N115 Cells Research Articles

Characterization of the rat light neurofilament (NF-L) gene promoter and identification of NGF and cAMP responsive regions.

We have isolated a genomic DNA clone covering the coding and 14 kb upstream region of the rat light neurofilament (NF-L) gene and sequenced 2.3 kb of its promoter. DNase I hypersensitive sites have been mapped in PC12 cells. For functional analysis of the NF-L promoter, constructs carrying 38, 97, 407, 564, 650, 1,099, 1,660, 2,003 base pairs (bp) upstream region in front of the chloramphenicol acetyltransferase (CAT) reporter gene were tested for their capability to direct CAT expression after transient transfection into various cell lines. Similar CAT activities were recorded both in rat pheochromocytoma (PC12) and mouse neuroblastoma N115 cells and also in several nonneural cell lines (HeLa, C127, NIH 3T3). Regions responsible for the basic promoter activity were located between -407 and +75 bp from the transcription initiation site. The NGF-responsive element was located between -38 and +75 bp, and sequence -97 to -38 was found to contain a functional cAMP-responsive element. In PC12 cells in which nerve growth factor (NGF) induces neurite outgrowth and NF-L transcription, NF-L promoter-driven CAT expression was stimulated up to 12-fold within three days of NGF treatment, whereas epidermal growth factor (EGF) had no effect. Rat NF-L promoter contained Sp1, AP-2 and CGCCCCCGC elements. In PC12 cells, NGF transiently induced the binding of transcription factors to the deoxyoligonucleotide probes containing the binding sites of these elements. The role of these factors in NF-L gene transcriptional induction by NGF in PC12 cells is discussed.

High molecular weight tau distribution and microtubule stability in neuroblastoma N115 cells.

The localization of high molecular weight (HMW) tau proteins in neuroblastoma N115 cells and of their transcripts was compared to that of non-tyrosinated and tyrosinated tubulin before and after treatment with depolymerizing drugs. Microtubules stained by tau antibodies were present both in a limited region of the cell center and in the cell processes, whereas tau transcripts were detected only in the cell body. The microtubules localized in the cell center and labeled by tau antibodies resisted colcemid treatment, whereas those in the neurites were completely depolymerized by the drug. Microtubules containing stable and unstable microtubule tracts were identified in the neurites after colcemid treatment. These composite microtubules were not labeled by tau antibodies. It is concluded that stable and unstable polymers--localized in the cell center and in the neurites, respectively--contain HMW tau proteins, whereas composite microtubules displayed in the cell processes do not. Microtubule stability in this cell line does not therefore seem to be related to the association of tau proteins to the polymers but, rather, to posttranslational modifications of the tubulin subunits.

Heterogeneity of the high molecular weight tau proteins in N115 neuroblastoma cells.

The sequence of a high molecular weight (HMW) tau cDNA cloned from a neuroblastoma N115 library contains, in addition to the C- and N-terminal and middle regions present in the low molecular weight mouse brain tau proteins, a 711-bp nonhomologous domain (exon 4a) and a region of 198 bp corresponding to exon 6 of the tau gene. Protein immunoblot analysis, performed with antibodies specific either for a sequence present in the N-terminal region of all the tau variants or for exon 4a revealed several bands suggesting that more than one tau form is expressed in this cell line. Northern blot experiments performed with a number of cDNA probes spanning domains common and uncommon to low molecular weight and HMW tau allowed the identification of four tau transcripts differing in the size of their coding and noncoding regions. All these transcripts contain the sequence encoded by exon 6, but two of them lack exon 4a. As shown by RNase protection assays, the N-terminal region of these transcripts is also variable and contains either exon 1, or exons 1 and 2, or exons 1-3. Yet all these HMW tau forms contain four homologous repeats in their C-terminal domain both in the differentiated and nondifferentiated cells, i.e., have adult characteristics. In conclusion, the data reported in this article demonstrate that several HMW tau variants are expressed in neuroblastoma N115 cells and that the transition between immature to mature tau forms occurring during brain development is not required for neurite outgrowth during morphological differentiation of this cell line.

Primary structure of high molecular weight tau present in the peripheral nervous system.

