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Retinoic acid-induced differentiation of F9 embryonal carcinoma cells
RETINOIC
S. LINDER,
ACID-INDUCED DIFFERENTIATION EMBRYONAL CARCINOMA CELLS IJ. KRONDAHL.
R. SENNERSTAM
OF F9
and N. R. RINGERTZ
Department of Medical Cell Genetics. Medical Nobel Institrrte. Krrrolinskrr Institutet, S-10401 Stockholm. Slc,rden
SUMMARY The ability of retinoic acid (RA) to induce differentiation in embryonal carcinoma (EC) cells was examined by growing mouse F9 cells in a medium containing 1 PM RA. The altered properties of the cells became apparent after a lag period of approx. 24 h and were fully expressed after 5 days. The RA-induced phenotype was characterized by changes in cell morphology, slowing of the rate of cell multiplication, reduced DNA and protein synthesis, altered pattern of polypeptide synthesis and changes in cell surface components. The slowing of cell multiplication and general reduction in the rate of protein synthesis was paralleled by changes in the relative rates at which different polypeptides were synthesized. Two-dimensional gel electrophoretic analysis of [%]methioninelabelled cell proteins showed an altered relative synthesis of at least fifty polypeptides. The relative rate of synthesis of two components of the cytoskeleton identified as vimentin and tropomyosin were shown to increase.
Embryonal carcinoma (EC) cells constitute the growing stem line in a group of tumors known as teratomas. An important characteristic of these tumors is that they also contain many types of differentiated tissues. Transplantation experiments with mouse EC cells have shown that the differentiated cells are formed from EC cells. Furthermore, biochemical and immunological studies have demonstrated enzymic and antigenic similarities between EC cells and cells present in the inner cell mass of blastocysts. The differentiated cell types in mouse teratomas are non-malignant and appear to be identical with cells found in tissues of adult animals. Mouse teratomas therefore represent an interesting model for in vitro studies of early embryonic development (for reviews see [ 1, 21). Several mouse EC lines have been estab-
lished. Under conditions which favor rapid multiplication these lines will consist of EC cells only. Differentiation is favored by cell crowding and aggregation. Many EC lines are described as ‘pluripotent’, i.e. capable of histotypic differentiation. Such lines produce nerve, muscle, cartilage, fat and other specialized cells in vitro. Some lines, among them the F9 line, have been considered unable (nullipotent) to form specialized cell types. While it is true that this cell line has a markedly reduced capacity to differentiate, it does, however, sometimes produce flat endoderm-like cells in small numbers [3]. Studies of in vitro differentiation of pluripotent EC lines are associated with technical difficulties due to the heterogeneity of the cell cultures. Therefore, the finding by Strickland & Mahdavi [4] that retinoic acid
454
Linder et al.
(RA) induced massive differentiation of F9 cells to endoderm-like cells may increase the usefulness of EC cells as an in vitro model of embryonic cell differentiation. In this report we have examined phenotypic changes which occur following RAinduced differentiation of F9 cells. Furthermore we have tried to relate the phenotypic changes to the rate of DNA and protein synthesis and to cell growth and multiplication. MATERIAL
AND METHODS
Cell cultures FY embryonal carcinoma cells [5] were obtained from Drs S. Grandchamp and B. Ephrussi. PYS-2 endodermal cells [6] were generously provided by Dr J. F. Nicolas, Pasteur Institute, Paris. All cells were cultivated in Dulbecco’s modified Eagle’s medium supplemented with non-essential amino acids, nucleosides, sodium pyruvate and 10% fetal calf serum (FCS) (growth medium). Cell cultures were mycoplasma-free as judged by the Hoechst staining method [7]. Cell culture reagents were obtained from Gibco Bio-Cult, Paisley, Scotland. Time-lapse recordings were made with a TV camera (National) and a videotape recorder (National). For induction of differentiation, FY cells were plated in plastic culture dishes or on glass slides to give a cell concentration of aoprox. 3.5-5X 103/cm2.One day after plating, 1~1 of an ethanol solution of retinoic acid (RA, obtained from Sigma Chemical Co.) was added oer ml culture medium to give a final concentration of 1 FM. The medium was subsequently changed every second day. Labelling with [35S]methionine (Radiochemical Centre, Amersham, UK) was carried out for 4 h using 200 &i/ml of the labelled amino acid in methioninefree growth medium.
Preparation of samples for 20 gel electrophoresis of proteins At the end of the labelling period, the cells were washed twice with phosphate buffered saline (PBS) and scraued from the culture dishes. The cells were then washed once with sonication buffer according to O’Farrell [8], freeze-thawed five times and incubated with DNase (50 LLg/ml) for 5 min on ice. Lysis buffer [8] was added followed by solid urea until saturation.
