scholarly journals Predominance of the acylation route in the metabolic processing of exogenous sphingosine in neural and extraneural cells in culture

1999 ◽  
Vol 338 (1) ◽  
pp. 147-151 ◽  
Author(s):  
Laura RIBONI ◽  
Rosaria BASSI ◽  
Alessandro PRINETTI ◽  
Paola VIANI ◽  
Guido TETTAMANTI
Keyword(s):  
Cultured Cells ◽  
Primary Cultures ◽  
Calf Serum ◽  
Neuroblastoma Cells ◽  
Cell Types ◽  
Total Radioactivity ◽  
Metabolic Fate ◽  
Pulse Period ◽  
Human Neuroblastoma ◽  
Sphingosine 1 Phosphate

The metabolic fate of exogenous [3H]sphingosine was investigated in five types of cultured cells: primary cultures of neurons and astrocytes, murine and human neuroblastoma cells and human skin fibroblasts. After administration of 40 nM [3-3H]sphingosine into a cell-conditioned medium containing fetal calf serum, all cell types rapidly and efficiently incorporated the long-chain base in a time-dependent fashion. In all cases, after a 120 min pulse, the amount of radioactivity taken up was in the range of the endogenous sphingosine content. However, unchanged [3H]sphingosine represented only a very minor portion of the label incorporated into cells throughout the pulse period (10–120 min), indicating rapid and efficient sphingosine metabolism in these cells. Most of the [3H]sphingosine taken up was metabolically processed, either by degradation (assessed as 3H2O release into the culture medium) or by N-acylation (mainly to radioactive ceramide, sphingomyelin, neutral glycolipids and gangliosides). [3H]Sphingosine 1-phosphate accounted for less than 2% of the total radioactivity incorporated in all cases. Throughout the pulse period and in all cell types, 3H-labelled organic metabolites largely prevailed over 3H2O, indicating that N-acylation is the major metabolic fate of sphingosine in these cells under apparently physiological conditions. These results are consistent with the notion that sphingosine has a rapid turnover in the cells studied, and indicate that regulation of the basal level of this bioactive molecule occurs mainly through N-acylation.

scholarly journals The tumour suppressor p53 regulates the expression of amyloid precursor protein (APP)

2009 ◽  
Vol 418 (3) ◽  
pp. 643-650 ◽  
Author(s):  
Ascensión Cuesta ◽  
Alberto Zambrano ◽  
María Royo ◽  
Angel Pascual
Keyword(s):  
Transcription Factor ◽  
Amyloid Precursor Protein ◽  
Dna Sequences ◽  
Primary Cultures ◽  
Neuroblastoma Cells ◽  
Dominant Negative ◽  
Precursor Protein ◽  
Human Neuroblastoma Cell ◽  
Dependent Manner ◽  
Human Neuroblastoma

The expression of the APP (amyloid precursor protein), which plays a key role in the development of AD (Alzheimer's disease), is regulated by a variety of cellular mediators in a cell-dependent manner. In this study, we present evidence that p53 regulates the expression of the APP gene in neuroblastoma cells. Transient expression of ectopic p53, activation of endogenous p53 by the DNA-damaging drug camptothecin or Mdm2 (murine double minute 2) depletion decreases the intracellular levels of APP in murine N2aβ neuroblastoma cells. This effect was also observed in primary cultures of rat neurons as well as in SH-SY5Y cells, a human neuroblastoma cell line. Transient transfection studies using plasmids that contain progressive deletions of the 5′ region of the gene demonstrate that p53 represses APP promoter activity through a mechanism that is mediated by DNA sequences located downstream of the transcription start site (+55/+101). Accordingly, expression of a dominant-negative p53 mutant significantly increases the transcriptional activity of the APP promoter. In addition, results obtained in gel mobility-shift assays show that p53 does not bind to the +55/+101 APP region, although it reduces binding of the transcription factor Sp1 (stimulating protein 1). Reduction of Sp1 binding after activation of p53 with camptothecin was also observed in chromatin immunoprecipitation assays. Altogether, our results strongly suggest a mechanism by which p53 precludes binding of Sp1 to DNA, and therefore the stimulation of the APP promoter by this transcription factor.