The tau proteins are a family of brain microtubule binding proteins that are required during axonal outgrowth and are found in neurofibrillary tangles in Alzheimer disease. A protein of higher molecular weight, immunologically related to tau, is expressed in the adult peripheral system and in cultured neuronal cell lines of neural crest origin. The predicted amino acid sequence of the high molecular weight tau from N115 cells has been determined from the sequence of its 2340-base-pair cDNA. High molecular weight tau contains an open reading frame encoding 733 amino acid residues. It contains sequences homologous to those present in the N-, middle, and C-terminal domains of adult brain tau proteins, including four homologous repeats, which are the tubulin binding sites, and an amino acid stretch, which is present only in the N-terminal domain of the mature brain variants. The middle region contains a previously unidentified nonhomologous stretch of 237 amino acid residues as well as a domain of 66 residues homologous to exon 6 of the bovine gene that is absent in all bovine, rat, and mouse tau cDNAs sequenced so far. A cDNA probe specific to the nonhomologous tau insert hybridizes to the 8- to 9-kilobase tau mRNA in N115 cells but not to the 6-kilobase tau mRNA in brain. Probes for the domains common to brain tau isoforms hybridize to both messages. The sequence of high molecular weight tau protein also suggests that it, like low molecular weight tau, is an elongated hydrophilic molecule. This cDNA should allow us to study the role of the domains specific to these tau forms in the specialization of the peripheral nervous system and for study of their expression in normal and pathological states.

Murine Neuroblastoma Cells Express Ganglioside Binding Sites on Their Cell Surface

The ability of S20Y cholinergic, and N115 adrenergic, murine neuroblastoma cells to adhere to immobilized gangliosides was studied. Viable S20Y cells adhered more strongly to GM1-coated plastic wells than to those coated with GM2, GD1a, or GT1b. The oligosaccharide portion of GM1 inhibited adherence of S20Y cells to GM1-coated wells, indicating that the carbohydrate moiety of GM1 bore the recognition site. Analysis of S20Y cell adherence to wells coated with derivatives of GM1 indicated that the cells did not adhere to asialo-GM1 and adherence to the methyl ester or de-N-acetyl derivatives was significantly reduced. Expression of the GM1 binding sites by S20Y cells appears to be density dependent; cells harvested at the confluent stage of growth were more adherent than those harvested at the preconfluent stage. Trypsin treatment of the S20Y and N115 cells resulted in a loss of binding to GM1-coated wells, suggesting that the cell surface GM1 binding site is a protein. In contrast, N115 cells showed no significant difference in their adherence to wells coated with GM1, GD1a, GT1b, Gal-Cer, asialo-GM1, or the methyl ester of GM1 when assayed under the same conditions as those imposed on the S20Y cells. The N115 cells did show a reduction in adherence to GM2-coated wells, suggesting that they recognized the terminal galactosyl moiety.

Bilirubin toxicity in a neuroblastoma cell line N-115: II. Delayed effects and recovery.

Clinical studies have suggested that the early stages of bilirubin encephalopathy are reversible. These phenomena are investigated at the cellular level using the neuroblastoma cell line N-115 as a model system. To determine whether the cell line N-115 can recover from bilirubin toxicity, and whether the cellular function remains intact after a short period of bilirubin exposure during which time no toxic effects are manifest, the cells are exposed to bilirubin at varying concentrations and varying bilirubin:albumin ratios for 1 and 2 h. The bilirubin is then washed out, and the cells are reincubated in fresh media with appropriate amounts of albumin. Mitochondrial function, [3H]thymidine uptake and L-[35S]methionine uptake are assessed at 2, 8, and 24 h of reincubation after the bilirubin washout. After the short-term exposure, the cells begin to demonstrate evidence of toxicity in all parameters measured 8-24 h after the bilirubin washout. After the 2-h exposure to bilirubin, the cells demonstrate significant toxicity within 2 h of the bilirubin washout. The degree of toxicity seems to depend on the bilirubin:albumin ratio and bilirubin concentration. In general, after bilirubin exposure of 1 h or longer, the N-115 cells develop evidence of toxicity which is progressive and irreversible.

Turnover of the carboxy-terminal tyrosine of alpha-tubulin and means of reaching elevated levels of detyrosination in living cells.

