Neuroblastoma Growth Factors Derived from Neurofibroma (NF1): Participation of Uridine in a Neuroblastoma Growth

Abstract: Human glioma cell extracts were found to elicit a marked growth-promoting activity on human neuroblastoma cells. This activity was also detected in the extracts of neurofibroma type 1 (NF1; von Recklinghausen neurofibromatosis) comprising aberrant Schwann cell growth. The purified substance from the NF1 extracts by HPLC on ODS columns was identical to a pyrimidine nucleoside, uridine, the chemical structure of which was identified by gas chromatography-mass spectrometry. The authentic uridine showed a strong growth-promoting activity on human neuroblastoma cells. Other purine or pyrimidine nucleotides, their derivatives, and ribose sources for their syntheses were employed to test the activity; a purine nucleoside, adenosine, showed a stronger activity than uridine. The current study raises the possibility that human neuroblastoma cells may be affected by dysfunctions of the de novo pathway of both purine and pyrimidine nucleotide biosyntheses.     

In vitro cytodifferentiation of perinatal rat islet cells within a tridimensional matrix of collagen

Summary Cell suspensions prepared by collagenase digestion of pancreases obtained from rat fetuses (21.5 d old) and newborns (2.5 d old) were mixed with a collagen solution and inoculated on a collagen base layer. At the onset of the culture, most acinar cells became necrotic, whereas other epithelial cells proliferated. Most of the cell clusters arranged themselves into simple polarozed structures composed of epithelial cells forming hollow spheres, and from these budded neoformed endocrine islets. Scarce fibroblasts were located close to these structures. Immunocytochemical localization of insulin and glucagon, as well as ultrastructural characteristics of the cell types revealed an intrainsular distribution similar to the in vivo localization. Tridimensional matrix of collagen offers, to perinatal pancreatic cells in culture, an environment close to the in vivo conditions: cells reorganize themselves in tissuelike structures and cell interactions concerned in the cytodifferentiation of pancreatic islets occur. This system allows for the study of undifferentiated epithelial cells—the presumed stem cells—differentiating and differentiated endocrine cells in the same preparation. B.A. is supported by a doctoral scholarship from the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture, Brussels. This work was supported by grants from the Fonds National de la Recherche Scientifique, Brussels, and from Petrofina S.A., Brussels.     

Established cell lines of mouse marrow adherent cells producing differentiation factor(s) for the granulocyte-macrophage lineage

Summary Two continuous cell lines derived from long-term cultures of AKR mouse bone marrow adherent cells were isolated. These cell lines release colony stimulating activity (CSA), a factor that induces in vitro differentiation of granulocyte-macrophage progenitor cells. The colony forming cells and cluster forming cells in mouse marrow responsive to CSA from cell line conditioned medium were compared with those responsive to CSA from mouse lung conditioned medium (MLCM). Colony forming cells were characterized by analysis of their density distribution after equilibrium centrifugation in density gradient. Cluster forming cells were characterized by analyzing the progeny of individual clusters after transfer to fresh semisolid culture medium containing MLCM. The results obtained indicate that the CSA from cell line conditioned medium closely compares with the CSA from MLCM in terms of the populations of colony and cluster forming cells stimulated. This work was supported by a research grant from the Institut National de la Santé et de la Recherche Médicale (CRL 802620), Paris, France.     

Differentiating cells of chick embryo hemispheres: Characterization and quantitative evaluation of the cell types and bulk preparation of enriched fractions

