Production of PDGF-like growth factors by embryonal carcinoma cells and binding of PDGF to their endoderm-like differentiated cells

DEVELOPMENTAL

BIOLOGY

110,

15-22 (1985)

Production of PDGF-like Growth Factors by Embryonal Carcinoma Ceils and Binding of PDGF to Their Endoderm-like Differentiated Cells ANGIE ‘Eppley

Institute

RIZZINO*

AND DANIEL

f&r Research in Cancer and Allied Diseases, University TDepartment of Pathology, School of Medicine, University Received March

F. BOWEN-POPE? of Nebraska Medical Center, Omaha, Nebraska of Washington, Seattle, Washington 981%

19, 1984; accepted in revised form

February

68105, and

5, 1985

In this report, we demonstrate that F9 and PC-13 embryonal carcinoma (EC) cells do not bind significant amounts of platelet-derived growth factor (PDGF), whereas the endoderm-like differentiated cells derived from EC cells do. The FS-differentiated cells exhibit approximately 8300 receptors per cell, with an apparent dissociation constant of 30 PM. Two endoderm-like cell lines, PSA-5E and PYS-2, also bind PDGF and exhibit approximately 4800 and 23,500 receptors per cell, respectively. The lack of PDGF binding by the parental EC cells is consistent with their release of a factor(s) that is closely related to PDGF. This factor(s) competes with PDGF for binding to membrane receptors and is recognized by antibodies raised against PDGF. However, this factor(s) does not appear to be antigenieally identical to PDGF. We also show that production of this PDGF-like factor(s) is reduced more than 90% when F9 EC cells differentiate into cells that bind PDGF. Thus, our findings indicate that EC cells release a factor(s) that should be capable of binding to their differentiated cells. This raises the possibility that PDGF, or a closely related factor, can influence cell proliferation and/or cell behavior of early embryonic cells. o 1985 Academic Press, IIIC. INTRODUCTION

of differentiated cells derived from EC cells (Rizzino et aZ., 1980; Isacke and Deller, 1983; Heath and Isacke, 1984), (2) support the establishment of pluripotent cell lines from early mouse embryos (Martin, 1981), (3) promote the anchorage-independent growth of nontransformed cells (Rizzino, 1982a, 1983b; Rizzino et ah, 1983; Heath and Isacke, 1983a), (4) exhibit the properties of an insulin-like growth factor (Heath and Isacke, 1983b), and (5) compete with human PDGF for binding to membrane receptors (Gudas et ah, 1983). At least in the third example, it appears that early mouse embryos produce similar factors (Rizzino, 1982a, 1983b). The study of EC cells in defined and serum-free media has determined that EC cell lines respond to relatively few hormones and growth factors (Rizzino and Crowley, 1980; Rizzino and Sato, 1978) and this may be related to the production of endogenous growth factors by EC cells. In contrast, differentiated cells derived from several different EC cell lines have been shown to exhibit receptors for, or respond to, a variety of hormones and growth factors, including: Epidermal growth factor (EGF) (Rees et aZ., 1979; Jetten, 1980; Salomon, 1981; Rizzino et al., 1983), insulin (Heath et al, 1981), fibroblast growth factor (FGF) (Rizzino, 1983a), and transforming growth factors (TGFs) (Rizzino et al., 1983). Thus far, the parental EC cells have not been shown to respond to these factors and apparently lack receptors for them. Only insulin appears to be an exception, and only in some cases. Certain EC cell lines, such as F9, respond to insulin (Rizzino and