Two-dimensional
gel electrophoresis
Isoelectric focusina and SDS slab eel electroohoresis was performed according to O’Far&l[8] as previously described [Y]. Second dimension gels consisted of Y20% exponential gradients or 10% uniform gels. Dried
gels were exposed to Kodak No-screen X-ray film for approx. 2x 1oRcpmlh. In order to quantitate 19lmethionine incorporation into individual-polypeptidesi spots were cut from the gels using radioactive inkdots as a guide. The radioactivity in each spot was determined by liquid scintillation counting. The relative synthesis was calculated as the activity in one spot divided by the total activity applied to the gel. No correction was made for quenching in acrylamide gels relative to Whatman filters.
Cell surface labelling gel electrophoresis
and SDS slab
Lactoperoxidase-catalysed iodination of external proteins was based on the procedure described bv Hynes [lo]. At the end of the labelling period, the cells were washed with PBS, scraped from the dishes and pelleted. SDS sample buffer (60 mM Tris/HCl, pH 6.8/2.3% SDS/5 % beta-mercaotoethanol/lO% elvcerol) was added and the sampies were boiled 6; 3 min. Iodinated proteins were separated on 10% SDSpolyacrylamide slab gels using the gel system described by Laemmli [ 1I].
Immunofluorescence For antibody staining of external structures, cells were fixed in 2% paraformaldehyde in PBS for 15 min. For additional staining of intracellular components, cells were fixed in methanol for 5 min. Immunofluorescence staining was by the indirect procedure using antihuman fibronectin serum (a gift from Dr Jorma Wartiovaara) as the first antibodv. The soecificitv of this antiserum and its interspecies cross-reactivity have been described elsewhere [12, 131. FITC-conjugated goat anti-rabbit IgG was purchased from Statens Bakteriologiska Laboratorium (Solna, Sweden) and used as the second antibody.
RESULTS Morphological administration
changes upon of RA
Under normal conditions, F9 cultures consisted of more than 99% embryonal carcinoma (EC) cells. The appearance of F9 cells under the phase contrast microscope can be seen in fig. la. The addition of 1 PM retinoic acid (RA) to the .F9 cultures caused the cells to differentiate massively, forming flat, more adherent cells. The morphology of the cells changed rather slowly. The cells started to flatten out 2-3 days after induction and the changes were
Retinoic acid-induced EC-cell differentiation
455
Fig. 1. Appearance of F9 cultures under the inverted phase contrast microscope. (a) Untreated F9 cells; (h-d) F9 cells at different time points after addition of
1 PM retinoic acid. (b) 24; (c) 72; (d) 144 h. Bar, 50 pm.
manifest after approx. 5 days (fig. 1b-d). If by day 4 the RA-containing medium was exchanged for normal medium the altered phenotype persisted for at least several weeks. For the first 3-4 days of RA treatment the changes in morphology appeared to be the same for all cells. As a result the cells became flat and epithelial-like with numerous thread-like extensions. After l-2 weeks of culture it became apparent, however, that RA treatment induced the formation of more than one non-EC phenotype. Most of these phenotypes have not yet been identified. One type of cell could be recognized as fat cells on the basis of morphology and Sudan black staining.
Effects on cell growth Under optimal culture conditions F9 cells have a population doubling time of less than 10 h. Feulgen microspectrophotometry of the DNA content of individual cells showed a large proportion of cells to be in S phase. The DNA histogram (fig. 2) showed no marked Gl or G2 peaks. Treatment of the cultures with RA decreased the proportion of cells in S phase and increased the number of cells in Gl (fig. 26). The reduction in DNA synthesis was also evident from measurements of the rate of [3H]thymidine incorporation into populations of cel!s (fig. 3). No decrease was observed during the first 24 h but with further culture the incorporation decreased to about 30% of the
456
Linder et al.
I
G? G?
2xG2
k LOI I I I I I
ae 30-
I I I I I I I I I I I I I
20-
lo-
6
12
I
ml I
18
2L
F9 +RA 170 H
times after RA addition. Mean values of three independent experiments expressed as % of untreated cells.
