Growth inhibitory factor diffusing from chick embryo fibroblasts

Cell Biology

International

GROWTH INHIBITORY

Reports,

Vol. 10, No. 72, December

FACTOR DIFFUSING

1986

947

FROM CHICK EMBRYO FIBROBLASTS

Ch., BLAT+ J VILLAUDY", G., DESAUTY+ A GOLDE" & L. HAREL+. 'Institut di Reiherches Scientifiques sir ii Cancer. 7, rue G. Mocquet. B.P.N"8. 94802 VILLEJUIF Cedex. 'Institut Curie. Pavillon Regaud. 26, rue d'Ulm.75005. PARIS (France). ABSTRACT : Density-dependent inhibition (DDI) of growth is assumed to be the result of diffusion in the medium of growth in.hibitory molecules. In this work, we demonstrate the presence of inhibitory molecules (IDF : chicken inhibitory diffusible factor) in the medium of chick embryo fibroblasts (CEF) cultures. IDF partially purified by Bio-Gel P150 chromatography followed by refierse phase FPLC. The dose-response curve showed that 250 nglml IDF inhibited 50% DNA synthesis. IDF was also able to inhibit thg growth of sparse cultures of CEFC; this inhibition was reversible. IDF was unable to prevent the DNA synthesis in cells transformed by'v-src gene expression. These results suggest that IDFc is involved in the DDI of CEF growth. INTRODUCTION The regulation of cell growth in vitro is poorly understood. Density-dependent inhibition (DDI) of growth has been assumed to be the result of diffusion in the medium of growth inhibitory molecules (Hare1 et al., 1978 and Blat et al., 1985). Growth inhibitory substances have been fractionated or purified from medium conditioned by dense cultures of 3T3 cells (Hare1 et al., 1983, Hare1 et al., 1985, Blat et al., 1986, Hsu and Wang, 1986) and other cell types (Halley et al., 1980, Wells and Malluci, 1983 Iwata et al., 1985). However, it was recently shown that a molecule of 25 Kd, inhibitory for BSC cells (Halley et al., 1980) was similar to B-TGF secreted by tlransformed and normal cells (Rbberts et al. 1981, Lawrence et al., 1985), and could be a bifunctional regulator of cell growth (Tucker et al., 1984, Roberts et al., 1985, Shipley et al., 1985). Previous data of this laboratory showed that cell metabolism was inhibited in dense cultures of chick embryo fibroblasts (CEF). Moreover, when CEF were infected by Ny68 virus, a mutant of Rous thermosensitive for transformation, they were sarcoma virus density-inhibited when multiplied at 41" C. The DDI was released 1984), a when they were transferred to 37" C (Jullien et al., permissive temperature for the expression of transformation. Therefore, it was of interest to verify whether inhibitory embryo substances diffused from dense cultures of chick fibroblasts, and whether these inhibitors might be involved in In this work, inhibitory molecule of CEF the DDI of growth. 0309-l

651/86/l

20947-08/$03.00/O

@ 1986 Academic

Press

Inc. (London)

Ltd.

948

Cell Biology

International

Reports,

Vol. IO, No. 12, December

1986

growth, chicken inhibitory diffusible factor (IDF ), was partially purified from medium conditioned by dense cult&-es of CEF. The inhibitory activity of IDFc was lost when it was tested on transformed CEF. MATERIALS AND METHODS c 11 Primary cuTfires of chick embryo fibroblasts were prepared YFomsiO-day old brown Leghorn chick embryos. CEF were cultivated at 37°C in Eagle's medium with a double concentration of amino acids and vitamins supplemented with antibiotics and 5% calf serum. Primary chick embryo cells were infected at a multiplicity of 0.5 FFU per cell with a thermosensitive mutant (Ny68) of the Rous sarcoma virus (Schmidt Ruppin strain,subgroup A). Infected cells were maintained at 37°C. R-TGF purified from human platelets was a gift from Dr. F. Godeau (Institut Pasteur, Paris). Assays of IDF activity. Secondary cultures of CEF were seeded at KTO cells/well in complete medium with 5% calf serum in 96-well culture plates ; 6 h later, medium was discarded and cells were maintained for 60 h in serum-free medium. Fractions to be tested were solubilized in culture medium (centrifuged if necessary to eliminate unsoluble material) and added on quiescent CEF culture in the presence of 100 g alpha globulin, a seric protein (Cohn Fraction IV), which tr as properties of growth factors (Michl and Spurna, 1974). DNA synthesis was determined, asl(escribed (Hare1 et al., 19851, by labelling the cells with C-thymidine (0.07 uCi/well) between 5 and 24 h after addition of the fractions and alpha globulin. The inhibitory activity of IDF was determined by the percent inhibition of stimulation by alpfja glovylin of DNA synthesis. In the absence of alpha globulin, C-thymidine incorporation into cells was between 15 and 25% of the incorporation in the presence of this growth factor. When 5cells were infected by Ny68 virus, cells were seeded at 1.2x10 cells/well at 37°C in complete medium with 8% calf serum. They were transferred to 41°C 5 h later and maintained at this temperature until 18 h prior to the saturation density ; experiment, the medium was replaced by serum-free medium and cells were maintained at 41°C. IDF was then tested on cells maintained at 41°C or14transferred to 37°C for 24 h in the presence or not of serum. C-thymidine was added between 5 and 24 h after addition of serum or transfer to 37°C. Isolation of IDF . CEF were seeded at 1.6X105 cells/cm2 in complete medium ; /2 h lCater, cells were stimulated by a change of medium for 24 h. After that, medium was removed and replaced by serum-free medium. medium was discarded, cells were washed Five hours later, with serum-free medium and then incubated at 37°C for 18 h in the presence of fresh serum-free medium. The culture was then slowly stirred for 15 min, and medium was removed and used as conditioned medium for the fractionation of inhibitory molecules.

