Gérard Géraud

Extracellular matrix-dependent differentiation of rabbit tracheal epithelial cells in primary culture

SummaryThe differentiation of tracheal epithelial cells in primary culture was investigated according to the nature of the extracellular matrix used. Cultures obtained by the explant technique were realized on a type I collagen substratum either as a thin, dried coating or as a thick, hydrated gel supplemented with culture medium and serum. These two types of substratum induced distinct cell morphology and cytokeratin expression in the explant derived cells. Where cells are less proliferating (from Day 7 to 10 of culture), differentiation was evaluated by morphologic ultrastructural observations, immunocytochemical detection of cytokeratins, and determination of cytokeratin pattern by biochemical analysis.The epithelium obtained on gel was multilayered, with small, round basal cells under large, flattened upper cells. The determination of the keratin pattern expressed by cells grown on gel revealed an expression of keratin 13, already considered as a specific marker of squamous metaplasia, that diminished with retinoic acid treatment. Present results demonstrated by confocal microscopy that K13-positive cells were large upper cells with a dense keratin network, whereas lower cells were positively stained with a specific monoclonal antibody to basal cells (KB37). Moreover, keratin neosynthesis analysis pointed out a higher expression of K6, a marker of hyperproliferation, on gel than on coating. All these data suggest a differentiation of rabbit tracheal epithelial cells grown on gel toward squamous metaplasia. By contrast, the epithelium observed on coating is nearly a monolayer of very large and spread out cells. No K13-positive cells were observed, but an increase in the synthesis of simple epithelium marker (K18) was detected. These two substrata, similar in composition and different in structure, induce separate differentiation and appear as good tools to explore the mechanisms of differentiation of epithelial tracheal cells.

Biological characterization of folic acid-conjugated poly(H2NPEGCA-co-HDCA) nanoparticles in cellular models.

The folate receptor (FR) is a highly selective tumor marker over expressed in many human cancers and it constitutes a useful target for tumor-specific drug delivery. Thus, the conjugation of folic acid to different drugs or drug carriers may enhance the delivery of the therapeutic agent to FR-positive tumor cells. The aim of this study was to investigate the interactions of folate-conjugated polyalkylcyanoacrylate nanoparticles with tumor cells overexpressing the FR. For this purpose, nanoparticles were prepared by nanoprecipitation of the poly[aminopoly(ethylene glycol) cyanoacrylate-co-hexadecyl cyanoacrylate] [poly(H2NPEGCA-co-HDCA)] copolymer and labeled with the hydrophobic fluorescent dye nile red. Nile red-loaded nanoparticles were then conjugated to folic acid via the PEG terminal amino groups. Four human cancer cell lines were then tested by western blot in order to evaluate the FR expression levels. KB3-1 cell line showed the higher expression level, while MCF-7 cells were taken as a control. After measuring the cytotoxicity of the nanoparticles on these two cell lines, fluorescent folate-nanoparticles were incubated with them and the cellular uptake was evaluated by confocal microscopy and flow cytometry. KB3-1 cells showed a greater nanoparticle internalization, when compared to MCF-7 cells.

Fibre‐type distribution and subcellular localisation of α and β enolase in mouse striated muscle

Enolase is a dimeric glycolytic enzyme exhibiting tissue specific isoforms. During ontogenesis, a transition occurs from the embryonic αα towards the specific αβ, and ββ isoforms in striated muscle. Immunocytochemical analyses on transverse sections of adult mouse gastrocnemius muscle, allowed us to compare the expression of α and β subunits to that of myosin heavy chain (MHC) isoforms. Levels of β immunoreactivity followed the order IIB > IIX > IIA > I. This gradient parallels the ATPase activity associated to MHC isoforms, indicating that the expression of β enolase in myofibres is finely regulated as a function of energetic requirements. By contrast, variations in α immunolabelling intensity appeared independent of fibre types.

