N18TG2 Neuroblastoma Research Articles

Entry and replication of Japanese encephalitis virus in cultured neurogenic cells

The entry mode and growth pattern of Japanese encephalitis (JE) virus in mouse neuroblastoma N18TG2 cells and mouse neuroblastoma x rat glioma NG108-15 hybrid cells were studied by electron microscopy. At two minutes after inoculation, JE virions adsorbed onto and directly penetrated through the plasma membrane of the hybrid cells, whereas virions did not adsorb nor entered the neuroblastoma cells. Correspondingly, the hybrid cells showed assembling progeny JE virions in the cistemae of rough endoplasmic reticulum (RER) 1 day postinoculation (p.i.) although virions were rarely found on the following days during the experiment. On the other hand, progeny virions did not assemble in the RER cisternae of the neuroblastoma cells throughout the experiment. The morphologic observations, therefore, suggest that (a) the hybrid cells express JE-virus receptors which facilitate the viral attachment onto and entry into the cells, while the neuroblastoma cells do not and (b) JE virus replicates very poorly after the entry into the hybrid cells while it does not replicate at all in the neuroblastoma cells. The virus titrations of the media of the neuroblastoma and hybrid cell cultures showed only titers indicative of residual virus of the inoculum that progressively decreased during the experiment. The present results show therefore that of the two neurogenic cell culture lines studied only the hybrid cell line can be used for the study of viral entry and replication, although it is not suited for virus production. Possible reasons for the poor replication of JE virus in the hybrid cells are discussed.

Novel cell lines display properties of nociceptive sensory neurons

Hybrid cell lines derived from neonatal rat dorsal root ganglia neurons fused with the mouse neuroblastoma N18Tg2 exhibit sensory neuron-like properties not displayed by the parental neuroblastoma. These properties include an inward (depolarizing) current with a conductance increase in response to activation of a bradykinin receptor, an inward (depolarizing) current with a conductance increase in response to the sensory excitotoxin capsaicin, the expression of sensory neuropeptides (substance P, CGRP and somatostatin), the expression of phosphatidylinositol-anchored molecules including adhesion molecules of the immunoglobulin superfamily that can be regulated in serum-free culture by nerve growth factor (N-CAM, F-3 and Thy-1), and low permissivity to herpes simplex virus infection. These lines thus provide appropriate models for the study of mechanisms involved in nociceptor activation and the regulation of expression of sensory-neuron specific markers including neuropeptides.

Neuronal properties and trophic activities of immortalized hippocampal cells from embryonic and young adult mice

The hippocampal formation elaborates trophic factors such as nerve growth factor (NGF) to support the cholinergic innervation it receives from the septal region. To further study the trophic interactions of this pathway, hippocampal cells from embryonic day 18 and postnatal day 21 mice were immortalized via somatic cell fusion to N18TG2 neuroblastoma cells. The hippocampal cell lines exhibit morphological and cytoskeletal features which are typical of their neuronal parents but which are not expressed by the neuroblastoma parent. When differentiated with retinoic acid, the hippocampal cell lines exhibit electrophysiological features similar to cultured hippocampal neurons. Many of the lines constitutively express high levels of NGF, and at least one cell line exerts a non-NGF trophic effect on the expression of choline acetyltransferase by septal neurons in vitro. These cell lines are potentially useful for investigating the neurochemical and excitable properties of hippocampal neurons and identifying novel trophic activities that promote the development and maintenance of the septohippocampal pathway.

Immortalized young adult neurons from the septal region: generation and characterization

Studies of the development of the central nervous system would be greatly facilitated by the ability to immortalize neuronal tissue from a broad range of ages. We have previously used somatic cell fusion techniques to generate neuronal cell lines from embryonic mice. To immortalize older neuronal cells, a cell isolation technique was developed to obtain viable septal cells from postnatal day 21 mice. The septal cells were fused to N18TG2 neuroblastoma cells and then cultured in selective medium to isolate septum × neuroblastoma cell lines. The hybrid nature of the lines was verified by chromosome analysis and electrophoretic analysis of glucosephosphate isomerase isozymes. The lines express phenotypes typical of differentiated septal neurons. Many lines morphologically resemble neurons and express the high molecular weight neurofilament protein. Several lines express high levels of choline acetyltransferase activity; others synthesize nerve growth factor. These results demonstrate that young adult neuronal tissue can be immortalized and that hybrid cells express properties of the neuronal parent.