FEULGEN-DNA.pg
Fig. 2. DNA content measured by Feulgen-microspectrophotometry in (a) untreated F9 cells; (b) F9 cells grown in presence of 1 PM retinoic acid for 170h.
initial value. Furthermore growth curves revealed a slowing of the rate of cell proliferation. Time-lapse cinematography clearly showed that cells with RA-induced morphology continued to divide but that the rate of multiplication was considerably slower than that of untreated EC cells. The mean cell
II 20
II I, 40 60 HOURS AFTER
HOURS
Fig. 4. [3H]Leucine incorporation per cell at different
II 60 ADDITION
1 100 OF RA
E‘ig. 3. [3H]Thymidine incorporation per cell at different times after RA addition. The values are mean values of four experiments expressed as % of untreated cells.
cycle time for 8 EC cells followed for 3-4 cell cycles was found to be 10 h. In one experiment cells were treated with RA for 5 days and then kept on RA-free medium for 2 days. Among 70 cells with a typical RA-induced phenotype 34 cells divided during an observation time of 24 h. Effects of retinoic acid on F9 protein synthesis Retinoic acid-treated and untreated F9 cells were labelled for 4 h with [35S]methionine and [3H]leucine. Measurements of the rate of [3H]leucine incorporation into protein showed a reduction in protein synthesis (fig. 4) which seemed to follow the same kinetics as the depression in DNA synthesis. [“5S]Methionine-labelled polypeptides were subjected to electrophoresis in two dimensions (fig. 5). Under our experimental conditions about 500 polypeptide spots could be resolved on one gel. Upon visual inspection it appeared that RA treatment lead to an increased synthesis of at least 26 polypeptides, whereas the synthesis of another 27 polypeptides was significantly decreased. Among the induced polypeptides were two components of the cytoskeleton, vimentin and tropomyosin. The changes in
Retinoic acid-induced EC-cell differentiation MW
457 10
-3
- 200
-92
.69
Fig. 5. Autoradiographs of 2D gels of (a) untreated F9 cells; (b) F9 cells grown in the presence of 1 PM RA for 120 h and labelled with [YS]methionine. Polypeptides marked v (vimentin, 10 nm filament subunit) and
l-3 were quantified (see fig. 6); tm, tropomyosin. Exponential gradient gels (9-20%) were used in the second dimension.
polypeptide synthesis over a 7 day period were determined by labelling F9 cultures for different time periods in RA medium. The 2D separations of F9 cells treated for 24 h were almost identical to those obtained with untreated cells. At 48 h of treatment significant changes in polypeptide synthesis had begun. The new pattern of polypeptide synthesis was stabilized by 120 h and did not change appreciably over the next few days (figs 6, 7). Fig. 6 shows the pattern of polypeptide synthesis in the tropomyosin region of the gels at different time points after the start of the RA treatment. Some of
the changes found in these gels (arrows in fig. 5a) were quantitated by scintillation counting. This analysis confirmed the visual impression that the rate of polypeptide synthesis remained unaltered for the first 24 h and then changed markedly (fig. 7). The synthesis of vimentin and the polypeptide marked “3” increased, whereas the synthesis of polypeptides “1” and “2” decreased. It should be pointed out that the increase in vimentin synthesis is overestimated, since two other spots appeared very close to vimentin after RA inducation (fig.
458
Linder et al.
Fig. 6. Changes in spot intensity in the mol. wt 30K
region. (a) Untreated F9 cells; (b) 24; (c) 48; (d) 72; (e) 120 h after RA addition. (a-e) Arrows pointing upwards indicate polypeptides with increased synthesis; downwards, with decreased synthesis.
. ‘;ii zY10-4 r [
5b). These spots could not be separated from the vimentin spot when the gels were cut. Effects of RA treatment on F9 cell surface components External cell surface components of untreated and RA-treated F9 cells were labelled by lactoperoxidase catalyzed iodination, and then separated by SDS slab gel electrophoresis (fig. 8). The most striking finding was the induction of a 220K molecular weight polypeptide in RA-treated cells. Other changes in the gel profiles were also found. Of these, the appearance of a 32K polypeptide and loss of a 96K component (indicated by arrows in fig. 8) represented the most reproducible changes induced by RA treatment of F9 cells. Since fibronectin is an external cell protein with approximately the same molecular weight as the large polypeptide induced by RA treatment, attempts were made to demonstrate fibronectin (LETS) by immunological methods. F9 embryonal carcinoma cells were fixed with paraformaldehyde and then stained with a rabbit antihuman fibronectin serum. Uninduced EC cells lacked extracellular deposits of immunoreactive material (fig. 9a-b). Some intracellular material could be detected in
-l
\
1 (
,
,
24
40
72
\
\
\
;---; 120
170 HOURS
Fig. 7. Determination of relative incorporation into ‘specific polypeptides at different times after RA polyadministration, O-O, \I, Vimentin; A---A, peptide 1; q - - -0, polypeptide 2; O-O, polypeptide 3. For the identity of these polypeptides, see fig. 5a. Mean values of two separate experiments.
