A web of imprinting in stem cells

Imprinted genes are the prototypical epigenetically regulated genes. On the basis of findings in adult lung stem cells, Zacharek et?al. (2011) suggest in this issue of Cell Stem Cell that epigenetic silencing of imprinted genes is a common requirement for maintaining self-renewal in adult stem cell populations.     

Proliferation of serum-deprived neuroblastoma cells

Both the mitotic index after colchicine block and the fraction of cells labeled by a short exposure to [³H]TdR indicate that the rate of proliferation in cultures of murine neuroblastoma cells (N18 clone) decreases only after several days of maintenance in serum-deficient medium. Viable cell counts remain constant because of a large increase in the number of cells which die and are shed into the medium. Therefore, the neurite formation and enzyme induction which occur under these conditions cannot be caused by inhibition of DNA synthesis.     

Insulin and insulin-like growth factor-I stimulate a common endogenous phosphoprotein substrate (pp185) in intact neuroblastoma cells

Mouse neuroblastoma N18 cells contain specific high affinity insulin and insulin-like growth factor-I (IGF-I) receptors. Insulin and IGF-I induce phosphorylation, in intact cells, of their respective receptor beta subunits. The insulin receptor beta subunit is represented by a 95-kDa phosphoprotein that is recognized by a specific antiserum (B10). The IGF-I receptor beta subunit is represented by two phosphoproteins of molecular mass 95 and 105 kDa. The hormone-induced phosphorylation was rapid and dose-dependent occurring on both phosphoserine and phosphotyrosine residues. In addition, both insulin and IGF-I induced phosphorylation of an endogenous protein of molecular mass 185 kDa (pp185). The rapidity and dose dependency of the phosphorylation of pp185 suggested that it may represent a common endogenous substrate for the insulin and IGF-I receptors in these neural-derived cells. Phosphorylation was primarily on phosphoserine and phosphotyrosine residues. pp185 did not absorb to wheat germ agglutinin-agarose and was not stimulated by either epidermal growth factor or platelet-derived growth factor. The finding of pp185 in these neural-related cells as well as in non-neural tissues suggests that it may represent a ubiquitous endogenous substrate for both the insulin and IGF-I receptor kinases.     

Insulin receptor substrate (IRS)-2, not IRS-1, protects human neuroblastoma cells against apoptosis

Insulin receptor substrates (IRS)-1 and -2 are major substrates of insulin and type I insulin-like growth factor (IGF-I) receptor (IGF-IR) signaling. In this study, SH-EP human neuroblastoma cells are used as a model system to examine the differential roles of IRS-1 and IRS-2 on glucose-mediated apoptosis. In the presence of high glucose, IRS-1 underwent caspase-mediated degradation, followed by focal adhesion kinase (FAK) and Akt degradation and apoptosis. IRS-2 expression blocked all these changes whereas IRS-1 overexpression had no effect. In parallel, IRS-2, but not IRS-1, overexpression enhanced IGF-I-mediated Akt activation without affecting extracellular regulated kinase signaling. While IRS-1 was readily degraded by caspases, hyperglycemia-mediated IRS-2 degradation was unaffected by caspase inhibitors but blocked by proteasome and calpain inhibitors. Our data suggest that the differential degradation of IRS-1 and IRS-2 contributes to their distinct modes of action and the increased neuroprotective effects of IRS-2 in this report are due, in part, to its resistance to caspase-mediated degradation.     

Genome imprinting in stem cells: A mini-review

Genomic imprinting is an epigenetic process result in silencing of one of the two alleles (maternal or paternal) based on the parent of origin. Dysregulation of imprinted genes results in detectable developmental and differential abnormalities. Epigenetics erasure is required for resetting the cell identity to a ground state during the production of induced pluripotent stem (iPS) cells from somatic cells. There are some contradictory reports regarding the status of the imprinting marks in the genome of iPS cells. Additionally, many studies highlighted the existence of subtle differences in the imprinting loci between different types of iPS cells and embryonic stem (ES) cells. These observations could ultimately undermine the use of patient-derived iPS cells for regenerative medicine.     

