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.).     

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).     

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.     

Malignant transformation of human keratinocytes during adaptation to autotrophy

The work was supported by grants from the Association pour la Recherche sur le Cancer and from the Fondation pour la Recherche Médicale.     

Comparative use of fructose and glucose in human liver and fibroblastic cell cultures

Summary The effect of fructose as a substitute for glucose in cell culture media was investigated in human skin fibroblast and liver cell cultures. Cells were grown for between 2 and 10 days in identical flasks in four different media, containing 5.5, mmol·1⁻¹ and 27.5 mmol·I⁻¹ glucose and fructose, respectively. In the presence of fructose, cell growth was stimulated, but less in liver cells than fibroblasts. At Day 6, increases were observed in [³H]thymidine incorporation, protein levels, and amino acid consumption, and a reduction was noted in ATP levels. In media containing 5.5, mmol·1⁻¹ glucose or fructose, consumption of fructose was four times lower than that of glucose at Day 3 and did not rise until Day 6. In fructose media, the lactate production was very low (four to five times less than that of glucose) and the pH values were always higher. Some findings were different for the fibroblasts and liver cells, owing to the specific characteristics of these two cell types in culture; this applied especially to the effects of glucose and fructose concentrations of 27.5 mmol·1⁻¹. Several possible explanation for the stimulation of cell growth in fructose medium were discussed. This work was supported by grants for the Institut National de la Santé et de la Recherche Médicale (ATP 82-79-114) and the Unité d'Enseignement et de Recherche, Le Kremlin-Bicêtre, Université Paris-Sud (C. R. 848).     

A method for separating cultured preadipocytes according to their density: Application to stromal cells from overfed suckling rats

Summary The stroma vascular fraction of adipose tissue consists of a heterogeneous cell population; not all the cells in this compartment undergo adipose conversion in primary culture. A density gradient centrifugation procedure was used to separate cultured cells on the basis of their triglyceride content. This method was applied to both stroma vascular cells from rat adipose tissue and to a 3T3 F442A preadipose cell line as a reference. Comparison of the results obtained from these two cell types suggests that this separation procedure can lead to a quantification of adipose differentiation in the heterogeneous stroma cell population. Separation procedures were applied to cultured stromal cells derived from young rats during the onset of nutritional obesity induced by overfeeding in early life. Results show that early overfeeding induced an increase in the stromal cell differentiation capacity which is expressed in vitro. This work was supported in part by Institut National de la Santé et de la Recherche Médicale (CRL n^o 82-70-22).     

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).     

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.     

Discussing epigenetics in Southern California: A report from the International Symposium on Epigenetic Control and Cellular Plasticity,UCI, December 15–16, 2011

With the goal of discussing how epigenetic control and chromatin remodeling contribute to the various processes that lead to cellular plasticity and disease, this symposium marks the collaboration between the Institut National de la Santé et de la Recherche Médicale (INSERM) in France and the University of California, Irvine (UCI). Organized by Paolo Sassone-Corsi (UCI) and held at the Beckman Center of the National Academy of Sciences at the UCI campus December 15–16, 2011, this was the first of a series of international conferences on epigenetics dedicated to the scientific community in Southern California. The meeting also served as the official kick off for the newly formed Center for Epigenetics and Metabolism at the School of Medicine, UCI (http://cem.igb.uci.edu).     

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.     

Precocious induction of arginase in primary cultures of fetal rat hepatocytes

Summary Fetal rat hepatocytes were isolated and cultured in primary culture to investigate activity changes of arginase under defined conditions. In hormone-free medium, cultured cells maintained the enzyme activity at levels equal to that of freshly isolated cells for at least 4 d. Arginase activity could be induced by dexamethasone in hepatocytes isolated from 16.5-d-old fetuses although cells were competent to respond to glucagon only at the stage of 18.5 d. The combination of the two hormones induced greater levels of arginase activity than the individual compounds. These findings indicate that glucocorticoid and glucagon receptors appear early and sequentially before birth and reveal that cultured fetal hepatocytes provide a suitable system for the investigation of the role of hormones in the initiation of enzyme synthesis. This work was supported by the Institut National Scientifique et de la Recherche Médicale through Grant 85.80.117.     

