Activity of a novel PDGF beta-receptor enhancer during the cell cycle and upon differentiation of neuroblastoma

PDGF acts as an autocrine and paracrine factor in certain tumors through upregulation of the PDGF beta-receptor expression. In order to elucidate the control mechanism for the receptor expression, we have isolated an enhancer from two P1 clones that together contain a 102 kb NotI region covering the entire human PDGFRB gene. They were partially digested with TspI and cloned into the PDGFRB enhancer trap vector to make a library for identification of enhancers. The digested DNA containing enhancer was identified by expression of GFP when transfected in PDGF beta-receptor expressing cells. One of the enhancer clones was further examined by making several deletion mutants in a luciferase vector. This enhancer was most active in neuroblastoma cells, IMR32 and BE2, but less active in hemangioma and in smooth muscle cell lines. Chip assay revealed that SP1, AP2, and GATA2 bound the enhancer in BE2 cells. Their interaction occurred dependently of the cell cycle and synchronously with their binding to the promoter. Transfection of GATA2 alone or with Ets, which binds adjacent to GATA, resulted in differentiation of BE2 cells in parallel with increased PDGF beta-receptor expression. Furthermore, over-expression of the PDGF beta-receptor in BE2 cells induced neurite extension.     

Lembehyne A, a spongean polyacetylene, induces neuronal differentiation in neuroblastoma cell.

Lembehyne A (LB-A), a spongean polyacetylene, induced neuronal cell differentiation in a neuroblastoma cell line, Neuro 2A. The LB-A treatment of Neuro 2A cells predominantly resulted in a morphological change with bipolar neurites. The acetylcholinesterase activity of Neuro 2A was also increased by the treatment of LB-A. Furthermore, the cell cycle of Neuro 2A cells was found to be specifically blocked at the G1 phase by LB-A. The structure-activity relationship study using the LB-A analogues revealed the importance of the terminal 1-yn-3-ol and unsaturated long-chain alkyl moieties for the neuronal differentiation activity of LB-A.     

Effect of cytidine analogs on cell growth and differentiation on a human neuroblastoma line

The cytidine analog 5-AZA-2'-deoxycytidine (5-AZA-CdR) has been demonstrated to induce cellular differentiation; on the other hand, induction of differentiation has been suggested as a possible form of therapy for leukemic cells. We have evaluated the possibility that the neuroblastoma malignant tumor growth could be controlled by treatment that promotes the differentiation of immature tumor cells. We have previously reported on differentiation of murine neuroblastoma cells (41A3) treated with 5-AZA-CdR. In this paper, we describe the effect of 5-AZA-CdR on human neuroblastoma cell line CHP-100. The drug-treated cells show some degree of differentiation, demonstrated by morphological and biochemical markers. A significant DNA hypomethylation and partial inhibition of DNA synthesis and cell proliferation is also observed. This effect is more stable than that caused by another cytidine analog, Cytosine-beta-D-Arabinofuranoside (ARA-C).     

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.     

掷孢酵母及其应用研究进展

掷孢酵母是一类能够弹射孢子(称为掷孢子)的酵母菌,主要由Bullera属、Sporobolomyces属和Sporidiobolus属组成,该类酵母分布广泛。在实验室培养过程中多以芽殖、掷孢子以及菌丝状生长,Sporidiobolus属的菌株经配对能够形成有性孢子。分子生物学手段的开展使掷孢酵母各类群间的系统发育关系更加明确。本文结合本实验室的研究结果,阐述了掷孢酵母存在的细胞分化现象,并且推测细胞分化有助于菌体抵抗逆境。因此,掷孢酵母可以作为一种潜在的模式生物对抗逆机制进行探究。在食品和环境问题备受瞩目的今天,掷孢酵母以其可自身积累酯类、色素、酶类等有益代谢产物的特点,及治理污染物特性越来越受到人们的关注。     

Rapid disappearance of statin, a nonproliferating and senescent cell- specific protein, upon reentering the process of cell cycling

