Persistent measles virus infection of a clonal line of neuroblastoma: Effect on neural differentiation

Murine neuroblastoma cells, chronically infected with measles virus were examined for changes in neural-specific function and structure as well as cellular growth and macromolecular synthesis. When exposed to neural differentiation-inducing culture conditions, neurite formation and acetylcholinesterase activity are significantly increased in control cultures but not in persistently infected cells. Infected cultures manifest a more rapid doubling time, but depressed RNA and protein synthesis at saturation densities. Higher concentrations of papavarine, a cyclic 3′–5′ AMP phosphodiesterase inhibitor, result in selective death of persistently infected cells.     

Persistent infection of a rat nephroma cell line with kilham rat virus

A rat nephroma cell line was found to be a carrier culture of the Kilham rat virus. Infectious virus was demonstrable only by passage of culture materials into other host systems.     

Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus

Duc-Nguyen, Huu (National Cancer Institute, Bethesda, Md.), Edith N. Rosenblum, and Robert F. Zeigel. Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus. J. Bacteriol. 92:1133-1140. 1966.-The propagation of a murine leukemia virus (Rauscher) in a kidney cell line, derived from a rat with lymphoid leukemia, was studied. A complement-fixing (CF) antigen reacting with Rauscher immune sera was detected at various passage levels, which correlated with the visualization by use of electron microscopy of viral buds and viral particles in different stages of maturation in all passages. Five-month-old monolayers continued to shed virus and to yield high CF antigen titers. The cell-free supernatant fluid from cultures of the 14th passage was shown to be infectious for a normal rat kidney cell line, as evidenced by the appearance of the CF antigen in this line. Interferon production was not demonstrated in infected cultures. The overall data indicated that rat kidney cells could be used to propagate Rauscher virus in a carrier state.     

Persistent natural infection of a Culex tritaeniorhynchus cell line with a novel Culex tritaeniorhynchus rhabdovirus strain

Culex tritaeniorhynchus rhabdovirus (CTRV) is a mosquito virus that establishes persistent infection without any obvious cell death. Therefore, occult infection by CTRV can be present in mosquito cell lines. In this study, it is shown that NIID‐CTR cells, which were derived from Cx. tritaeniorhynchus, are persistently infected with a novel strain of CTRV. Complete genome sequencing of the infecting strain revealed that it is genetically similar but distinct from the previously isolated CTRV strain, excluding the possibility of contamination. These findings raise the importance of further CTRV studies, such as screening of CTRV in other mosquito cell lines.     

Extracellular matrix effects on a neuroblastoma cell line

Cells of various lines assume similar shapes when grown attached to substrates like coverslips. In contrast, cells cultured in a collagen and/or laminin matrix often assume a relatively normal morphology in comparison with their in situ counterparts. During investigations of neuroblastoma SH-SY5Y cells, an attempt was made to identify culture conditions which would cause the cells to assume a more regular shape. SH-SY5Y cells cultured on bare coverslips, on coverslips coated with rat-tail collagen, and in approximately 1 mm thick gels containing extracellular matrix components were compared. Striking differences were apparent when comparing the gel-cultured cells with cells cultured on coverslips. Cells grown in the gel formed ganglia-like clusters which generated bundles of neurites which targeted other 'ganglia'. The same cells grown on coverslips, whether or not they were collagen-coated, appeared unaware of the presence of other cells, and did not cluster, nor did they generate neurites.     

Identification and characterization of a leptin-responsive neuroblastoma cell line

The adipocyte-derived hormone leptin plays a critical role in a variety of physiological and pathological actions. As such the determination of leptin signal transduction pathways are important both for understanding the molecular mechanisms of leptin action and for identifying sites for possible therapeutic intervention. Since the hypothalamus is the primary site of leptin action, we sought to identify a neuronal-derived human cell line containing the long form of the leptin receptor (OBRb). To this end, we screened several neuroblastoma cell lines and isolated a sub-line of SH-SY5Y cells, which we designated as SH-OBRb, for further studies. We characterized the transduction pathways induced by leptin in SH-OBRb cells and demonstrated that OBRb mediates tyrosine phosphorylation of STAT3, phosphorylation of ERK1/2, but not SAPK/JNK and p38 MAPK, in a dose and time dependent fashion. In addition, Akt appears to be phosphorylated in the basal state and to be insensitive to further activation by leptin. In summary, we have isolated a unique cell line that can be utilized as a model for use in the study of leptin action and molecular mechanisms.     

