3T3 cells to PDGF by growth factors, transformation, and tumor promoters

Cell, Vol. 36, 279-285,

February 1984, Copyright

(D 1984 by MIT

Alteration of the Chemotactic Response of NIH/3T3 Cells to PDGF by Growth Factors, Transformation, and Tumor Promoters Gary R. Grotendorst Laboratory of Development Biology and Anomalies National institute of Dental Research National Institutes of Health Bethesda, Maryland 20205

Summary The platelet-derived growth factor (PDGF) is a potent chemoattractant for cells that respond to PDGF as a mitogen. The chemotactic response of these cells to PDGF is inversely related to their rate of proliferation, with quiescent cells exhibiting a 25fold greater chemotactic response than exponentially growing cells. Factors that stimulate the growth of quiescent cells (EGF, FGF, PDGF, and serum) decrease the cells’ migratory response to PDGF but not to fibronectin, suggesting that the decreased migration is not due to a general paralysis of cell motility. Transformed lines of NIH/3T3 cells lose their ability to respond to PDGF as a chemoattractant but can still migrate in response to fibronectin. Similarly, after treatment of 3T3 cells with the tumorpromoter phorbol myristate acetate, which induces a transformation-like phenotype, the cells no longer respond to PDGF as a chemoattractant but retain their migratory response to fibronectin. Thus it appears that the growth state of the cells can alter their migratory response to PDGF. These data suggest that growth factors, transformation, and tumor promoters specifically alter the cells’ ability to respond to the PDGF-mediated chemotactic signal. It appears that both transformation and tumor promoters accomplish this by altering PDGF-binding to the cell surface. Introduction Cellular migrations occur during various biological processes including tissue formation, wound healing, and inflammation. It is likely that different chemical factors regulate the migration of the cells to specific locations by acting as chemoattractants. Some well characterized examples of directed cell movement (chemotaxis) in response to specific attractants are the movement of the cellular slime mold myxamebae toward CAMP (Bonner, 1967) and of leukocytes toward bacterial products (Ward et al., 1968; Schiffmann et al., 1975) and toward complement-derived factors (Ward and Newman, 1970; Snyderman et al., 1970). Fibroblasts can respond to other chemoattractants, such as lymphokines (Postlethwaite et al., 1976) complement-derived peptides (Postlethwaite et al., 1979) collagenous peptides (Postlethwaite et al., 1978) and fibronectin (Gauss-Muller et al., 1980; Postlethwaite et al., 1981). Chemotaxis may be involved in neovascularization and angiogenesis since endothelial cells have been shown to

migrate to extracts made from adult tissues (Glaser et al., 1980) and to tumor-derived factors (Zetter, 1980). Recently, we have shown that the platelet-derived growth factor (PDGF), a major mitogenic factor in serum (Ross et al., 1974; Kohler and Lipton, 1974; Antoniades et al., 1975; Westermark and Wasteson, 1976) is a potent chemoattractant for smooth-muscle cells (Grotendorst et al., 1981, 1982) and fibroblasts (Seppa et al., 1982). This is not a general property of mitogens since other growth factors, including fibroblast growth factor (FGF), epidermal growth factor (EGF), and insulin, are not chemoattractants for these cells. PDGF has been called a “competence” factor as it appears to enable cells to respond to other plasma-derived growth factors called “progression” factors (Pledger et al., 1977; Vogel et al., 1978). EGF, insulin, and somatomedins can substitute for plasma as mitogens for PDGF-treated cells (Stiles et al., 1980). In contrast, PDGF is active alone in inducing chemotaxis and progression factors do not enhance this response (Grotendorst et al., 1982). Physiologically, the chemotactic activity of PDGF may enhance its function as a wound hormone, stimulating wound repair by attracting connective tissue cells to the site and then causing them to proliferate. Because the migratory response of cells to PDGF occurs prior to the stimulation of DNA synthesis and does not require additional mitogenic factors, chemotaxis may serve as a direct measure of the initial activity of PDGF. Using the Boyden chamber assay for chemotaxis, I have investigated factors that alter the migratory response of cells to PDGF. These studies show that the growth state of the cells, as well as transformation, alters the cells’ chemotactic response to PDGF. Furthermore, it appears that other growth factors and tumor promoters, which do not themselves possess chemotactic activity, alter the migratory response of cells to PDGF. Both transformed cells and phorbol myristate acetate (PMA)-treated NIH/3T3 cells show reduced binding of PDGF, suggesting that changes in the PDGF receptor may be involved in the loss of the cells’ chemotactic response.