Monoclonal antibodies specific for either the tyrosinated (Tyr) or the detyrosinated (Glu) form of alpha-tubulin were elicited with synthetic peptides spanning the carboxy-terminal sequences of the two forms. While almost all microtubules (MTs) are usually of the Tyr-tubulin type (Tyr-rich MTs) some MTs containing noticeable amounts of Glu-tubulin (Glu-rich MTs) were found in many but not all cell lines studied. Glu-rich MTs seemed absent from proliferating CHO and N115 neuroblastoma cells. When differentiation of these cells was initiated by the addition of forskolin for CHO, or by serum deprivation for N115, elevated levels of microtubular Glu-tubulin were observed. In differentiated N115 cells Glu-tubulin was restricted to MT of elongated cell processes and was not found in growth cones and many MT of the cell body. Elevated levels of Glu-tubulin were also characteristic of other differentiated cell types, including neurones and myotubes but were not found in glial cells, astrocytes and fibroblasts in the same primary cultures. Additional experiments suggested that the restricted distribution of Glu-tubulin is the result of MT subsets with different stabilities. Results with mitotic drugs indicated that detyrosination occurs on MTs rather than on soluble tubulin and that stabilization of MTs usually favours the detyrosination process. Evidence for a functional alpha-tubulin cycle involving an inherent carboxypeptidase and a recharging ligase was apparent in 3T3 cells from the preponderance of Glu-rich MTs induced by taxol treatment or the micro-injection of certain antibodies either protecting the detyrosinated form (Glu-tubulin antibodies) or inhibiting retyrosination (ligase antibodies). As the same treatment of CHO cells resulted in comparable arrays of Glu-rich MTs only when forskolin was also present, different cell types may differ in the level of active carboxypeptidase. The results are discussed in terms of possible functions of the tyrosination/detyrosination cycle of alpha-tubulin. While most results can be explained on the basis of 'older' and, consequently, more detyrosinated MTs, others raise the possibility that cyclic-AMP-dependent events and certain environmental influences known to induce either a morphological transformation or a differentiation event may influence the carboxypeptidase inherent in the alpha-tubulin cycle.

Bilirubin toxicity in neural cell lines N115 and NBR10A.

The toxicity of bilirubin was investigated in 2 neural cell lines NBR10A and N115 using a quantitative dye assay 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium biomide (MTT) as a measure of cell viability and [3H]thymidine incorporation as a measure of DNA synthesis. Short exposures (up to 2 h) to bilirubin, even up to a bilirubin-albumin molar ratio of 1.5, yielded no evidence of toxicity using these assays. At longer exposure times (24 h) a decrease in cell viability and [3H]thymidine incorporation was detected at a molar ratio of 0.8 when the bilirubin concentration was 0.1 mM or higher, whereas lower bilirubin levels at this molar ratio showed no deleterious effect. The effect of bilirubin is more pronounced at a molar ratio of 1.5 with longer incubation periods. The MTT assay showed the N115 cells appeared to be more resistant to bilirubin cytotoxicity than NBR10A cells, a finding which was not obtained from [3H]thymidine incorporation studies. This discrepancy can be explained by the fact that we are measuring two different variables; the MTT assay estimates the number of viable cells at the end of the experiment by measuring mitochondrial function whereas the [3H]thymidine assay measures the rate of DNA synthesis during the last 2 h of the experiment. The concentration effect of bilirubin is evident from the [3H]-thymidine studies in that at a molar ratio of 1.5 and bilirubin concentration of 0.075 mM or higher, there is both cell kill (decrease in DNA) and inhibition of [3H]thymidine incorporation (decrease in specific activity).(ABSTRACT TRUNCATED AT 250 WORDS)

1518 3H BILIRUBIN UPTAKE IN NEUROBLASTOMA CELLS (N115)