Cyst Fluids of Malignant Human Brain Tumors Contain Substances That Stimulate the Growth of Cultured Human Gliomas of Various Histological Type

Neurosurgery ◽  
1989 ◽  
Vol 25 (2) ◽  
pp. 196-201 ◽  
Author(s):  
Manfred Westphal ◽  
Hildergard Nausch ◽  
Hans-Dietrich Herrmann
Keyword(s):  
Cell Lines ◽  
Cultured Cells ◽  
Culture Media ◽  
Primary Cultures ◽  
Calf Serum ◽  
Needle Aspiration ◽  
Acoustic Neurinoma ◽  
Response Patterns ◽  
Human Gliomas ◽  
Cyst Fluids

Abstract The contents of 14 cysts that were located within human intracranial tumors were obtained at surgery by needle aspiration. These tumor cyst fluids (TCFs) were mostly derived from glial tumors (10 cases). TCFs from one metastasis from a mammary carcinoma, one cystic meningioma, one hemangioblastoma, and a cystic acoustic neurinoma were also included. These TCFs were added to primary cultures of human gliomas, established human glioma cell lines, and normal human arachnoid cells in culture. The presence of proliferation-promoting factors in all cyst fluids could be demonstrated. On the basis of the response patterns of the cultures, it was possible to distinguish different levels of growth autonomy and growth factor sensitivity among these cultures and to speculate about varying degrees of cellular autocrine activation. The TCFs appear to contain factors that are not normally present in fetal calf serum, which is a regular constituent of most cell culture media. Some primary cultured cells as well as cell lines react in an oversensitive manner to the addition of TCFs.

Characteristics of papillary collecting duct cells in primary culture

1988 ◽  
Vol 255 (6) ◽  
pp. F1160-F1169 ◽  
Author(s):  
R. F. Husted ◽  
M. Hayashi ◽  
J. B. Stokes
Keyword(s):  
Cultured Cells ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Calf Serum ◽  
Morphological Characteristics ◽  
Short Circuit Current ◽  
Papillary Collecting Duct ◽  
Collecting Duct Cells ◽  
Na Uptake

We examined the electrophysiological and Na+ transport characteristics of rat papillary collecting duct (PCD) cells grown in primary cultures. Grown as monolayers on polycarbonate filters, the cells displayed similar morphological characteristics to native epithelia. They also bound Dolichus biflorus lectin, a property shared by native cells. Monolayers developed a peak electrical resistance of 100-200 omega.cm2 and a transmonolayer voltage of less than 2 mV. Similar values were measured in the perfused, native PCD of the same species as well as PCD cells cultured from rabbit and bovine kidneys. Hamster cells did not readily develop confluent monolayers under the same conditions. Exposure of the cultured cells to 10% fetal calf serum for 24 h caused the Na+ uptake across the apical membrane to double, an effect not reproduced by indomethacin, insulin, vasopressin, aldosterone, dexamethasone, or hexamethylene bisacetamide (an inducer of differentiation). Amiloride (1 mM) inhibited Na+ uptake by 50-80%. The measured short-circuit current did not correlate with Na+ uptake and was clearly dissociated by exposure to serum. The results suggest that there is more than one mechanism of ion transport by the rat PCD.

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

1987 ◽  
Vol 88 (2) ◽  
pp. 185-203
Author(s):  
J. Wehland ◽  
K. Weber
Keyword(s):  
Primary Cultures ◽  
Neuroblastoma Cells ◽  
Cell Types ◽  
Morphological Transformation ◽  
Alpha Tubulin ◽  
Tubulin Antibodies ◽  
Cell Processes ◽  
Carboxy Terminal ◽  
Almost All ◽  
Different Cell Types

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.