Concanavalin A affects β-tubulin mRNA expression during neuritic processes of mouse neuroblastoma N18TG2 cells in a different manner from colchicine

Addition of concanavalin A (Con A) to mouse neuroblastoma N18TG2 cells cultured with dibutyryl-cAMP which can stimulate neurite outgrowth, stopped the neuritic processes effectively. The extended neurites showed a gradual retraction for at least 8 hrs after addition of Con A, while addition of colchicine caused rapid retraction of the neurites. Immunocytochemistry showed that the addition of Con A did not disorganize the microtubules but the addition of colchicine did. The increase in β-tubulin mRNA expression which was observed after cell culture and after stimulation by dB-cAMP was suppressed by the addition of Con A. Con A did not affect the β-tubulin mRNA expression when the cells had already been cultured, while colchicine drastically decreased it. Thus, Con A appeared to affect the β-tubulin mRNA expression in a different manner from colchicine, probably through inhibition of cell movement.

Neurite outgrowth from mouse neuroblastoma and cerebellar cells induced by the protein kinase inhibitor H-7

The protein kinase inhibitor H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) or staurosporine was found to induce neurite outgrowth from mouse neuroblastoma N18TG2 cells and cultured cerebellar cells by light microscopy. Examination by electron microscopy revealed some morphological differences between the neurites of N18TG2 cells induced by H-7 and dB-cAMP, in which H-7 formed longer and thinner neurites and more abundant localized varicose structures along neurites than dB-cAMP. These results indicate that protein kinase inhibitor itself can act as a potent stimulus for inducing neurite outgrowth in a different manner from dB-cAMP.

Increased N-Myc Mrna Expression Associated with Dibutyryl Cyclic amp Induced Neuroblastoma Differentiation

The N-myc cellular oncogene is frequently amplified and expressed at a high level in neuroectodermal tumor cells such as neuroblastoma and retinoblastoma. We examined N-myc expression in NCB-20 hybrid (N18TG2 neuroblastoma x embryonic Chinese Hamster brain) cells. After five days of culture, cells treated with 1 mM db cAMP show extensive neurite outgrowth and secrete acetylcholinesterase into the media at a level three times higher than untreated control. In situ hybridizations, dot blots, and Northern analyses reveal four- to eight-fold higher levels of N-myc mRNA in the treated, differentiated cells than in the untreated, undifferentiated controls. Our results show that the highly differentiated state is not incompatible with a high level of N-myc mRNA.

A high performance liquid chromatography assay for glutamine synthetase

A sensitive method for assaying glutamine synthetase activity is described. This enzyme produces γ-glutamylhydroxamate in the presence of l-glutamic acid and hydroxylamine as substrates. This amino acid hydroxamate was separated and quantified by high performance liquid chromatography on an ion-exchange resin column using post-column derivatization with o-phthalaldehyde for detection. As little as 50 pmol of γ-glutamylhydroxamate was detected in assays using cell-free extracts from fish retina, rat clonal C6 glioma cells, mouse clonal NIE 115 and N18TG2 neuroblastoma cells, whose specific activity measured was 1.0, 0.03, 0.01 and 0.01 μmol of γ-glutamylhydroxamate produced per 30 min per milligram protein, respectively.

Structure-binding relationship of quinuclidinyl benzilate analogs on N4TG1 neuroblastoma muscarinic receptors.

By Scatchard plot analysis of [3H]QNB (quinuclidinyl benzilate) binding, there are 2 x 10(5) muscarinic sites/cell with a KD about 10 nM in N4TG1 neuroblastoma cells. We have now examined a group of compounds structurally related to aprophen and QNB for their ability to compete with the binding of QNB to the muscarini receptor. Using this structure-inhibition relationship, the functional groups of the muscarinic ligand necessary for binding were partially characterized. It was found that the quinuclidinyl ring structure of QNB can be substituted by either alkane, H, or pyrrolidine at the N without loosing their ability to bind. The addition to the quinuclidinyl ring increases the bulk of the structure and decreases binding. Like the benzilate in QNB, a similar hydrophobic structure is apparently required for the binding.