Summary A method of fractionation of neuroblasts and spongioblasts perikarya from developing chick embryo cerebral hemispheres is described. The tissue, treated by a mixture of acetone-glycerol-water is dissociated through a nylon sieve. Differential centrifugation on sucrose 1.3 M and 1.8 M has been used to obtain fractions enriched in neuroblasts (80% purity) and spongioblasts (90% purity). The duration of the whole operation is two hours.The evolution and distribution of the cells was studied on dissociated tissue from the 6th day of embryonic life to hatching. The proportion of neuroblasts varies from 13% at 6 days to 36% at 12 days. At the same period the proportion of the sum of indifferent cells and spongioblasts decreases from 81 to 58%, whereas the ependymal cells show little variation (6 to 9%). At hatching these proportions have not changed greatly: neuroblasts, spongioblasts and ependymal cells represent respectively 33, 65 and 2% of the total cells.The cellular differentiation proceeds from the 6th day to hatching. The size of neuroblasts increases and many of them become multipolar. The number of spindle-like cells and small indifferent cells whose origin is probably the germinal layer, decreases together with the thickness of this layer. Early spongioblasts appear only at 10 days and, later on, differentiate into oligodendroblasts and astroblasts.This study is a part of the Doctorat ès-Sciences thesis of C. Judes (Attaché de Recherche au C. N. R. S.).Chargée de Recherche au C. N. R. S.Maître de Recherche au C. N. R. S.     

Effect of sodium butyrate on the hepatoma cell cycle: Possible use for cell synchronization

Summary Exposure of HTC cells to sodium butyrate caused inhibition of growth. The site of growth inhibition was studied by time-lapse cinematography and [³H]thymidine incorporation studies. Evidence is presented that sodiunm butyrate affected the cell cycle at a specific point immediately after mitosis. Inasmuch as it does not modify the interphase duration after its removal, butyrate may be used for HTC synchronization. This work was supported by l'Institut Nationale de la Santé et de la Recherche Médicale and la Centre Nationale de la Recherche Scientifique (L. T. and J. K.).     

Growth of preadipocyte cell lines and cell strains from rodents in serum-free hormone-supplemented medium

Summary Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described. This work was supported by the “Centre National de la Recherche Scientifique” (Grant 1208-Biochimie du Développement and Grant 4162-Endocrinologie), by the “Ministère de la Recherce et de la Technologie” (Grant 81-L-1322), by the “Fondation pour la Recherche Médicale,” by NATO (Grant 1704), and by the “Institut National de la Santé et de la Recherche Médicale” (Grant 827006).     

Structural basis for restriction-site polymorphism at the albumin locus in inbred strains of rats

Two types of variant EcoRI restriction enzyme patterns of albumin-gene DNA fragments are found in different inbred strains of rats and reflect allelic polymorphism. The structural basis of the two allelic forms has been analyzed by mapping the EcoRI fragments using cloned albumin cDNA probes corresponding to the 5 or 3 end of the rat albumin mRNA and different genomic subclones. Additional restriction fragment length polymorphism has been detected using the restriction endonucleases HindIII and MspI. The results suggest that the two allelic variants differ from each other by multiple cleavage-site variations (base-pair substitutions) and by an insertion or deletion of DNA sequences. An extensive DNA sequence variation appears to exist between the two forms of the albumin gene; we have estimated that as much as 4% of the nucleotides in this region varied between the two alleles. All of this genetic variation is found in the intervening sequences of the gene and has no phenotypic manifestation.This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (Contract 7940 222) and the Association pour le développement de la Recherche sur le Cancer (Contract 6109). A. Gal was supported by a fellowship from the Institut National de la Santé et de la Recherche Médicale.On leave of absence from Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.     

A brief history of genome research and bioinformatics in France

The development of in silico genomics has progressed slowly in France for a number of political reasons. Two administrative organizations, the Groupement de Recherche sur les Génomes (GREG) and the Groupement de Recherche 1029 (GDR 1029) of the Centre National de la Recherche Scientifique (CNRS) have been established. These organizations have created the dynamics that hopefully will place France (which coordinated consortia that completed several of the first large microbial genomes) among the developed nations that support Large-Scale Biology.     