The role of hormones and growth factors during the early stages of mammalian development is poorly defined. One approach to this problem has been the use of mouse embryonal carcinoma (EC’) cells. These cells are recognized as a useful model system, since they are able to closely mimic, both morphologically and biochemically, important stages of early mammalian development (Martin, 1980; Solter and Damjanov, 1979; Graham, 197’7). In the case of F9 EC cells, exposure to retinoic acid (RA) in either serum-containing media (Strickland and Mahdavi, 1978; Hogan et al., 1981) or in defined media (Rizzino and Crowley, 1980) induces differentiation. The endoderm-like cells that form exhibit many of the properties of extraembryonic endoderm, which is one of the first cell types to form during early mammalian development. The study of EC cells under various culture conditions has established that EC cells condition their culture medium. Although the factors involved have not been adequately characterized, it appears that many factors are produced, including several endogenous growth factors. Thus far, several different activities have been identified, including those that: (1) promote the growth ’ Abbreviations used: EC, embryonal carcinoma; PDGF, plateletderived growth factor; RA, retinoic acid; EGF, epidermal growth factor; FGF, fibroblast growth factor; TGFs, transforming growth factors; DME, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; ICM, inner cell mass. 15

0012-1606/85 $3.00 Copyright All rights

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

16

DEVELOPMENTAL BIOLOGY

Sato, 1978; Rizzino and Crowley, 1980) and exhibit insulin receptors (Nagarajan and Anderson, 1982), whereas others, such as PC-13, exhibit little or no response to insulin and exhibit few insulin receptors (Heath et al, 1981). To better understand the role of growth factors during early development, this study has examined the binding of PDGF to two EC cell lines and to endodermlike cells derived from EC cells. Human PDGF is a 30,000-Da protein present in blood platelets (Raines and Ross, 1982; Antoniades et al., 1979; Heldin et al., 1979; Deuel et ah, 1981) and it is one of the major mitogens in serum. PDGF is active in stimulating the growth of cultured connective tissue cell types and has been proposed to play a role in stimulating cell proliferation in vivo at sites of vascular damage (Ross and Glomset, 1976). Our findings indicate that EC cells do not bind exogenously added PDGF but do release a PDGF-like growth factor(s). In direct contrast, endoderm-like cells derived from EC cells do not release this PDGF-like factor(s) and differentiation to endoderm-like cells is accompanied by a marked increase in ability to bind exogenously added PDGF. We discuss these findings in terms of their possible relationship to the production of TGFs and to the expression of oncogenes by EC cells. MATERIALS

AND

METHODS

Cells. Stock cultures of F9 EC cells (Bernstine et ak, 1973) and PC-13 EC cells, clone lA4 (Bernstine et ah, 1973), were cultured on gelatin-coated surfaces. F9 cells were grown in Dulbecco’s modified Eagle’s medium (DME-GIBCO 430-2100) containing 15% fetal calf serum (FCS-Reheis). PC-13, PSA-5E (Adamson et al., 1977), and PYS-2 (Lehman et al, 1974) were grown in a-medium (GIBCO 410-1900) containing 10% FCS. Human diploid foreskin fibroblasts were grown in DME plus 5% calf serum. All cells were cultured at 37°C in a humidified atmosphere of 95% air and 5% COZ.

Preparation and use of factors fin- media supplementation. Insulin, transferrin, RA, FGF, and EGF were prepared for use in culture as described previously (Rizzino and Crowley, 1980; Rizzino, 1983a; Rizzino et aZ., 1983). Highly purified human PDGF, prepared as described elsewhere (Raines and Ross, 1982), was a gift of R. Ross and E. Raines (Univ. of Washington, Seattle, Wash.). Highly purified FGF was obtained from Toyobo Limited, Osaka, Japan. Preparation of conditioned media. Conditioned media were prepared from F9, PC-13, FS-differentiated cells, PSA-5E, and PYS-2. In each case, the cells were plated on lOO- or 150-mm dishes in serum-containing medium (the same as that used for stock cultures). After 48 hr,

