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Aims In this study, we evaluated the ability of differentiating embryonic chick DRG neurons to release and respond to acetylcholine (ACh). In particular, we investigated the neuronal soma and neurites as sites of ACh release, as well as the mechanism(s) underlying this release. Main methods ACh release from DRG explants in the Campenot chambers was measured by a chemiluminescent assay. Real-time PCR analysis was used to evaluate the expression of ChAT, VAChT, mediatophore and muscarinic receptor subtypes in DRGs at different developmental stages. Key findings We found that ACh is released both within the central and lateral compartments of the Campenot chambers, indicating that ACh might be released from both the neuronal soma and fibers. Moreover, we observed that the expression of the ChAT and mediatophore increases during sensory neuron differentiation and during the post-hatching period, whereas VAChT expression decreases throughout development. Lastly, the kinetics of the m2 and m3 transcripts appeared to change differentially compared to the m4 transcript during the same developmental period. Significance The data obtained demonstrate that the DRG sensory neurons are able to release ACh and to respond to ACh stimulation. ACh is released both by the soma and neurite compartments. The contribution of the mediatophore to ACh release appears to be more significant than that of VAChT, suggesting that the non-vesicular release of ACh might represent the preferential mechanism of ACh release in DRG neurons and possibly in non-cholinergic systems.

The effects of cyclic nucleotide phosphodiesterase (PDE) inhibitors on cell proliferation of SK-N-MC human neuroepithelioma cell line was studied. Clonal density experiments in the presence of 100 microM rolipram and zaprinast showed respectively 27% and 91% inhibition. The effects of PDE inhibitors were then investigated on crude cell extracts; the calculated IC50 were 32 microM for zaprinast and 16 nM for DC-TA-46; the latter inhibitor was used instead of rolipram for its higher efficacy. Dose-response experiments in clonal density conditions showed IC50 of 5 microM and 1.8 microM in the presence respectively of zaprinast and rolipram. These data show that both inhibitors are effective in reducing cell growth, although the response was quantitatively different.

Neuroblastoma cultures provide an experimental model for the study of neuronal differentiation. Modifications of the culture conditions, in fact, induce the immature neuroblast to form fibres and under appropriate conditions to form synapses with muscle fibres. This system can provide clues to the understanding of the molecular basis of cellular differentiation.

The possible occurrence of genetic damage arising from exposure of human cells to visible laser light has been evaluated in PHA-stimulated human lymphocytes. Aneuploidy and chromosome aberations have been observed after exposure to an argon laser. These findings appear of special interest in view of the possible role of these chromosome alterations in carcinogenesis.

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.

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). 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-β-D-Arabinofuranoside (ARA-C). We also tested oncogene expression in CHP-100 cells, and 5-AZA-CdR does not affect the myc and fos levels under the reported experimental conditions. © 1989 Humana Press Inc.

Ribosomal RNA synthesis and processing was investigated in monolayer and suspension cultures of neuroblastoma cells. After uridine labelling, the specific activity of UTP and the initial rate of total RNA synthesis were the same in suspension and monolayer cells. The rate of synthesis of 45S preribosomal RNA, measured by methionine labelling, was also the same in the two cell types. Similarly, ribosomal RNA was found to decay with similar rates in the two conditions of growth. On the other hand, kinetic studies of cytoplasmic labelling showed a much lower rate of ribosomal RNA appearance in the cytoplasm of suspension cells.

Separation of nucleic acid derivatives by thin-layer electrophoresis on a microcrystalline cellulose, Avicel, is reported. The present paper describes: 1. 1. Separation of the four 2′, 3′-ribonucleotides from an RNA alkaline hydrolysate. 2. 2. Separation of the four deoxynucleotides and the four deoxynucleosides. 3. 3. Separation of deoxynucleoside mono-, di- and triphosphates. 4. 4. Separation of ten dinucleotides from a deoxyribonuclease digest. 5. 5. Two-dimensional separation of 13 bases occurring in nucleic acids.

14-3-2, a nervous system specific protein, has been studied in mouse neuroblastoma cells grown in culture by means of the microcomplement fixation assay. The experiments reported indicate that 14-3-2 protein is rapidly accumulated during the early phase of cell growth.

Publisher Summary This chapter discusses the changes occurring during neuronal maturation in neuroblastoma (NB) and dorsal root ganglia (DRG) in culture. The general properties and advantages of the NB system are reviewed. One may envisage that the sequence of events in the course of neuronal maturation would be an external change in the environment produces a change in the cell surface architecture, resulting in the activation of adenylate cyclase and, therefore, in an increase in the intracellular cAMP level. This increase in cAMP induces global physiological changes, including the induction of neurospecific enzymes. During development, examples of changes in the surface carbohydrate-containing molecules are numerous, and glycocomponents have been implicated in many processes including specific cell interaction and recognition. NB cultures have been widely used as a model system in approaching a number of problems related to developmental neurobiology. The possibility of culturing undifferentiated and differentiated cells has allowed the study of various molecular events occurring during differentiation. Differentiated NB cells are often characterized by neurites, a higher RNA and protein content, and a higher activity of neurospecific enzymes. All these molecular events are regulated at a posttranscriptional level, but the mechanisms have not been clarified.