Only Pyrimidinoceptors Are Functionally Expressed in Mouse Neuroblastoma Cell Lines

The ability of UTP, UDP, ATP, and ADP to influence inositol phospholipid hydrolysis in neuroblastoma origin cell lines was assessed. The mouse neuroblastoma lines N1E 115, Neuro 2a, and NB4 1A3 and the rat glioma/mouse neuroblastoma hybrid line NG108-15 gave robust responses to both UTP and UDP, which were essentially equipotent. Thus a range of cell lines of mouse neuroblastoma origin express a pyrimidine-selective P2Y receptor. The NG108-15 cells were the only cell type tested at which ATP and ADP displayed activity with EC₅₀ values of greater than 100 μM, compared with values of 0.58 and 1.25 μM for UTP and UDP, respectively. In contrast to the cell lines derived from mouse neuroblastoma, the human neuroblastoma lines SH-SY5Y and SK-N-SH did not respond to any nucleotides, although both responded well to carbachol.     

Organ culture of suckling rat intestine: Comparative study of various hormones on brush border enzymes

Summary Jejunal mucosa of 6 d-old rats were cultured for 24 and 48 h in the presence of thyroxine, insulin, pentagastrin, glucagon, epidermal growth factor (EGF) or dibutyryl-A-3:5-MP cyclic with or without dexamethasone (DX). The enzymes were assayed on the purified brush borders. The various agents added alone to the basic culture medium had no effect with the exception of DX on the levels of enzyme activities. Dexamethasone alone induced sucrase, stimulated maltase, and protected other brush border enzyme activities (aminopeptidase, lactase, and alkaline phosphatase). When added to DX-supplemented medium, only the following factors modified the levels of enzymatic activities observed with DX alone. Insulin (10⁻⁶ M) increased maltase, alkaline phosphatase, and lactase activity to a greater extent than DX at 24 h culture, the effect being maintained at 48 h on alkaline phosphatase only. At 48 h culture, both EGF (10⁻⁸ M) and dbcAMP (10⁻³ M) decreased DX-induced sucrase activity. The latter agent also depressed DX-stimulated aminopeptidase activity. This work was supported by the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and a grant 79.7.1243 from the Délégation Générale a la Recherche Scientifique et Technique. P. M. S. is a recipient of a grant from Fondation de la Recherche Médicale (France).     

Enhancement of T suppressor activity in mice by high doses of BCG

Summary The spleen cells from mice injected 2 weeks previously with high doses of BCG have a lower reactivity as shown when they are engaged in a graft versus host reaction or in a mixed lymphocyte reaction. This suppression is an active and nonspecific phenomenon, at least partly attributable to the T-cell population.This work was supported by CRAMP and Action urgente DGRST 74-7-11-85, by CRL 74.5.015.01 from the Institut National de la Santé et de la Recherche médicale.     

Erythroid precursors cultured from adult mice are sensitive to H2O2 toxicity

Summary The growth of late erythroid precursors (CFU-Es) from adult bone marrow is inhibited when Iscove's modified Dulbecco's medium supplied in liquid form is used. Catalase and other H₂O₂ destroying compounds restore the capacity of culture medium to support colony development. However early precursors from adult bone marrow and fetal liver CFU-Es were resistant to H₂O₂. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale and the Fédération Nationale des Centres de Lutte contre le Cancer.     

Organotypic culture, a powerful model for studying rat and mouse fetal testis development