Cytotoxicity of acrylamide and related compounds to mouse neuroblastoma and rat schwannoma cells.

Cytotoxicity of acrylamide and related compounds to mouse neuroblastoma N18TG-2 and rat Schwannoma RT4 cells was studied. Of nine test chemicals, acrylamide and N-hydroxymethylacrylamide were most toxic compared with others on the basis of ED50 value. Observation under phase-contrast microscopy revealed that in N18TG-2 acrylamide produced both degeneration of the cells and inhibition of cell growth, and that in RT4 it produced only inhibition of cell growth without causing any marked morphological changes. Dibutyryl cyclic AMP (dBcAMP) reduced the cytotoxicity of acrylamide to both types of cells on the basis of the dose-effect curve. Studies on the subcellular distribution of 14C-acrylamide incorporated showed that more than 90% of the total radioactivity was incorporated in the 15,000 g supernant fraction. Kinetics of acrylamide uptake by N18TG-2 cells showed that in the cells untreated with dBcAMP there were two binding sites with high and low affinity, and that after treatment with dBcAMP the site of high affinity disappeared. The situation was true for RT4 cells, the results indicating that the immature cells are more vulnerable to acrylamide than the mature cells.

A novel enhancement of SV40 enhancer activity by treatment of mouse neuroblastoma N18TG2 with protein kinase inhibitor H-7.

To determine the role of protein phosphorylation in transcription regulation, we have treated mouse neuroblastoma N18TG2 cells with the protein kinase inhibitor H-7 and tested its effect on transcription. After the preculture and transfection in the presence of H-7, the cell preparation was divided in half and cultured with and without H-7. The level of CAT expression of pSV2-CAT was found to be higher in the cells cultured in the absence of H-7 than in those cultured in the presence of H-7. This difference was observed only after pretreatment of the cells with H-7, suggesting that withdrawal of H-7 from the culture medium after preculture with H-7 gave an enhancing effect on CAT expression. This phenomenon was also observed with transformants that expressed the CAT gene of pSV2-CAT stably. The 72 base-pair (bp) repeat of SV40 DNA was responsible for this difference in CAT expression. A similar effect of H-7 on the SV40 enhancer activity was observed in mouse neuroblastoma x rat glioma hybrid NG108-15 cells, but not in rat glioma C6-BU-1 cells.

Cell-free synthesis of opiate binding sites

A procedure is described for the detection of opiate binding sites synthesized during in vitro translation of various mRNA preparations. RNA were isolated from membrane bound polysomes which were prepared from NG 108-15 hybridoma, C6BU1 glioma cells, as well as from N18TG2, NB2aAg and NB41A3 neuroblastoma cells. Polyadenylated [poly(A)(+)] RNA were purified, translated in vitro in a rabbit reticulocyte lysate and the translation products assayed for their ability to bind [(3)H] bremazocine. Bound and free ligands were separated by column chromatography. After translation of poly(A)(+) RNA obtained from NG 108-15 cells we demonstrated a stereospecific, saturable binding of [(3)H]bremazocine (displaced by levorphanol and not by dextrorphan) with a K(d) of 2.4 +/- 1.0 nM. The total amount of opiate binding sites synthesized was 6.2 +/- 0.5 fmol per ?g of poly(A)(+) RNA. Opiate binding sites were undetectable at zero time and a plateau was reached after translation had proceeded for 20 min. Five time less opiate binding sites were synthesized when the poly(A)(+) RNA purified from N18TG2 neuroblastoma cells were used under the same experimental conditions. There was no detectable binding of opiate ligands with poly(A)(+) RNA obtained from C6BU1 glioma cells, NB2aAg or NB41A3 neuroblastoma cells.