VOLUME 110, 1985

the medium was removed and the cells were washed twice with serum-free medium for 1 hr. Both washes were discarded and the cells were fed a third time with serum-free medium. After 24 hr, the conditioned medium was collected and clarified by centrifugation (100,OOOg for 45 min). The medium was then concentrated (lo- to 30-fold) by ultrafiltration using an Amicon YM-5 membrane. The concentrated conditioned medium was then stored at -20°C until used. The serum-free medium used to prepare the conditioned medium was a 1:l mixture of DME and Ham’s F-12 medium, supplemented with 25 nM selenous acid, 15 mM Hepes buffer (pH 7.4), 1 pg/ml insulin, and 1 pg/ml transferrin. This serum-free medium is a modification of the defined medium, EM-3, which is used to culture F9 EC cells in the absence of serum (Rizzino and Crowley, 1980; Rizzino, 198413). PDGF binding. The binding of PDGF to EC cells and to their differentiated cells was determined as described previously (Bowen-Pope and Ross, 1982). Briefly, the culture medium was removed and the cells (cultured in 16-mm wells) were washed three times with binding rinse (PBS with 0.25% BSA) at 4°C. The last rinse was removed and binding medium (Hepesbuffered Ham’s F-12 medium with 0.25% BSA, pH 7.4) containing ‘251-PDGF (sp act ranged from 800 to 2000 dpm/fmole) was added. The cells were incubated at 4°C with gentle shaking for 4 hr. Binding was terminated by washing the cells four times with binding rinse. The cell-bound lz51-PDGF was solubilized with 1% Triton X-100 and quantitated by gamma counting. Nonspecific binding was measured in samples to which 10 pg/ml of partially purified PDGF (10% pure) was added in addition to the 1251-PDGF. Saturation binding plots were carried out as described above and the concentration of PDGF was varied from 0.125 to 6 ng/ ml. The values obtained from the saturation binding plot, plus the levels of unbound PDGF, were used to plot the data according to the method of Scatchard (1949). In the experiment to examine the time-dependent appearance of receptors for PDGF (Table 1) and in the experiment to examine the ability of other growth factors to influence the binding of ‘251-PDGF (Table 3), a single concentration of ‘251-PDGF (0.5 ng/ ml) was used. Measurements of PDGF-like growth factors in conditioned media. The levels of PDGF-like growth factors in the conditioned media were determined by a radioreceptor assay using human diploid fibroblasts as described previously (Bowen-Pope and Ross, 1985). Briefly, the assay was performed as follows: cultures of diploid human fibroblasts were rinsed once with cold PBS, incubated with 1 ml test solution in binding medium for 3 hr at 4°C with continuous oscillatory mixing,

RIZZINO AND BOWEN-POPE

PDGF and Embymal

rinsed once with cold PBS, then incubated with 1 ml binding medium containing 0.5 rig/ml lz51-PDGF for 1 hr at 4°C with continuous oscillatory mixing. The cultures were rinsed four times with cold binding rinse and the cell-bound lz51-PDGF was solubilized with 1% Triton X-100. Standard curves were obtained using pure PDGF standards (0.025 to 0.8 rig/ml) and 20 ng/ ml pure PDGF to determine nonspecific binding (lo20% of total binding). In each case, three or more different dilutions of the conditioned media were used to determine the level of the PDGF-like growth factor present in the conditioned media. The values from each dilution were averaged to arrive at the concentration reported. It should be noted that for these assays (Tables 4, 5) a sequential assay was used, rather than a simultaneous assay in which iz51-PDGF and the competitor are added at the same time. Although a simultaneous assay can be used, the sequential assay avoids the complications that can result from the presence of PDGF-binding proteins in the competitor sample to be tested. Such binding proteins have been found in plasma (Huang et al., 1983; Raines et al., 1984; Bowen-Pope et al., 198413)and have been shown to yield “false positive” results (Bowen-Pope et al, 198413).