Induction of K-channel expression in a neuroblastoma cell line

Whole-cell currents were examined in mouse neuroblastoma cells of the N2AB-1 line. In standard culture medium, N2AB-1 cells exhibited large voltage-dependent Na currents but no discernible K currents. Treatment of N2AB-1 cells with either dimethylsulfoxide (DMSO) in low-serum medium or with retinoic acid (RA) caused the expression of delayed rectifier K currents. Currents from two types of K channel with single channel slope conductances of 15.0 pS and 6.4 pS were observed in outside-out patches from cells of both treatment groups. Thus, while N2AB-1 cells did not exhibit K currents under standard culture conditions, they did possess the gene(s) encoding K channels. The treatments caused other changes that were not directly linked to K-channel expression. RA treatment caused neurite extension in most, but not all, N2AB-1 cells; however, all RA-treated cells, including those without neurites, expressed K currents. RA treatment did not suppress cell division or cause hypertrophy. In contrast, treatment with DMSO/low serum suppressed cell division and caused cellular hypertrophy, but did not cause long neurites to form. Thus, the regulation of K channels was not coupled in a simple fashion to properties that have been associated with a differentiated neuronal phenotype: neurite elaboration, changes in cell size, and inhibition of cell division. These results suggest that N2AB-1 cells may be a good model system for investigating the processes regulating K-channel expression.     

Sigma1 binding in a human neuroblastoma cell line

Behaviorally, sigma₁ agents modulate opioid analgesia. To examine possible mechanisms responsible for these interactions, we have identified a cell line containing both sigma₁ and opioid receptors. [³H](+)-pentazocine binding in BE(2)-C human neuroblastoma cells is high affinity (K_D 3.4±0.7 nM) and high density (B_max 2.98±0.14 pmol/mg protein). Competition studies reveal a selectivity profile similar to that of sigma₁ sites in guinea pig brain. (+)-Pentazocine has no effect upon either basal or forskolin-stimulated cyclase in the BE(2)-C cells, but cAMP accumulation is inhibited by the morphine, DPDPE and naloxone benzoylhydrazone. (+)-Pentazocine at concentrations as high as 10 μM does not affect this opioid effect, implying that sigma₁/opioid interactions are not mediated at the level of the cell. This suggests that their behavioral interactions result from interacting neural circuits. Although (+)-pentazocine is without effect in the cyclase system, it does block carbachol-stimulated phosphoinositol turnover (IC₅₀ 6.5±1.14 μM). The specificity of the effect is confirmed by the ability of haloperidol (1 μM) to shift the IC₅₀ value of (+)-pentazocine 2-fold to the right. Special issue dedicated to Dr. Eric J. Simon.     

Characterization of L-glutamine transport by a human neuroblastoma cell line

This study characterized theNa⁺-dependent transport of L-glutamine by ahuman neuroblastoma cell line, SK-N-SH. The Na⁺-dependentcomponent represented >95% of the total glutamine uptake. Kineticstudies showed a single saturable high-affinity carrier with aMichaelis constant (K_m) of 163 ± 23 µMand a maximum transport velocity (V_max) of13,713 ± 803 pmol · mgprotein¹ · min¹. Glutamine uptakewas markedly inhibited in the presence of L-alanine, L-asparagine, and L-serine. Li⁺ didnot substitute for Na⁺. These data show thatL-glutamine is predominantly taken up through systemASC. Glutamine deprivation resulted in the decrease of glutamine transport by a mechanism that decreasedV_max without affectingK_m. The expression of the system ASC subtypeASCT2 decreased in the glutamine-deprived group, whereas glutaminedeprivation did not induce changes in system ASC subtype ASCT1 mRNAexpression. Adaptive increases in Na⁺-dependent glutamate,Na⁺-dependent 2-(methylamino)isobutyric acid, andNa⁺-independent leucine transport were observed underglutamine-deprived conditions, which were completely blocked byactinomycin D and cycloheximide. These mechanisms may allow cells tosurvive and even grow under nutrient-deprived conditions.

     

稳定表达β-synuclein的SH-SY5Y细胞株的构建

目的构建稳定表达β-synuclein的SH-SY5Y细胞株。方法利用脂质体转染技术将质粒pcD-NA3.1-β-synuclein转染SH-SY5Y细胞,通过Zeocin进行抗性筛选。采用原位免疫荧光、免疫组织化学染色及Western blot杂交检测β-synuclein在SH-SY5Y细胞中的表达情况;通过MTT比色法检测β-synuclein稳定表达对SH-SY5Y细胞增殖的影响。结果原位免疫荧光、免疫组织化学染色及Western blot检测结果显示β-synuclein在SH-SY5Y细胞中的表达水平较对照组明显升高;稳定表达β-synuclein的细胞增殖程度较对照组明显增高(P<0.05)。结论β-synuclein在SH-SY5Y细胞中稳定表达,为后续的研究提供有用的细胞模型。为进一步研究β-sy-nuclein对帕金森病发病神经保护作用的研究奠定了良好的基础。     

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.     