Characterization of a new cell line,XL2, obtained fromXenopus laevis and determination of optimal culture conditions

Summary A new amphibian permanent cell line is described. It is called XL2 and was initiated from Stage 35 tadpoles ofXenopus laevis. The cell line has an epithelioid morphology and most cells can be classified into two populations with respective chromosome modal numbers 36 and 74. Contact inhibition is low. Its growth is vigorous in L15 or MEM medium supplemented with fetal bovine serum. The mean doubling time is 39 hr and the saturation density is 700,000 cells/cm² at 25°C. The absolute plating efficiency is about 70%. Cell line XL2 is unable to grow in L15 medium containing a macromolecular fraction of fetal bovien serum. Growth is restored if the latter medium is supplemented with 10 μg/ml of hypoxanthine. Optimal conditions for the dye exclusion test, for harvesting the cells, and for cloning in petri dishes are described. This work was supported by Grant 3,4514,79 of the Fonds de la Recherche Scientifique Médicale (Belgium) and by a grant of the Fonds de Développment Scientifique of the Catholic University of Louvain. The authors are indebted to Mrs. M. S. Denis, Mr. F. Desneux, and Ms. J. Janssens for competent technical assistance. Smith Kline-RIT, Genval (Belgium), is acknowledged for the generous gift of antibiotics and for performing the cultures for mycoplasma detection.     

How to induce non-polarized cells of hepatic origin to express typical hepatocyte polarity: generation of new highly polarized cell models with developed and functional bile canaliculi

Few in vitro models expressing complex hepatocyte polarity are available. We used the unpolarized rat Fao cell line to isolate the polarized WIF-B line. These complex rat-human hybrid cells form functional simple bile canaliculi. To obtain Fao-derived polarized models with a simpler chromosome content and developed bile canaliculi, we employed two approaches. Partial success was achieved with monochromosomal hybrids. As shown by the immunolocalization of apical, basolateral, and tight-junctional proteins, monochromosomal hybrid 11-3 cells were polarized. They formed simple functional bile canaliculi and transiently expressed the typical polarity of simple epithelial cells. One subclone blocked in this polarity state was isolated. A more robust approach was provided by spheroid culture, a three-dimensional system that strengthens cell-cell contacts. Transient spheroid culture induced irreversible polarization of Fao cells. This induction occurred in most spheroids (approximately 1% of the cells). From populations enriched in stably polarized cells, we generated new polarized cell models, designated Can. Can 3-1 cells formed simple functional bile canaliculi when plated at high density. Regardless of plating density, Can 9 and Can 10 cells formed long tubular branched canaliculi competent for vectorial transport of organic anions and bile acids, and involving several dozen adjacent cells. Thus, we have generated new cell models stably expressing typical hepatocyte polarity. Among these models, Can 9 and Can 10 are the first capable of forming functional, highly developed bile canaliculi similar to those formed in vivo. This work was supported by grants from the Association pour la Recherche sur le Cancer (no.6551), the Institut Curie (PIC Signalisation Cellulaire, no. 914) and the Institut National de la Santé et de la Recherche Médicale (contract PRISME 98-09).     

Antagonistic effects of laminin and fibronectin in cell-to-cell and cell-to-matrix interactions in MCF-7 cultures

Summary During morphogenesis, tumor progression and metastasis, cell adhesion, dissociation, and migration result from a complex balance between cell-to-cell and cell-to-matrix interactions. Two different organization patterns of MCF-7 cells were induced by different extracellular matrix proteins. When plated on plastic or polymeric type I collagen gel used as a model of interstitial matrix, MCF-7 cells spread and grew in monolayer. When cultured on a solid gel of basement membrane (BM) proteins (85% laminin) used as a model of BM, cells formed clusters attached to the matrix. Matrix proteins regulated these two types of cell organization by preferentially promoting cell-to-cell or cell-support interactions. On plastic in the presence of soluble laminin or on laminin-coated dishes, cells also formed clusters. Addition of soluble fibronectin induced spreading of the cells, suggesting that laminin and fibronectin have competitive antagonistic effects on MCF-7 cell morphology. Antilaminin antibodies inhibited cluster formation and attachment, emphasizing the important role of this glycoprotein not only in promoting cluster attachment but also in cell-to-cell contact formation. Such effects of extracellular matrix proteins could play significant roles in tumor progression and metastasis. This work was supported by grants 3.4512.85 and 3.4514.85 from the Belgian Fonds de la Recherche Scientifique Médicale and the Fonds Cancérologique de la CGER.     

Ability of mammalian fibroblasts to grow in synthetic medium containing neither serum nor exogenously added macromolecules

