It can be concluded that the degradation rate of the more basic Go alpha isoform is not a characteristic of the protein itself but depends on the state of the cell differentiation. The comparison between the t1/2 for degradation of the more acidic Go alpha isoform is differentiated neuroblastoma cells (51 +/- 6 h, n = 3) with that of cerebellar granule cells (154 +/- 22 h, n = 6) suggests that there is also a decrease in the degradation rate of the more acidic Go alpha isoform during differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

A Role for the Putative Tumor Suppressor Bin1 in Muscle Cell Differentiation

Bin1 is a Myc-interacting protein with features of a tumor suppressor. The high level of Bin1 expression in skeletal muscle prompted us to investigate its role in muscle differentiation. Significant levels of Bin1 were observed in undifferentiated C2C12 myoblasts, a murine in vitro model system. Induction of differentiation by growth factor withdrawal led to an upregulation of Bin1 mRNA and to the generation of higher-molecular-weight forms of Bin1 protein by alternate splicing. While Bin1 in undifferentiated cells was localized exclusively in the nucleus, differentiation-associated isoforms of Bin1 were found in the cytoplasm as well. To examine the function of Bin1 during differentiation, we generated stable cell lines that express exogenous human Bin1 cDNA in the sense or antisense orientation. Cells overexpressing Bin1 grew more slowly than control cells and differentiated more rapidly when deprived of growth factors. In contrast, C2C12 cells expressing antisense Bin1 showed an impaired ability to undergo differentiation. Taken together, the results indicated that Bin1 expression, structure, and localization are tightly regulated during muscle differentiation and suggested that Bin1 plays a functional role in the differentiation process.     

Expression and DNA-binding activity of MYCN/Max and Mnt/Max during induced differentiation of human neuroblastoma cells

Amplification of MYCN is one of the most important prognostic markers for neuroblastoma and is correlated with rapid tumor progression and poor prognosis. MYCN belongs to the Myc/Max/Mad/Mnt network of proteins that regulate proliferation, apoptosis, and differentiation. It is well established that MYCN is downregulated during induced differentiation of neuroblastoma cells carrying an amplified MYCN gene, but very little is known about other components of the network, i.e., the Max, Mad, and Mnt proteins, during this process. In this study we show that Mad and Mnt expression was only modestly regulated in differentiating SK-N-BE(2) neuroblastoma cells, while MYCN was rapidly downregulated. This downregulation was reflected in a decreased MYCN/Max DNA-binding activity while the Mnt/Max binding did not change during differentiation. In parallel experiments we also analyzed the Myc/Max/Mad expression and DNA binding capacity during induced differentiation in the MYCN single copy neuroblastoma cell line SH-SY5Y. In this cell line only modest changes in expression of the components of the MYCN/Max/Mad/Mnt network was detected, but since the cell line expresses relatively low levels of MYCN and c-Myc, these changes might be of functional significance. Cell cycle analyses of SK-N-BE(2) demonstrated an increase in the G1-phase fraction after RA-treatment. These data show that the decreased MYCN expression and MYCN DNA-binding is correlated with retarded cell cycle progression. Furthermore, when Mad1 or Mnt was overexpressed in SK-N-BE(2) cells they retained the capacity to differentiate, underscoring the notion that MYCN downregulation, and not changes in Mad/Mnt expression, is essential for neuroblastoma cell differentiation.     

Down-regulated RPS3a/nbl expression during retinoid-induced differentiation of HL-60 cells: a close association with diminished susceptibility to actinomycin D-stimulated apoptosis

The efficacy of anticancer agents significantly depends on the differential susceptibility of undifferentiated cancer cells and differentiated normal cells to undergo apoptosis. We previously found that enhanced expression of RPS3a/nbl, which apparently encodes a ribosomal protein, seems to prime cells for apoptosis, while suppressing such enhanced expression triggers cell death. The present study found that HL-60 cells induced to differentiate by all-trans retinoic acid did not undergo apoptosis following treatment with actinomycin D whereas undifferentiated HL-60 cells were highly apoptosis-susceptible, confirming earlier suggestions that differentiated cells have diminished apoptosis-susceptibility. Undifferentiated HL-60 cells highly expressed RPS3a/nbl whereas all-trans retinoic acid -induced differentiated cells exhibited markedly reduced levels, suggesting that apoptosis-resistance of differentiated cells could be due to low RPS3a/nbl expression. Down-regulation of enhanced RPS3a/nbl expression was also observed in cells induced to differentiate with the retinoid 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1- propenyl]benzoic acid without any significant induction of cell death. While down-regulation of RPS3a/nbl expression during differentiation did not apparently induce apoptosis, RPS3a/nbl antisense oligomers triggered death of undifferentiated HL-60 cells, but not of retinoid-induced differentiated cells. It therefore seems that while down-regulation of enhanced RPS3a/nbl expression can induce apoptosis in undifferentiated cells, down-regulation of enhanced RPS3a/nbl expression during differentiation occurs independently of apoptosis, and could be regarded as reverting the primed condition to the unprimed (low RPS3a/nbl) state.     