Muscarinic binding sites in a catecholaminergic human neuroblastoma cell line

Tyrosine hydroxylase (TH) a characteristic enzyme activity for the catecholaminergic clonal cell line LA-N-1 and choline acetyltransferase (ChAT) a characteristic enzyme activity for the cholinergic clonal cell line LA-N-2 were previously shown to be increased in these cells exposed to 10^–5 M retinoic acid (RA) as differentiating agent. An investigation of the receptor characteristics suggests a complementarity between the two cell lines. The binding of QNB, a muscarinic ligand, was undetectable with the LA-N-2 cells but was present in the LA-N-1 cells and possessed a kD of 1.8 nM and 2.2 nM and a B_max of 0.56 and 0.68 for control and RA grown cells respectively. There was a gradual increase in QNB binding to LA-N-1 cells from 2 days in vitro (DIV) until 6 DIV in both control and RA grown cells. An IC₅₀ of 2.5×10^–8 M and 0.9×10^–8 M for atropine inhibition was obtained for the control and RA grown cells respectively. The corresponding values for carbachol inhibition were 7×10^–2 M and 3×10^–2 M respectively. The inhibition by the agonist oxotremorine is comparable to that of carbachol and 1 mM pilocarpine inhibited the binding by 21%. QNB binding showed a low affinity for pirenzepine and for AF-DX-116 but was inhibited with a rather high affinity by 4-DAMP (IC₅₀:110 M) thus suggesting the presence of an M₃ receptor. Acetylcholine (100 M) plus eserine (50 M) and BW284c55 (1 M), an acetylcholinesterase inhibitor, reduced the binding of QNB by approximately 25%. Nicotine (1 mM) caused a 36% reduction of binding and hemicholinium-3 (HC-3) (1 M), an inhibitor of choline uptake, inhibited the binding by 53%. There was a down regulation of QNB binding observed with cells grown for 24 hours with either the antagonist atropine or the agonists carbachol or oxotremorine. Low amounts of -bungarotoxin (-BgTx) binding sites were barely detectable in both LA-N-1 and LA-N-2 cells. The LA-N-1 muscarinic receptor is coupled to polyphosphoinositide hydrolysis without increased cyclic AMP formation further suggesting its being an M₃ receptor.     

Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line

The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRES trans-acting factor polypyrimidine tract binding protein (PTB) to the 5′-UTR of insulin mRNA. For this purpose, human islets were incubated for 2 h in the presence of low (1.67 mM) or high glucose (16.7 mM). Nitrosative stress was induced by addition of 1 mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5′-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67 mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7 mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7 mM glucose but not at 1.67 mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5′-UTR in vitro, and that this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40–100% of all insulin biosynthesis during conditions of nitrosative stress. These data suggest that the pancreatic β-cell is able to uphold basal insulin synthesis at conditions of starvation and stress via a cap- and eIF4A-independent mechanism, possibly mediated by the binding of PTB to the 5′-UTR of the human insulin mRNA.     

Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line

Prolyl oligopeptidase (POP) is a post-proline cleaving enzyme, which is widely distributed in various organs, with high levels in the brain. In this study, we investigated the effects of a selective POP inhibitor, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746), on the growth of NB-1 human neuroblastoma cells. SUAM-14746 treatment for 24–72 h suppresses the growth of NB-1 cells without cell death in a dose-dependent manner (10–60 μM). Similar suppressive effects were observed with another POP inhibitor benzyloxycarbonyl-thioprolyl-thioprolinal. The SUAM-14746-induced growth inhibition in NB-1 cells was associated with pronounced G₀/G₁ arrest and reduced levels of phosphorylated retinoblastoma protein (pRb), cyclin E, and cyclin dependent kinase (CDK) 2, and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. SUAM-14746 also induced transient inhibition of S and G₂/M phase progression, which was correlated with retardation of the decrease in the levels of cyclins A and B. Moreover, RNAi-mediated knockdown of POP also led to inhibition of NB-1 cell growth and the effect was accompanied by G₀/G₁ arrest. These results indicate that POP is a part of the machinery that controls the cell cycle.     

