JOURNAL OF CELLULAR PHYSIOLOGY 142:108-116 (1990)

Appearance of Basic Fibroblast Growth Factor Receptors Upon Differentiation of Rat Mammary Epithelial to Myoepithelial-Like Cells in Culture DAVID C. FERNIG,* JOHNA. SMITH, AND PHILIP S. RUDLAND

Cancer and Polio Research Fund Laboratories, Department of Biochemi,try, Unwersity ot Liverpool, Liverpool L69 35X, England The binding of ['251]-epidermalgrowth factor (EGF) and ["51]-basic fibroblast growth factor (bFGF) to a number of single-cell cloned rat mammary cell lines was measured using a saturation assay. Similar numbers of high-affinity ['LSI]-EGF binding sites (K,, 1 . 3 nM) were found i n epithelial and myoepithelial-like cell lines. In contrast, high-affinity (K, 35-276 pM) ["'I]-bFGF binding sites were present on fibroblastic and myoepithelial-likecell lines h u t were not detectable on epithelial cell lines. A series of cell lines representing stages in the differentiation pathway of epithelial cells to an elongated myoepithelial-like morphology showed a graded increase in the number of bFCF receptors. The sensitivity of a cell line to stimulation of DNA synthesis by bFGF correlated with the level of expression of bFGF receptors on the cellular surface. Complexes of cell surface receptors affinity-cross-linked to 11251]-bFGFwere analysed by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE). In each case two distinct complexes having apparent molecular weights of 180 kDa and 160 kDa were observed The growth and development of the mammary gland is controlled not only by levels of circulating hormones (Lyons et al., 1958; Nandi, 1958) but also by other trophic agents (Kano-Sueoka, 1983). One approach to the elucidation of the nature of such agents has involved the use of single-cell cloned rat mammary cell lines that represent the different cell types that are found in the mammary gland: the epithelial cell, the elongated myoepithelial cell, and the fibroblastic cell (Rudland, 1987). Using such a system two trophic agents from the pituitary have been identified: a novel mammary growth factor, active only on the epithelial cell lines, and fibroblast growth factor (FGF), active only on the myoepithelial-like cell lines and the fibroblastic cell lines (Smith et al., 1984a).Prostaglandin E, (PGE,) and, more recently, transforming growth factor alpha (aTGF) have been identified a s possible local trophic agents that are released predominantly by the fibroblastic cell lines and the myoepithelial-like cell lines, respectively. The former stimulates the growth of the rat mammary epithelial cell lines, whereas the latter stimulates the growth of all the rat mammary cell lines tested (Rudland et al., 1984; Smith et al., 1989). One of the pituitary factors, basic fibroblast growth factor (bFGF), is a prototypic member of a family of growth factors and oncogenes that has five known members at present; bFGF, acidic FGF, and the oncogenes hstiKS53, int-2, and FGF5 (Thomas, 1988). bFGF elicits mitogenic and other responses in vitro in a wide spectrum of cells of mesodermal and neuroectodermal origin (Thomas and Gimenez-Gallego, 1986; G 1990 WILEY-LISS, INC

Thomas, 1988), but its role in vivo is unclear. In our rat mammary cell lines, bFGF stimulates the growth of the rat mammary fibroblastic cell line Rama 27 and the elongated myoepithelial-like cell line Rama 29, but has no effect on three different cuboidal epithelial cell lines, Rama 25, Rama 600, and Rama 704 (Smith et al., 1984a). The epithelial cell line Rama 25 (Bennett et al., 1978) can convert in culture to elongated myoepithelial-like cells (Rudland et al., 1982b). Single-cell cloned cell lines with morphologies intermediate between cuboidal epithelial and elongated myoepithelial-like cells have been isolated (Rudland et al., 1986). Biochemical analysis of these cell lines has shown that they also have intermediate levels of proteins and mRNAs characteristic of the elongated myoepithelial-like cell lines (Barraclough et al., 1987; Rudland e t al., 1982b, 1986). Thus these intermediate cell lines may represent different stages of differentiation to myoepithelial-like cells in vitro in a n analogous fashion to the stages seen a t ductal termini in vivo (Ormerod and Rudland, 1984). To investigate further the possible role of bFGF and epidermal growth factor 1EGF)-related molecules in mammary gland growth and development, we have used quantitative binding methods to characterize the EGF and bFGF receptors in the different rat mammary clonal cell lines. We have also char-

Received June 2, 1989; accepted September 1, 1989.