Results Chemotactic Response of Epithelial and Fibroblastic Cells to PDGF Because PDGF acts as a mitogen only for fibroblastic cells (Ross et al., 1979) the ability of PDGF to act as a migration factor for various cell types was investigated. As seen in Table 1, cells whose growth is stimulated by PDGF, including, NIH/3T3, NRK, smooth muscle, and skin fibroblasts, migrate in a dose-dependent manner to PDGF. Several epithelial cell lines, including MDCK, TERA, and PAM 212 cells, as well as aortic endothelial cells (which do not show a PDGF-dependent growth response), are not stimulated to migrate toward PDGF. These results indicate that the chemotactic response to PDGF is a property shared by cells that respond to PDGF as a mitogen.

Cell

280

Table 1. Ability of Various Cell Types to Respond and as a Chemoattractant Chemotactic No Additions

Cell Type

Cells Responsive

-

to PDGF as a Mitogen

Response’

2 rig/ml PDGF

IO rig/ml PDGF

to PDGF as a Mitogen

Bovine Aortic Smooth-Muscle Human Skin Fibroblasts NIH/3T3 Cells NRK Cells

Cells

8 15

4 5

to PDGF as a Mitogen Bovine Aortic Endotheliai Cells 6 MDCK Cells 4 TERA Cells 3 PAM 212 Cells 7

31

64

27 40 18

55 76 40

8 5 4 6

9 3 5 8

Cells Not Responsive

’ Data are expressed as cells/field. All assays were performed as described

under Experimental

Procedures.

Chemotactic Response of Transformed Cells to PDGF Transformed 3T3 cells arising spontaneously or by viral infection lose their PDGF growth requirement and are no longer restricted to low-density growth in culture (Scher et al., 1978). Here I compared the chemotactic responsiveness of normal 3T3 cells with that of cells transformed with a DNA virus (SV40), an RNA virus (Kirsten sarcoma virus), and spontaneously transformed cells. None of the transformed cells exhibited any directed migration toward PDGF (Figure 1A). Unexpectedly, the virally transformed lines were observed to have a higher level of random migration than either the normal or the spontaneously transformed 3T3 cells. The decreased response was not due to an inability to migrate, since the transformed cells showed a normal chemotactic response to fibronectin (Figure 1 B). The migration of normal and transformed connective tissue cells prepared from adult mouse skin was also determined. Adult connective tissue cells (ACT) spontaneously transform in culture at approximately the eighth to tenth passage and from then on will produce tumors when injected into nude mice (Liotta et al., 1978; Vembu et al., 1979). The ACT cells exhibit a positive chemotactic response to PDGF, whereas their transformed counterparts (TACT) showed a decreased ability to migrate toward PDGF (Table 2). The TACT cells, like the virally transformed cells, showed an elevated level of random migration. These data indicate that transformed cells lose their ability to migrate in response to PDGF.

Effect of Growth State on the Chemotactic Response of 3T3 Cells to PDGF The effect of the growth state of normal 3T3 on their chemotactic response to PDGF was investigated. NIH/3T3 cells were plated at densities ranging from 1 to 40 X lo3 cells/cm2. After culture for 72 hr, the cells had proliferated to densities ranging from 4-80 x IO3 cells/cm’. The cells at the highest densities (60-80 x lo3 cells/cm2) were growth-arrested, whereas those at the lower densities (4-

Figure 1. Effect of Transformation 3T3 Cells to PDGF and Fibronectin

on the Chemotactic

Response

of NlH/

(A: upper panel) The chemotactic response of NIH/3T3 (0) SWO-transformed NIH/3T3 (0) Kirsten sarcoma virus-transfoned NIH/3T3 (0) and spontaneously transformed NIH/3T3 cells (A) to various concentrations of PDGF was measured. Cell migration was measured using the Soyden chamber chemotactic assay as described under Experimental Procedures. The migratory response of the SV40/3T3 and K/3T3 cells in the absence of attractant was 35 cells/field and was 5 cells/field for the NIH/3T3 and SP/3T3 cells. (8: lower panel) The chemotactic response of NIH/3T3 (0) SW-transformed NIH/3T3 (0) Kirsten sarcoma virus-transformed NIH/3T3 (Cl), and spontaneously transformed NIH/3T3 cells (A) to various concentrations of fibronectin was measured as described under Experimental Procedures. Data are expressed as the difference between background and fibronectin-stimulated migration.