Muscarinic pharmacology of the inhibition of adenylate cyclase in N18TG2 neuroblastoma cells

This study characterizes the muscarinic cholinergic receptors associated with the inhibition of adenylate cyclase on N18TG2 neuroblastoma cell membranes. Agonists could be divided into two classes: oxotremorine, acetylcholine, carbachol and arecoline exerted the most efficacious and potent inhibition, while McN-A343, bethanechol and AHR-602 were partial agonists. Both quinuclidinyl benzilate and atropine maximally antagonized the inhibitory effect of McN-A343, carbachol and oxotremorine. Pirenzepine was almost as potent as atropine in reversing the inhibitory effect of McN-A343, but was 300 times less potent than atropine or quinuclidinyl benzilate in antagonizing the effects of either carbachol or oxotremorine. Gallamine was ineffective as an antagonist at concentrations up to 1 mM. These results suggest that the receptors that modulate this inhibition are of the M(2) type, since they were activated by carbachol, acetylcholine and oxotremorine, but much less by McN-A343 and AHR-602 (both M(1) selective agonists). The full agonists were blocked by atropine and quinuclidinyl benzilate but not by low concentrations of pirenzepine (M(1) selective antagonist).

An assessment of the role of opioid receptors in the response to cannabimimetic drugs.

Cannabimimetic drugs have been shown to inhibit adenylate cyclase activity in N18TG2 neuroblastoma cells. This investigation examines the possible role of opioid receptors in the cannabimimetic response. Opioid receptors of the delta subtype were found on N18TG2 membranes using [3H]D-Ala2-D-Leu5-enkephalin. No mu or kappa receptors were detected using selective ligands for these sites. The delta binding affinity and capacity were unaltered by cannabimimetic drugs. To test if cannabimimetic drugs may modulate opioid effector mechanisms, cyclic AMP metabolism was determined in intact cells and in membranes. N18TG2 adenylate cyclase was inhibited by the cannabimimetic drugs delta 9-tetrahydrocannabinol and desacetyllevonantradol, and by the opioid agents morphine, etorphine, and D-Ala2-Met5-enkephalinamide. The opioid inhibition was reversed by naloxone and naltrexone; however, the cannabimimetic response was unaffected. Both cannabimimetic and opioid drugs decreased cyclic AMP accumulation in intact cells, but opioid antagonists blocked the response only to the latter. Thus, cannabimimetic effects are observed even though opioid receptors are blocked by antagonist drugs. The interaction between desacetyllevonantradol and etorphine was neither synergistic nor additive at maximal concentrations, suggesting that these two drugs operate via the same effector mechanism. Other neuronal cell lines having an opioid response were also examined. The cannabimimetic inhibition of cyclic AMP accumulation in NG108-15 neuroblastoma X glioma cells was not as great as the response in N18TG2. N4TG1 neuroblastoma cells did not respond to cannabimimetic drugs under any conditions tested. Thus, the cannabimimetic inhibition of adenylate cyclase is not universally observed, and the efficacy of the cannabimimetic response does not correlate with the efficacy of the opioid response.

Somatic cell genetic analysis of human cell surface antigens: chromosomal assignments and regulation of expression in rodent-human hybrid cells.

The expression of 13 newly defined human cell surface antigens identified by monoclonal antibodies was studied in a panel of reduced rodent-human somatic cell hybrid clones. For each antigenic system the segregation of antibody reactivity was concordant with the segregation of a specific human chromosome, permitting the chromosomal assignment of 13 gene loci determining antigen expression. The antigens can be placed in four groups on the basis of their patterns of control in the hybrid cells. (i) Presence of a single human chromosome is necessary and sufficient for antigen expression; L230 (assigned to chromosome 2), AJ425, K15 (chromosome 3), SR84 (chromosome 5), JF23, Q14 (chromosome 11), SV13 (chromosome 15), and F10 (chromosome 19). (ii) AJ2 (chromosome 10) and J143 (chromosome 17); two antigens coded for by separate human chromosomes but associated as a molecular complex on the surface of AJ2+/J143+ human cells. (iii) F8 (chromosome 19); antigen expression dependent on the growth characteristics of hybrid cells: substrate-adherent cells are F8+, whereas cells growing in suspension are F8-. (iv) AO122 and F23 (chromosome 15); antigen expression controlled by the permissive/inducing vs. nonpermissive/noninducing nature of the rodent fusion partner. Hybrids derived from both antigen-positive and antigen-negative human cells can express AO122 and F23 but only when specific rodent cell types are used for hybridization: N4TG-1 neuroblastoma and L cells, but not RAG renal carcinoma cells, permit AO122 expression, whereas RAG and L cells, but not N4TG-1 cells, permit F23 expression. The rapidly expanding list of monoclonal antibodies defining human cell surface molecules provides a range of markers to probe the genetic regulation of antigen diversity in somatic cells.