methanol-fixed cells (fig. 9c-d). Cells in RA-treated F9 cultures (one week) showed extensive extracellular networks of fibronectin-positive material. Practically all cells showed the immunofluorescence pattern illustrated in fig. 9f. DISCUSSION It was first reported by Strickland & Mahdavi [4] that F9 embryonal carcinoma cells can be induced to differentiate by treatment with low doses of retinoic acid (RA). The phenotype of RA-induced F9 cells was tentatively identified as parietal endoderm. This conclusion is supported by several observations of enzymic and antigenie markers [4, 14-161. In addition to retinoic acid, hexamethylene bisacetamide (HMBA [17, 181) and plating at low cell density [ 191appear to induce differentiation of EC cells. It is not certain, however, that the same phenotype is obtained with all
Retinoic acid-induced EC-cell differentiation
Fin. 8. External surface polypeptides labelled by lactoperoxidase catalysed iodin&on. (a) F9 EC c&s; (b) F9 cells grown in presence of RA for 7 days. The polypeptides were separated on a 10% SDS slab gel. The most reproducible differences between the cell types are marked by arrows.
9. Fibronectin immunofluorescence staining of (a, d) F9 embryonal carcinoma cells; (e, f, F9 cells treated with retinoic acid for 8 days. The cells were
Fig.
459
these treatments. Furthermore there are indications that different EC lines may react differently to the same induction procedure [20, 211. We find that the morphological alteration takes place simultaneously in practically all cells. One week after RA addition the cultures consist of predominantly one cell type. Our biochemical data therefore reflect a more or less synchronous phenomenon which takes place in virtually all cells. It is of some importance for the interpretation of the biological effects of RA, however, that at later time points other cell types do appear. Cultures analysed several weeks after an initial 4-day induction period contain differentiated cells with a phenotype which is neither EC nor the RA-induced ‘endodermlike’ phenotype. The RA-treated F9 cultures, however, never reach the high level of differentiation observed with multipotent EC lines. The RA-induced effects on cell growth do
fixed with 2 % paraformaldehyde (a, b, e. J) or methanol (c, d). Bar, IO pm.
460
Linder et al.
not occur immediately after addition of RA but seem to manifest themselves after a lag period of some 24 h. Parallel to the reduction in protein synthesis there is also a change in the pattern of polypeptides synthesized. We found a relative increase in the synthesis of at least 26 polypeptides and a significant decrease in the synthesis of some 27 other polypeptides. Our data show that RA induces a major change in gene expression and that this change affects cytoplasmic as well as cell surface components. Also the kinetics of the change in the pattern of polypeptide synthesis appeared to be the same for almost all polypeptides involved, similar to what has been reported for myoblast differentiation [22]. Among the polypeptides which increased were two components of the cytoskeleton: tropomyosin and vimentin. This is of interest in view of previous findings [ 18,231 indicating that spontaneous and HMBA-induced formation of differentiated cells from EC cells is associated with reorganization and altered synthesis of cytoskeletal components. Analysis of cell surface components by the technique of lactoperoxidase-induced iodination showed that RA treatment leads to marked changes in the molecular architecture of the cell periphery. These observations are of interest both in relation to the identity of the RA-induced phenotype and to questions concerning the loss of malignancy when EC cells differentiate into specialized cell types.
This investigation was supported by grants from the Swedish Medical Research Council and Karolinska Institutet.