Table 2. Chemotactic Response of Normal and Spontaneously Transformed Mouse Adult Connective Tissue Cells Chemotactic Cell Type ACT (Normal) TACT (Transformed) Assays

were performed

No Additions

Response

to PDGF (Cells/Field)

2 rig/ml

10 rig/ml

4

55

73

15

25

as described

under Experimental

27 Procedures.

20 x lo3 cells/cm) were still growing exponentially. Cells taken from the density-arrested cultures show a 25-fold higher chemotactic response to PDGF than the exponentially growing cells (Figure 2). The response of the cells at intermediate densities was proportional to cell density. These data suggest that quiescent cells respond to PDGF as an attractant and that growing cells are much less responsive. This decreased response to PDGF as a chemoattractant does not appear to be due to changes in the cells’ ability to bind PDGF. PDGF-binding studies on both low- and high-density cultures indicate that cells at low

PDGF-Induced 281

Chemotaxis

1

CULTURE DENSITY WlS/Crn~ x 10-3)

Figure 2. Effect of Cell Density on the Chemotactic Cells to PDGF

Response

of NIH/3T3

Cells at various culture densities were harvested and their chemotactic response to PDGF was determined as described under Experimental Procedures. (A: left panel) Chemotactic response of NIH/3T3 cells at the indicated culture density to various concentrations of PDGF. The nonstimulated migration in the absence of PDGF ranged from no cells/field at the lowest culture densities to IO cells/field at the highest densities. (B: right panel) The chemotactic response of NIH/3T3 ceils to F’DGF (IO rig/ml) as a function of culture density.

Table 3. Effect of Cell Density on PDGF Binding by NIH/3T3 Cells

Figure 3. Effect of Serum-Stimulation Response to PDGF

of NIH/3T3 Cells on their Chemotactic

Cells in density-arrested cultures containing 10% fetal calf serum were stimulated to divide by increasing the concentration of serum to 20% at the indicated times prior to their preparation for the chemotaxis assay. The chemotactic response of the cells to 10 rig/ml of PDGF was measured as described under Exparimental Procedures.

Table 4. Effect of Growth Factors and Serum Treatment of NIH/3T3 on the Chemotactic Response to PDGF and to Fibronectin’

Pretreatment of Density-Arrested

Cells

Cells

PDGF (10 ml ml)

% Control

Fibronectin (20 sg/ml)

% Control

Culture Density” (Cells/cm* X 10e3)

fmoles of PDGF-Bound/l@

None

70

100

55

100

67

210

20% Serum

20

17

44

75

63

234

PDGF (50 tlg/ml)

24

23

46

80

49

232

FGF (50 a/ml)

31

35

48

84

34

273

EGF (50 rtglml)

35

40

50

89

18

264

a Density-arrested cultures of NIH/3T3 cells were incubated with the indicated components for 3 hr prior to trypsinization and preparation for the chemotactic assay. The chemotactic response of the cells to both PDGF and fibronectin was performed as described under Experimental Procedures. The background migration with no attractant was 10 cells/field. Data are the average of duplicate experiments.

’ Culture dens@ was determined cultures. b PDGF binding was determined dures

Cell?

using an electrimic cell counter on parallel as described

under Experimental

Proce-

density actually bind 26% more PDGF than cells at confluent densities (Table 3). Next it was determined if cells at high culture density change their chemotactic responsiveness to PDGF when stimulated to grow again with serum. Within 3 hr after exposure to serum, the chemotactic response of the NIH/ 3T3 cells decreased to approximately 20% of the nonstimulated control cells (Figure 3). To determine if the decreased chemotactic response was due to a unique feature of certain growth factors, the migration of growth factor-treated, density-arrested cells was measured. Density-arrested cultures in DMEM/iO% fetal calf serum were stimulated to grow by the addition of either EGF (50 ng/ ml), FGF (50 rig/ml), PDGF (50 rig/ml), or by increasing the serum concentration to 20% (v/v). Three hours after the addition of these factors to the cells, the cells were removed with trypsin and their chemotactic responses to PDGF and fibronectin were determined. The chemotactic response to PDGF of 3T3 cells after exposure to EGF, FGF, or PDGF was decreased significantly (260%; Table 4). However, the ability to migrate toward fibronectin was

not altered (~25%; Table 4). These data indicate that proliferating 3T3 cells are less responsive to PDGF as a chemoattractant than quiescent cells and that the mechanism of this decreased migratory response is not through a general paralysis of the motility machinery since the cells maintain their ability to migrate toward fibronectin.

