74 Biology of the Cell

THE AMOUNT OF THE TWO MAJOR AG-NOR I’ROTEINS, NUCLEOLIN AND PROTEIN 823 IS-CELL-CYCLE DEPENDENT.

SIRRI Vaientina, ROUSSEL Pascal, GENDRON Mane-Cl,lutic and HERNANDEZ-VERDUN Dani+

Institfrt ]ncyJtes Monad, Purrs, Pranct~

IMMORTALIZATION OF RABBIT ARTKULAR CHONDROCYTES BY SV40 LARGE T-ANTIGEN UNDER

THE CONTROLOF~TYPE II COLLAGEN R-E~ULATOtiY SEQUENCES : PHENOTYPE STUDY OF~TNE

CHONDROCYTES -CULTURED IN 3 DIMENSfONS.

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 Hernandez- Verdun 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 serurn- deprived 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 phasc- specific 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

The study of the chonclrocyte, the unique ceU type of cartilage, could allow ro understand the disregulations leading to cartilage disorders, such as osteoarthritis and a&u%. The establishment of an immonalitied c1londrocyt.e cell line would be of advantage for physiological or pharmaco-tuxicobgicat studies.

We immortalized rabbit articulax chondrocytes with a vector carrying the SV40 large T antigen under the control of the promoter and enhancer sequences of-type Ilcollagen, to obtain differentiated clones which express the large T antigen. The phenotypic characterization of these cell lines showed nevertheless a dediffetentiation with subcultures in monoIayer, -like normal chondrocytes. In order to know if the differentiated phenotype couid be restored, Gnfture ir! 3 dimensions was developed.. The 2116 cel1 line was cultured either in alginate beads;shown to fa&i rhe redifferentiation of normatchondrocytes dedifferentiated witi subculture (Lemare F.. Demignot S., Arlofphe~M., in preparation), or in aggregates. in which ceil-cell contact are fighter than in alginate beads. After I month in alginate beads or in aggregates, rhe 21 I6 clone synthesized essentially type I and type III collagens (study of 3H. ProIine labelled collagen, c4iVed by CNEr, and separated by- SDS-PAGE). However, an immunofluorescence staining of. type If collagen- showed an increase of collagen II-stained cells, mare important for cells.cuItured iff aggregates than for cells in a!ginate beads. In sitrc hybridization studies seemed to cofifirm rhar mRNA encoding type II collagen were expressed in numerous cells within aggregates. Moreover, the extrticellular matrix synthesized by cells irt aggrkgates contained cartilage proteoglycans as determined with alcian blue-staining. mRNA for type I and type II collagen as well as for aggtecwj (major constituent of cartilage proteoglycans) will be studied by in situ hybridization, in order to know if the extracellular matrix genes are expressed differently it: tilese two models of 3 dimensions culture.

CONFORMATION OF RIBOSOMAL GENES AND DISTRIBUTION OF THE TRANSCRH’TION FACTOR UBF ARE

CELL CYCLE DEPENDENT JUNBRA’ I-l. Roberte , MASSON] Claude, C&AUDI GCrard,

SUJA2 Jose AND HERNANDEZ-VERDLJN’ Daniille ~lr~st.~~cq~~es Monad, 2 plncr~ Jussir~r. 75251 Pm> C&r 05.

Frauw

DECREASED EXPRESSION OF THE ANTI-APOPTO’I‘ZC PROTOUNCONGENE PRODUCT, Bet-2, IN

HYPERTROF@IC CHOM)R#CYTES OF THE EPIPHYSEA. PLATE CARTILAGE OF THE RAT

2 U~lJdRd dr 8iOlO@J Ce~Lhr, ~!&~artnlrlrlltO do’ i?JO:OgJJJ, bhji~J~ de Biologin, Universidad Atrtonomn dr Madrid, 28049 Madrid

WANG Ying, TQURY R nt?e HAUCHECORNE Mlchell% EALeMAiN Nicole

iNSERM U. 120, Hopital R. Debri, 48 Boulevard SCrurier 75019 PARIS FRANCE

The most extenslveiy- studled iorm oi programmed ccii &at!-, 1 PCDI. aoootosis. is characterized bv morohologic alterations. biochemicai

The in sitll distribution of the ribosomal genes (rDNA) and their transcription factor (UBF) correlatively with their RNA transcripts was investigated in PtKl cells during the cell cycle. In nucleoli, rDNA is distributed with alternate sites of clustered genes and dispersed genes. UBF was found associated with some but not all clustered genes and proportionally even more with dispersed genes. UBF was distributed in the nucleoli in several foci more numerous and heterogeneous in size during G2 than GI. The cellular amount of UBF increased during S-phase. The unravelling of the transcriptional units by DRB treatment indicated a similar amount of UBF per transcription unit and consequently ihe heterogeneous size of lhe UBF foci is proposed to represent a variable amount of transcription units active or associated with UBF. Therefore the amount of UBF is not only linked to the number of rDNA genes but also seems to depend on active or repressed sites in the same locus. In conclusion, UBF is associated to rDNA during the cell cycle in variable amounts relative to rDNA distribution and activitv. After visualization of the transcripts together with rDNA or with UBF, three types uf rDNA can be discriminated: 1) clustered genes without UBF, 2) clustered genes with UBF some of them associated with transcription and, 3) dispersed genes with UBF and transcription.

modifr&ohs an& changes in the eipresslon levels of the protooncogene products c-Myc and Bcl-2. However. there are PCD urocesses that do not &ludeall tbe;e phenomena (Green DR., Mahboubi A.; Nishioka-W,, C$ S., Echeverri F., Shi Y.. Glynn J., Ashwell J., Bissonnette R. (1994): Imm. Rev, 142, 321-342) (Linette G.P. Kosmeyer S.J. (1994) Cur. Opin. Cell Biol, 6, 809-815). Althopgh the hypertrophic chundrocyles at their terminal stage of developmentdo not show the morphoiogicai features of apoptosis, they do over produce c-Myc (Wang Y., Toury R. Huuchecorne M., Balmain N.(1996), Cell. Mol. Biol, (in press)).We have now determined whether another characteristic of apoptosis, a decrease in Bcl-2 expression, is occurs during chondrocyte PCD.

Longitudinal ultrathin se&ions of the tibial growth camiage ot ? 1 &y-old rats were incubatedwith a specific anti-Bcl-2 antibody-(Santa Cruz CA) and exauiined under the electron microscope. Bcl-2 immunoreactivity was intense in the early chondrocytes of the prolifemtive zone, decreased m those of the mature zone, and very iow in the fuliy-differentiared hypertrophic cells of the calcifying zone. Inununolabeli~g W;IS mtinly located in the cytoplasm, with a particularly high concentration within the mitochondria. There was little &l-2 immunolabeting scattered th:roughout the chromatin without any specific location in the nucleus of all chondrocytes, whatever the cell developmental stage.

These results indicate that chondrocyte PCD involves a decretise in Bcl-2 production. This protooncogeoe product seems to regulate a set of events implicated in chondrocyte survival. these occur in the cytoplasm of chondrocytes an&particularly within their mitochondria