Increase in apoptosis with neural differentiation and shortening of the lifespan of P19 cells overexpressing tau

Apoptosis or programmed cell death is considered to be involved in neurodegenerative disorders including Alzheimer's disease (AD). AD is characterized by intracellular aggregates of hyperphosphorylated tau, a microtubule-associated protein. To investigate the effect of the overexpression of tau in P19 cells, we engineered P19 wild-type cells (P19wt) stably expressing human tau441 (P19tau). When P19tau cells were induced to undergo neural differentiation by treatment with retinoic acid (RA), a remarkable increase in apoptosis was observed. However, in the undifferentiated state, there was no notable difference of phenotype between P19wt and P19tau cells. Additionally, we found that tau dissociated from microtubules, and co-localized with the RA receptor (RAR) at nucleoli. Further, the lifespan of the differentiated P19tau cells was shorter than that of P19wt cells, and the re-treatment of differentiated P19wt cells with RA resulted in a reduction of lifespan. These observations suggested that tau affects RA signaling in apoptosis and lifespan during the neural differentiation induced by RA treatment.     

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.     

Marked change in microRNA expression during neuronal differentiation of human teratocarcinoma NTera2D1 and mouse embryonal carcinoma P19 cells

MicroRNAs (miRNAs) are small noncoding RNAs, with a length of 19-23 nucleotides, which appear to be involved in the regulation of gene expression by inhibiting the translation of messenger RNAs carrying partially or nearly complementary sequences to the miRNAs in their 3' untranslated regions. Expression analysis of miRNAs is necessary to understand their complex role in the regulation of gene expression during the development, differentiation and proliferation of cells. Here we report on the expression profile analysis of miRNAs in human teratocarcinoma NTere2D1, mouse embryonic carcinoma P19, mouse neuroblastoma Neuro2a and rat pheochromocytoma PC12D cells, which can be induced into differentiated cells with long neuritic processes, i.e., after cell differentiation, such that the resultant cells look similar to neuronal cells. The data presented here indicate marked changes in the expression of miRNAs, as well as genes related to neuronal development, occurred in the differentiation of NTera2D1 and P19 cells. Significant changes in miRNA expression were not observed in Neuro2a and PC12D cells, although they showed apparent morphologic change between undifferentiated and differentiated cells. Of the miRNAs investigated, the expression of miRNAs belonging to the miR-302 cluster, which is known to be specifically expressed in embryonic stem cells, and of miR-124a specific to the brain, appeared to be markedly changed. The miR-302 cluster was potently expressed in undifferentiated NTera2D1 and P19 cells, but hardly in differentiated cells, such that miR-124a showed an opposite expression pattern to the miR-302 cluster. Based on these observations, it is suggested that the miR-302 cluster and miR-124a may be useful molecular indicators in the assessment of degree of undifferentiation and/or differentiation in the course of neuronal differentiation.     

Modification of the indolamine content in neuroblastoma × glioma hybrid NG108-15 cells upon induced differentiation

1. The neuroblastoma x glioma hybrid NG108-15 cell line has been widely studied as a neuronal model for its serotonergic, cholinergic, and peptidergic properties. 2. The catecholamine and serotonin content and that of their major metabolites have been determined by high-performance liquid chromatography with electrochemical detection (HPLC-EC) in NG108-15 cells under differentiated and undifferentiated conditions. 3. Cellular contents of L-DOPA, norepinephrine, (NE), L-epinephrine (EPI), and dopamine (DA) in differentiated cells, induced by 1 mM dibutyryl cyclic AMP (dBcAMP), are 149, 40, 129, and 124%, respectively, higher than those in undifferentiated cells. 4. 3,4-Dihydroxyphenethylacetic acid (DOPAC), the major metabolite of DA, is detectable only in differentiated cells. Similarly, DOPAC is present only in culture medium from differentiated cells, and not that of undifferentiated cells. 5. Serotonin (5-HT) is detectable only in undifferentiated cells; and the level of 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of 5-HT, is also 12.7% higher is undifferentiated cells. 6. Comparative analyses of differentiated and undifferentiated cells in monolayer cultures and undifferentiated cells cultured in the presence of 1 mM dBcAMP under suspension conditions suggest that change in the indolamine content is due to cellular changes upon morphological differentiation. 7. The clonal NG108-15 cell line is also catecholaminergic, in addition to cholinergic and serotonergic; and a shift of neurotransmitter pattern from serotonin to dopamine production occurs during morphological differentiation.     