Effect of Phorbol-Ester Tumor Promoter on the Migratory Response of NIH/3T3 Cells The tumor-promoter phorbol myristate acetate (PMA) has been shown to have a wide variety of effects on cells in culture. PMA treatment induces a transformed like phenotype in fibroblasts that is reversible after the removal of the promoter (Sivak and Van Duuron, 1967; Gottesman and Sobel, 1980). PMA has also been shown to act synergistically with various growth factors to stimulate the growth of 3T3 cells (Frantz et al., 1979). After treatment of NlH/ 3T3 cells with PMA (20 rig/ml) for 2 hr. the cells’ migratory response to PDGF was abolished (Figure 4). In contrast, the migratory response of the cells to fibronectin was only

Cdl 232

either a lack of receptors or blocking of receptors by a cell-derived product but not increased internalization and degradation, as binding experiments performed at 4% show no binding by SV40-NIH/3T3 cells and decreased binding by PMA-treated cells (data not shown). Thus both PMA treatment and viral transformation may alter the binding of PDGF to the cell surface.

NON-TREATED

Discussion

1.25

2.5

5

PDGF

I nglml)

Figure 4. Effect of Short-Term sponse of NIH/3T3 Cells

10

5

10 FIBRONECTIN

PMA Treatment

20

50

lrgimli

on the Chemotactic

Re-

Density-arrested cultures of NIH/3T3 cells were incubated with PMA (20 rig/ml) for 2 hr prior to preparation for the chemotaxis assay. The chemotactic response of the cells to PDGF (left) and fibronectin (right) was determined as described under Experimental Procedures.

O&l I 0

5

10

I 15

PDGF ADDED nM

Figure 5. Binding of PDGF to PMA-Treated 3T3 Cells

and SV40-Transformed

NlH/

PDGF binding was measured using ‘zsI-FDGF and various concentrations of nonlabeled PDGF to NIH/3T3 cells (0) PMA-treated NIH/3T3 cells (A), and SV40.transformed NIH/3T3 cells (0) as described under Experimental Procedures.

slightly decreased be chemotactic.

(Figure 4). PMA itself was found not to

Alterations in ‘%PDGF Binding to PMA-Treated and SV40-Transformed 3T3 Cells The biological effects of PDGF are initiated by its binding to specific cell-surface receptors (Heldin et al., 1981b; Bowen-Pope et al., 1982; Huang, et al., 1982). It is therefore possible that the loss of the cells’ chemotatic response to PDGF may be due to alterations in PDGF binding to its receptor. In order to detect such alterations in PDGFbinding, the binding of ‘251-labeled PDGF to normal NIH/ 3T3 cells, to NIH/3T3 cells treated with PMA, and to SV40NIH/3T3 cells was determined (Figure 5). The results of these studies suggest that after PMA treatment (50 rig/ml for 90 min) there is a decreased binding of PDGF by the NIH/3T3 cells. Further, it appears that SV40-NIH/3T3 cells lack the ability to bind PDGF. This appears to be due to