REFERENCES 1. Martin, G R, Cell 5 (1975) 229. 2. Sherman, M I & Solter, D (ed), Teratomas and differentiation. Academic Press, New York (1975). 3. Sherman, M I & Miller, R A, Dev biol 63 (1978) 27. 4. Strickland, S & Mahdavi, V, Cell 15 (1978) 393. 5. Bernstine, E G, Hooper, M L, Grandchamp, S & Enhrussi, B, Proc natl acad sci US 70 (1973) 3899. 6. Lehman, J M, Speers, WC, Swartzendruber, D E & Pierce, G B, J cell physiol 84 (1974) 13. 7. Chen, T R, Exp cell res 104(1977) 255. 8. O’Farrell. P, J biol them 250 (1975) 4007. 9. Linder, S, Brzeski, H & Ringertz; N R, Exp cell res 120 (1979) 1. 10. Hynes, R, Proc natl acad sci US 70 (1973) 3170. 11. Laemmli. U K. Nature 227 (1970) 680. 12. Stenman; S, Wartiovaara, J & Vaheri, A, J cell biol 74 (1977) 453. 13. Kuusela, P, Ruoslahti, E, Engvall, E & Vaheri, A. Immunochemistrv 13 (1976) 639. 14. Solter, D, Shevinsky, L,‘Knowles, B B & Strickland, S. Dev biol70 (1979) 176. 15. Oshima, R & Linney, E; Exp cell res 126 (1980) 485. 16. Strickland, S & Sawey, M J, Dev biol 78 (1980) 76. 17. Jakob, H, Dubois, P, Eisen, H &Jacob, F, Compt rend acad sci 286 (1978) 109. 18. Paulin, D, Perreau, J, Jakob, H, Jacob, F & Yaniv, M, Proc natl acad US 76 (1979) 1891. 19. Adamson, E D, Gaunt, S J & Graham, C F, Cell 17 (1979) 469. 20. Hogan, B L M, Dev bio176 (1980) 275. 21. Jetten, A M, Jetten, M E R & Sherman, M I, Exp cell res 124 (1979) 381. 22. Devlin. R B & Emerson. C P. Cell 13 (1978) 599. 23. Paulin, D, Nicolas, J F, Yaniv, MI Jacob, F, Weber, K & Osborn, M, Dev biol66 (1978) 488.
Received October 12, 1980 Accepted November 21, 1980
Printed
in Sweden
S. LINDER,
ACID-INDUCED DIFFERENTIATION EMBRYONAL CARCINOMA CELLS IJ. KRONDAHL.
R. SENNERSTAM
OF F9
and N. R. RINGERTZ
Department of Medical Cell Genetics. Medical Nobel Institrrte. Krrrolinskrr Institutet, S-10401 Stockholm. Slc,rden
SUMMARY The ability of retinoic acid (RA) to induce differentiation in embryonal carcinoma (EC) cells was examined by growing mouse F9 cells in a medium containing 1 PM RA. The altered properties of the cells became apparent after a lag period of approx. 24 h and were fully expressed after 5 days. The RA-induced phenotype was characterized by changes in cell morphology, slowing of the rate of cell multiplication, reduced DNA and protein synthesis, altered pattern of polypeptide synthesis and changes in cell surface components. The slowing of cell multiplication and general reduction in the rate of protein synthesis was paralleled by changes in the relative rates at which different polypeptides were synthesized. Two-dimensional gel electrophoretic analysis of [%]methioninelabelled cell proteins showed an altered relative synthesis of at least fifty polypeptides. The relative rate of synthesis of two components of the cytoskeleton identified as vimentin and tropomyosin were shown to increase.
Embryonal carcinoma (EC) cells constitute the growing stem line in a group of tumors known as teratomas. An important characteristic of these tumors is that they also contain many types of differentiated tissues. Transplantation experiments with mouse EC cells have shown that the differentiated cells are formed from EC cells. Furthermore, biochemical and immunological studies have demonstrated enzymic and antigenic similarities between EC cells and cells present in the inner cell mass of blastocysts. The differentiated cell types in mouse teratomas are non-malignant and appear to be identical with cells found in tissues of adult animals. Mouse teratomas therefore represent an interesting model for in vitro studies of early embryonic development (for reviews see [ 1, 21). Several mouse EC lines have been estab-
lished. Under conditions which favor rapid multiplication these lines will consist of EC cells only. Differentiation is favored by cell crowding and aggregation. Many EC lines are described as ‘pluripotent’, i.e. capable of histotypic differentiation. Such lines produce nerve, muscle, cartilage, fat and other specialized cells in vitro. Some lines, among them the F9 line, have been considered unable (nullipotent) to form specialized cell types. While it is true that this cell line has a markedly reduced capacity to differentiate, it does, however, sometimes produce flat endoderm-like cells in small numbers [3]. Studies of in vitro differentiation of pluripotent EC lines are associated with technical difficulties due to the heterogeneity of the cell cultures. Therefore, the finding by Strickland & Mahdavi [4] that retinoic acid
454
Linder et al.