Inspite of an understanding of the biochemistry of bilirubin and the clinical picture associated with hyperbilirubinemia, no studies have defined the target of bilirubin at the cellular level. To determine this, 3H-bilirubin is prepared biosynthetic-ally in the rat by injection of 3H-δ aminolevulinic acid. 3H-bilirubin extracted from the pooled bile of 6 rats had specific activity of 3.6 × 106cmp/mMol. The N115 cells seeded at a concentration of 105 cells in 2 cm tissue culture dishes are adapted to grow in a protein free media. To determine the effect of time and temperature on 3H-bilirubin uptake, 0.2μCi/24nM of 3H-bilirubin was added to the media at bilirubin-albumin molar ratio (B/A) of 0-1.5 and incubated from 0 min to 24 hrs at 37°C, 24°C and 4°C. The amount of bilirubin uptake plateaus at 3hrs, is affected by the concentration of albumin, but is not sharply temperature dependent. The bilirubin uptake at 3hrs in the absence of albumin was 89.3cpm/μg DNA, decreased to 42.5cpm/μg DNA at B/A of 2.5 and to 31.2cpm/μg DNA for a B/A of 0.8. To determine the effect of bilirubin concentration on uptake, 3H-bilirubin ranging from 0.005μCi to 0.040μCi were added to the media and incubated for 3 hrs at 37°C in the absence of albumin. Uptake is linearly related to the concentration of 3H-bilirubin added. The results show 3H-bilirubin uptake peaks by 3hrs, is concentration dependent and there is no sharp temperature dependence. Although the uptake mechanism has not been determined, it is limited by the amount of albumin present, and is probably a passive process.

Role of glycoproteins in neuronal differentiation: Inhibition of neurite outgrowth and the major cell surface glycoprotein of murine neuroblastoma cells by a purified tunicamycin homologue

Mouse neuroblastoma cells in culture can be induced to differentiate morphologically by serum deprivation or by dibutyryl cyclic AMP (db-cAMP), e.g. they appear flattened, adhere more firmly to the culture substratum and extend long neuritic processes, and thus represent a widely used model system for neuronal cells. This differentiation is accompanied by modulation of cell surface components, such as the induction of a high molecular weight (HMW) glycoprotein (200 kD). We have studied the role of glycoproteins in the process of neuronal differentiation, using a purified homologue of the antibiotic tunicamycin (Al-tunicamycin) and neuroblastoma N115 cells grown in culture. Al-tunicamycin markedly inhibited (up to 60–75%) the incorporation of radioactively labelled sugars into cellular proteins of differentiating neuroblastoma cells. Concomitantly, the cells altered their morphology, they became rounded and less adhesive and retracted their neurites. Changes in the appearance, glycosylation and electrophoretic mobility of several cellular and secreted glycoproteins were observed, when cells were incubated in the presence of Al-tunicamycin. The most striking effect of Al-tunicamycin on the composition of cellular glycoproteins was the marked reduction in appearance of the 200 kD glycoprotein. The findings suggest that glycoproteins and in particular the neuron-specific 200 kD glycoprotein, are related to morphological differentiation processes, mainly to cellular adhesion and neurite outgrowth.

Density-dependent changes in gangliosides and sialidase activity of murine neuroblastoma cells.

Density-dependent changes in ganglioside composition, Vibrio cholerae neuraminidase (VCN)-susceptible sialyl residues, and membrane-associated sialidase activity were determined for the cholinergic murine neuroblastoma cell line S20Y. A decrease in total ganglioside sialic acid and VCN-releasable sialic acid was observed with increasing cell density. GM3 was the major ganglioside component of preconfluent S20Y cells, whereas GD1a was predominant in postconfluent cells. Sialidase activity increased in confluent and postconfluent cells and may account for the reduction in total ganglioside sialic acid observed with increasing cell density. In contrast, while adrenergic N115 cells showed a decrease in VCN-susceptible sialic acid residues with increasing cell density, there was no significant change in ganglioside composition or ganglioside sialic acid levels.

Mitosis in a cell with multiple centrioles.

N115 mouse neuroblastoma cells possess a large number of microtubule organizing centers (MTOCs) which can be identified ultrastructurally as single centrioles. The distribution and activity of these organizing centers can be followed through all stages of the cell cycle by labeling microtubules with anti-tubulin and chromatin with the Hoechst dye, Bisbenzimid. We have found that multiple MTOCs persist and continue to organize microtubules during mitosis. They exhibit a well-defined sequence of movements, starting from a loose cluster during interphase, proceeding to a widely and evenly dispersed arrangement in prophase, gathering into small clusters and chains during prometaphase, and residing in two ring-shaped groups at the mitotic poles during metaphase and anaphase. Despite their large number of centrioles, virtually all N115 cells show a normal bipolar mitosis, but often with unequal numbers of centrioles at the two poles. Such observations bring into question the importance of the centriole in establishing bipolar division in this cell type.