Previous work established that PDGF was chemotactic for smooth-muscle cells and fibroblasts (Grotendorst et al., 1981, 1982; Seppa et al., 1982). Here, a concentration gradient of PDGF stimulates target cells to migrate toward the higher concentrations of PDGF. Chemotaxis was directly demonstrated by checkerboard analysis as described by Zigmond and Hirsch (1973) using the Boyden chamber assay and by observing the orientation (Zigmond, 1977) of these cells towards a source of PDGF (unpublished observations). Migration of the cells occurred prior to the initiation of DNA synthesis and occurred when DNA synthesis was blocked. However, this does not mean that the chemotactic response of PDGF occurs by a mechanism entirely separate from its mitogenic action. As shown here, only cells that respond to the mitogenic effects of PDGF exhibit a chemotactic response. Further, the magnitude of the chemotactic response of 3T3 cells changes markedly with the growth state of the cells and is low in rapidly proliferating cultures and high in stationary cells. The quiescent cells show a decreased chemotactic response when stimulated to reenter the cell cycle by a variety of mitogens including EGF, FGF, PDGF, and serum. Thus cells that are committed to enter the cell cycle lose their ability to migrate in response to PDGF. Transformed 3T3 cells (Scher et al., 1978) as well as many malignant tumor cells (Currie, 1981) are not dependent on PDGF for growth in culture. The transformed lines of 3T3 cells, as well as transformed adult mouse skin fibroblast, no longer respond to PDGF as a chemotactic factor. Virally transformed lines appear to have a higher inherent rate of migration than either the nontransformed 3T3 or the spontaneously transformed 3T3 line. This could mean that the migratory system is under constant stimulation in such cells. It is unlikely that transformation alters a central process required for cell movement, since the transformed cells respond in a chemotactic fashion to a factor in SV40-3T3cellconditioned media (Bleiberg and Grotendorst, unpublished observations) and to fibronectin, suggesting that the alteration may be in some specific process involving PDGF. The “Y-PDGF-binding experiments suggest that the cells are unable to detect PDGF either because of a lack of surface receptors, or because of the blocking of the PDGF receptors by an endogenous factor. It is interesting to note that Dicker et al. (1981) have reported that SV40transformed BHK cells produce a factor that shares many biological and physical properties with PDGF. Heldin et al. (1981 b) and Nister et al. (1982) have also reported tumor-derived growth factors, which appear

PDGF-Induced 283

Chemotaxis

to bind to the PDGF receptor. These data indicate that transformation eliminates the response of cells to both the mitogenic and chemotactic effects of PDGF-probably through the same mechanism. It is possible that the chemotactic response is largely controlled by the availability of PDGF receptors, since only cell types possessing the receptors are responsive to PDGF (Heldin et al., 1981 b; Bowen-Pope and Ross, 1982; Huang et al., 1982). Treatment of 3T3 cells with PMA for 2 hr also caused a decrease in the cells’ chemotactic response to PDGF. Since the PMA-treated cells maintain their ability to migrate toward fibronectin, a general inhibition of migration is not likely to be the mechanism. This is supported by the PDGF-binding studies reported here, which indicate that the PMA-treated cells exhibit a decreased binding of PDGF. These results are similar to those reported by Lee and Weinstein (1978) and Shoyab et al. (1979) where PMA treatment resulted in decreased EGF binding. In addition, it has been shown that incubation of fibroblasts with PDGF (Wrann et al., 1980; Heldin et al., 1982) or with a PDGF-like factor isolated from SV40transformed BHK-cell-conditioned media (Rozengurt et al., 1982) causes a similar decrease in EGF binding. Investigations directed at the mechanism that causes this decrease in the ligand binding affinity of surface receptors, and at whether both EGF and PDGF receptors are regulated by similar mechanisms, may give some insight as to how cells detect chemical gradients during chemotaxis. The ability of other mitogens (EGF) or the growth state of the cells to depress the chemotactic response without altering PDGF binding suggests that some cytoplasmic events may also play important regulatory steps. The chemotactic response of cells to PDGF is dependent on RNA and protein synthesis (Grotendorst et al., 1981, 1982; Seppa et al., 1982) suggesting that PDGF may stimulate the synthesis of new proteins that are required for cell migration. The finding that transformation (Gottesman, 1978) PMA treatment (Gottesman and Sobel, 1980) and PDGF treatment (Scher et al., 1982) all stimulate the synthesis of an identical protein (major excreted protein; MEP) strongly indicates that these agents induce similar changes in cellular metabolism. PDGF appears to be unique among the growth factors tested with respect to its chemoattractant activity (Grotendorst et al., 1981, 1982; Seppa et al., 1982). Since PDGF acts via specific ceil-surface receptors (Heldin et al., 1981 b; Bowen-Pope and Ross, 1982; Huang et al., 1982) it is likely that activation of these receptors induces a specific set of events that leads to cell migration. The data presented here indicate that the PDGF-induced chemotactic response may be altered through changes in apparent receptor number or receptor affinity, as well as by cytoplasmic events that do not alter PDGF binding. Experimental