"To whom reprint requestskorrespondence should be addressed.

EGF AND bFGF RECEPTORS IN RAT MAMMARY CELL LINES

109

TABLE 1. Origins of the mammary cell lines Mammary tissue Normal rat, fast-sticking fraction Sprague-Dawley outbred rat, benign 7,12 dimethylbenz[alanthracene (DMBA) tumor

Cell line Rama 27

Identity Fibroblastic

Reference Rudland et al., 1984 Bennett et al., 1978 Rudland et al., 1986 Rudland et al., 1986 Rudland et al., 1986 Bennett et al., 1978 Rudland et al., 1982a

Furth-Wistar inbred rat, benign DMBA syngeneic tumor

Rama 37CL-A3 Rama 37-EX

Epithelial Cells intermediate between epithelial Rama 25 and rnyoepithelial-like Rama 29 Myoepithelial-like Myoepithelial-like, tumorigenic Epithelial Myoepithelial-like

acterized the bFGF receptor in the cell lines intermediate between cuboidal epithelial and elongated myoepithelial-like to find out a t what stage in the differentiation of Rama 25 cells to a myoepithelial-like morphology the bFGF receptor is expressed at the cell surface and when the intermediate cells become sensitive to bFGF.

MATERIALS AND METHODS Cells The derivation, routine culture, and detailed characterization of the single-cell cloned mammary cell lines used in this study have been reported previously and are summarized in Table 1.

Preparation of growth factors Bovine pituitary-derived bFGF was isolated using ammonium sulphate precipitation and sequential chromatography on CM Trisacryl and heparin-sepharose under the conditions described by Gospodarowicz et al. (1984). It was further purified by affinity HPLC on a heparin-5PW column (7.5 cm x 7.5 mm i.d.; Tosoh Corp., Yamaguichi-Ken, Japan), as illustrated in Figure 1.Preparations from 400 g bovine pituitary glands yielded 60-80 pg bFGF with a n ED,, of 0.04 nglml for Rama 27 cells (not shown). EGF was prepared from mouse submaxillary glands a s previously described (Smith et al., 1984b). Growth factor iodination bFGF was iodinated using IODOGEN (Pierce and Warriner, Chester, UK) a s the oxidant. Briefly, 5 pg IODOGEN dissolved in 50 pl chloroform was added to a microfuge tube and the chloroform was evaporated by hand-warming. To this tube, 20 p10.5 M phosphate, pH 7.4, and 3 pg bFGF in 50 p12 M NaC1,20 mM Tris-HCI (pH 7.4) were added, followed by 0.3 nmoles carrierfree [lZ51]-NaI(Amersham International, AmershamUK). After 10 min the reaction mixture was stopped by removal and was added to a tube containing 5 pl 100 mM Na2S205,5 pl 100 mM KI, and 20 p1 1.1M NaC1, 10 mM Tris-HC1 (pH 7.4), and 0.2% gelatin (wiv). EGF was iodinated by the chloramine T method (Hollenberg and Cuatrecasas, 1975). Free ['"I]-NaI was removed by centrifugal desalting (Christopherson, 1983) on a 1 ml Sephadex G25 column. Analysis of the biological activity of the iodinated growth factors indicated that 30% of the original EGF activity and 100% of the orig-

Dunnington et al., 1983 Dunnington et al., 1983

inal bFGF activity was recovered. bFGF and EGF were iodinated to specific activities of 1.5-26 pCiipg and 10-110 pCiipg, respectively. [1"51]-EGFwas stored frozen a t -20°C and used within a week. 11251]-bFGFwas used immediately.