Protein synthesis in reuroblastoma cells has been studied in a cell-free system. The activity of lysates from cells grown insuspension and monolayer has been compared. A higher level of activity has been found in monolayer cells. The activity of some components of the lysate that are involved in protein synthesis has been analyzed.The data suggest that the controlling step of protein synthesis in this system might be in the initiation process. The correlation between activation of protein synthesis and neurite outgrowth in monolayer cultures is discussed.

Changes in carbohydrate composition of the cell surface related to neuronal maturation have been studied on neuroblastoma and embryonic dorsal root ganglia (DRG) cultures by using fluorescein conjugated lectins. In neuroblastoma cells, it has been found that the surface of the fibers differs from that of the cell body as shown by concanavalin A (Con A) and WGA binding. In primary cultures of embryonic DRG, lectin binding has also shown that the neuron surface undergoes changes during maturation. In fact, lectin binding which is absent at early stages (5--6 day old embryos) becomes first detectable at the 7th day and then increases progressively. At day 7, the Con A binding pattern resembles that observed in neuroblastoma cells. The possibility of correlating these surface changes with cell adhesive properties and cell differentiation is discussed.

Synthesis of nuclear HnRNA in monolayer and suspension culture of neuroblastoma cells has been studied by uridine-labelling after inhibition of ribosomal RNA synthesis by low doses of Actinomycin D. Although the initial rate of nuclear HnRNA synthesis did not change in the two culture conditions, accumulation of HnRNA was observed in monolayer cells after long labelling times. The rate of cytoplasmic labelling in the presence of low-level of Actinomycin D was the same in suspension and monolayer cultures.

Two mouse neuroblastoma cell lines were analyzed for their permissivity for polyoma virus growth. One (N18) is fully permissive for polyoma replication, the other (41A3) shows limited permissivity and the viral genome persists, without noticeable cell death. Virus persistence does not seem to alter the cells' ability to differentiate in vitro and leads to selection of viral mutants altered in the untranscribed regulatory region of the genome. The mutant types obtained appear to be related to the degree of host cell differentiation. Nucleotide sequence analysis of the restriction fragment covering the regulatory region shows that duplications are present in all mutants, while deletions in the non-duplicated segment are only present in mutants selected from less differentiated cells. These alterations involve both domains of the regulatory region that are considered to be essential for DNA replication and for enhancer activity. Mixed infections with polyoma wild type show that the selected mutants have cis-advantage in replication in neuroblastoma cells and not in 3T6 cells. Mutants carrying the deletion in the non-duplicated segment of the enhancer show a selective advantage in replication over the undeleted one in mixed infection. This advantage is much stronger in neuroblastoma cells in suspension (less-differentiated stage) than in monolayer cells (more-differentiated stage). An interpretation of the overall structure of the regulatory enhancer region, based on the observed differences between the mutants selected at different stages of differentiation in neuroblastoma and previously described mutants selected in undifferentiated cells, is discussed.

Study of the molecular mechanism of differentiation has been hampered by, among other difficulties, the complexity of the systems investigated, where several changes occur at the same time and overlap each other. The possibility of selecting a homogeneous cellular population in which only a limited step of cellular maturation occurs may provide a useful system for studying cell differentiation.

The morphological alterations and the effects on the metabolism of DNA, RNA, protein, and sulfate, caused by amantadine on developing sea urchin embryos have been investigated. Amantadine causes formation of exogastrulae. This phenomenon might be related to alterations of the morphogenetic movements occurring at the late blastula stage and which lead to the invagination of the endoderm. The incorporation of dTR, uracil, amino acids, and 35S-sulfate by the developing embryos is inhibited by amantadine. The uptake of amino acids and 35S-sulfate is also inhibited. It is suggested that the main effect of amantadine on the sea urchin embryos is an alteration of the cellular membranes.

Publisher Summary This chapter discusses a cell culture approach to the study of the site of action of hormones and a cell culture system for studying differentiation. The procedures are designed to obviate the selective advantages of fibroblasts in culture and owe much to enrichment culture techniques, which have been successful in microbiology. With these methods, several cell lines are developed, which are clonally derived, permanently established in culture, and persistent in performing the functions of the tissue of origin. These strains include ACTH responsive, steroid-secreting adrenal cortex cells, ACTH-secreting pituitary cells, growth hormone-secreting pituitary cells, cyclic AMP-responsive and steroid-secreting mouse Leydig cells, hormone-secreting rat Leydig cells, glucocorticoid-responsive rat glial cells, neuroblastoma cells capable of synthesizing epinephrine and acetylcholine, and teratoma cells that maintain their multipotent capacity for differentiation. These cell cultures are hormone-secreting, hormone-responsive, or potentially hormone-responsive systems.