Pracedures

CdlS

NIH/3T3. SV40transfonad NIH/3T3 rus-transformed NIH/3T3 (K-NIH/3T3),

(SV40-NIH/3T3). Kirsten sarcoma viand spontaneously transformed NIH/

3T3(SP-NIH/3T3) were obtained in earfy passage from S. Aaronson (NIH, Bethesda, Maryland). New ampules of ceils were thawed when needed so that only early-passage cells were used. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and gentamicin (50 #g/ml) and maintained at 37°C in an atmosphere of 90% air/IO% COZ. Stock cultures were passaged weekly and were not allowed to reach a high density. Density-inhibited cell cultures were initiated by plating exponentially growing cells at 3 x 103 cells/cm* in Falcon 25 cm2 tissue cutture flasks in DMEM containing 10% fetal calf serum and gentamicin (50 As/ml). Wahin 7 days, the cultures reached a maximal cell density and ceased division. Density-inhibited cultured cells were serumstimulated to divide with DMEM containing 20% FCS. All other chemotactic assays were performed on density-inhibited cultured cells unless other&a stated. Pam 212 cells, a differentiating epidermal cell line (Yuspa et al., 1980) were obtained from Dr. J. Stanley (USUHS. Bethesda, MD). TERA-1 cells, a human tumor cell line with epithelial morphdogy (Fogh and Trempe, 1975) and ACT and TACT cells (Liotta et al., 1978; Vembu et at., 1979) were obtained from L. Liotta (NCI, Bethesda, MD). MDCK cells were obtained from C. Scher (Univ. of Pennsylvania). Human skin fibroblasts (CRL 1475) were obtained from American Type Culture Collection (Rockville, MD). Both bovine aortic endothelial cells and bovine aortic smoothmuscle cells were prepared from fetal aortas as described previously (Glaser et al., 1980; Grotendorst et al., 1981, 1982). Confluent cultures were used for all chemotactic assays.

Preparation of Collagen and Fibmnectin Type I collagen was prepared from isdated rat-tail tendons by acid extraction and NaCl precipitation as described by Bomstein and Piez (1986). Fibronectin was purified from human plasma using gelatin-affinity chromatography (Engvatl and Ruoslahti, 1977).

Growth Factors PDGF was purified using a modification of established methods (Antoniades et al., 1975; Heldin et al., 198la; Deuel et al., 1981; Raines and Ross, 1982). Outdated platelets were obtained from a local blood bank and washed twice with 0.10 M NaCI, 0.05 M Tris-HCI (pH 7.4) containing 0.901 M EDTA. The washed platelets were resuspended in 0.1 M NaCI, 0.05 M Tris-HCI (pH 7.4) using 2 ml of buffer/unit of platelets and the suspension was frozen and thawed three times. The extracts were then incubated at 100°C for 10 min. Insoluble material was removed by centrifugation at 10,000 g for 15 min and the pellets were reextracted with 1 M NaCI. The supernatant fractions were combined and dialyzed against 0.08 M NaCI. 0.01 M Tris-HCI (pH 7.4) and applied to a CM-cellulose column (CM-52 Whatman) (2.5 x 30 cm) equilibrated with the same buffer. PDGF was eluted with a linear gradient of NaCl from 0.08 to >I .O M with the active material eluting at approximately 0.3 M NaCI. These fractions were pooled and chromatographed on a phenyl-sepharose (Pharmacia) column (1.5 x 5 cm). The column was washed with 1 M NaCI, 0.05 M Tris-HCI (pH 7.4). and then with 0.15 M NaCI, 0.05 M Tris (pH 7.4) containing 30% ethylene glycol. PDGF was then eluted with a solution of 0.15 M NaCI, 0.05 M Tris (pH 7.4) containing 50% ethylene glycol. The active fractions were pooled and dialyzed against 0.5 N acetic acid and lyophilized. The lyophilized powder was dissolved in a small vdume of 1.O N acetic acid adjusted to pH 3.5 with NH,OH and run on a sphercgel-TSK 2000 column (Beckman) (0.75 x 60 cm) equilibrated in the same solvent. The column was run at a flow rate of 1 .O ml/min and the active PffiF fractions were pooled and stored at -20°C in small aliquots. FDGF prepared in this manner is 95% pure as judged by SDS-acrylamide gel electrophoresis after staining with Coomassie Blue R-250. Purified fibroblast growth factor (FGF) was a gift from D. Gospodarowicz (University of California, San Francisco). EGF was purchased from Collaborative Research (Waltham, MA). All growth factors were assayed for mitogenic activity using methods established for Balb/c3T3 cells and PDGF (Antoniades et at., 1975) using cell numbers instead of ?--thymidine rncorporation as a measure of activity. One unit PDGF activity is the amount required to stimulate 50% of the density-arrested NIH/3T3 cells to undergo cell division in 5% plasma-derived serum after 38 hr and corresponds to 2-5 rig/ml of the HPLC-purified PDGF.