17

Carcinoma Cells

TABLE 1 THE TIME-DEPENDENTAPPEARANCEOF PDGF RECEPTORS ONEC CELL-DERIVEDDIFFERENTIATEDCELLS Cells F9 FS-differentiated (Day 3) FS-differentiated (Day 6) PC-13 PC-13-differentiated (Day 3) PC-13-differentiated (Day 6)

‘%I-PDGF bound (cpm/106 cells) 49

Binding relative to parental EC cells 1

1430

29.3

4134

84.7

234

1

701

3

4089

17.5

Note. The binding of 0.5 rig/ml ‘%I-PDGF to the cells was carried out as described under Materials and Methods. The values given are the average of triplicate samples, which usually varied by less than 10% and in all cases by less than 15%. The F9 and PC-13 EC cells were plated 48 hr prior to measuring ‘251-PDGF binding. The differentiated cells were plated in serum-containing medium plus RA for 48 hr, at which time the medium was removed and replaced with fresh serum-containing medium without RA either for 24 hr (Day 3 cells) or for 96 hr, with one medium change after 48 hr (Day 6 cells).

of the Scatchard plot at the low concentrations of bound lz51-PDGF is unclear. However, a similar, though less pronounced, curvature has been observed in the Binding of PDGF to EC Cells and to Their binding of PDGF to other cells, and may result from Diflerentiated Cells positive cooperativity or artifactual inactivation of The ability of lz51-PDGF to bind to EC cells and PDGF at low concentrations (Bowen-Pope and Ross, their differentiated cells was initially examined with 1982). By similar analyses, two endoderm-like cell F9 EC cells and their RA-induced differentiated cells. lines, PYS-2 and PSA-5E, were found to exhibit apThe F9 cells bound very little PDGF (Table l), whereas proximately 23,500 and 4800 receptors per cell, respectheir endoderm-like differentiated cells bound signifitively, whereas the F9 EC cells bound fewer than 200 cant amounts of PDGF. In the latter case, the amount molecules of PDGF per cell (Table 2). bound was dependent on the number of days that had In a related experiment, the ability of other factors elapsed after differentiation was induced. At the end to interfere with the binding of PDGF was examined. of the 48-hr exposure to RA (Day Z), the differentiated Except in the case of PDGF, the factors were used at cells bound very little PDGF (data not shown), but by concentrations that optimally stimulated growth of Day 3 the differentiated cells bound approximately 30 the differentiated cells. EGF, insulin, and transferrin times more PDGF than the F9 cells, and by Day 6 did not interfere with the binding of PDGF to the F9they bound greater than 80 times more (Table 1). differentiated cells, and FGF slightly increased the Similar, though less dramatic, results were observed binding of PDGF (Table 3). with PC-13 EC cells treated with RA (Table 1). Although the differentiated cells derived from PC-13 cells EC Cells Release a Factor Related to PDGF under these conditions have not been completely characterized, they are known to exhibit some of the The failure of F9 and PC-13 EC cells to bind significant amounts of PDGF and the ability of conditioned properties of endoderm (Adamson et al., 1979). The binding of PDGF was examined more closely by media prepared from EC cells to stimulate the growth both Scatchard and saturation binding plots. By both of their differentiated cells (Rizzino et al, 1980; Isacke methods, the FS-differentiated cells (6 days after RA and Deller, 1983) were consistent with the report that treatment) were found to have approximately 8300 PSA-1 EC cells do not bind PDGF but do release a receptors per cell, with an apparent dissociation con- PDGF-like factor (Gudas et al, 1983). Consequently, serum-free media conditioned by F9 and PC-13 EC stant of 30 pM (Fig. 1). The reason(s) for the curvature RESULTS

18

DEVELOPMENTAL BIOLOGY

VOLUME 110, 1985 TABLE BINDINGOF~~I-PDGF

Cells

2 ‘251 PDGF4nglml)

6

B

I 125f PDGF Bound (fmole/106cell) FIG. 1. Binding of PDGF to FS-differentiated cells. (A) Saturation binding plot. The binding of PDGF to FS-differentiated cells was determined as described under Materials and Methods. The differentiated cells were derived from F9 EC cells (in 16-mm wells) cultured in serum-containing medium plus RA (5 X 1Om6M) for 48 hr. The medium was changed at the end of Days 2 and 5 with serumcontaining medium lacking RA. PDGF binding was measured 24 hr later, on Day 6. (B) Scatchard plot. The values obtained from the saturation binding plot (plus the unbound PDGF) were plotted according to the method of Scatchard (1949).