The key role of the fetal testis in the masculinization of genital organs has been known for a long time. More recently, the observed increases in male reproductive disorders has been postulated to be the result of changes in fetal and neonatal testis development in response to increasing environmental pollution. However, few tools are available for studying fetal testis development and the effects of physiological or toxic substances. We have developed an organ culture system in which rat fetal testis is grown on a filter floating on a synthetic medium containing no serum, hormones or biological factors. In this study, we have compared the long-term morpho-functional development of the various testicular cell types in this system with that observed in vivo and have extended this system to the mouse. Rat Leydig, Sertoli and germ cells and macrophages develop normally over a period of 1–2 weeks in this system. Fewer cells are produced than in vivo but the level of differentiated function is similar. Germ cells, which are difficult to culture in vitro, resume mitosis after a quiescent period, at the same time as in vivo. Similar results have been obtained with mouse fetuses, except that Leydig cells dedifferentiate in vitro if the testis is explanted after 13.5 days post conception. Testicular architecture and intercellular communication are sufficiently preserved for the development of the main fetal and neonatal testicular cell types in vitro with no added factors. Our floating-filter organotypic culture system in synthetic medium therefore allows the morpho-functional development of somatic and germ cells in fetal testis explants taken at all developmental stages in rat and at early stages in mouse. This method is potentially useful for studies of the effects of various factors, and of xenobiotics, in particular.This work was supported by the Université Paris 7, Commissariat à l’Energie Atomique (CEA) and the Institut National de la Santé et de la Recherche Médicale (INSERM) and by grant Agence Francaise de Sécurité Sanitaire et Environnementale (AFSSE). G. Delbes is the recipient of a fellowship from the Ministère de la Recherche et de la Technologie and from the Association pour la Recherche contre le Cancer.This work is dedicated to the memory of José Maria Saez in recognition of his helpful and constant encouragement and discussions over the last decade.     

Regulation of cyclic GMP levels by neurotensin in neuroblastoma clone N1E115

The binding of 125I-labeled [monoiodo-Tyr3]neurotensin to intact neuroblastoma N1E115 cells and the effect of neurotensin on the intracellular concentration of cyclic nucleotides were studied at 37 degrees C and under physiological conditions of pH and ionic strength. The radiolabeled neurotensin analogue bound specifically to differentiated cells with a dissociation constant of 0.75 nM and a maximal binding capacity of 45 fmol/10(6) cells. Incubation of neuroblastoma cells with neurotensin in the presence of calcium ions resulted in a transient increase of 10 fold over basal level of the intracellular cyclic GMP concentration. Half-maximal stimulation was obtained with 2 nM neurotensin. Under identical conditions the cyclic AMP concentration only decreased by 20-30%. These results suggest that cyclic GMP is a second messenger of neurotensin in neuroblastoma clone N1E115.     

Correlative immunocytochemical and electron microscopic studies: Identification of (Entero)glucagon-somatostatin- and pancreatic polypeptide-like-containing cells in the human colon

Summary Correlative immunocytochemical and electron microscopic studies, using the semi thin-thin technic, were performed to identify the (entero) glucagon, somatostatin and pancreatic polypeptide-like immunoreactive cells of the human colonic mucosa. Mean granule diameter for each cell type was estimated according to two methods and histograms showing the granule size distribution were constructed. A total of 139 immunostained cells identified at the ultrastructural level were analyzed. Mean granule diameter for (entero)glucagon-containing cells was 318±11 nm but a reduction of granule size with age was noteworthy: granules were larger in the fetus (mean diameter 350±15) than in adults (mean diameter 310±10 nm). Somatostatin-containing cells, very rare in adults, were present in the fetal distal colon. Their general mean granule diameter was 354±18 nm but many cells had a mean granule diameter of more than 400 nm. A pancreatic polypeptidelike immunoreactivity was found only in (entero)glucagon-containing cells, pointing out the possible occurrence of both peptides (or of similar sequences) in the same cells. Previous ultrastructural studies dealing with a tentative classification of the human colonic endocrine cells were compared with the present data.This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM).     

Changes in proteoglycans of cultured pig aortic smooth muscle cells during subculture

Summary Smooth muscle cells were cultured from pig aorta. Changes in both the growth and the properties of sulfated proteoglycans were observed during passage. The population doubling time during log phase growth was 34 h from Passages 3 to 7–8 but 20 h at the Passage 11, and the cell density at the stationary phase, was 86 000 and 136 000 cells/cm² at Passages 3 and 11, respectively. Structural characteristics of sulfated proteoglycans secreted into the medium were investigated after metabolic labeling with [³⁵S]-sulfate. Significant differences were observed with age in vitro: a) [³⁵S]proteoglycan complexes were in a greater amount at Passage 10 than at Passage 3; b) the hydrodynamic size of at least 45% of subunits and about 90% of monomers decreased with in vitro aging; c) this decrease in the size of proteoglycans was partly due to a decrease in the size of their glycanic chains; d) an increase of 15% in the proportion of dermatan sulfate was observed when cells were subjected to 10 passages. This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM, U. 181) and the Fondation pour la Recherche Médicale.     