Binding assays Cells were plated at about 5 x lo4 cells per 1.5 cmdiameter multiwell (24 well plates; Sterilin, Hounslow, UK) in 1 ml Dulbecco's modified Eagle's medium (DEM) containing 5% fetal calf serum (FCS) (Gibco, Paisley, UK), 50 ngiml insulin, and 50 ngiml hydrocortisone. Twenty-four hours after plating, the cell monolayers were washed twice with phosphate-buffered saline (PBS) and 500 pl DEM containing 0.1% bovine serum albumin (BSA) (wiv) (Sigma, Poole, UK) was added. After 18 h the cells were washed three times with PBS, once with binding medium (M199 [Sigma] supplemented with 10 mM HEPES, pH 7.4, and 0.1% BSA [wivl a t PC), and then 200 pl of binding medium was added. Increasing concentrations of [lziiI1growth factor were added to the cells; the nonspecific binding was determined by the inclusion of a 200-fold excess of unlabelled growth factor. Cells were incubated at 4°C on an orbital shaker until the binding of [1251]-gro~th factor had reached equilibrium. Maximal binding was attained within 90 min for [lZ5I1-EGFand 120 min for ['251]-bFGF. After incubation at 4°C the cell monolayers were washed twice with 500 p1binding medium and three times with 500 pl PBS a t 4°C. Cellassociated radioactivity was solubilized with 0.2 M NaOH and determined in a Wilj gamma counter. Results were analysed both graphically and with the LIGAND program (Munson and Rodbard, 1980). Crosslinking of [1251]-bFGFto its receptor Cells (10") were plated in 5.5 cm-diameter dishes. After 24 h the cells were prepared for binding to [1251]bFGF as described above, except that the binding medium was replaced with 1.5 ml PBS supplemented with 0.1% BSA (w/v). [lZ5I]-bFGFwas added to each dish a t a final concentration of 20 ng/ml and binding was carried out for 2 h at 4°C on a rotary shaker. The cells were then washed once with PBS (4°C) before adding 2 ml PBS (4°C). Cross-linking was carried out by adding freshly prepared 20 mM disuccinimidyl suberate (Pierce and Warriner, Chester, UK) in dimethyl sulphoxide to the cells a t a final concentration of 1 mM

110

FERNIG ET AL

2

Conc. o f 1

NaCl (MI 0

0

5

1c

15

20

25

E l u t i o n T i m e (mins) Fig. I . Analysis of bFGF by HPLC affinity chromatography. Bovine bPGF derived from 400 kg pituitary glands was eluted from heparinsepharose in 2 M NaCI, diluted 1:4 in 10 mM Tris-HC1 (pH 6.5), and

(Pilch and Czech, 1979). The reaction was continued for 15 min at 4°C and then quenched by the addition of 3 ml binding medium. Nonspecific cross-linking was determined by including 800 ngiml unlabelled bFGF in the binding reaction. The cells were washed three times with 4 ml binding medium and three times with PBS; they were then collected by scraping in 200 ~1 of sample buffer (Laemmli, 1970) and processed for SDSPAGE. Cells in sample buffer containing 5% SDS and 5% P-mercaptoethanol were sonicated for 1 min a t minimum power in a Dawe soniprobe and were then incubated for 2 h at 37°C. Samples were run on 7.5% SDScontaining polyacrylamide gels, which were then stained and fixed (Laemmli, 1970). Gels were autoradiographed for 1 to 2 weeks at -70°C with Kodak-Xomat film and intensifying screens (Dupont, Stevenage, UK).

applied to a heparin-5PW column. bFGF was eluted with a gradient of NaCl as indicated. The fraction eluting between 22 and 24 ml was used as purified bFGF for receptor-binding studies.