Nerve growth factor (NGF) induced differentiation of human neuroblastoma cells

1. Nerve growth factor (NGF) induced differentiation of human neuroblastoma cell line. 2. The differentiated cells had a relatively high activity of adenylate cyclase and cyclic AMP phosphodiesterase, and a high intracellular level of cyclic AMP. 3. These cells synthesized a higher amount of met5-o-enkephalin than undifferentiated cells. 4. Undifferentiated cells bound less met5-enkephalin than differentiated cells. The maximum number of [3H]met5-enkephalin receptor sites per mg of membrane protein increased more in differentiated cells. 5. Previous observations taken together with our results suggests that increased intracellular levels of cyclic AMP after treatment with NGF induced differentiation of human neuroblastoma cells. Reversal of undifferentiated tumor cells into the differentiated changes the capacity of synthesis of met5-enkephalin and its interaction with receptors.     

Immunolocalization of secretogranin II, chromogranin A, and chromogranin B in differentiating human neuroblastoma cells.

In order to obtain further insights into the expression of the known markers of secretory neuroendocrine dense core organelles, secretogranin II (SgII), chromogranin A (CgA), and chromogranin B (CgB) during neuronal differentiation, the immunolocalization of these proteins was studied by means of double immunofluorescence in both undifferentiated and retinoic acid-differentiated SH-SY5Y human neuroblastoma cells. The majority of undifferentiated cells was not immunolabeled for all three proteins. In the majority of differentiated cells, a clearly punctate SgII immunolabeling indicative of the presence of secretory organelles was present in the Golgi region, at the cell periphery, along the neurites and in growth cones. Only relatively few of the SgII-immunolabeled cells were also immunolabeled for CgA and CgB, and in a single cell the three proteins were not always present in the same organelles. These results, obtained in a cultured cell line, confirm the not necessarily parallel distribution of SgII, CgA, and CgB observed in different neuroendocrine tissues and suggest that SgII may be the best marker of human neuroblastoma cell differentiation.     

Antisense inhibition of BCL-2 expression induces retinoic acid-mediated cell death during differentiation of human NT2N neurons

Changes in expression of the proto-oncogene Bcl-2 are well known in the developing brain, with a high expression level in young post-mitotic neurons that are beginning the outgrowth of processes. The physiological significance of the Bcl-2 up-regulation in these neurons is not fully understood. We used a differentiation model for human CNS neurons to study the expression and function of Bcl-2. NT2/D1 human neuronal precursor cells differentiated into a neuronal phenotype in the presence of 10 microM retinoic acid for 3-5 weeks. This concentration of retinoic acid was not toxic to undifferentiated NT2/D1 cells but was sufficient to up-regulate the BCL-2 protein in 6 days. The BCL-2 levels increased further after 3 weeks, i.e. when the cells started to show neuronal morphology. Inhibition of the accumulation of endogenous BCL-2 with vectors expressing the antisense mRNA of Bcl-2 caused extensive apoptosis after 3 weeks of the retinoic acid treatment. The loss of neuron-like cells from differentiating cultures indicated that the dead cells were those committed to neuronal differentiation. Death was related to the presence of retinoic acid since withdrawal of retinoic acid after 16 days of treatment dramatically increased cell surviving. The ability of BCL-2 to prevent retinoic acid-induced cell death was also confirmed in undifferentiated NT2/D1 cells that were transfected with a vector containing Bcl-2 cDNA in sense orientation and exposed to toxic doses (40-80 microM) of retinoic acid. Furthermore, down-regulation of BCL-2 levels by an antisense oligonucleotide in neuronally differentiated NT2/D1 cells increased their susceptibility to retinoic acid-induced apoptosis. These results indicate that one function of the up-regulation of endogenous BCL-2 during neuronal differentiation is to regulate the sensitivity of young post-mitotic neurons to retinoic acid-mediated apoptosis.     

Sodium and calcium currents in neuroblastoma x glioma hybrid cells before and after morphological differentiation by dibutyryl cyclic AMP

Sodium and calcium inward currents (INa and ICa) were measured in neuroblastoma X glioma hybrid cells of clones 108CC5 and 108CC15 by a single suction pipette method for internal perfusion and voltage clamp. Morphologically undifferentiated, exponentially growing cells were compared with cells differentiated by cultivation with 1 mmol/l dibutyryl cyclic AMP. Outward currents were eliminated by perfusing the cells with a K+-free solution. Voltage dependence and ion selectivity as well as steady state inactivation characteristics of INa and ICa resembled those of differentiated mouse neuroblastoma cells, clone N1E-115 (Moolenaar and Spector 1978, 1979). These parameters were identical in undifferentiated and differentiated cells of both clones. After differentiation the average density of the peak sodium and calcium currents was increased two and four-fold, respectively, in both cell lines. Our data indicate that exponentially growing, morphologically undifferentiated 108CC5 and 108CC15 neuroblastoma X glioma hybrid cells possess functional Na+ and Ca2+ channels undistinguishable from those of non-proliferating cells of these clones differentiated morphologically by treatment with dibutyryl cyclic AMP. That Na+ and Ca2+ spikes were not detected by other authors in these cells prior to morphological differentiation by dibutyryl cyclic AMP may be attributed to the fact that at the low resting membrane potential measured the Na+ and Ca2+ channels are inactivated.