Insulin binding and fluid-phase endocytosis stimulation in the mouse neuroblastoma cell line 41A3

As well as many other hormones and growth factors, insulin is known to influence several processes in the CNS; its specific effects, however, are still poorly understood. Neuroblastoma cell lines represent a useful experimental system for the analysis of the insulin-specific effect on neurons, in the absence of possible regulatory mechanisms elicited by other neuronal/glial cells and/or soluble factors. The expression and the binding properties of insulin receptors, as well as the insulin effects on both membrane fluidity and cell surface architecture, have been investigated in 41A3 mouse neuroblastoma cells, by radioligand-binding fluorescence spectroscopy and scanning electron microscopy, respectively the same cells, insulin-induced modifications on cytoskeletal organisation also have been studied. Binding studies were performed using 125I-insulin, while the cationic fluorescent probe trimethylammonium 1,6-diphenyl-1,3,5-hexatriene was used for biophysical investigations. The results presented in this paper provide evidence that insulin interacts with 41A3 neuroblastoma cells through a receptor-mediated mechanism and that, in these cells, insulin binding modifies the cell surface morphology and stimulates endocytosis.     

Control of expression of differentiated functions in neuroblastoma cell hybrids

Cell hybrids have been extensively utilized for gene mapping; more than 50 enzymes and nonenzyme proteins have been assigned to individual human chromosomes. Hybrids have also been used in the study of differentiation; fusions involving mouse or human neuroblastoma cells and various nonneuronal lines resulted in hybrid cells that continued to express neuronspecific functions. The expression of the differentiated state is, however, not an all-or-none phenomenon: One neuronal trait may be evident in such hybrids, in the absence of others. The potential usefulness of the human neuroblastoma hybrids for the assignment of genes involved in the expression of differentiated functions to specific chromosomes is discussed.     

Schwann cell-conditioned medium promotes neuroblastoma survival and differentiation

Neuroblastomas are histopathologically heterogeneous, ranging from immature malignant tumors to benign ganglioneuromas. The amount of Schwann cell stroma greatly increases with neuroblastoma differentiation, and these Schwann cells appear to be normal cells that infiltrate the tumor. To determine whether Schwann cells influence neuroblast differentiation, four human neuroblastoma cell lines were cultured in the presence or absence of human Schwann cell-conditioned medium for 7 days. Neuroblastoma cell survival, as determined by a colorimetric assay, more than doubled in three of the four neuroblastoma cell lines in the Schwann cell-conditioned medium. There was a corresponding reduction in apoptosis as measured by a nick-end labeling assay, with little change in mitotic rate. Schwann cell-conditioned medium induced extensive neurite outgrowth in all of the neuroblastoma cell lines, and these processes contained mature neurofilament in three of the cell lines. These results indicate that Schwann cells produce soluble substances capable of supporting survival and differentiation in neuroblastoma cell lines. This may represent a biological mechanism responsible for neuronal differentiation in stroma-rich neuroblastomas.     

Neuroblastoma and Alzheimer's disease brain cells contain aromatase activity

Human brain steroidogenic mechanisms, particularly aromatase, have been investigated in healthy and diseased conditions. Aromatase activity was measured in differentiated and undifferentiated neuroblastoma cell lines from mouse (TMN) and human (5H SY5Y) and in human post mortem brain samples. Neuroblastomas show much higher aromatase activity than human brain samples. Homogenates of adult human male and female cortex and frontal and temporal areas of both Alzheimer's and control patients all show considerably lower activity. The temporal area has significantly higher aromatase activity than the frontal. Aromatisation activity in differentiated neuroblastoma cells of both species is lower than in undifferentiated cells. These results are consistent with an inverse relationship between brain estrogen formation and stage of neuronal differentiation and the hypothesis that aromatase may be involved in the early stages of neuronal growth. Significant but variable activities of other androgen-metabolising enzymes, such as 5 alpha-reductase, 3 alpha/beta-hydroxysteroid dehydrogenases, and 17 beta-hydroxysteroid dehydrogenase, which generate a spectrum of regulatory molecules, are also found.     

Induction of apoptosis in human neuroblastoma cells by abrogation of integrin-mediated cell adhesion

The survival, proliferation and differentiation of neuroblastoma (NB) cells are largely dependent on adhesion to extracellular matrix (ECM) proteins. Integrin occupancy seems to play a primary role. To elucidate the role of integrin heterodimers during neuronal cell death, we have analysed the changes in integrin expression in 2 human NB cell lines which represent different stages of neuronal maturation. Retinoic acid (RA) had different effects on the 2 NB cell lines: on LAN-5 cells it acted as a differentiation-promoting agent, while it had an anti-proliferative effect on GI-LI-N cells, driving them to apoptosis. Indeed, this occurrence was evidenced by the visualization of a "DNA ladder" on gel electrophoresis, by propidium iodide staining, and by DNA flow cytofluorimetric analysis. RA treatment rapidly and drastically decreased integrin expression and cell adhesion on GI-LI-N cells. These findings were also obtained by treating both NB cell lines with the apoptotic agent fenretinide. Furthermore, treatment of NB cells with anti-sense oligonucleotides to beta 1 integrin chain specifically induced chromatin condensation and nucleosomal DNA laddering. Moreover, blocking cell-matrix interactions by means of perturbing antibody against beta 1 subunit resulted in the induction of typical features of apoptotic cells. In conclusion, these findings indicate that abrogation of cell adhesion through down-modulation of integrin receptors plays a crucial role in the induction of neuroblastoma programmed cell death.     

Expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion neurons: caveolin-2 is up-regulated in response to cell injury

Caveolae are cholesterol/sphingolipid-rich microdomains of the plasma membrane that have been implicated in signal transduction and vesicular trafficking. Caveolins are a family of caveolae-associated integral membrane proteins. Caveolin-1 and -2 show the widest range of expression, whereas caveolin-3 expression is restricted to muscle cell types. It has been previously reported that little or no caveolin mRNA species are detectable in the brain by Northern blot analyses or in neuroblastoma cell lines. However, it remains unknown whether caveolins are expressed within neuronal cells. Here we demonstrate the expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion (DRG) neurons by using mono-specific antibody probes. In PC12 cells, caveolin-1 expression is up-regulated on day 4 of nerve growth factor (NGF) treatment, whereas caveolin-2 expression is transiently up-regulated early in the differentiation program and then rapidly down-regulated. Interestingly, caveolin-2 is up-regulated in response to the mechanical injury of differentiated PC12 cells; up-regulation of caveolin-2 under these conditions is strictly dependent on continued treatment with NGF. Robust expression of caveolin-1 and -2 is also observed along the entire cell surface of DRG neurons, including high levels on growth cones. These findings demonstrate that neuronal cells express caveolins.     

IFN-tau increases PGE2 production and COX-2 gene expression in the bovine endometrium in vitro

Autologous peripheral blood stem cells, obtained by CD34+ stem cell selection, are being used with increasing frequency for transplantation in patients with neuroblastoma. Here, we examined the surface membrane antigens of neuroblastoma cells with a panel of hematopoietic monoclonal antibodies (mAbs), including anti-CD34 mAbs, by flow cytometric analysis. We found stronger binding of anti-CD34 mAbs to clonogenic, less differentiated, non-adherent neuroblastoma cells than to adherent neuroblastoma cells. Moreover, the majority of neuroblastoma cell lines shared hematopoietic-associated antigens with all blood cells. Because of these cross-reactions, especially found with the anti-CD34 mAbs 12.8 and ICH3, we have demonstrated that there is a potential risk of cell harvest contamination by circulating neuroblastoma cells during CD34+ stem cell selection.     

Cytotoxicity of aluminum silicates in primary neuronal cultures

To study their cytotoxicity, clays containing aluminum silicates were added to cultures of primary murine spinal cord neurons and differentiated N1E-115 neuroblastoma cells. Bentonite (0.1 mg/ml) and montmorillonite (0.1 mg/ml) rapidly associated with the outer membrane of both N1E-115 and neuronal cells. Erionite (0.1 mg/ml) was randomly distributed throughout the culture. Both bentonite and montmorillonite caused complete cell lysis in the neuronal cultures within 60 min following addition. Erionite had no effect. None of the clays appeared to be cytotoxic to the differentiated N1E-115 cells even though bentonite and montmorillonite were closely associated with the cell membrane. N1E-115 cell lysis did not occur up to 18 h after addition of the clay. Aluminum silicate-containing clays caused a rapid lysis of primary neuronal cells. Differentiated N1E-115 neuroblastoma cells were not susceptible to clay-induced lysis, suggesting that the lytic mechanism is not a general phenomenon that affects all cell types equally.     

Deletion of the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5 from the herpes simplex virus type 1 genome substantially impairs productive viral infection in cell culture and pathogenesis in the rat central nervous system

A distinctive feature of the genetic make-up of herpes simplex virus type 1 (HSV-1), a human neurotropic virus, is that approximately half of the 81 known viral genes are not absolutely required for productive infection in Vero cells, and most can be individually deleted without substantially impairing viral replication in cell culture. If large blocks of contiguous viral genes could be replaced with foreign DNA sequences, it would be possible to engineer highly attenuated recombinant HSV-1 gene transfer vectors capable of carrying large cellular genes or multiple genes having related functions. We report the isolation and characterization of an HSV-1 mutant, designated d311, containing a 12 kb deletion of viral DNA located between the L-S Junction a sequence and the U(S)6 gene, spanning the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5. Replication of d311 was totally inhibited in rat B103 and mouse Neuro-2A neuroblastoma cell lines, and was reduced by over three orders of magnitude in human SK-N-SH neuroblastoma cells compared to wild-type (wt) HSV-1 KOS. This suggested that the deleted genes, while nonessential for replication in Vero cells, play an important role in HSV replication in neuronal cells, particularly those of rodent origin. Unlike wt KOS which replicated locally and spread to other regions of brain following stereotactic inoculation into rat hippocampus, d311 was unable to replicate and spread within the brain, and did not cause any apparent local neuronal cell damage. These results demonstrate that d311 is highly attenuated for the rat central nervous system. d311 and other mutants of HSV containing major deletions of the nonessential genes within U(S) have the potential to serve as useful tools for gene transfer applications to brain.     