Viologen-phosphorus dendrimers exhibit minor toxicity against a murine neuroblastoma cell line

Dendrimers containing viologen (derivatives of 4,4′-bipyridyl) units in their structure have been demonstrated to exhibit antiviral activity against human immunodeficiency virus (HIV-1). It has also recently been revealed that novel dendrimers with both viologen units and phosphorus groups in their structure show different antimicrobial, cytotoxic and hemotoxic properties, and have the ability to influence the activity of cholinesterases and to inhibit α-synuclein fibrillation. Since the influence of viologen-phosphorus structures on basic cellular processes had not been investigated, we examined the impact of such macromolecules on the murine neuroblastoma cell line (N2a). We selected three water-soluble viologen-phosphorus (VPD) dendrimers, which differ in their core structure, number of viologen units and number and type of surface groups, and analyzed several aspects of the cellular response. These included cell viability, generation of reactive oxygen species (ROS), alterations in mitochondrial activity, morphological modifications, and the induction of apoptosis and necrosis. The MTT assay results suggest that all of the tested dendrimers are only slightly cytotoxic. Although some changes in ROS formation and mitochondrial function were detected, the three compounds did not induce apoptosis or necrosis. In light of these results, we can assume that the tested VPD are relatively safe for mouse neuroblastoma cells. Although more research on their safety is needed, VPD seem to be promising nanoparticles for further biomedical investigation.     

Cytogenetic and molecular characterization of a newly established neuroblastoma cell line LS

Summary A new human neuroblastoma cell line (LS) that originated from an abdominal tumor of a 16-month-old girl is presented; it was classified, according to Evans, as being stage III. Morphological (dense-core particles) and biochemical characteristics (dopamine--hydroxylase, acetylcholinesterase, neuron-specific-enolase) confirmed the diagnosis. In addition to a slightly variable modal chromosome number of 48 or 49 (because of marker-chromosomes and autosomal trisomies), cytogenetic analysis revealed two constantly appearing chromosomes with homogeneously stained regions (HSR's). The karyo-type remained constant over 50 passages in vitro [49,XX, –12,+der5, + 17,+mar1,+mar2]. Double minutes were a rare phenomenon and appeared only in a few metaphases. In situ hybridization showed that some of the HSR's consisted of amplified N-myc copies. The distribution of the N-myc copies according to in situ hybridization signals along the HSR's was compared with the data of Southern and Northern blotting analyses.     

Persistent infection with herpes simplex virus type 1 in an Ia antigen-positive murine macrophage cell line

The interaction of herpes simplex virus type 1 (HSV-1) with murine macrophage cell lines was examined. The cell lines appeared to be moderately permissive for HSV-1 replication, though the yield of the virus was limited compared with that in Vero cells. Furthermore, the murine macrophage cell line SL-1, bearing Ia antigen, was persistently infected with HSV-1 for over one year, and was designated SL-1/KOS. Persistent infection could not be established in an Ia antigen-negative macrophage cell line, SL-4. In the SL-1/KOS culture, there was a small number of infected cells as revealed by infectious center assay. Treatment with monoclonal antibody against HSV-1 cured the persistent infection. Therefore maintenance of the persistent infection is considered to be due to a carrier culture consisting of a minority of infected cells and a majority of uninfected cells. In the SL-1/KOS cultures a low level of interferon (IFN) was found. When a large amount of exogenous recombinant murine IFN-beta (10(5)-10(6) international units/ml) was added to the culture, virus production diminished to undetectable levels. These results suggest that IFN plays an important role in the maintenance of persistent infection. In long-term persistently infected cultures, syncytium formation appeared and the virus from such cultures had a different DNA structure from that of the virus originally used for infection as revealed by restriction endonuclease analysis.     

NMB: a human neuroblastoma cell line with specific opiate binding sites

The human neuroblastoma cell line designated NMB (Brodeur et al., 1977, Cancer 40: 2256) has been shown to have specific opiate binding sites. These sites are highly stereospecific. Two characteristic delta specific peptides, D-Ala2-D-Leu5 enkephalin and D-Thr2-D-Thr6 enkephalin, have high affinity for the binding sites. Morphine binds specifically but with a much lower affinity. Dextrorphan and the mu specific peptide morphiceptin (Tyr-Pro-Phe-Pro-CO-NH2) do not bind to the site. The binding sites are heat and trypsin sensitive. Sodium ions specifically lower agonist binding to the sites. Approximately 14,000 binding sites per cell are found. The binding characteristics of these sites are very similar to those of the delta sites characterized on mouse neuroblastoma cell lines.