cells were examined to determine whether they contain factors able to compete with human ‘%I-PDGF for binding to membrane receptors on human diploid fibroblasts. Both EC cell lines were found to release a factor related to PDGF (Table 4). In the case of F9 cells, approximately 240 pg of PDGF-competing activity were released by lo6 cells in a 24-hr period. In contrast, the FS-differentiated cells appeared to release approximately lo-fold less competing activity. To test the possibility that this could be due to the release of a PDGF binding protein, such as that found in plasma (Huang et al., 1983; Bowen-Pope et al, 198413;Raines et

Apparent dissociation constant

Receptors/cell

F9 FS-differentiated PYS-2 PSAdE

0.18-

2

TOFU EC CELLSANDEC CELL-DERIVED ENDODERM-LIKECELLS

<200 8,300 23,500 4,800

ND 3 x lo-” 5 x 10-I’ 5 x lo-”

Note. The binding of ‘%I-PDGF was determined as described under Materials and Methods. The number of receptors per cell and the apparent dissociation constants were derived by plotting the data according to the method of Seatchard. The actual plots are shown only for the FS-differentiated cells (Fig. 1). In the cases of F9, PYS-2, and PSAdE, the cells were cultured in serum-containing medium for 48 hr prior to measuring the binding of ‘%I-PDGF. The FS-differentiated cells were cultured as described in Table 1 and binding was measured on Day 6. The apparent dissociation constant for the F9 cells was not determined (ND) due to the limited binding of ‘%I-PDGF to these cells.

aL, 1984), conditioned medium prepared from F9 EC cells was mixed with conditioned medium prepared from their differentiated cells. Our findings indicate that medium conditioned by FS-differentiated cells does not contain components that prevent detection of the PDGF-like material in medium conditioned by F9 EC cells (Table 5). Thus, the differentiation of these EC cells is accompanied by a large reduction in the production of the PDGF-like factor and by acquisition of the ability to bind exogenously added PDGF. The similarity between PDGF and the PDGF-like factor(s) released by EC cells was examined using TABLE

3

EFFECTSOFGROWTH-PROMOTING FACTORSONTHEBINDING OFPDGF TO F9-DIFFERENTIATED CELLS

Factor

added

None PDGF (1 rig/ml) EGF (10 rig/ml) FGF (100 rig/ml) Insulin (5 Kg/ml) Transferrin (5 @g/ml)

?-PDGF (cpm/lO’ 382 120 374 460 356 357

bound cells)

% Change 0 -69 -2 +21 -7 -6

Note. The FS-differentiated cells (2.9 X 106 cells per well) were derived from F9 cells by a 48-hr exposure to RA. At that point, the cells were fed with fresh serum-containing medium. After 3 days, the cells were refed with fresh serum-containing medium and the specific binding of ‘%I-PDGF was determined 24 hr later, as described under Materials and Methods. In this experiment, the indicated competing factors were added at the same time as the ?-PDGF. The values shown are the average of three samples, which varied by less than 10%.

RIZZINO

TABLE PRODUCTION

AND

BOWEN-POPE

PDGF

4

OF PDGF-LIKE FACTORS BY EC CELLS AND RELATED CELLS

PDGF-competing activity (pg)

Cells

and

Embryonal

Carcinoma

Cells

19

F9 conditioned medium (Fig. 2). Thus, it appears that the PDGF-like growth factor(s) released by mouse EC cells is antigenically very similar to mouse PDGF, but not identical to it. DISCUSSION

FS FS-Differentiated

240 16

PC-13 PSA-5E PYS-2

280 20 3

Note. Conditioned media were prepared from the indicated cells as described under Materials and Methods. The level of PDGFcompeting activity was determined by a radioreceptor assay for PDGF, as described under Materials and Methods. The values given are for the amounts released into 1 ml of medium by lo6 cells during a 24-hr period. In each case, three or more dilutions of the concentrated conditioned medium were used to determine the amount of the PDGF-like growth factor present. The values shown represent the average of the amounts measured at the different dilutions after correction for the dilution factor.