Statin, a 57,000-D protein characteristically found in nonreplicating cells, was identified by a monoclonal antibody produced by hybridomas established from mice injected with extracts of in vitro aged human fibroblasts (Wang, E., 1985, J. Cell Biol., 100:545-551). Fluorescence staining with the antibody shows that the expression of statin disappears upon reinitiation of the process for cell replication. The rapid de-expression is observed in fibroblasts involved in the in vitro wound-healing process, as well as in cells that have been subcultured after trypsinization and replated from a confluent culture. Kinetic analysis shows that 50% of the cell population lose their statin expression at 12 h after replating, before the actual events of mitosis. Immunogold labeling with highly purified antibodies localizes the protein at the nuclear envelope in nonreplicating cells, but not in their replicating counterparts. Immunoblotting analysis confirms the disappearance of statin in cells that have reentered the cycling process. Using the technique of flow cytometry to examine the large number of nonreplicating fibroblasts in confluent cultures, we have found that statin is mostly expressed in those cells showing the least amount of DNA content, whose growth is blocked at the G0/G1 stage of the cell cycle. This close correlation is rapidly altered once the cells are released from the confluent state. These results suggest that the expression of statin may be regulated by a fine mechanism controlling the transition from the nonreplicating to the replicating state, and that the protein is structurally associated with the nuclear envelope.     

Induced differentiation of a neuroblastoma

Neurite formation in a cloned tissue culture line of mouse neuroblastoma C1300 can be rapidly induced by plating cells in serum-free or conditioned media. This induced differentiation, defined here in terms of neurite formation and a change in macromolecular synthesis, is not initiated by the inhibition of cell division, but requires a strong interaction between the cell and the surface of the culture dish. This interaction is inhibited by several proteins and is enhanced by one or more dialyzable cell metabolites. Neurite formation is reversible, and microtubule formation in neurites is dependent on protein synthesis.     

Surface glycosaminoglycans as a differentiation cofactor in neuroblastoma cell cultures.

The possible role of surface glycosaminoglycans (GAGs) in neuronal maturation occuring in neuroblastoma cultures has been investigated. GAGs of neuroblastoma cells, grown in suspension and monolayer, were labelled with 3H-glucosamine and 35S-sulfate. Neuron maturation, following cell adhesion to culture dishes, is accompanied by an increased ability of the cells to retain heparan sulfate (HS) on their surface, which is otherwise lost into the culture medium. The role of surface HS as a cofactor of cellular differentiation is discussed.     

Rapid disappearance of translatable actin mRNA during cell differentiation in Naegleria

Actin, the major protein of Naegleria gruberi, is selectively not synthesized during the differentiation of amebae to flagellates. When RNA extracted from cells at intervals during differentiation is translated in the wheat germ cell-free system, a major translation product with the electrophoretic mobility of actin is seen to disappear with time during differentiation. This translation product is shown to be actin by its electrophoretic mobility, copolymerization with rabbit actin, peptide map, and immunoprecipitation by antibodies specific to Naegleria actin. Multiple isoforms of actin are synthesized in the cell-free system. Quantitative immunoprecipitation of translation products was employed to measure the relative amount of actin mRNA. Translatable actin mRNA begins to decrease in abundance within 7 min after the initiation of differentiation and thereafter decreases with a half-life of about 25 min. The selective disappearance of this major translatable mRNA provides a favorable opportunity to dissect the rules governing the half-life of a specific mRNA.     

Effects of 50 Hz electromagnetic field exposure on apoptosis and differentiation in a neuroblastoma cell line

Experiments were carried out to assess whether a magnetic field of 50 Hz and 1 mT can influence apoptosis and proliferation in the human neuroblastoma cell line LAN-5. TUNEL assays and poly-ADP ribose polymerase (PARP) expression analysis were performed to test apoptosis induction, and the WST-1 assay was used to calculate the proliferation index in a long term exposure. No alterations were found in cellular ability to undergo programmed cell death, but a small increase in the proliferation index was evidenced after 7 days of continuous exposure. Also, a slight and transient increase of B-myb oncogene expression was detected after 5 days of exposure. Combined exposures of cells to EMF and to chemical agents which interfere with proliferation, such as the differentiative agent retinoic acid and the apoptotic inducer camptothecin, showed an antagonistic effect of magnetic fields against the differentiation of the LAN-5 cells and a protective effect towards apoptosis.     

Pyridoacridine alkaloids inducing neuronal differentiation in a neuroblastoma cell line,from marine sponge Biemna fortis

A new and three known pyridoacridine alkaloids were isolated from the Indonesian marine sponge Biemna fortis as neuronal differentiation inducers against a murine neuroblastoma cell line, Neuro 2A. The chemical structure of the new compound, labuanine A (1), was determined by spectroscopic study and chemical conversion. These pyridoacridine alkaloids induced multipolar neuritogenesis in more than 50% of cells at 0.03-3 micro M concentration. Compound 3, which showed the strongest neuritogenic activity among them, also induced increase of acetylcholinesterase, a neuronal marker in Neuro 2A and arrested cell cycle at the G2/M phase.     