acterized in a series of saturation experiments. The specific binding of ['"II-bFGF to the fibroblastic cell line Rama 27 and the myoepithelial-like cell line Rama 29 is shown in Figures 3A,B. Analysis of the data showed that there were 347,000 high-affinity receptors on the surface of Rama 27 cells with a K, of 46 pM (Table 3). Rama 29 cells possessed a similar number of high-affinity receptors for bFGF but with a Kn of 276 pM. In contrast, the epithelial cell line Rama 25 did not appear to have any high-affinity cell surface bFGF receptors as bound [1251]-bFGFwas not displaced from this cell line with excess nonradioactive bFGF (Table 3). All three cell lines intermediate in character between the epithelial Rama 25 and the elongated myoepithelial-like Rama 29-Rama 25-12, Rama 25-11, and Rama 25-14-bound displaceable ['2511-bFGF(Fig. 3CE, Table 3). Rama 25-12, the intermediate cell line closest to the epithelial cell line Rama 25, had only 13,000 bFGF receptors (Fig. 3E, Table 3). Itama 25-11 and Assay for r3H]DNA synthesis Rama 25-14 cells had a higher number of bFGF recepThe stimulation of incorporation of [3H1-thymidine tors, but still not as many a s were found on the myo(Amersham) into DNA was carried out exactly a s de- epithelial-like Rama 29 cells (Fig. 3C,D, Table 3). However, the tumorigenic transformant of Rama 29 cells, scribed by Smith et al. (1984a). Rama 521, had a greatly reduced number of high-afRESULTS finity receptors for bFGF (26,000) when compared to its parent cells (Fig. 3F, Table 3). Consistent with the abBinding of EGF sence of bFGF receptors on the surface of Rama 25 [ 1251]-EGFbound specifically to both the myoepithe- cells, no bFGF receptors were detected on another inlial-like Rama 29 and the epithelial Rama 25 cell lines, dependently isolated rat mammary epithelial cell line, producing saturable binding curves a s shown in Figure Rama 37CL-A3 (Table 3). I n contrast the myoepithe2. Analysis of such curves indicated that Rama 25 and lial-like cell line Rama 37-E8, derived from Rama Rama 29 cells had a single high-affinity binding site 37CL-A3, had 46,000 receptors per cell with a K, of 72 for [lZ5I1-EGF(Fig. 2A,B insets). Table 2 shows that pM (Fig. 3G, Table 3). Low-affinity binding site. At high concentrations these cell lines expressed a similar number of EGF of [1251]-bFGFthe shape of the Scatchard plots (Fig. 3 , receptors a t their cell surface, with a K, of 1.3 nM. insets) suggested the presence of a low-affinity binding Binding of bFGF site in Rama 27, Rama 29, Rama 521, and the interHigh-affinity binding site. The binding of [1251]- mediate cell lines (Fig. 3 insets, Table 3). A two-site bFGF to the different r a t mammary cell lines was char- model fitted the data considerably better than a one-

111

E G F AND b F G F RECEPTORS IN RAT MAMMARY CELL LINES

TABLE 2. K, and number of EGF receptors on myoepithelial-like and eDithelia1 rat mammary cell lines

I

A

Cell line Rama 29 myoepithelial-like Rama 25 epithelial 'Mean

/

i

K., (nM)' 1.3 + 0.5

ReceDtors oer cell 21 + 8

1.3 ? 0.5

23

?

(X

13

SE of values calculated from a t least three independent experiments.

4 ,

TABI,E 3. K, and number of bFGF receptors on fibroblastic, myoepithelial-like, and epithelial rat mammary cell lines

0

I

: . . , . . ( . . I . . , . . , . ;

2

0

4

6 8 B (frnol)

10

12

I

I

I

f

10

20

30

40

[1251]-EGF (nglml)

63 i

Cell line Rama 27, fibroblasts Rama 29, myoepithelial-like Rama 25-14, intermediate myoepithelial-epithelial Rama 25-11, intermediate myoepithelial-epithelial Rama 25-12, intermediate myoepithelial-epithelial Rama 25, epithelial Rama 521, myoepitheliallike, tumorigenic Rama, 3 7 C L A 3 epithelial Rama, 37-E8 myoepithelial-like

High-afinitv receDtors Receptors per cell K, (pM)' ( x 10 - 3 ) 1 3 4 7 ? 49 46 2 12

Low-affinity receptors Yes2

276

2

80

343 2 69

Yes

46

?

27

140 2 46

Yes

229

Yes

115 + 33

?

42

80 + 59

13 ? 6

Yes

N.D.3 35 + 32

N.D. 26 ? 12

N.D. Yes

N.D. 72 + 26

N.D. 46

2

10

N.D.