PDGF-specific antibodies prepared against human PDGF. Previous studies have shown that this antiserum does not react with other growth factors, including EGF and FGF (Bowen-Pope et aL, 1984a), and we have determined that the preimmune serum does not react with PDGF in mouse serum or with the PDGF activity released by F9 EC cells (Fig. 2). In contrast, the antiPDGF neutralized all of the PDGF activity in mouse serum and, under these conditions, 90% of the PDGFlike activity in medium conditioned by EC cells. HOWever, more anti-PDGF was needed to neutralize the activity released by EC cells. The amount of antibody needed to neutralize 50% of mouse PDGF was 15 pg/ ml compared to 150 pg/ml for the PDGF activity in TABLE LACK

5

OF INTERACTION BETWEEN FACTORS RELEASED AND THEIR DIFFERENTIATED CELLS

FS CM FS-differentiated CM FS CM and FS-differentiated

0.237 CM

IgG,pglml

BY EC CELLS

PDGF-competing activity (rig/ml)

Sample

The data presented here demonstrate that the endoderm-like cells derived from EC cells bind exogenously added human PDGF, whereas the parental EC cells (F9 and PC-13) do not. The endoderm-like cells derived from F9 EC cells have approximately 8300 PDGF receptors per cell and the endoderm-like cell lines, PYS-2 and PSA-5E, exhibit approximately 23,500 and 4800 receptors per cell, respectively. The failure of F9 and PC-13 to bind significant amounts of PDGF is consistent with the finding that these mouse EC cell lines release a factor(s) that: (1) competes with human PDGF for binding to membrane receptors, and (2) is antigenically very similar, though apparently not identical, to mouse PDGF. In addition, we have determined that differentiation of F9 EC cells results in a significant reduction in the production of this PDGF-like factor(s). In this regard, since a small proportion of the parental

0.02 0.235

Note. Concentrated conditioned medium (CM) from FS cells and FS-differentiated cells, either alone or together, were incubated overnight at 4°C. The following day, the level of PDGF-competing activity was determined by a radioreceptor assay for PDGF, as described under Materials and Methods. In each case, several dilutions were used to determine the amount of PDGF-competing activity present. The values shown represent the average of the amounts measured at the different dilutions after correction for the dilution factor.

FIG. 2. Neutralization of PDGF-like activity in FS-conditioned medium by antibody against PDGF. FS-conditioned medium was concentrated by ultrafiltration and diluted with binding medium to give a final concentration of 0.29 ng of PDGF-like activity per milliliter (squares). Mouse whole blood was drawn from the retroorbital plexus, clotted overnight at room temperature, centrifuged to remove the clot and the serum diluted with binding medium to give a final concentration of 2% (0.19 rig/ml of mouse PDGF activity) (circles). These solutions were incubated for 1 hr at 4°C with the concentrations of goat anti-PDGF IgG (closed symbols) or goat nonimmune IgG (open symbols) indicated on the abscissa, then assayed for PDGF activity by radioreceptor assay as described under Materials and Methods. The results were normalized to the amount of PDGF-like activity observed in the absence of antibody. The results are plotted as the mean plus or minus the range of two experiments. For clarity, the ranges for the nonimmune IgG are not shown, but were usually within 10% of the mean and always within 20%.