Laminin provides a better substrate than fibronectin for attachment,growth, and differentiation of 1003 embryonal carcinoma cells

Summary Culture of cells in hormonally defined media has allowed (a) the demonstration of physiological responses from cells usually unable to express them in vitro and (b) the study of the effects on growth and differentiation of diffusible factors and attachment factors. The embryonal carcinoma line 1003 forms multidifferentiated tumors in vivo but is unable to differentiate in vitro when grown in serum-containing medium. In a defined medium containing insulin, transferrin, selenium, and fibronectin as attachment factors, 1003 cells grow for several generations and differentiate into neurons and embryonic mesenchyme (Darmon et al., 1981, Dev. Biol. 85: 463–473). In the present work the effects of fibronectin and laminin were compared. In the presence of laminin the cells attached and spread better, grew faster, and could be plated at lower densities. Neurite extension was also better under these conditions and most importantly, it was found that laminin induced an important formation of muscular tissue when the cells had been seeded at low densities. Multinucleated myotubes could be stained with antibodies directed against embryonic muscular myosin. Coating the dishes with polylysine or adding FGF or serum-spreading factor to the medium allowed growth of low-density cultures with fibronectin instead of laminin but muscular differentiation was not detected under these conditions. Addition of fibronectin to laminin-containing medium did not inhibit muscular differentiation. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This research was supported in part by grants from the “Centre National de la Recherche Scientifique” (LA 269), the “Délégation Générale à la Recherche Scientifique et Technique,” the Fondation pour la Recherche Médicale Fran?aise,” the “Institut National de la Santé et de la Recherche Medicale,” the “Ligue Nationale Fran?aise centre le Cancer,” and the “Fondation André Meyer.” This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.     

Phosphofructokinase (PFK) isozymes in man

Summary Isozymic heterogeneity of human phosphofructokinase was investigated by means of ATP inhibition, immunoneutralization by antihuman muscle-type and antiliver-type phosphofructokinase antisera, solubility in (NH₄)₂SO₄ solutions, and starch gel and polyacrylamide slab gel electrophoresis. The enzymes studied by these methods were purified from various normal and malignant human adult tissues by chromatography on blue Dextran Sepharose 4 B columns. From the results of these studies we suggest that three basic phosphofructokinase isozymes could exist: muscle-type, fibroblast-type, and liver-type isozymes.Muscle-type isozyme is the single form found in adult muscle, and is involved in the enzymes from heart, brain, red cell, and testis.Fibroblast-type isozyme is found mainly in the placenta, fibroblasts, kidney, and some malignant tissues.Liver-type phosphofructokinase seems to be very definitely the predominant form in mature polymorphonuclear cells, platelets, and liver.Testis and red cell phosphofructokinase enzymes definitely include muscle-type and liver-type subunits, associated in various hybrid forms.With the technical assistance of R. KernempUnité 129 de l'Institut National de la Santé et de la Recherche Médicale, laboratoire associé 85 au Centre National de la Recherche Scientifique     

Parasitology in France: some aspects of the present

Numerous organizations participate and cooperate on parasitological research in France including the Institut national de la Santé et de la Recherche Médicale (INSERM), the Centre national de la Recherche Scientifique (CNRS), the Institut Pasteur, the Institut Fran?ais de Recherche Scientifique pour le Développement en Coopération (ORSTOMM), the Institut national de la Recherche Agronomique (INRA), the Muséum national d'Histoire naturelle (MNHN), the Universities, the Collège de France, the Ecole Pratique des Hautes Etudes (EPHE) as well as various commercial firms. Exchanges and collaborations with foreign workers are continuous and essential to the success of research on tropical diseases. Here, in their own words, Odile Bain, Daniel Camus and Jacques Prod'hon highlight some aspects of current parasitological research in France.