Nerve growth factor-induced differentiation of human neuroblastoma and neuroepithelioma cell lines

A series of neuroepithelioma and neuroblastoma cell lines were screened for nerve growth factor (NGF)-induced differentiation. All three neuroepithelioma cell lines and all nine neuroblastoma cell lines with amplified N-myc oncogene did not show any apparent NGF-induced differentiation. However, neurite extension was observed for three of six neuroblastoma cell lines with single-copy N-myc oncogene. The three responsive lines had a neuronal phenotype (short processes) which was enhanced by the addition of NGF. The three nonresponsive cell lines were flat without any processes. The addition of NGF to the responsive cell lines resulted in an up-regulation of neurofilament mRNA expression. Peripherin and synapsin, two markers of terminal neuronal differentiation, were not induced. There was little effect of NGF on the rate of cell growth or colony formation on soft agar. Binding of NGF to eight of the cell lines was analyzed by the method of Scatchard. Two responsive neuroblastoma cell lines and one nonresponsive neuroepithelioma cell line expressed both low- and high-affinity binding sites. Two nonresponsive neuroblastoma cell lines expressed only a small number of high-affinity binding sites, and two other nonresponsive neuroblastoma cell lines did not detectably bind NGF. Hence, NGF-induced differentiation is confined to a particular class of neural-related tumors, and, even for these cell lines, differentiation is incomplete.     

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67^phox, one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67^phox membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation.     

Silencing of miR-124 induces neuroblastoma SK-N-SH cell differentiation, cell cycle arrest and apoptosis through promoting AHR

Neuroblastoma is the most common extracranial solid tumor in children. We investigate whether miR-124, the abundant neuronal miRNA, plays a pivotal role in neuroblastoma. Knockdown of miR-124 promotes neuroblastoma SK-N-SH cell differentiation, cell cycle arrest and apoptosis. Further miR-124 is predicted to target aryl hydrocarbon receptor (AHR) which may promote neuroblastoma cell differentiation. We validate that miR-124 may suppress the expression of AHR by targeting its 3'-UTR. These results suggest that miR-124 could serve as a potential therapeutic target of neuroblastoma.     

Biochemical and electrophysiological differentiation profile of a human neuroblastoma (IMR-32) cell line

A human neuroblastoma cell line (IMR-32), when differentiated, mimics large projections of the human cerebral cortex and under certain tissue culture conditions, forms intracellular fibrillary material, commonly observed in brains of patients affected with Alzheimer's disease. Our purpose is to use differentiated IMR-32 cells as an in vitro system for magnetic field exposure studies. We have previously studied in vitro differentiation of murine neuroblastoma (N1E-115) cells with respect to resting membrane potential development. The purpose of this study was to extend our investigation to IMR-32 cells. Electrophysiological (resting membrane potential, V(m)) and biochemical (neuron-specific enolase activity [NSE]) measurements were taken every 2 d for a period of 16 d. A voltage-sensitive oxonol dye together with flow cytometry was used to measure relative changes in V(m). To rule out any effect due to mechanical cell detachment, V(m) was indirectly measured by using a slow potentiometric dye (tetramethylrhodamine methyl ester) together with confocal digital imaging microscopy. Neuron-specific enolase activity was measured by following the production of phosphoenolpyruvate from 2-phospho-d-glycerate at 240 nm. Our results indicate that in IMR-32, in vitro differentiation as characterized by an increase in NSE activity is not accompanied by resting membrane potential development. This finding suggests that pathways for morphological-biochemical and electrophysiological differentiations in IMR-32 cells are independent of one another.     

Hydroxyl free radicals induce cell differentiation in SK-N-MC neuroblastoma cells

The SK-N-MC cell line is frequently used as a model of neuronal differentiation induced by 5-bromodeoxyuridine (BrdU). In this study, the differentiation properties of this cell line were investigated under hydroxyl free radical generation, and compared to BrdU treatment. Hydroxyl free radicals were generated in the cultures by the Fenton reaction, i.e. by simultaneous addition of ADP-Fe2+ complex and H2O2. Microscopic morphological signs, as well as the acetylcholinesterase and ganglioside sialidase activities were considered as markers of neuronal differentiation of this cholinergic neuroblastoma cell line. Apart from the altered morphological appearance, the marker enzymes displayed significant increases after both types of intervention. We suggest that hydroxyl free radicals can induce in vitro cell differentiation. They apparently play a more complex role in cell physiology than simply causing oxidative damage.