Claude Verger

Nature of the iron requirement for chick embryo cells cultured in the presence of horse serum

Chick or bovine transferrin induces proliferation of chick embryo cells cultured in the presence of horse serum, which cannot itself assure their multiplication. Cell growth can also be induced by iron salts and iron complexes such as hemoglobin or hemin, but also by biliverdin which has no iron atom in its molecule.

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

SummaryWe have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagles minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.

Stimulatory effects of protein synthesis inhibitors on the spreading rate of 3T3 cells

Three protein synthesis inhibitors, puromycin, cycloheximide and anisomycin were found to increase the rate of spreading of Swiss-3T3 cells after 1 h incubation in the presence of low concentrations of serum. Data from experiments with anisomycin suggest that this effect is roughly proportional to the extent of inhibition of protein synthesis.

Isolement de “ RNA messager ” de haut poids moléculaire à partir de polysomes de cellules animales

Summary High molecular weight polysomal « messenger RNAwas isolated from various animal cells. Polysomes from various animal cells were prepared in high ionic strength buffer, in order to minimize the polysomal messenger RNA degradation. A comparative study of the sedimentation behaviour of the mRNA recovered from polysomes prepared either in isotonic TKM buffer or hypertonic TKNM buffer was performed whith mouse ascitic tumour cells labelled for 15 min with [ 3 H] Uridine. 30 p. cent of the mRNA extracted from polysomes isolated in TKM buffer sedimented faster than 18 S. In contrast, when the polysomes were prepared in TKNM buffer 65 p. cent of the mRNA sedimented faster than 18 S. Using TKNM buffer, heavy polysomal mRNA was also isolated from [ 3 H]pulse labelled chicken cells. 40 p. cent of the mRNA from cultivated embryonic cells and 60 p. cent of the mRNA from leukaemic myeloblasts infected with Avian Myeloblastosis Virus, sedimented ahead of 28 S. The possibility that heavy mRNA recovered from cells lysed in TKNM buffer represents nuclear RNA was examined. Unlabelled cells were homogenized in TKNM buffer with pulse labelled nuclei prepared in TKM buffer and the heavy polysomes were sedimented from the postnuclear supernatant. Very small amounts of labelled RNA sedimenting faster than 18 S were recovered. This suggests that the heavy mRNA extracted from the « TKNM polysomesdoes not originate from discrupted nuclei. The isolation of « heavy mRNAfrom the « TKNM polysomescould be explained by an efficient inhibition of the RNase action after disruption of the cells. In addition, the proportion of heavy mRNA seems increased by the elimination of the fragmented polysomes (polysomes no more engaged in protein synthesis). A « protectiveeffect due to a modification of the mRNA structure in high salt concentration is also discussed. The method we have described seems a very convenient and reproducible procedure for the isolation of undegraded polysomal mRNA from various animal cells.

Stimulatory and inhibitory effects of serum and conditioned medium on cell spreading

This work deals with the effects of coating culture dishes with chick embryo fibroblast conditioned medium (CM) and 1% fetal calf serum (FCS) on the rates of attachment and spreading of chick embryo fibroblasts. It appeared that a FCS coating over a CM precoating exerted a remarkable promoting cooperative effect on cell spreading whereas a CM coating over a FCS precoating had a very marked inhibitory effect. Precoating with cobalt-protoporphyrin IX (CoPP) was also performed to serve as a substrate for FCS or CM coating. This CoPP precoating was found to exert a promoting effect for FCS coating only.

The inhibitory effect of serum on cell attachment can be prevented by cobalt-protoporphyrin IX

The rate of attachment of chick embryo fibroblasts (CEF) incubated for 1 h at pH 6 in the absence of serum, was equal to about 90% with 2% of spreading cells. The addition of 1% horse serum (HS) decreased to about 30% the rate of attached cells but increased to about 30% the proportion of spreading cells. We found that 3 microM cobalt-protoporphyrin IX (CoPP) added for 3 min before the addition of 1% HS increased both the rate of cell attachment (about 60%) and the proportion of cell spreading (about 50%).

Infection par le virus de la myéloblastose aviaire, biosynthèse de RNA lourd polysomal rapidement marqué et différenciations cellulaires

Summary Secundary cultures of normal chick embryos were treated with the incubation medium of avian leukemic cells (AMV transformed myeloblasts kept at 4°C for 1 h or 24 h in Eagles medium) which in all cases contains numerous virions bound to macromolecular complexes (apparently deoxyribonucleoproteins). On the first day the treatment very markedly increases the radioactivity of pulse labelled RNA ( 3 H Uridine, 15 min) associated with the polysomes with a peak of radioactivity between 30 and 40S. This increase (up to 16 times the level of control cultures) is not due to stimulation of the cellular uptake of uridine. It cannot be attributed to heavy molecular weight RNA or ribonucleoproteins of nuclear origin, as demonstrated by zonal centrifugation of polysomes after treating with EDTA, or equilibrium sedimentation in Cs Cl gradient after formaldehyde fixation. The early stimulation of polysomal RNA synthesis is followed by abundant virus production on the 2 d day and cell differentiation, both muscular and epithelial, start appearing on the 3 d day. The early stimulation of RNA biosynthesis may, to a great extent be dissociated from both the later virus production or cell differentiations. The supernate of the 1 h incubation medium made virus free by filtering through Millipore membranes, retains the capacity to induce cell differentiation, without virus production, although the effect on polysomal RNA synthesis is small or negligible. Besides in embryonic cells infected by purified AMV, no marked effect is observed on the labelling of polysomal RNA, but virus is subsequently produced in the absence of noticeable cell differentiation. In contrast a mixture of purified virus and 55 000 g supernate of the 1 h incubation medium has the same enhancing effect on RNA synthesis, as the total incubation medium of leukemic cells. Finally no similar effect was observed in serially passaged cells consisting of fibroblasts. Different interpretations of these results are discussed.

In Vitro and In Vivo Cytostatic Effect of Antiribosome FLS Antisera on Mouse Ascitic Tumour Cells FLS

I would like to present the results of experimental work carried out with Cl. Verger and E. Nahon in the laboratory of Immunology of the Gustave Roussy Institute and also to discuss our findings which indicate that both growth and proliferation of mouse ascitic tumour cells can be modified by an immunological process.