The VIP-receptor system in neuroblastoma cells

Neuroblastoma (NB), the most common extracranial tumor during childhood arises from the embryonic sympathetic nervous system. Remarkably, NB can spontaneously regress, even after metastasis, leading to complete remission. Subpopulations of neuroblastic (N-type) and nonneuronal cells coexist in NB. Expression of the high-affinity nerve growth factor (NGF) TrkA receptor in NB is correlated with good prognosis, while MYCN amplification is associated with advanced stages of disease. N-type cells undergo differentiation when treated with different compounds, such as retinoids, phorbol esters, growth and neurotrophic NGF and neuropeptides, especially vasoactive intestinal peptide (VIP). These substances stabilize proliferation, leading to a more mature neuronal phenotype, neurite outgrowth and induction of expression of sympathetic neuronal markers. Therefore, receptors for these substances and their associated signalling pathways, appear like promising targets for the development of novel NB therapeutics. The aim of the present review is to summarize the quite considerable array of data, concerning production of VIP and related peptides, expression of their receptors in NB and the key regulation exerted by the VIP-receptor system in the control of NB cell behaviour.     

Tubulin pools in differentiating neuroblastoma cells

The distribution of tubulin in soluble, reversibly stabilized (assembled) and insoluble forms has been determined in neuroblastoma cells undergoing microtubule-dependent neurite elongation. Procedures were developed to obtain reproducible tubulin fractions and to assay total tubulin. Radioimmunoassays showed that both differentiated and nondifferentiated cell contained approximately 4 pg of tubulin per cell, of which 3-10% was in an insoluble, particulate form. The amount of tubulin assembled in differentiated cells was four to five times greater than in nondifferentiated cells, constituting 48-63% and 11-16% of the total tubulin pool in the respective cell types. Calculation of the concentration of soluble tubulin indifferentiated cells (approximately 0.8 mg/ml) and nondifferentiated cells (approximately 1.6 mg/ml) indicates that a critical concentration of subunits probably does not limit the induction of microtubule formation during neurite elongation.     

Branched polyamines cure prion-infected neuroblastoma cells

Branched polyamines, including polyamidoamine and polypropyleneimine (PPI) dendrimers, are able to purge PrP(Sc), the disease-causing isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture (S. Supattapone, H.-O. B. Nguyen, F. E. Cohen, S. B. Prusiner, and M. R. Scott, Proc. Natl. Acad. Sci. USA 96:14529-14534, 1999). We now demonstrate that exposure of ScN2a cells to 3 microg of PPI generation 4.0/ml for 4 weeks not only reduced PrP(Sc) to a level undetectable by Western blot but also eradicated prion infectivity as determined by a bioassay in mice. Exposure of purified RML prions to branched polyamines in vitro disaggregated the prion rods, reduced the beta-sheet content of PrP 27-30, and rendered PrP 27-30 susceptible to proteolysis. The susceptibility of PrP(Sc) to proteolytic digestion induced by branched polyamines in vitro was strain dependent. Notably, PrP(Sc) from bovine spongiform encephalopathy-infected brain was susceptible to PPI-mediated denaturation in vitro, whereas PrP(Sc) from natural sheep scrapie-infected brain was resistant. Fluorescein-labeled PPI accumulated specifically in lysosomes, suggesting that branched polyamines act within this acidic compartment to mediate PrP(Sc) clearance. Branched polyamines are the first class of compounds shown to cure prion infection in living cells and may prove useful as therapeutic, disinfecting, and strain-typing reagents for prion diseases.     

Cultured neuroblastoma cells in biochemical studies

Neuroblastoma is a spontaneous tumour of animals and people. Tumour strains maintained as transplanted tumours and constant cell lines in suspension and monolayer cultures in vitro are obtained. The neuroblastoma cells are able to spontaneous and induced differentiation. The differentiated neuroblastoma cells have many specific properties of nature neurons and are a rather valuable object for solution of many problems in neurochemistry and general cell biology. It is neuroblastoma that has been used to show first the reversibility of neoplastic transformation and to carry out numerous studies to elucidate the mechanisms of cell differentiation. The neuroblastoma cell cultures are a convenient object for studying the ion channels and specifying the role of membrane lipids in their work.     