(RA) induced massive differentiation of F9 cells to endoderm-like cells may increase the usefulness of EC cells as an in vitro model of embryonic cell differentiation. In this report we have examined phenotypic changes which occur following RAinduced differentiation of F9 cells. Furthermore we have tried to relate the phenotypic changes to the rate of DNA and protein synthesis and to cell growth and multiplication. MATERIAL
AND METHODS
Cell cultures FY embryonal carcinoma cells [5] were obtained from Drs S. Grandchamp and B. Ephrussi. PYS-2 endodermal cells [6] were generously provided by Dr J. F. Nicolas, Pasteur Institute, Paris. All cells were cultivated in Dulbecco’s modified Eagle’s medium supplemented with non-essential amino acids, nucleosides, sodium pyruvate and 10% fetal calf serum (FCS) (growth medium). Cell cultures were mycoplasma-free as judged by the Hoechst staining method [7]. Cell culture reagents were obtained from Gibco Bio-Cult, Paisley, Scotland. Time-lapse recordings were made with a TV camera (National) and a videotape recorder (National). For induction of differentiation, FY cells were plated in plastic culture dishes or on glass slides to give a cell concentration of aoprox. 3.5-5X 103/cm2.One day after plating, 1~1 of an ethanol solution of retinoic acid (RA, obtained from Sigma Chemical Co.) was added oer ml culture medium to give a final concentration of 1 FM. The medium was subsequently changed every second day. Labelling with [35S]methionine (Radiochemical Centre, Amersham, UK) was carried out for 4 h using 200 &i/ml of the labelled amino acid in methioninefree growth medium.
Preparation of samples for 20 gel electrophoresis of proteins At the end of the labelling period, the cells were washed twice with phosphate buffered saline (PBS) and scraued from the culture dishes. The cells were then washed once with sonication buffer according to O’Farrell [8], freeze-thawed five times and incubated with DNase (50 LLg/ml) for 5 min on ice. Lysis buffer [8] was added followed by solid urea until saturation.
Two-dimensional
gel electrophoresis
Isoelectric focusina and SDS slab eel electroohoresis was performed according to O’Far&l[8] as previously described [Y]. Second dimension gels consisted of Y20% exponential gradients or 10% uniform gels. Dried
gels were exposed to Kodak No-screen X-ray film for approx. 2x 1oRcpmlh. In order to quantitate 19lmethionine incorporation into individual-polypeptidesi spots were cut from the gels using radioactive inkdots as a guide. The radioactivity in each spot was determined by liquid scintillation counting. The relative synthesis was calculated as the activity in one spot divided by the total activity applied to the gel. No correction was made for quenching in acrylamide gels relative to Whatman filters.
Cell surface labelling gel electrophoresis
and SDS slab
Lactoperoxidase-catalysed iodination of external proteins was based on the procedure described bv Hynes [lo]. At the end of the labelling period, the cells were washed with PBS, scraped from the dishes and pelleted. SDS sample buffer (60 mM Tris/HCl, pH 6.8/2.3% SDS/5 % beta-mercaotoethanol/lO% elvcerol) was added and the sampies were boiled 6; 3 min. Iodinated proteins were separated on 10% SDSpolyacrylamide slab gels using the gel system described by Laemmli [ 1I].
Immunofluorescence For antibody staining of external structures, cells were fixed in 2% paraformaldehyde in PBS for 15 min. For additional staining of intracellular components, cells were fixed in methanol for 5 min. Immunofluorescence staining was by the indirect procedure using antihuman fibronectin serum (a gift from Dr Jorma Wartiovaara) as the first antibodv. The soecificitv of this antiserum and its interspecies cross-reactivity have been described elsewhere [12, 131. FITC-conjugated goat anti-rabbit IgG was purchased from Statens Bakteriologiska Laboratorium (Solna, Sweden) and used as the second antibody.
RESULTS Morphological administration
changes upon of RA
Under normal conditions, F9 cultures consisted of more than 99% embryonal carcinoma (EC) cells. The appearance of F9 cells under the phase contrast microscope can be seen in fig. la. The addition of 1 PM retinoic acid (RA) to the .F9 cultures caused the cells to differentiate massively, forming flat, more adherent cells. The morphology of the cells changed rather slowly. The cells started to flatten out 2-3 days after induction and the changes were
Retinoic acid-induced EC-cell differentiation
455
Fig. 1. Appearance of F9 cultures under the inverted phase contrast microscope. (a) Untreated F9 cells; (h-d) F9 cells at different time points after addition of
1 PM retinoic acid. (b) 24; (c) 72; (d) 144 h. Bar, 50 pm.
manifest after approx. 5 days (fig. 1b-d). If by day 4 the RA-containing medium was exchanged for normal medium the altered phenotype persisted for at least several weeks. For the first 3-4 days of RA treatment the changes in morphology appeared to be the same for all cells. As a result the cells became flat and epithelial-like with numerous thread-like extensions. After l-2 weeks of culture it became apparent, however, that RA treatment induced the formation of more than one non-EC phenotype. Most of these phenotypes have not yet been identified. One type of cell could be recognized as fat cells on the basis of morphology and Sudan black staining.