Cell 204

Chemotexis

Assey

The chemotactic response of the various cells was assayed using the modified Boyden chamber (Zigmond and Hirsch, 1973; Postlethwafte et al., 1978; Grotendcrst et al., 1981). The test substance was diluted in DMEM containing bovine serum albumin (BSA) (1 mg/ml) and added to the lower well of the blind-well chamber. The lower well was then covered with a collagen-coated polycarbonate filter (Nucleopore, 8 fi dieter pores), ensuring a continuous contact between the lower surface of the filter and the solution. The upper well was then fixed in place and cells (3 X 10r’ cells/assay) prepared from the tissue-culture flasks were added to the upper well in DMEM containing BSA. After the chambers were incubated 4-6 hr at 37°C in an atmosphere of 90% air/lo% Ca, the filters were removed from the chambers and the cells were fixed and stained for light microscopy using Dif Quick stain (Harfeco). The filters were placed on glass microscope slides with the lower surface of the filter adjacent to the glass slide. The upper-surface cells were removed by gently scraping with a rubber policeman, and the chemotactic response was analyzed by counting the number of cell nuclei/microscopic field (400X) on the lower surface. Each assay was performed in duplicate and the cell numbers varied by less than 10%. All data are expressed as the mean of duplicate experiments, each of which is the average cell number in ten microscopic fields on two filters. Each experiment was repeated at least three times with similar results.

PDGF-Binding

Studies

Pure PDGF (5 pg) was iodinated by the chloromine T procedure (Hunter and Greenwood, 1962) as described by Heldin et al. (1981 b). The labeled protein was separated from free iodine by gel-filtration chromatography on a PD.1 0 column (Pharmacia) equilibrated in 1 M acetic acid adjusted to pH 3.5 with NH,OH containing 1 mg/ml BSA. The ‘S-PDGF-containing fractions were pooled and stored at -20°C. The binding activity of this preparation remained unchanged over a period of 2 months. The specific activity of this material was 20,COO cpm/ng or 0.25 atoms of iodine per molecule of PDGF. Receptor-binding studies were performed as described by Heldin et al. (1981 b). Briefly, cells were cultured for 5 days in 24-well costar dishes in DMEM containing 10% FCS. The NIH/3T3 cells were densitygrowtharrested at this time. Labeled PDGF (5 ng/iOO,COO cpm) was mixed with various amounts of nonlabeled PDGF in binding media (PBS containing 2 mM CaCI,, 1 mM MgSO,, and 1 mg/ml BSA). The cells were washed twice with binding media and incubated with the labeled PDGF for 90 min at 25°C in air. The cell layers were then washed four times with washing media (PBS containing 2 mM CaC12, 1 mM MgSO, and 1% fetal calf serum). The specifically bound PDGF was then extracted with lysing media (20 mM HEPES (pH 7.4) containing 1% Triton X-100, 10% glycerol, and 1 mg/ml BSA). The amount of bound ‘?PDGF was assayed using a Beckman model 9030 gamma counter. All binding assays were performed in duplicate and varied by less than f 10%. Scatchard analysis (Scatchard, 1949) indicated that the NIH/3T3 cells possessed 250.000-300,000 PDGF binding sites/cell with a Kd of 2.0 x lO+ M, which is comparable to reports by others (Heldin et al.. 1981b; Bowen-Pope and Ross, 1982; Huang et al., 1982). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

April 8, 1983; revised

November

2, 1983

Antoniades, H. N.. Stathakos, D., and Scher, C. D. (1975). lsdation of a cationic polypeptide from human serum that stimulates the growth of 3T3 cells. Proc. Nat. Acad. Sci. USA 72. 2603-2605. Antoniades, H. N.. Scher. D. C., and Stiles, C. M. (1979). Purification of human platelet-derived growth factor. Proc. Nat. Acad. Sci. USA 76, 18091813.

Bomstein,

P., and Piez, K. (1966). The nature of the intramolecularcrosslinks

New

of peptides 5, 34603473.

from

the

Bowen-Pope, D. F., and Ross, R. (1982). Platelet-derived growth factor Specific binding to cultured cells. J. Biol. Chem. 257, 5161-5171. Currie, G. A. (1981). Platelet-derived growth-factor profiferation of normal and malignant mesenchymal 335343.

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