NO'

'Mean ? SE of values calculated from at least three independent experiments. 'A two-site model fitted the saturation binding data considerably better for Rama 27, Rama 29, Rama 521, Rama 25-14, Rama 25-11, and Rama 25-12 cells than a one-site model ( P c: 0.001) when analyzed with the LIGAND program (Munson and Rodhard, 1980). 'Not determined, since no specific [i2511-bFGFbinding was observed. 4No evidence for a low-affinity site was found for this cell line when the data were analysed with the LIGANU program (Munson and Rodbard, 19801.

4

0

8

12

B (fmol) 0 0

I

I

I

1

10

20

30

40

[1251]-EGF (nglml)

Fig. 2. Binding of [12511-EGFto Rama 25 and Rama 29 cell monolayers. A Specific binding of [I2'I1-EGF to Rama 25 cells. B: Specific binding of ["'Il-EGF to Rama 29 cells. Insets, Scatchard analysis of the data. Specific binding of 112511-EGFwas determined as described in Materials and Methods. Nonspecific binding did not exceed 5% of the total. Data are expressed as the mean i S D of quintuplet samples.

site model in these cell lines (P < 0.001, Table 3). The apparent K, of the low-affinity site was of the order of 100 nM. However this site was not fully characterized, because the accurate determination of the number of low-affinity receptors and their K would require the use of higher concentrations of ["I]-bFGF and unlabelled bFGF (Klotz, 1982).

bFGF stimulation of r3H]-thymidine incorporation into DNA bFGF stimulated the synthesis of DNA, a s measured by the incorporation of F3H1-thymidineinto DNA, in all cell lines that expressed a detectable number of bFGF receptors a t their cell surface (Table 4).The cell lines expressing more than 100,000 receptors per cell (Table 3) were all stimulated to synthesize ["]-DNA by bFGF to the same level as that achieved by 5% FCS. However, bFGF was unable to stimulate maximally ['HIthymidine incorporation into DNA in cell lines such as Rama 25-12, Rama 621, and Rama 37-E8, which expressed a relatively lower number (less than 50,000; Table 3) of bFGF receptors. Maximal incorporation of L3Hl-thymidine into DNA was observed a t 0.3-1 ng/ml bFGF in all cases (not shown). At higher concentrations bFGF began to inhibit ['HI-DNA synthesis in a concentration-dependent manner (not shown). No stimulation of ['HI-thymidine incorporation into DNA was observed in the epithelial cell lines Rama 25 and Rama 37CL-A3 (Table 4). Affinity cross-linking of [1251]-bFGF to cell surfaces The bFGF receptor was further characterized by affinity cross-linking experiments using some of the

112

FERNIG ET AL. 400 -

300

A 300 u)

P 0

200

.

201-I' 100

cn

n

0

100

m 0

60

0

120

B (frnol)

0

0

100

200

0

300

0

conc bFGF (ng/rnl)

100

300

200

[1251]-bFGF (nglrnl)

30

-

20

-

m n

0

10

i"' 60

1

I

.

LL

3m 0

0

0

0 0

100

200

0

300

[1251]-bFGF (ng/rnl)

100

k

j

20 40 B (frnol) I

I

200

300

[1251]-bFGF (nglrnl)

Fig. 3 A-D.

above cell lines. Analysis of complexes of [lZ5I]-bFGF specifically cross-linked to its receptor on SDS gels indicated that these complexes existed in a high 180 kDa and a lower 160 kDa molecular weight form in all the cell lines examined (Fig. 4).Assuming that a single ['2511-bFGFof molecular weight 18 kDa binds to a single bFGF receptor, this result suggests an apparent molecular weight of 160 kDa and 140 kDa for the bFGF receptor. [lZ5I]-bFGF specifically cross-linked to such molecules only in those cell lines in which bFGF receptors were identified in the radioligand binding experiments (Fig. 3, Table 3).