Effects on cell growth Under optimal culture conditions F9 cells have a population doubling time of less than 10 h. Feulgen microspectrophotometry of the DNA content of individual cells showed a large proportion of cells to be in S phase. The DNA histogram (fig. 2) showed no marked Gl or G2 peaks. Treatment of the cultures with RA decreased the proportion of cells in S phase and increased the number of cells in Gl (fig. 26). The reduction in DNA synthesis was also evident from measurements of the rate of [3H]thymidine incorporation into populations of cel!s (fig. 3). No decrease was observed during the first 24 h but with further culture the incorporation decreased to about 30% of the
456
Linder et al.
I
G? G?
2xG2
k LOI I I I I I
ae 30-
I I I I I I I I I I I I I
20-
lo-
6
12
I
ml I
18
2L
F9 +RA 170 H
times after RA addition. Mean values of three independent experiments expressed as % of untreated cells.
FEULGEN-DNA.pg
Fig. 2. DNA content measured by Feulgen-microspectrophotometry in (a) untreated F9 cells; (b) F9 cells grown in presence of 1 PM retinoic acid for 170h.
initial value. Furthermore growth curves revealed a slowing of the rate of cell proliferation. Time-lapse cinematography clearly showed that cells with RA-induced morphology continued to divide but that the rate of multiplication was considerably slower than that of untreated EC cells. The mean cell
II 20
II I, 40 60 HOURS AFTER
HOURS
Fig. 4. [3H]Leucine incorporation per cell at different
II 60 ADDITION
1 100 OF RA
E‘ig. 3. [3H]Thymidine incorporation per cell at different times after RA addition. The values are mean values of four experiments expressed as % of untreated cells.
cycle time for 8 EC cells followed for 3-4 cell cycles was found to be 10 h. In one experiment cells were treated with RA for 5 days and then kept on RA-free medium for 2 days. Among 70 cells with a typical RA-induced phenotype 34 cells divided during an observation time of 24 h. Effects of retinoic acid on F9 protein synthesis Retinoic acid-treated and untreated F9 cells were labelled for 4 h with [35S]methionine and [3H]leucine. Measurements of the rate of [3H]leucine incorporation into protein showed a reduction in protein synthesis (fig. 4) which seemed to follow the same kinetics as the depression in DNA synthesis. [“5S]Methionine-labelled polypeptides were subjected to electrophoresis in two dimensions (fig. 5). Under our experimental conditions about 500 polypeptide spots could be resolved on one gel. Upon visual inspection it appeared that RA treatment lead to an increased synthesis of at least 26 polypeptides, whereas the synthesis of another 27 polypeptides was significantly decreased. Among the induced polypeptides were two components of the cytoskeleton, vimentin and tropomyosin. The changes in
Retinoic acid-induced EC-cell differentiation MW
457 10
-3
- 200
-92
.69
Fig. 5. Autoradiographs of 2D gels of (a) untreated F9 cells; (b) F9 cells grown in the presence of 1 PM RA for 120 h and labelled with [YS]methionine. Polypeptides marked v (vimentin, 10 nm filament subunit) and
l-3 were quantified (see fig. 6); tm, tropomyosin. Exponential gradient gels (9-20%) were used in the second dimension.
polypeptide synthesis over a 7 day period were determined by labelling F9 cultures for different time periods in RA medium. The 2D separations of F9 cells treated for 24 h were almost identical to those obtained with untreated cells. At 48 h of treatment significant changes in polypeptide synthesis had begun. The new pattern of polypeptide synthesis was stabilized by 120 h and did not change appreciably over the next few days (figs 6, 7). Fig. 6 shows the pattern of polypeptide synthesis in the tropomyosin region of the gels at different time points after the start of the RA treatment. Some of
the changes found in these gels (arrows in fig. 5a) were quantitated by scintillation counting. This analysis confirmed the visual impression that the rate of polypeptide synthesis remained unaltered for the first 24 h and then changed markedly (fig. 7). The synthesis of vimentin and the polypeptide marked “3” increased, whereas the synthesis of polypeptides “1” and “2” decreased. It should be pointed out that the increase in vimentin synthesis is overestimated, since two other spots appeared very close to vimentin after RA inducation (fig.
458
Linder et al.
Fig. 6. Changes in spot intensity in the mol. wt 30K
region. (a) Untreated F9 cells; (b) 24; (c) 48; (d) 72; (e) 120 h after RA addition. (a-e) Arrows pointing upwards indicate polypeptides with increased synthesis; downwards, with decreased synthesis.