Summary Rous sarcoma virus transformed Chinese hamster fibroblasts, clone CHR1-3, were established at high temperature, then subcloned. Six subclones with round and flat morphology harboring undeleted and partially deleted RSV proviruses, respectively, were seeded into serum-free synthetic medium with no macromolecular additives, and maintained for 2 mo. One flat subclone no. 14, fully designatedsfCHR1-3.14 for itsserum-free phenotype, was further propagated in the same medium. The cells grew exponentially in loosely attached monolayers and dould be serially passaged on bare polystyrene with an average population doubling time of 46 h. Cell attachment could be improved by using collagen-coated polystyrene or by adding a methionine supplement to the culture medium. Furthermore, thesfCHR1-3.14 cells could be subcloned and further grown in nonselective medium. The reversion rate of thesf phenotype was estimated to be 1 to 2%/cell generation. Evidence for an autocrinal stimulation was obtained by cloning efficiency assays showing a requirement for a threshold cell density. Slight growth stimulation could also be detected in assays using conditioned medium fromsfCHR1-3.14 cells and serum-restrictedwild-type (wt)NIH3T3, but notwtCHR1-3.14, cells as indicator cells. Finally,wtNIH3T3 cells used in these assays were assayed for serum-free growth and found to be able to develop their ownsf phenotype; in this respect they resemble the previously establishedsfCHR1-3.14 cells. Supported by grants from CNRS, INSERM, contract 852012, Association pour la Recherche sur le Cancer, and Fondation pour la Recherche Médicale. V. K. was supported by the Association pour la Recherche sur le Cancer and by the Fédération Nationale des Centres de Lutte contre le Cancer.     

Zytophotometrische Bestimmung des Histon- und Gesamtproteingehalts von Zellen des lymphatischen Gewebes

Résumé L'étude au microscope électronique a révélé la présence de particules B dans le thymus de la souris NZB, adulte et saine. Les particules se forment dans un type de cellule thymique bien défini, la cellule réticulo-épithéliale à vacuoles et naissent par bourgeonnement des membranes plasmatiques limitant les vacuoles. L'élaboration de particules B dans lethymus d'animaux apparemment sains est un fait nouveau dont la signification n'est pas encore connue.

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Summary An electron microscopical study revealed the presence of virus-like particles in the thymus of adult healthy NZB-mice. The particles are found in the vacuoles of reticuloepithelial cells and are elaborated by a budding process from the membranes surrounding the vacuoles. The formation of B-particles in the thymus of apparently healthy animals is a so far unknown fact the significance of which remains to be determined.

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Avec l'aide du Centre National de la Recherche Scientifique et de l'Institut National de la Santé et de la Recherche Médicale, et la collaboration technique dévouée de M^lle J. Patry et de Mr. B. Fontaine.     

Modulation of sulfated glycosaminoglycan and small proteoglycan synthesis by the extracellular matrix

Cell culture in collagen lattice is known to be a more physiological model than monolayer for studying the regulation of extracellular matrix protein deposition. The synthesis of sulfated glycosaminoglycans (GAG) and dermatan sulfate (DS) proteoglycans by 3 cell strains were studied in confluent monolayers grown on plastic surface, in comparison to fully retracted collagen lattices. Cells were labelled with³⁵S-sulfate, followed by GAG and proteoglycan analysis by cellulose acetate and SDS-polyacrylamide gel electrophoresis, respectively. The 3 cell strains contracted the lattice in a similar way. In monolayer cultures, the major part of GAG was secreted into culture medium whereas in lattice cultures of dermal fibroblasts and osteosarcoma MG-63 cells but not fibrosarcoma HT-1080 cells, a higher proportion of GAGs, including dermatan sulfate, was retained within the lattices. Small DS proteoglycans, decorin and biglycan, were detected in fibroblasts and MG-63 cultures. They were preferentially trapped within the collagen gel. In retracted lattices, decorin had a higher M_r than in monolayer. Biglycan was detected in monolayer and lattice cultures of MG-63 cells but in lattice cultures only in the case of fibroblasts. In this last case, an up regulation of biglycan mRNA steady state level and down regulation of decorin mRNA was observed, in comparison to monolayers, indicating that collagen can modulate the phenotypical expression of small proteoglycan genes.Supported by a fellowship from the Centre National de la Recherche Scientifique     

Discussing epigenetics in Southern California: A report from the International Symposium on Epigenetic Control and Cellular Plasticity,UCI, December 15–16, 2011

With the goal of discussing how epigenetic control and chromatin remodeling contribute to the various processes that lead to cellular plasticity and disease, this symposium marks the collaboration between the Institut National de la Santé et de la Recherche Médicale (INSERM) in France and the University of California, Irvine (UCI). Organized by Paolo Sassone-Corsi (UCI) and held at the Beckman Center of the National Academy of Sciences at the UCI campus December 15–16, 2011, this was the first of a series of international conferences on epigenetics dedicated to the scientific community in Southern California. The meeting also served as the official kick off for the newly formed Center for Epigenetics and Metabolism at the School of Medicine, UCI (http://cem.igb.uci.edu).     

Groupe de prise en charge clinico-biologique en Assitance Médicale à la Procréation (AMP) des Patients à Risque Viral

La prise en charge en Assitance Médicale à la Procréation concerne de plus en plus de couples à risque viral. Cette prise en charge est aujourd’hui le seul moyen efficace de réduire une transmission viral mère-enfant ou entre partenaires séro-différents. Elle met en jeu une importante activité pluridisciplinaire où travail en commun et concertation entre les différentes équipes médicales clinico-biologiques sont les deux points clefs.     

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.