Characterisation of a sphingosine 1-phosphate-activated Ca2+ signalling pathway in human neuroblastoma cells

2003 ◽  
Vol 73 (2) ◽  
pp. 215-226 ◽  
Author(s):  
Israel Ramos Villullas ◽  
Alison J. Smith ◽  
Robert P. Heavens ◽  
Peter B. Simpson
Keyword(s):  
Neuroblastoma Cells ◽  
Signalling Pathway ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma ◽  
Sphingosine 1 Phosphate

scholarly journals Steviol Glycosides Modulate Glucose Transport in Different Cell Types

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Benedetta Rizzo ◽  
Laura Zambonin ◽  
Cristina Angeloni ◽  
Emanuela Leoncini ◽  
Francesco Vieceli Dalla Sega ◽  
...  
Keyword(s):  
Glucose Uptake ◽  
Glucose Transport ◽  
Molecular Mechanisms ◽  
Neuroblastoma Cells ◽  
Stevia Rebaudiana ◽  
Cell Types ◽  
Akt Pathway ◽  
Steviol Glycosides ◽  
Human Neuroblastoma

Extracts fromStevia rebaudianaBertoni, a plant native to Central and South America, have been used as a sweetener since ancient times. Currently,Steviaextracts are largely used as a noncaloric high-potency biosweetener alternative to sugar, due to the growing incidence of type 2 diabetes mellitus, obesity, and metabolic disorders worldwide. Despite the large number of studies onSteviaand steviol glycosidesin vivo, little is reported concerning the cellular and molecular mechanisms underpinning the beneficial effects on human health. The effect of four commercialSteviaextracts on glucose transport activity was evaluated in HL-60 human leukaemia and in SH-SY5Y human neuroblastoma cells. The extracts were able to enhance glucose uptake in both cellular lines, as efficiently as insulin. Our data suggest that steviol glycosides could act by modulating GLUT translocation through the PI3K/Akt pathway since treatments with both insulin andSteviaextracts increased the phosphorylation of PI3K and Akt. Furthermore,Steviaextracts were able to revert the effect of the reduction of glucose uptake caused by methylglyoxal, an inhibitor of the insulin receptor/PI3K/Akt pathway. These results corroborate the hypothesis thatSteviaextracts could mimic insulin effects modulating PI3K/Akt pathway.

GPR40 activation leads to CREB and ERK phosphorylation in primary cultures of neurons from the mouse CNS and in human neuroblastoma cells

Hippocampus ◽  
2014 ◽  
Vol 24 (7) ◽  
pp. 733-739 ◽  
Author(s):  
Marta Zamarbide ◽  
Iñigo Etayo-Labiano ◽  
Ana Ricobaraza ◽  
Eva Martínez-Pinilla ◽  
María S. Aymerich ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Neuroblastoma Cells ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma ◽  
Erk Phosphorylation

scholarly journals Retinoic acid stimulates IGF binding protein (IGFBP)-6 and depresses IGFBP-2 and IGFBP-4 in SK-N-SH human neuroblastoma cells

1998 ◽  
Vol 159 (2) ◽  
pp. 227-232 ◽  
Author(s):  
D Chambery ◽  
B de Galle ◽  
S Babajko
Keyword(s):  
Retinoic Acid ◽  
Transcriptional Activity ◽  
Biological Fluids ◽  
Neuroblastoma Cells ◽  
Cell Types ◽  
Human Neuroblastoma ◽  
Proliferation And Differentiation ◽  
Igf Binding ◽  
The Stability ◽  
Igf Binding Protein