Cell Biology

International

Reports,

Vol. 10, No. 12, December

1986

949

a

2

o~O- b

Fig.lIsolation of IDF . mApproximately c35 mg of macromolecules concentrated from mooned medium and solubilized in acetic acid 1 M were chromatographed over a "Bio-Gel P 150" column (22x2.5 cm), packed in 1M acetic acid. Fractions of 2.7 ml were collected. An aliquot of lyophilired fractions was tested for inhibitory activity on DNA synthesis in CEF. Fig.lbThe lyophilized fractions (15-16-17-18) from "Bio-Gel P 150"romatography were pooled. About 600 pg of proteins were solubilized in TFA 0.05% injected on a ProRPC-HR 512 column (Pharmacia) and eluted as described in "Materials and Methods". Fractions (0.3 ml) were lyophilized and aliquots were tested in sparse culture of CEF (1 OD280 (-----I.

950

Cell Biology

International

Reports,

Vol. 70, No. 12, December

1986

About 2 liters of conditioned medium were centrifuged (10,000 RPM, 15 mini, and the supernatant was concentrated by ultrafiltration on membrane Amicon YM 10. The concentrate was acidified by glacial acetic acid (1M final concentratidn) and the acid-soluble macromolecules were fractionated over a Bio-Gel P 150 column packed in acetic acid 1 M as described previously (Hare1 et al., 1985). The lyophilized fractions containing IDF activity were pooled, solubilized in 0.05% TFA (Trifluoroacetic acid), then ajusted to 17% acetonitrile 0.05% TFA and fractionated by reverse phase FPLC on Pro RPC-HR 512 column (Pharmacia). Elution was achieved with a linear lo-min gradient of 17 to 24% acetonitrile 0.05% TFA, followed by a 30-min gradient of 24 to 34% acetonitrile 0.05% TFA, a 13min gradient of 34 to 45% acetonitrile 0.05% TFA, and finally a 12 min gradient of 45 to 100% acetonitrile 0.05% TFA. Flow rate was 0.3 mllmin. SDS-PAtiE. Lyophilized fractions from FPLC were solubilized in SDS sample buffer (glycerol lo%, SDS 2.3%, Tris-HCl, 0.0625 M pH 6.8). SDS polyacrylamide gel electrophoresis was performed with 10% acrylamide according to the technique of Laemmli (19701. After electrophoresis, proteins were revealed by silver staining (Sammons et al., 1981). Bovine serum albumin (67K), ovalbumin (43K1, carbonic anhydrase (30K) and trypsin inhibitor (20K1, were used as molecular weight markers. RESULTS ISOLATION OF INHIBITORY MOLECULE. Macromolecules (soluble in acetic acid 1 M1 f medium conditioned by dense cultures of CEF were fractionate: by Bio-Gel P 150 chromatography and the lyophilized fractions were tested on sparse cultures of CEF stimulated to as indicated in "Materials and proliferate by alpha globulin, Methods". Fractions 15.16.17.18., which eluted like ovalbumin (43 K) inhibited DNA synthesis (fig la). These fractions were pooled In each and used for further purification by reverse phase FPLC. experiment, (fig lb), one peak of inhibitory activity was observed The results of SDS-PAGE of the which eluted at 27% acetonitrile. different fractions (fig 21 showed that the inhibitory activity coincided with the appearance of 30-Kd proteins (lane 24). Figure 3 After FPLC purification, about 250 shows the dose-response curve. nglml of fraction 25 decreased the stimulation of DNA synthesis by 50% in sparse cultures of CEF. It is of interest to note that IDF was different from BTGF which 19851. In is known to be secreted by CEF ?Lawrence et al., in gel filtration on Bio-Gel in acidic conditions, Bparticular, TGF eluted with an apparent molecular weight of 16 Kd and on reverse phase chromatography (FPLC) between 33 and 37% acetonitrile Furthermore, RTGF was stimulatory of DNA (Lawrence et al., 19851. synthesis in CEF (table I) when it was tested in the same conditions as IDFc.