Inverse expression pattern of REST and synapsin I in human neuroblastoma cells

The zinc finger protein REST is a repressor of neuronal genes in nonneuronal tissues. We have analyzed the expression of REST, together with the expression of a REST target gene, encoding synapsin I, in human neuroblastoma cells. It was found that REST and synapsin I are coexpressed in neuroblastoma cell lines, although the expression of REST was inversely proportional to the levels of synapsin I mRNA. Thus, increased expression of synapsin I was directly correlated with decreased expression of REST. These expression data are in excellent correlation with synapsin I promoter activity measured in neuroblastoma cells showing that an increase in the REST concentration switched off synapsin I promoter activity. We conclude that the concentration of REST determines the expression level of neuronal genes such as the synapsin I gene.     

Evidence that the modulation of membrane-associated protein kinase C activity by an endogenous inhibitor plays a role in N1E-115 murine neuroblastoma cell differentiation

Murine neuroblastoma cells, N1E-115, were induced to differentiate into neuron-like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro "direct" assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane-associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane-associated PKC activity in cells and thereby modulate PKC-related neuronal functions.     

Differential effects of physostigmine and organophosphates on nicotinic receptors in neuronal cells of different species

The effects of the carbamate physostigmine and of the organophosphates (OPs) parathion, paraoxon and phenyl saligenin cyclic phosphate (PSP) were examined on different subtypes of neuronal nicotinic acetylcholine receptors (nAChR). Stimulation with 1 mM ACh induced transient nicotinic inward currents in mouse N1E-115 and human SH-SY5Y neuroblastoma and in locust thoracic ganglion cells. All four acetylcholinesterase (AChE) inhibitors reduced the nicotinic currents in a concentration-dependent manner. Parathion is about 50 times more potent in blocking nAChR, compared to its active AChE inhibiting metabolite paraoxon. The relative blocking potency of the different AChE inhibitors was the same in all cell types, and followed the order parathion > physostigmine > PSP > paraoxon. In N1E-115 cells the IC50 values of block amounted to 2 microM, 30 microM, 39 microM and 96 microM for parathion, physostigmine, PSP and paraoxon, respectively. In all cell types, the nicotinic currents were equally blocked by parathion. Human nAChR in SH-SY5Y cells appeared more sensitive to block by physostigmine, PSP and paraoxon, while these AChE inhibitors similarly inhibited nicotinic currents in insect cells and in mouse neuroblastoma cells. The observation that the concentration-dependence of block is different from that of AChE inhibition, indicates a distinct interaction of AChE inhibitors with nAChR. Only in locust cells physostigmine induced a non-desensitizing inward current, that appeared to originate from nAChR activation. Occasionally, the OPs were able to activate slow ionic currents in mouse, but not in human and locust cells. As the OP-induced agonistic activity in mouse cells was not associated with the blocking action, the target site appeared to be distinct from nAChR. These results show that AChE inhibitors block nAChR with different potencies, dependent on the compound and the receptor subtype, and may activate distinct ion currents in neuronal cells of different species origin.     

CD44H expression by human neuroblastoma cells: relation to MYCN amplification and lineage differentiation

The human CD44 cell surface glycoprotein has been involved in a variety of functions including lymphocyte homing, extracellular cell matrix attachment, and tumor metastasis. Due to the alternative splicing of the single gene, a large family of different variants or isoforms is generated. Several reports have indicated an up-regulation of CD44 variant (v) isoforms in malignant process, conferring metastatic potential to non-metastatic cells. Neuroblastoma is a tumor characterized by an aggressive and metastatic behavior in advanced stages with amplification of the MYCN protooncogene. In this report we show that the CD44 standard molecule is highly expressed in 100% of stage I-III, IVs neuroblastomas and ganglioneuromas but only in a subset of stage IV tumors. In contrast, no expression of CD44 was detected on MYCN amplified stage IV tumors, thus demonstrating a highly significant negative relationship between MYCN amplification and CD44 expression in neuroblastoma. The expression of CD44 on neuroblastoma cultured cell lines was not shown to be related to MYCN amplification but rather linked to the S-type, schwann/glial differentiation lineage. Immunochemical analysis of tumor samples with anti-CD44v3 and -v6 antibodies and Northern blot analysis of mRNA from cell lines with probes spanning exons 4-10 did not reveal any expression of splice variants on neuroblastomas of all stages and cell lines, thus ruling out a major role of these isoforms in neuroblastoma progression and metastasis.     