Cytotoxic action of prostaglandin D2 on mouse neuroblastoma cells.

Addition to the culture medium of prostaglandin (PG) D2 resulted in the degeneration in a dose- and time-dependent manner of N18TG-2 cells cloned from mouse neuroblastoma. The ED50 for PGD2-induced cytotoxicity was about 10 microM. The degenerative changes were irreversible when the cells were exposed for more than 10 h. Scanning and transmission electron microscopic examination revealed that treatment with PGD2 resulted in appearance of numerous blebs of various sizes along the cell surface and also in destruction of surface membrane and of cytoplasmic organelles. Tumor weight of N18TG-2 neuroblastoma inoculated subcutaneously on the backs of A/J mice was about 35-70% less than that of controls after 14 days of single daily i.p. or s.c. injections of 0.5-1 mg/kg of PGD2. The results indicate that PGD2 has growth-inhibitory effects on mouse neuroblastoma cells in vitro and in vivo.

Modulation in the levels and localization of plasminogen activator in differentiating neuroblastoma cells

Variations in the specific activity and localization of plasminogen activator (PA) were examined prior to and during the differentiation of mouse neuroblastoma and rat glioma cells in culture, in an attempt to reveal whether PA is actively involved in neurite extension and development in these cells. Following differentiation induced by dibutyryl cyclic AMP, the level of PA increased by 10 to 20-fold in neuroblastoma N18TG-2 cells and in the hybrid cell line NG108-15, obtained by fusion of mouse neuroblastoma N18TG-2 and rat glioma C6BU-1 cells, but was very low in the glioma parental cells throughout differentiation. Plasminogen-dependent proteolysis was found not to be an obligatory requirement for the differentiation of the hybrid cells in culture, since the extension of neurites, the appearance of toxin-activated Rb +-efflux and the eletrical properties were largely unaffected by eliminating and blocking this activity. Confluent cells from several clones derived from mouse neuroblastoma C1300 cells displayed significant intracellular and secreted PA activities, and the axon-minus neuroblastoma NIA-103 cells were found to secrete the highest amounts of PA. Upon gel electrophoresis, all mouse neuroblastoma cell lines examined displayed a major molecular form of active PA with an apparent molecular weight of 80,000 and a minor one of 50,000. Only the larger enzyme form was expressed by the rat glioma or the hybrid cells, and the electrophoretic migration pattern of the active enzyme species remained unchanged upon differentiation of the various cell lines. Distribution of PA activity on the surface of differentiating neuroblastoma N1E-115 cells and hybrid NG108-15 cells was visualized by high resolution autoradiography of [ 125I]fibrin degradation products, cross-linked to the cell surface. Particularly intense labeling was observed along membranes of cell bodies in both developing and mature cells, around branching zones, along individual neurite extensions and in part, but not all, of the growth cones. These findings suggest that PA plays a role in the metabolism of mature cells of neuronal origin, and imply its involvement in the development and/or the functions of specific neuronal processes and growth cones.

Characterization of an adenylate cyclase-linked serotonin (5-HT1) receptor in a neuroblastoma X brain explant hybrid cell line (NCB-20).