Lanthanum binding to murine neuroblastoma cells

The binding of lanthanum to murine neuroblas-toma cells (clone N1E-115) was studied by means of electron microscopy and x-ray microanalysis. Lanthanum bound only to cellular membranes, in a nonuniform manner. This lanthanum binding was reduced by pharmacological agents that affect muscarinic receptors of these cells or the function of the receptors. These results suggest that this binding of lanthanum is to sites closely related to muscarinic receptors in the cells.     

Staurosporine-induced morphological differentiation of human neuroblastoma cells

SH-SY-5Y human neuroblastoma cells rapidly elaborated an extensive network of neuritic processes following treatment with staurosporine, an inhibitor of protein kinase C. These neurites were retracted within 24hr following removal of inhibitor. Another inhibitor of protein kinase C, H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], also induced rapid, reversible neurite outgrowth. However, neurites induced by these two inhibitors were morphologically distinct: staurosporine-treated cells elaborated a branching neuritic network adjacent to cell bodies, with some longer, unbranching neurites extending out of this network, while H7-treated cells elaborated only long, unbranching neurites. HA-1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide], which inhibits of cAMP- and cGMP-dependent protein kinases but not protein kinase C, did not induce neuritogenesis. Staurosporine-induced neurite outgrowth did not require protein synthesis but did require microtubule assembly, suggesting that cells contained the necessary components for neuritogenesis, and that alterations in protein phosphorylation alone was sufficient to initiate neurite outgrowth by rearrangement of existing structures or cytoskeletal precursors. These results implicate phosphorylation in the regulation of neuronal differentiation and neuritogenesis.     

Sublethal irradiation promotes invasiveness of neuroblastoma cells

Neuroblastoma is the most frequent extracranial solid tumour of childhood. Despite multiple clinical efforts, clinical outcome has remained poor. Neuroblastoma is considered to be radiosensitive, but some clinical studies including the German trial NB90 failed to show a clinical benefit of radiation therapy. The mechanisms underlying this apparent discrepancy are still unclear. We have therefore investigated the effects of radiation on neuroblastoma cell behaviour in vitro. We show that sublethal doses of irradiation up-regulated the expression of the hepatocyte growth factor (HGF) and its receptor c-Met in some neuroblastoma cell lines. The increase in HGF/c-Met expression was correlated with enhanced invasiveness and activation of proteases degrading the extracellular matrix. Thus, irradiation at sublethal doses may promote the metastatic dissemination of neuroblastoma cells through activating the HGF/c-Met pathway and triggering matrix degradation.     

Free nucleotides in bromodeoxyuridine-treated neuroblastoma cells

Free nucleotides in bromodeoxyuridine-treated neuroblastoma cells were investigated. The separation and purification of nucleotides were carried out by anion exchange and paper chromatography. After bromodeoxyuridine treatment, the distribution of free nucleotides with the same base, when referred to total nucleotides, shifted toward a distribution pattern closer to that of mature neuronal cells. There was an enhancement of adenylic nucleotides and a decrease of uridylic and cytidylic nucleotides. The guanylic nucleotides displayed no changes. This nucleotide distribution is in agreement with other biochemical data from differentiated neuroblastoma cells.Dr. Ciesielski-Treska is stagiaire de Recherche à l'INSERMDr. Wintzerith is Chargée de Recherche au CNRSThis paper is part of the Doctorat d'Etat thesis of A. Dierich.     

Phenotypic characteristics of heat-resistant murine neuroblastoma cells

One-step selection of murine neuroblastoma (N18TG2 cell line) for the resistance to injuring action of higher temperature resulted in obtaining cell line NTR1 stably capable of proliferating at 40 degrees C. The thermoresistance is shown to be coupled with the changes in some phenotypic features of NTR1 cells: the multiplication rate, saturation density of cells, morphological features, inducibility of long neurite-like processes and adhesion. A possible significance of plasma membrane changes in genetically stable thermoresistance of NTR1 neuroblastoma cells is discussed.