. ‘;ii zY10-4 r [
5b). These spots could not be separated from the vimentin spot when the gels were cut. Effects of RA treatment on F9 cell surface components External cell surface components of untreated and RA-treated F9 cells were labelled by lactoperoxidase catalyzed iodination, and then separated by SDS slab gel electrophoresis (fig. 8). The most striking finding was the induction of a 220K molecular weight polypeptide in RA-treated cells. Other changes in the gel profiles were also found. Of these, the appearance of a 32K polypeptide and loss of a 96K component (indicated by arrows in fig. 8) represented the most reproducible changes induced by RA treatment of F9 cells. Since fibronectin is an external cell protein with approximately the same molecular weight as the large polypeptide induced by RA treatment, attempts were made to demonstrate fibronectin (LETS) by immunological methods. F9 embryonal carcinoma cells were fixed with paraformaldehyde and then stained with a rabbit antihuman fibronectin serum. Uninduced EC cells lacked extracellular deposits of immunoreactive material (fig. 9a-b). Some intracellular material could be detected in
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Fig. 7. Determination of relative incorporation into ‘specific polypeptides at different times after RA polyadministration, O-O, \I, Vimentin; A---A, peptide 1; q - - -0, polypeptide 2; O-O, polypeptide 3. For the identity of these polypeptides, see fig. 5a. Mean values of two separate experiments.
methanol-fixed cells (fig. 9c-d). Cells in RA-treated F9 cultures (one week) showed extensive extracellular networks of fibronectin-positive material. Practically all cells showed the immunofluorescence pattern illustrated in fig. 9f. DISCUSSION It was first reported by Strickland & Mahdavi [4] that F9 embryonal carcinoma cells can be induced to differentiate by treatment with low doses of retinoic acid (RA). The phenotype of RA-induced F9 cells was tentatively identified as parietal endoderm. This conclusion is supported by several observations of enzymic and antigenie markers [4, 14-161. In addition to retinoic acid, hexamethylene bisacetamide (HMBA [17, 181) and plating at low cell density [ 191appear to induce differentiation of EC cells. It is not certain, however, that the same phenotype is obtained with all
Retinoic acid-induced EC-cell differentiation
Fin. 8. External surface polypeptides labelled by lactoperoxidase catalysed iodin&on. (a) F9 EC c&s; (b) F9 cells grown in presence of RA for 7 days. The polypeptides were separated on a 10% SDS slab gel. The most reproducible differences between the cell types are marked by arrows.
9. Fibronectin immunofluorescence staining of (a, d) F9 embryonal carcinoma cells; (e, f, F9 cells treated with retinoic acid for 8 days. The cells were
Fig.
459
these treatments. Furthermore there are indications that different EC lines may react differently to the same induction procedure [20, 211. We find that the morphological alteration takes place simultaneously in practically all cells. One week after RA addition the cultures consist of predominantly one cell type. Our biochemical data therefore reflect a more or less synchronous phenomenon which takes place in virtually all cells. It is of some importance for the interpretation of the biological effects of RA, however, that at later time points other cell types do appear. Cultures analysed several weeks after an initial 4-day induction period contain differentiated cells with a phenotype which is neither EC nor the RA-induced ‘endodermlike’ phenotype. The RA-treated F9 cultures, however, never reach the high level of differentiation observed with multipotent EC lines. The RA-induced effects on cell growth do
fixed with 2 % paraformaldehyde (a, b, e. J) or methanol (c, d). Bar, IO pm.
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Linder et al.
not occur immediately after addition of RA but seem to manifest themselves after a lag period of some 24 h. Parallel to the reduction in protein synthesis there is also a change in the pattern of polypeptides synthesized. We found a relative increase in the synthesis of at least 26 polypeptides and a significant decrease in the synthesis of some 27 other polypeptides. Our data show that RA induces a major change in gene expression and that this change affects cytoplasmic as well as cell surface components. Also the kinetics of the change in the pattern of polypeptide synthesis appeared to be the same for almost all polypeptides involved, similar to what has been reported for myoblast differentiation [22]. Among the polypeptides which increased were two components of the cytoskeleton: tropomyosin and vimentin. This is of interest in view of previous findings [ 18,231 indicating that spontaneous and HMBA-induced formation of differentiated cells from EC cells is associated with reorganization and altered synthesis of cytoskeletal components. Analysis of cell surface components by the technique of lactoperoxidase-induced iodination showed that RA treatment leads to marked changes in the molecular architecture of the cell periphery. These observations are of interest both in relation to the identity of the RA-induced phenotype and to questions concerning the loss of malignancy when EC cells differentiate into specialized cell types.
This investigation was supported by grants from the Swedish Medical Research Council and Karolinska Institutet.
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Received October 12, 1980 Accepted November 21, 1980
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