Betulinic acid induces apoptosis in human neuroblastoma cell lines

Neuroblastoma has long been recognized to show spontaneous regression during fetal development and in the majority of stage 4s infants < 1 year of age with disseminated disease. Stage 4s disease regresses with no chemotherapy in 50% of the patients. The mechanism by which this occurs is not understood but may be programmed cell death or apoptosis. Betulinic acid (BA) has been reported to induce apoptosis in human melanoma with in vitro and in vivo model systems. Melanoma, like neuroblastoma, is derived from the neural crest cell. We hypothesised that neuroblastoma cells have the machinery for programmed cell death and that apoptosis could be induced by betulinic acid. Nine human neuroblastoma cell lines were treated in vitro with BA at concentrations of 0-20 micrograms/ml for 0-6 days. Profound morphological changes were noted within 3 days. Cells withdrew their axonic-like extensions, became non-adherent and condensed into irregular dense spheroids typical of apoptotic cell death (ED50 = 14-17 micrograms/ml). DNA fragmentation analysis showed ladder formation in the 100-1200 bp region in 3/3 neuroblastoma cell lines treated with BA for 24-72 h. Thus, apparently BA does induce AP in neuroblastoma in vitro. This model will be utilised to investigate the role of apoptosis-related genes in neuroblastoma proliferation and to determine the therapeutic efficacy of BA in neuroblastoma in vivo.     

Immunohistochemistry of synapsin I and synaptophysin in human nervous system and neuroendocrine tumors. Applications in diagnostic neuro-oncology

Synapsin I is a phosphoprotein localized to the cytoplasmic surface of synaptic vesicles and is one of the best characterized neuron-specific proteins. Synaptophysin is an integral membrane glycoprotein, also located on presynaptic vesicles, which has been shown to be a useful immunohistochemical marker for neuroendocrine/neuronal differentiation in tumor diagnosis. The sensitivity and specificity of immunohistochemical staining for these two proteins in formalin-fixed, paraffin-embedded tissues was studied in a series of 67 neuroectodermal, neuroendocrine, and non-neural tumors. Intense immunoreactivity for both synapsin I and synaptophysin was observed in tumors containing well-differentiated neurons (gangliocytoma, ganglioglioma, neurocytoma). In these tumors, immunostaining was primarily concentrated along the outer surface of the cell membrane of the neuronal cells. Primitive neuroectodermal tumors (PNETs) (cerebral PNET, medulloblastoma, neuroblastoma) and most neuroendocrine tumors generally showed less intense and more variable immunoreactivity for these proteins. In most cases, immunostaining for synapsin I was sharper and often more intense than for synaptophysin. Some PNETs and neuroendocrine tumors that were immunoreactive for synapsin I did not stain for synaptophysin. We conclude that synapsin I is a reliable, sensitive immunohistochemical marker for neuronal/neuroendocrine differentiation in human neoplasms and may offer some advantages over synaptophysin when applied to formalin-fixed, paraffin-embedded tissues, particularly in the evaluation of primitive neuroectodermal tumors and neuroendocrine tumors.     

2F1 antigen, the mouse homolog of the rat "mast cell function-associated antigen", is a lectin-like type II transmembrane receptor expressed by natural killer cells

Inhibitory lectin-like receptors expressed on the surface of hematopoietic cells are critically involved in regulation of their effector functions. Here we report that a novel mAb specific for mouse NK cells, 2F1, recognizes the mouse homolog of the mast cell function-associated antigen (MAFA), an inhibitory lectin-like transmembrane receptor expressed on rat mast cells. The 2F1 antigen (2F1-Ag) and rat MAFA are structurally highly conserved and contain a cytoplasmic motif similar to the immunoreceptor tyrosine-based inhibitory motif that is presumably utilized for inhibitory signaling. We also identified a human homolog that is closely related to the rodent MAFA/2F1-Ag proteins. Like rat MAFA, 2F1-Ag is probably encoded by a single gene, which exhibits relatively little polymorphism. Strikingly, while rat MAFA is considered a mast cell antigen, we have been unable to detect cell surface expression of 2F1-Ag by mouse mast cell lines, bone marrow-derived mast cells, or peritoneal mast cells. Furthermore, mouse bone marrow-derived mast cells were devoid of 2F1-Ag mRNA. Instead, we find that approximately 40% of mouse NK cells express 2F1-Ag. Thus, MAFA/2F1-Ag may modulate immunological responses on at least two different cell types bridging the specific and innate immune system.