Clonal cell line NCB-20 (a hybrid of mouse neuroblastoma N18TG2 and Chinese hamster 18-day embryonic brain explant) expressed both high- (KD 180 nM) and low-affinity (greater than 3000 nM) binding sites for [3H]serotonin (5-HT) which were absent from the parent neuroblastoma. The low-affinity binding site was eliminated by 1 microM spiperone. The order of drug potency for inhibition of high-affinity [3H]5-HT binding was consistent with a 5-HT1 receptor (5,6 - dihydroxytryptamine = 5-HT = methysergide = 5-methoxytryptamine greater than cyproheptadine = clozapine = mianserin greater than spiperone greater than dopamine = morphine = ketanserin = norepinephrine). [3H]5-HT binding was inhibited by guanine nucleotides (e.g., GTP and Gpp(NH)p), whereas antagonist binding was not; ascorbate was also inhibitory. A 30-min exposure of cells to 1-2 microM 5-HT or other agonists produced a three- to fivefold stimulation of cyclic AMP levels. The order of potency for 5-HT agonist stimulation of basal cyclic AMP levels and 5-HT antagonist reversal of agonist-stimulated levels was the same as the order of drug potency for inhibition of high-affinity [3H]5-HT binding, suggesting linkage of the 5-HT1 receptor to adenylate cyclase in NCB-20 cells.

Cytotoxic action of retinoidal 4-ylidenebutenolides on mouse neuroblastoma and rat glioma cells

Proliferation and death were measured in cultures of mouse neuroblastoma N18TG-2 and rat glioma C6BU-1 cells during treatment with up to 100 uM retinoidal 4-ylidenebutenolides (RYB 1-3), whose chemical structures are already reported (Heterocycles, 19, 1385, 1982). The number of viable cells were measured after 2-days incubation with various concentrations of the compounds, among which RYB-3 (figure) was the most potent one to destroy the cells. IC50 of RYB-3 was about 0.5 uM for both types of cells, which was 80-fold more susceptible than that of retinoic acid. The degenerative changes of RYB-3 on C6BU-1 cells were irreversible even when the cells were exposed for 2 hrs. Tumor weights of N18TG-2 cells inoculated subcutaneously on the backs of A/J mice were 30-40% less than those of untreated controls after 14 days of single daily i.p. injections of RYB-3 at the dose of 100 mg/kg of the body weight. The results indicate that RYB-3 is cytotoxic on the tumor cells originating from the nervous system and has a inhibitory effect on neuroblastoma tumor growth in mice.

Expression of spectrin in nonerythroid cells

spleen cells to the T-cell mitogens concanavalin A and phytohemagglutimin. The ,f3-endorphin effect was dose-dependent at concentrations similar to those found in rat plasma. ,&Endorphin had no effect on resting, unstimulated spleen cells. Recently, Schweigerer et al. (Nature 296, 572-574, 1982) reported specific nonopiate binding of &-endorphin to both terminal complexes of human complement: the cytolytic, membrane-derived C5b-9(m) complex, and the cytolytically inactive, serum-derived SC5b-9 complex. These observations suggest that ,L?-endorphin may play an important role in the immune system. For the last few years, more than 60 /I-endorphin analogs have been synthesized by the solid-phase method and characterized. Their biological activities have been assessed by opiate receptor binding, inhibition of electrically stimulated contraction of guinea pig ileum preparation and mouse tail-flick analgesic and radioimmunoreactivity assays. These data, recently reviewed (Li, Hormonal Proteins and Peptides 10, l-34, 19811, suggest the presence of three binding sites in the /I-endorphin molecule, as shown in Figure 2. The first site (15 receptors) is located in the Met-enkephalin segment, and the second site (p receptor) resides in the COOH-terminal segment. The middle segment is the antibody-binding site. Among the synthetic ,&endorphin analogs, /I-endorphin-(6-31) (an analog without the Met-enkephalin segment) exhibits interesting properties. It possesses minimal analgesic potency and ileal opiate activity. As mentioned above, P-endorphin-(6-31) binds to the opiate receptor in the neuroblastoma N18TG2 cells. When P-endorphin-(6-31) is injected together with P-endorphin, it inhibits ,&endorphin-induced analgesia. It may be recalled that a segment of human ACTH [a,,-ACTH-(6-3811 is an inhibitor of ACTH action (Li et al., PNAS 75, 4306-4309, 1978). The concept that a peptide segment inside the amino acid sequence of a hormone is an inhibitor of the hormone itself may turn out to be a general phenomenon in biologically active polypeptides.