Cell Biology

International

Reports,

Vol. 10, No. 12, December

1986

951

Fig.2Silver stained SDSmof inhibitorv fractions. Ab t 3 pg of p;oteins f FPFF fractions (23 to !$? were applied on the gel. In the lane 0 the proteins (6 pgl of fraction 16 from BioGel P 150 chromatography were deposited.

Fig.3Dose-response curve. After reverse-phase l-PLC inhibitory fraction 25 wa; tested at different concentrations on sparse cultures of CEF in conditions described in Materials and Methods.

Inhibition of cell Fig.4m (typical results). -were seeded at 8~10~ cellslwell in complete medium +5% calf serum. The medium was changed 5 h later and replaced by serum-free medium ; 60 h later (time 01,cultures were stimulated by addition of 1% serum in the absence or presence (0-o 1 ( .--*I The medium of 260 ng IDF . was changed 26 h later and replaced by fresh medium + 1% serum. In experiment (n----a), IDF was added at time 0 and left for 72 h without a change of medium. 'Cell number was determined on a pool of 3 culture wells.

952

Table

Cell Biology

I :

OTGF effect

International

Reports,

on DNA synthesis

cpm CONTROL t B TGF

24 hours 1900 t 30 2890 + 75

Vol. 70, No. 72, December

1986

in CEF.

14C-Thymidine

in DNA 48 hours 470 !: 69 2080 2 118

At time 0 quiescent CEF were stimulated by 100 g alpha-globuline in the presence or not of BTGF (3ng). Cells wer P labelled for 19 h between 5 and 24 h (time 24) or 29 and 48 h (time 48) after the addition of growth factors. INHIBITION OF CELL GROWTH. IDF was able to inhibit growth of sparse cultures of Ctt stimulate&by serum (fig 4). The inhibition was reversible : when IDFc was added for 24 h in CEF (in conditions where DNA synthesis was 90%-inhibited), and the medium was inhibition was changed and replaced by fresh medium with 1% serum, released and cells resumed their growth (fig 4). EFFECT OF IDFr ON TRANSFORMED CELLS. CEF infected by Ny68 virus were either niaintained at 41" C and stimulated with serum, or transferred to 37" C (the permissive temperature for the expression of transformation) in the absence or presence of serum (fig 5). DNA IDF . The synthesis was determined in the absence or presence of stimulation by serum of DNA synthesis in cells maintained atc41" C was inhibited in the presence of IDF . By contrast, the stimulation of DNA synthesis due to the expressi& of transformation at 37°C in the absence of serum or the stimulation of DNA synthesis due to the addition of serum and the expression of src gene at 37°C was not inhibited by IDFc (fiq 5). Effect of IDF on Fig.5lNv68nfected cells (tvSical 37-c results) Cells were mblti~ plied and maintained at 41°C as described in "Materials and Methods". Then, one part of the culture was maintained at 41°C and stimulated by serum in the presence or ab~ sence of 60 ng IDF . Another part of the cflture was transferred to 37°C either in or presence of the absence IDF was added at the Sfig"of traksfer to 37°C. DNA fqnthesis was determined by C-thymidine incorporation between 5 and 24 h after adx SERWI dition of serum or transfer 111 in cells maintained at to 37°C. The incorporation of "C-thymidine 41°C in the absence of serum was substracted from the results.

Cell Biology

International

Reporls,

Vol. 70, No. 12, December

1986

953

DISCUSSION Our results show the presence of inhibitory molecule in medium conditioned by dense cultures of CEF. This is the first demonstration of the secretion by CEF of growth inhibitory macromolecule active at a concentration of 250 nglml (lower than 8nM). Indeed, it was reported (Yaoi, 1984) that a mixture of surface glycopeptides (obtained by digestion with pronase E of CEF) was inhibitory of CEF growth. However, the concentration used was of medium). large (equivalent to 10 g sugarlml IDF45 purified from meP ium conditioned by 3T3 cell cultures was also able to inhibit DNA synthesis in CEF and lost its inhibitory activity when it was tested on RSV transformed CEF (Blat et al., 1985). Taken together, these results suggest that such molecules The decreased sensitivity of are involved in DDI of growth. transformed cells to inhibitory molecules would explain the loss of densitydependent inhibition of growth after v-src transformation (Blat et al., 1985). ACKNOWLEDGEMENTS ne thank Dr. f- GODEAU for the gift of B-TGF, the Association le developpement de la Recherche sur le Cancer and C.N.R.S. N"9590391, which supported this work.