DISCUSSION The measurement of [Iz5I]-EGFbinding t o the epithelial Rama 25 and myoepithelial-like Rama 29 cells (Fig. 2, Table 2) indicates that these cell lines behave similarly in saturation binding experiments. They are also both stimulated to grow by similar concentrations of EGF (Smith et al., 1984a). Thus the stimulation of cell growth is probably due to the specific interaction of EGF with its receptor. Rama 27 cells show a greater increase in cell numbers in response t o EGF than Rama 25 and Rama 29 cells (Smith et al., 1984a). This

113

EGP AND bFGF RECEPTORS IN RAT MAMMARY CELL LINES 40

E

100

-

80

-

60

-

F

30

v)

n 0

lo

n -

20

0

40

-

10

4 8 B (fmol)

0 0 0

100

200

12 i

300

100

0

[1251]-bFGF (nglml)

200

300

[1251]-bFGF (ng/ml)

=:r-Ll 30

-

-

cn

30

0

20

-

. m LL

10

-

0

0

1

2

3

4

B (fmol)

"

I

0

100

200

300

[1251]-bFGF (nglml)

Fig. 3. Binding of T'251]-bFGFto rat mammary cell monolayers. Speeific binding of I'2511-bFGF to fibroblastic Rama 27 (A);myoepitheliallike Rama 29 (B); cells intermediate between epithelial and myoepithelial-like, Rama 25-14 (C); Rama 25-11 (D); Rama 25-12 (E); myoepithelial-like Rama 521 (F); myoepithelial-like Rama 37-E8 (G).

Insets, Scatchard analysis of the data. Specific binding of P2'I1-bFGF was determined as described in Materials and Methods. Nonspecific binding did not exceed 30% of the total. Data are expressed as the mean f SD of quintuplet samples.

may result from the presence of nearly three times as many high-affinity receptors on these cells (Smith et al., 1989) than on the cells with an epithelial origin (Table 2). The actual agent stimulating the growth of these cells through the EGF receptor may not be EGF itself, but a closely related molecule. For instance, aTGF exerts its biological activity through the EGF receptor (Massague, 1983a,b). aTGF is secreted by Rama 29 and Rama 25 cells in culture in sufficient amounts to stimulate their growth in an autocrine - aTGF is fashion (Smith et al., 1989).- Furthermore, present in the rat mammary gland (Smith et al., 1989), and the growth of the mammary gland is stimulated by

implants containing either EGF or aTGF (Vonderhaar, 1987). These observations have been used to suggest that aTGF could act as a local trophic agent for the cells of the rat mammary gland. However, since the number and K, of the EGF receptors observed on Rama 25 and Rama 29 cells are similar, the EGF receptor is unlikely to be modulated in any way during the differentiation of epithelial cells to myoepitheliallike cells in culture. In contrast to EGF, bFGF is mitogenic for both myoepithelial-like . -. __ cell._ lines Rama 29 and Rama 37-E8 and the fibroblastic cell line Rama 27, but it has no comparable activity toward the epithelial cell lines Rama

114

FERNIG ET AL.