20

DEVELOPMENTALBIOLOGY

EC cells does not differentiate under the conditions used in this study, there may, in fact, be complete cessation in the production of this factor(s) by the differentiated cells. At present, it is unclear why EC cells do not bind exogenously added PDGF. It is possible that the cells do not produce receptors for PDGF or that the receptors are occupied and down regulated by the PDGF-like growth factor(s) released by the EC cells. If the latter is the case, then EC cells may not respond to exogenously added PDGF because they produce all the PDGF-like activity that they require, as has been proposed for other systems in which autocrine growth control may function (De Larco and Todaro, 1978; Todaro et al, 1981; Seifert et al, 1984). In this regard, it is noteworthy that PDGF elicits a small but consistent growth response from the endoderm-like cells derived from EC cells, which exhibit receptors for PDGF, whereas the EC cells do not respond mitogenically to exogenously added PDGF (unpublished results). In an earlier study, it was reported that the multipotent EC cell line, PSA-l-G, releases a high level of a PDGF-like factor (approximately 4-7 ng per lo6 cells per 24 hr) (Gudas et ak, 1983). In the same study, it was reported that F9 EC cells release less of the factor but its concentration was not quantitated. The data presented here demonstrate that the F9 and PC-13 cells release 20- to 25fold less of the PDGF-like factor than is reported for PSA-1-G EC cells. The reason for this remains unclear, but the lower levels released by F9 and PC-13 may reflect their limited ability to differentiate into many cell types (Rizzino, 1982b). In contrast to the study by Gudas et al. (1983), which reported only a small increase (less than 2-fold by Day 3) in the binding of PDGF by the FS-differentiated cells, our data indicate that the differentiation of F9 EC cells results in a large increase in the binding of PDGF (nearly 30-fold by Day 3 and over 80-fold by Day 6). Part of the explanation for this discrepancy may lie in the conditions used to assay the binding of PDGF to the cells (e.g., a 2-hr assay versus the 4-hr assay used here), but this seems unlikely. Alternatively, since the receptors for PDGF are slow to appear on the differentiated cells, and cannot be detected on Day 2 (unpublished results), the different results reported in the two studies may reflect different rates of differentiation, perhaps due to the use of different concentrations of RA (50 versus 500 nM used in our study). However, it is not known whether the concentration of RA can influence the rate of differentiation. Thus, a satisfactory explanation is not readily apparent at this time.

VOLUMEHO,1985

The PDGF-like activity released by the EC cells may be related to another activity released by these cells. Previous studies have shown that EC cells release a factor(s) that promotes the anchorage-independent growth of normal rat kidney (NRK) fibroblasts (Rizzino, 1982a; Rizzino et al, 1983). Factors with this property are currently referred to as transforming growth factors or TGFs (reviewed by De Larco, 1983). However, the TGF-like factor(s) released by EC cells differs (Rizzino, 1983b) from the two TGFs (TGF-a and TGFp) that have been purified thus far (reviewed by Roberts et aZ., 1983). As in the case of the PDGF-like activity, differentiation of EC cells results in a large reduction in the release of the TGF-like activity (Rizzino, 1982a). Thus, the expression of these activities may be coordinately controlled. Alternatively, it is possible that one factor is responsible for both activities. Some evidence for the latter possibility is provided by the recent finding that highly purified human PDGF can induce the soft agar growth of NRK cells in a serumfree medium developed to assay TGFs (Rizzino, 1984b,c). Studies are underway to resolve this question by determining whether the PDGF-like and TGF-like activities released by EC cells copurify and exhibit the same biological and chemical properties. There is another interesting aspect to the production of a PDGF-like growth factor by EC cells and by a number of different transformed cell lines (Bourne and Rozengurt, 1976; Heldin et aZ.,1980; Graves et al, 1983; Singh et ak, 1983; Bowen-Pope et ah, 1984a). Recent studies have demonstrated that the oncogene, v-sis, isolated from simian sarcoma virus codes for a polypeptide whose amino acid sequence is very similar to that of PDGF (Doolittle et al, 1983; Waterfield et ah, 1983). As expected, cells transformed by simian sarcoma virus have been shown to produce a factor that is closely related to PDGF (Deuel et al., 1983; Owen et al., 1984). In addition, it appears that human c-sis codes for at least the A chain of PDGF (Huang et ah, 1984; Chiu et aZ., 1984). Consequently, the possibility exists that EC cells express c-sis, or a related gene, and that this gene is repressed when EC cells differentiate to endoderm-like cells. In this regard, it is noteworthy that several other oncogenes appear to be differentially expressed during murine and human development (Muller et al., 1982, 1983). It is clear from this discussion that many questions concerning the nature and the function of the PDGFlike factor(s) released by EC cells remain to be answered, including the possibility that the same factor is produced by the cells of the inner cell mass (ICM) during the early stages of mammalian development. Although this possibility cannot be examined readily