Insulin-like growth factors (IGF-I and IGF-II) stimulate proliferation and differentiation in many cell types. In biological fluids, they associate non-covalently with high-affinity binding proteins (IGFBPs) which control their bioavailability and modulate their action. We previously demonstrated that IGFBP-2, -4 and -6 are intimately involved in the growth of cells derived from human neuroblastomas. Here, we have investigated the effects of retinoic acid (RA), which induces differentiation in these cells, on the expression of IGFBPs secreted by SK-N-SH neuroblastoma cells. Analysis of transcriptional activity of the IGFBP-2, -4 and -6 genes in isolated nuclei (run-on experiments) showed that RA increased the transcriptional activity of the IGFBP-6 gene, reduced that of the IGFBP-4 gene and had no effect on that of the IGFBP-2 gene. Northern blot analysis following treatment with actinomycin D showed that RA increased the stability of IGFBP-6 mRNA by a factor of 2.6, decreased that of IGFBP-2 mRNA by a factor of 2.3 and failed to affect IGFBP-4 mRNA. Treatment of cells with cycloheximide indicated the involvement of labile proteins in the stabilization of these mRNAs the expression of which could be under the control of RA. The transcriptional and/or post-transcriptional mechanisms by which RA regulates each of the IGFBPs produced by SK-N-SH cells are therefore different. Such regulation may also reflect the state of differentiation of the neuroblastoma cells. With RA-induced differentiation, IGFBP-6 is strongly stimulated, whereas IGFBP-2 and IGFBP-4 are severely depressed, which would suggest that each IGFBP plays a specific role. Moreover, this regulation seems tissue-specific because it is different in other cell types.

scholarly journals Expression of the amplified domain in human neuroblastoma cells.

1984 ◽  
Vol 4 (11) ◽  
pp. 2370-2380 ◽  
Author(s):  
R W Michitsch ◽  
K T Montgomery ◽  
P W Melera
Keyword(s):  
Cell Line ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Human Tumor ◽  
Cell Types ◽  
Neuroblastoma Cell Line ◽  
Specific Gene ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
Myc Oncogene

Screening of a partial cDNA library prepared from the human neuroblastoma cell line BE(2)-C with genomic DNA probes containing sequences representative of the amplified domain of that cell line allowed us to identify cloned transcripts from an active gene within the domain. The gene BE(2)-C-59 is amplified ca. 150-fold and encodes a 3.0- and a 1.5-kilobase RNA transcript, both of which are overproduced in BE(2)-C cells. A survey of a large variety of human tumor cell types indicated that this gene is amplified to varying degrees in all neuroblastoma cell lines and a retinoblastoma cell line that exhibit obvious cytological manifestations of DNA sequence amplification, i.e., homogeneously staining regions and double-minute chromosomes. The BE(2)-C-59 gene is not amplified, however, in other nonrelated tumor types, even those containing amplified DNA. Although the functional significance of this specific gene amplification in neuroblastoma cells remains unknown, an indication that it may relate to the malignant phenotype of these cells follows from the remainder of our data which show that the amplified BE(2)-C-59 gene shares partial homology with both the second and third exons, but not the first exon, of the human c-myc oncogene.

scholarly journals Tumor-Derived Cell Culture Model for the Investigation of Meningioma Biology

2021 ◽  
Author(s):  
Erik J Uhlmann ◽  
Rosalia Rabinovsky ◽  
Hemant Varma ◽  
Rachid El Fatimy ◽  
Ekkehard M Kasper ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Vascular Endothelial Cells ◽  
Vasogenic Edema ◽  
Primary Cultures ◽  
Calf Serum ◽  
Mass Effect ◽  
Central Nervous System Tumor ◽  
Tissue Samples ◽  
High Throughput Drug Screening ◽  
Free Media

Abstract Meningioma is the most common primary central nervous system tumor. Although mostly nonmalignant, meningioma can cause serious complications by mass effect and vasogenic edema. While surgery and radiation improve outcomes, not all cases can be treated due to eloquent location. Presently no medical treatment is available to slow meningioma growth owing to incomplete understanding of the underlying pathology, which in turn is due to the lack of high-fidelity tissue culture and animal models. We propose a simple and rapid method for the establishment of meningioma tumor-derived primary cultures. These cells can be maintained in culture for a limited time in serum-free media as spheres and form adherent cultures in the presence of 4% fetal calf serum. Many of the tissue samples show expression of the lineage marker PDG2S, which is typically retained in matched cultured cells, suggesting the presence of cells of arachnoid origin. Furthermore, nonarachnoid cells including vascular endothelial cells are also present in the cultures in addition to arachnoid cells, potentially providing a more accurate tumor cell microenvironment, and thus making the model more relevant for meningioma research and high-throughput drug screening.

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