belled by [l2’1]-bFGF (Fig. 4) may represent some form of experimental artefact. bFGF exerts its maximal mitogenic effect on the rat % Maximum stimulation of L3H1-thymidine incorporation mammary cell lines within the range of the K, of the Cell line into DNA’.2 high-affinity binding site (not shown). Low-affinity re117 ? 213 Rama 27, fibroblastic ceptors for 112511-bFGFwere observed on Rama 27, 85 2 14 Rama 29, myoepithelial-like Rama 29, Rama 521, and the intermediate cell lines 101 ? 10 Rama 25-14, intermediate Rama 25-14, Rama 25-11, and Rama 25-12 (Table 3). myoepithelial-epithelial However, there was no evidence for a low-affinity bind90 zk 10 Rama 25-11, intermediate myoepithelial-epithelial ing site on the myoepithelial-like cell line Rama 37-E8, 26 2 8 Rama 25-12, intermediate although these cells have a normal response to bFGF myoepithelial-epithelial as measured by the incorporation of [’HI-thymidine 0 Rama 25, epithelial into DNA (Table 4). Thus it appears unlikely that the Rama 521, myoepithelial-like 26 2 8 tumorigenic low-affinity binding site is utilised for promoting the Rama 37CL-A3, epithelial 0 growth response triggered by bFGF. In other systems, 47 k 26 Rama 37-E8, myoepithelial-like cells and tissues often express both high- and low-af’The maximum incorporation was that observed in the presence of 5% FCS. finity binding sites for bFGF (Neufeld and Gospo‘Stimulation of l3H1-thymidine incorporation was measured between 1 p g h l darowicz, 1985; Olwin and Hauschka, 1986; Moscatelli, and 10 ng/ml bFGF, and dose-response curves were constructed. Maximal x t i m ulation was obscrved between 0.3 nglml and 1 ng/ml bFGF (not shown). 1987; Vigny et al., 1988). In bovine capillary endothe‘Mean t SD uf triplicate determinations. lial cells (Moscatelli, 1987) the low-affinity site is not involved in the biological response to bFGF. In these cells the low-affinity site appears to be a molecule with 25 and Rama 37Cl-A3 (Table 4). The saturation bind- heparin-like properties and is distinct from the highing experiments (Table 3) indicate that those cell lines affinity site (Moscatelli, 1987; Saskela et al., 1988). that are responsive to the growth-promoting effects of Furthermore, in the Englebreth Holm Swarm sarcoma bFGF possess 13,000-347,000 high-affinity bFGF re- the low-affinity site is located on the basement memceptors per cell, while the growth-unresponsive cell brane (Vigny et al., 1988). Since rat mammary myoeplines, Rama 25 and Rama 37Cl-A3, do not have a de- ithelial-like cells synthesize basement membrane comtectable number of cell surface receptors. Because ponents in culture (Barraclough et al., 1987; Rudland Rama 25 and Rama 29 cells have about the same num- et al., 198213, 1986), it is possible t h a t the low-affinity ber of EGP receptors per cell, this lack of detectable site is located in the basement membrane. However i t FGF receptors on Rama 25 cells is unlikely to be due to remains to be ascertained whether these observations a simple experimental artefact, but may be due to made on other cell systems and tissues are also true in Rama 25 cells not expressing the bFGF receptor at the rat mammary cell lines. their cell surface. Thus the failure of bFGF to stimulate The KD for bFGF of the high-affinity site ranged DNA synthesis in the epithelial cell lines Rama 25 and from 35 pM in Rama 521 to 276 pM in Rama 29 cells Rama 37CL-A3 (Table 4)is probably due to a n absence (Fig. 3 and Table 4) and is consistent with that meaof accessible cell surface receptors rather than the fail- sured in other cell lines (Neufeld and Gospodarowicz, ure a t a subsequent step in the DNA synthetic path- 1985; Moscatelli, 1987).The differences in K, for bFGF way. observed between the different rat mammary cell lines Affinity cross-linking experiments show that the are significant, but there does not appear to be any bFGF receptor binds to two components of different consistent pattern (Table 3). Although Rama 29 cells molecular size in all the cell lines examined (Fig. 4). have the highest K, there is no progressive increase in Assuming a molecular weight of 18 kDa for bFGF, the K, from Rama 25-12, the most epithelial-like of the bFGF receptor exists in two forms with molecular intermediate cell lines, to Rama 24-14, the most myoweights of 160 kDa and 140 kDa. The relationship be- epithelial-like intermediate cell line (Rudland et al., tween these two forms of the bFGF receptor is un- 1986). The level of glycosylation of the bFGF receptor known, although they have been observed in other sys- has been proposed to be responsible for changes in its tems (Neufeld and Gospodarowicz, 1985; Schweigerer affinity for bFGF (Feige and Baird, 1988).Because the et al., 1987). It has been suggested that the bFGF re- molecular weight observed for affinity cross-linked ceptor is heavily glycosylated (Feige and Baird, 19881, bFGF receptors is identical in the cell lines examined and thus the observed differences in molecular weight (Fig. 4), despite a sixfold increase in KD from Rama may result from either differential glycosylation or 25-14 to Rama 29 cells (Table 31, another explanation is consequent differential proteolytic processing of a sin- more likely. Although each of the intermediate cell lines was sepgle bFGF receptor polypeptide. Alternatively, two distinct bFGF receptor polypeptides may exist. Although arately isolated from the epithelial Rama 25 cells, they artefactual processing of the receptor in our experi- form a graded series between the epithelial and myoments cannot be excluded, it would seem to be unlikely epithelial-like cells in the order Rama 25, Rama 25-12, to account for the presence of two forms of the bFGF Rama 25-11, Rama 25-14, Rama 29 (Rudland et al., receptor for the following reason. No differences are 1982b, 1986; Barraclough et al., 1987). It is likely that observed in the relative intensities of the two compo- the Rama 25-12, Rama 25-11, and Rama 25-14 cell lines nents when the solubilized cells are incubated for 2 h a t represent true intermediate stages in the differentia37°C or boiled for 2 min prior to SDS-PAGE (not tion of the myoepithelial-like cells not only in vitro shown). However, the intermittent finding of addi- (Paterson and Rudland, 1985; Rudland et al., 1986; tional lower molecular weight bands, specifically la- Barraclough et al., 1987) but also in the development of TABLE 4. Stimulation of DNA synthesis in rat mammary cell lines by bFGF