RIZZINO AND BOWEN-POPE

PDGF

at this time, there are two reasons for suspecting that this may occur. First, EC cells and the cells of the ICM, which give rise to the embryo proper, share numerous properties (reviewed by Martin, 1980; Graham, 1977). Second, both EC cells (Rizzino et ab, 1983) and the cells of the ICM (or its derivatives) have been found to produce TGF-like factors (Rizzino, 1982a). If the cells of the ICM do produce a PDGF-like growth factor (an embryonic form of PDGF), this could provide a mechanism for the ICM to directly influence the growth of early embryonic cells derived from it, which would mean that paracrine secretion (Dockray, 1979) begins to play a role relatively early during mammalian embryogenesis. We thank Linda Dorman for technical assistance and Heather Rizzino for editorial assistance. We also thank E. Raines and R. Ross for the generous gifts of purified PDGF and anti-PDGF antibodies. Part of this research (A.R.) was supported by, and performed at, the National Cancer Institute, Frederick, Md. and by grants (SO7 RR05391 and 22-271-732) from the University of Nebraska Medical Center and a grant (CA 36727) from the National Cancer Institute. This research was also supported (D.B.-P.) by NIH Grant HL 18645, plus grants from R. J. Reynolds, Inc. and the Life and Health Insurance Medical Research Fund. REFERENCES ADAMSON, E. D., EVANS, M. J., and MAGRANE, G. G. (1977). Biochemical markers of the progress of differentiation in cloned teratocarcinoma cell lines. Eur. J. Biochem. 79, 607-615. ADAMSON, E. D., GAUNT, S. J., and GRAHAM, C. F. (1979). The differentiation of teratocarcinoma stem cells is marked by the types of collagen which are synthesized. Cell 17,469-476. ANTONIADES, H. N., SCHER, C. D., and STILES, C. D. (1979). Purification of human platelet-derived growth factor. Proc. NatL Acad Ski. USA 76, 1809-1813. BERNSTINE, E. G., HOOPER, M. L., GRANDCHAMP, S., and EPHRUSSI, B. (1973). Alkaline phosphatase activity in mouse teratoma. Proc. NatL Acad Sci. USA 70, 3899-3903. BOURNE, H. R., and ROZENGURT, E. (1976). An 18,000 molecular weight polypeptide induces early events and stimulates DNA synthesis in cultured cells. Proc. Natl. Acad. Sci. USA 73, 45554559. BOWEN-POPE, D. F., MALPASS, J. W., FOSTER, D. M., and Ross R. (1984b). Platelet-derived growth factor in vivo: Levels, activity, and rate of clearance. Blood 64, 458-469. BOWEN-POPE, D. F., and Ross, R. (1982). Platelet-derived growth factor: II. Specific binding to cultured cells. J. Biol. Chew. 257, 5161-5171. BOWEN-POPE, D. F., and Ross, R. (1985). The platelet-derived growth factor receptor. In “Methods in Enzymology,” Vol. 109, “Peptide Hormones” (L. Birnbaumer and B. W. O’Malley, eds.), Academic Press, New York. BOWEN-POPE, D. F., VOGEL, A., and Ross, R. (1984a). Production of platelet-derived growth factor-like molecules and reduced expression of PDGF receptors accompanies transformation by a wide spectrum of agents. Proc. NatL Acad Sci, USA 81, 2396-2400. CHIU, I-M., REDDY, E. P., GIVOL, D., ROBBINS, K. C., TRONICK, S. R., and AARONSON, S. A. (1984). Nucleotide sequence analysis identifies

and Embryonal

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