Relative distribution of rDNA and proteins of the RNA polymerase I transcription machinery at chromosomal NORs

Using confocal and immunofluorescence microscopy the relative distribution of the ribosomal chromatin and some proteins of the RNA polymerase I transcription machinery such as upstream binding factor (UBF), RNA polymerase I and DNA topoisomerase I was analyzed on chromosomal nucleolus organize regions (NORs) of PtK1 cells. Staining with various DNA fluorochromes revealed that the ribosomal chromatin may be found at the axial region of the NOR and also at lateral expansions around the axis that can also be detected by in situ hybridization. It was observed that the transcription machinery shows a crescent-shaped distribution around the axial ribosomal chromatin at the NOR of metaphase and anaphase chromatids. An ultrastructural analysis of serially sectioned NORs supports this crescent-shape organization. Taking into account previous and present results and the loop/scaffold model of chromosome structure, we propose a model of NOR organization. The model proposes that ribosomal genes that were inactive in the preceding interphase would be present as condensed short Q-loops occupying the axial region of the NOR. Ribosomal genes previously active during interphase would be undercondensed as large R-loops associated with the transcription machinery, which is distributed in a crescent-shaped fashion around the previously active ribosomal DNA.

Revealing nucleolar architecture by low ionic strength treatment

The internal nucleolar architecture of HeLa cells was revealed after a short hypotonic treatment. The response of nucleoli to a gradual reduction in the ionic strength of incubation buffer was assessed by immunofluorescence, confocal microscopy using human autoimmune sera monospecific for antigens present in different nucleolar components, and electron microscopy. The granular component dispersed first, followed by the dense fibrillar component, leaving distinct fibrillar centers remaining. This demonstrates differential sensitivity to low ionic strength treatment in the transcription and the maturation territories of the nucleoli. The changes described develop in only a few minutes and this approach can reveal momentary in situ intranucleolar arrangements. We suggest that the fibrillar centers provide a structural support for RNA-polymerase I complexes and are possibly also attached to a nuclear skeleton. The evidence presented implicates the fibrillar centers as the core elements of nucleoli and that functional nucleoli arise around them.

CONFORMATION OF RIBOSOMAL GENES AND DISTRIBUTION OF THE TRANSCRIPTION FACTOR UBF ARE CELL CYCLE DEPENDENT

The amount of Ag-NOR proteins has a prugnostic value m human cancers. Ag-NOR proteins are a set of argyrophihc nucleolar proteins that accumulate in highly proliferating celis whereas their expression is very low in non-proliferating cells. To know the biological basis allowing the use of Ag-NOR protein expression as proliferation marker in human malignancies, tht) relationship between cell cycle and amaunt of Ag-NOR protein was analyzed. The quantification of the two major Ag-NOR proteins (Roussel P, Belenguer P, Amairic F ;G Hernandez. Verdun D (1992), Ely. CEI[ Res. 203: 259-269; Roussel P Pr Hernandez-Verdun D (19Y4) Erp. C?II iies 274: 465-4721, nucleolin and protein B23 was performed m exponentially growing, serum-deprived and cell-cycle stimulated cells. The quantification was performed on Western blots as already described (Sirri V, Roussel I’, TrerP D, Derenzini M Br HernandezVerdun D (1995) 1. Histochen~ Cytochrn~ 43: 887-893). Expression of nucleolin was low in serum-deprived cells dnd increased mostly in S phase during cell-cycle stimulation. Conversely, expression of protein B23 was slightly repressed in serurndeprived cells, and increased progressively until C2 phase during cell-cycle stimulation. The accumulation of nucleolin and protein 823 in G2 compared to CI was demonstrated using sorted phascspecific cells. In Cg cells, sorted according tcl their very low RNA content, the amount of Ag-NOR proteirts was half elf that founil in Gt cells, nucieolin being only weaklv detectable. Therefore the expression of nucleolin Increased between G()-G [ and G 1-S phases. These data support the hypothesis that quantification of Ag-NOR reflects the percentage of cells at each cell cycle phase, it IS high iz S-C2 and low in G1 phases. STEIMBERG Natha& THENET-GAUCI Sophie, ADOLPHE Monique hborutoire de Phatmacologie CeNuLaire de I’EPHE 1.5 rue de I’ Ecole de hf&ecine 75006 Pot-is France