EGF AND bFGF RECEPTORS IN RAT MAMMARY CELL LINES

Fig. 4. SDS-PAGE of bFGF receptor affinity cross-linked to r12511bFGF, as described in Materials and Methods. Lanes 1-6: 20 ngiml [ T I - b F G F in PBS supplemented with 0.1% BSA (wiv) was crosslinked to cell surfaces with disuccinimidyl suberate. Lanes 7-12: 800 ngiml unlabelled bFGF was included in the incubation to determine

the mammary gland in vivo (Ormerod and Rudland, 1984). Table 3 shows that the more elongated the morphology of the intermediate cells, the higher the number of bFGF receptors. Thus it appears that bFGF receptors are first expressed at the cell surface at an early step in the differentiation pathway of Rama 25 to myoepithelial-like cells and this step precedes the stage represented by Rama 25-12 cells (Table 3). The number of receptors for bFGF increases again in going from Rama 25-12 to Rama 25-11 and then stabilizes before a final increase from Rama 25-14 to Rama 29. Moreover, Rama 521 cells, a spontaneous transformant of Rama 29, possess fewer myoepithelial characteristics (Rudland et al., 1982a) and also possess fewer receptors for bFGF. Thus the bFGF receptor may be an early developmental marker of the myoepithelial cells in vivo, alihough whether the bFGF receptor is expressed in the mammary gland in a similar cell-specific fashion as observed in vitro remains to be established. In an entirely independent rat mammary epithelial cell system (Dunnington et al., 19831,the epithelial cell line Rama 37CL-A3 also lacks receptors for bFGF, while the derivative myoepithelial-like cell line Rama 37-E8 possesses them (Fig. 3G, Table 3). This result indicates that the appearance of bFGF receptors on Rama 29 cells is not a chance event coinciding with the generation of this cell clone from the parental Rama 25 cells, but that the appearance of the bFGF receptor is a characteristic of the differentiation pathway of myoepithelial cells. Since cells that progress along this differentiation pathway express higher levels of bFGF receptors at the cell surface, bFGF or a related molecule may play a significant role in stimulating the growth of

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nonspecific cross-linking. Lanes 1,7: Rama 27; lanes 2,8: Rama 25-12; lanes 3, 9: Rama 25-11; lanes 4, 10: Rama 25-14; lanes 5 , l l : Rama 29; lanes 6,12: Rama 25. RG, start of the resolving gel. Molecular weight markers are indicated by arrows.

the myoepithelial component during the development of the mammary gland in vivo.

ACKNOWLEDGMENTS We thank Sarah Bottomley and John Cruise for expert technical assistance and the Cancer Research Campaign for generous financial support.

LITERATURE CITED Barraclough, R., Kimbell, R., and Rudland, P.S. (1987) Differential control of mRNA levels for Thy-1 antigen and laminin in rat mammary epithelial and myoepithelial-like cells. J. Cell. Physiol., 131: 393-401. Bennett, D.C., Peachy, L.A., Durbin, H., and Rudland, P.S. (1978) A possible mammary stem cell line. Cell, 15:283-298. Christopherson, R.I. (1983) Desalting protein solutions in a centrifuge column. Methods Enzymol., 91t278-281. Dunnington, D.J., Hughes, C.M., Monaghan, P., and Rudland P.S. (1983) Phenotypic instability of rat mammary tumour epithelial cells. JNCI, 71t1227-1240. Feige, J.-J.,and Baird, A. (1988) Glycosylation of the basic fibroblast growth factor receptor. J. Biol. Chem., 263:14023-14029, Gospodarowicz, D., Cheng,