pour (ATP

REFERENCES Blat,

Ch., Villaudy, J., Desauty, G., Golde, A., & Harel, L. (1985) Modifications induced by v-src expression on the regulation of cell proliferation. Hypothesis and preliminary results. Comptes Rendus de 1'Academie des Sciences 1 Paris). 301. III. 9, 417-422. Blat, Ch., Chatelain, G., Desauty, G., & Harel, L. (1986) Inhibitory20d3iff;si2ble factor IDF45, a Gl phase inhibitor. FEBS 0 175-180. Letters. Harel, L., Jullyen, M:,& De Monti, M. (1978) Diffusible factor(s) controlling density inhibition of 313 cell growth : a new approach. Journal of Cellular Physiology. 96,327-332. Harel, L., Blat, Ch., & Chatelain, G. (1983) Density-dependent inhibition of growth : fractionation of inhibitory diffusible factor(s) release by dense cultures of 3T3 cells. Biology of the Cell. 48.1. 11-16. Harel, L., Blat, Ch., & Chatelain, G.(1985) Regulation of cell proliferation inhibitory and stimulatory factors difffused by 3T3 cultured cells. Journal of Cellular Physiology. 123,139143. Holley, R. W., Bbhlen, P., Fava, R., Baldwin, J. H., Kleeman, G., (1980) Purification of kidney epithelial & Armour, R. cell growth inhibitors. Proceedings of the National Academy of Sciences. USA. 77, 10, 5989-5992.

954

Cell Biology

International

Hsu,

Reports,

Vol. 10, No. 12, December

1986

Y. M., & Wang, J. L. (1986) Growth control in cultured 3T3 fibroblasts. V. Purification of an Mr 13,000 polypeptide responsible for growth inhibitory activity. The Journal of Cell Biology. 102, 2, 362-368. Iwata, K. K., Fryling, C. M., Knott, W. B., & Todaro, G. J. (1985) Isolation of tumor cell growth-inhibiting factors from a human rhabdomyosarcoma cell line. Cancer Research. 45, 2689-2694. Jullien, M., Harel, L., Golde, A., Villaudy, J. & Pugnet, P. (1984) Early release of the density-dependent inhibition of phosphate uptake and ATP synthesis after src gene expression in chick embryo fibroblasts. Experimental Cell Research. 152, 390-401. Cleavage of structural proteins during the Laemmli, U. K. (1970) assembly of the head of bacteriophage T4. Nature. 227, 680-685. P. (1985) Conversion of a Lawrence, D. A., Pircher, R., & Jullien, high molecu ar weight-latent B-TGF from chicken embryo fibroweight active B-TGF under acidic blasts into a low molecular conditions. Biochemical and Biophysical Research Communications . 133 1026-1034. Michl, J., & Spurna, V. (1974) Growth-promoting alpha-globulin. Experimenta 1 Cell Research. 84, 56-62. Roberts, A. B., Anzano, M. A., Lamb, L. C., Smith, J. M., & Sporn, M. 8. (1981) New ciass of transforming growth factors Isolation from nonpotentiated by epidermal growth factor : neoplastic tissues. Proceedings of the National Academy of Sciences. USA. 78, 9, 5339-5343. Roberts, A. B., Anzano, M. A., Wakefield, L. M., Roche, N. S., Stern, D. F., & Sporn, M. B. (1985) Type I3 transforming growth factor : A bifunctional regulator of cellular growth. Proceedings of the National Academy of Sciences. USA. 82, 119-123. Sammons, D. W., Adams, L. D., & Nishizawa, E.E. (1981) Ultrasensitive silver-based color staining of polypeptides in polyacrylamide gels. Electrophoresis. 2, 135- 141. Shipley, G. D., Tucker, R. F., & Moses, H. L. (1985) Type Btransforming growth factor/growth inhibitor stimulates entry of monolayer cultures of AKR-2B cells into S phase after a prolonged prereplicative interval. Proceedings of the National Academy of Sciences. USA. 82, 4147-4151. Tucker, R. F., Shipley, G. D., Moses, H. L., & Holley, R. W. (1984) Growth inhibitor from BSC-1 cells closely related to platelet type 0 transforming growth factor. Science.226, 705707. & Malluci, L. (1983) Properties of a cell growth Wells, V., Journal of inhibitor produced by mouse embryo fibroblasts. Cellular Physiology. 117, 148-154. Yaoi, Y. (1984) Growth-inhibitory glycopeptides obtained from the cell surface of cultured chick embryo fibroblasts. Experimental Cell Research. 154, 147-154.

Received

7.8.86

Accepted

17.10.86