Proc. Natl. Acad. Sci. USAVol. 81, pp. 6757-6761, November 1984Cell Biology

Specific binding to cultured cells of "25I-labeled type j3 transforminggrowth factor from human platelets

(radioreceptor assay/soft agar growth/autocrine)

RONALD F. TUCKER, EARL L. BRANUM, GARY D. SHIPLEY, ROBERT J. RYAN, AND HAROLD L. MOSES*Department of Cell Biology, Mayo Clinic/Foundation, Rochester, MN 55905

Communicated by Robert W. Holley, July 16, 1984

ABSTRACT Purified type f3 transforming growth factorfrom human platelets (TGFI3) radioiodinated with 1251-labeledBolton and Hunter reagent was found to bind to a variety ofcultured cells of both epithelial and mesenchymal origin, in-cluding normal human fibroblasts and keratinocytes. TGF.3binding sites have also been found on three mouse embryo-derived fibroblast-like cell lines with lower levels of TGFJ3binding on the chemically transformed derivatives of these celllines. A variety of human tumor cell lines was shown to havean inverse correlation between their level of TGF/3 bindingand their ability to form colonies in soft agar. The mouse em-bryo-derived AKR-2B (clone 84A) cells reached maximal bind-ing of 1251-labeled TGF(3 after 2 hr at 220C. Scatchard analysisof the equilibrium binding of TGFf8 to AKR-2B (clone 84A)cells gives a Kd of 33 pM with 10,500 binding sites per cell.This Kd for TGFI3 binding to AKR-2B (clone 84A) cells agreedwell with the ED50 of 40 pM for stimulation of colony forma-tion of these cells by TGF(3. The TGFP binding sites on theAKR-2B cells were shown to be specific for TGF,( with nosignificant competition with epidermal growth factor, fibro-blast growth factor, or insulin and only a small level of compe-tition with high concentrations of platelet-derived growth fac-tor. Partially purified preparations with TGFf8-like activityfrom mouse embryos and medium conditioned by mouse em-bryo-derived cells competed effectively for binding to theTGFB receptor.

Transforming growth factors (TGF) are operationally de-fined as polypeptides that stimulate the anchorage-indepen-dent proliferation in soft agar of normally anchorage-depen-dent nontransformed cells. Two types of TGF have been pu-rified, TGFa and TGFP. TGFa, also called TGF type 1, hasbeen purified from culture medium conditioned by retrovi-rus-transformed fibroblasts and human cancer cells (1). Themolecule has been completely sequenced, has 50 amino ac-ids with three disulfide bonds (Mr 5516), shows significanthomology with mouse and human epidermal growth factor(EGF), binds to the EGF receptor, and has biological activi-ty very similar to EGF (2). TGFa has been identified in me-dium conditioned by a variety of transformed cells in culture(1), in human placenta (3), and in mouse embryos (4).The other type of TGF, termed TGFP, is far more ubiqui-

tous than TGFa and has been identified in a variety of non-transformed and transformed cells in culture as well as innormal and neoplastic tissue in vivo (5-11). TGFl3 requiresadded EGF (or TGFa) to be active in stimulating soft agargrowth of NRK cells (rat kidney-derived) (12), but other celltypes such as the mouse AKR-2B are stimulated to prolifer-ate in soft agar by TGFJ without added EGF (9). TGFB alsocauses morphologic transformation of AKR-2B cells inmonolayer culture (13). TGF, has been purified from bovine

kidney and human placenta (14, 15). It has a Mr of -25,000with two apparently identical subunits. Partial NH2-terminalsequence data of the TGFJ3 from bovine kidney shows nosignificant homology with other known growth factors (14).TGF, is present in platelets and serum, but not plasma (16).Platelets have been shown to be a rich source of TGFJ3 byAssoian et al. (17), who also described a simplified schemefor obtaining pure TGFf3 in reasonable quantities from hu-man platelets.

In the present study we show that radioiodinated TGFf3isolated from human platelets by a modification of the proce-dure of Assoian et al. (17) has specific, saturable bindingsites on responsive cells. The high affinity of the binding onmouse AKR-2B cells correlates with the dose required forstimulation of growth in soft agar of these same cells, indi-cating that the binding is responsible for the biological activi-ty. Numerous other cell types in culture, both epithelial andmesenchymal, are shown to have receptors for TGFB.

MATERIALS AND METHODSCell Culture. The characteristics of the three pairs of non-

transformed:chemically transformed mouse cell lines-theAKR-2B:AKR-MCA (18, 19), the C3H/lOT1/2:MCA-58 (19,20), and the BALB/3T3:BP-3T3 (21, 22) cell lines-havebeen described. The EGF-receptorless Swiss 3T3 cell line,NR-6, was kindly provided by H. R. Herschman (23). Therat NRK (49F) cells were kindly provided by J. E. DeLarco(24). The A204 human rhabdomyosarcoma, A431 human epi-dermoid carcinoma, A498 human kidney carcinoma, andA549 human bronchioloalveolar carcinoma were a generousgift from G. J. Todaro and J. E. DeLarco and were de-scribed previously (25). The human kidney carcinoma celllines, CAKI-2 (26) and 1072-F (27), were kindly provided byM. M. Lieber. The green monkey kidney epithelial cells,BSC-1, were kindly provided by R. W. Holley (28).Human embryonic lung fibroblasts, W138 cells (29), were

kindly provided by V. J. Cristofalo. Flow 2000 cells, anotherhuman embryonic lung fibroblast, were obtained from FlowLaboratories (McLean, VA). Human foreskin fibroblasts,HFF cells, were obtained by collagenase treatment of neona-tal foreskins in our laboratory. Human neonatal foreskin ker-atinocytes, NF272, were provided by R. E. Scott and weregrown in serum-free MCDB 153 medium supplemented withbovine pituitary extract, EGF, insulin, hydrocortisone,phosphoethanolamine, and ethanolamine as described byBoyce and Ham (30). Stock cultures of all of the cells exceptthe keratinocytes were maintained in McCoy's 5a mediumsupplemented with 5% (vol/vol) fetal bovine serum. All ex-periments were performed within 10 passages of the frozenstocks from which the cells were recovered periodically.

Abbreviations: EGF, epidermal growth factor; FGF, fibroblastgrowth factor; PDGF, platelet-derived growth factor; TGFa andTGF,3, type a and p transforming growth factors.*To whom reprint requests should be addressed.

6757

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad Sci. USA 81 (1984)

These stock cells have been shown to be free of mycoplasmacontamination by staining with Hoechst no. 33258 stain (31).

Soft Agar Colony Stimulation Assay. The assay for thestimulation of colony formation of anchorage-dependentcells suspended in soft agar was performed as described (9)by using AKR-2B (clone 84A) cells as indicators and usingthe Quantimet 800 image analyzer (Cambridge Instruments,Monsey, NY) to quantitate the colony formation.

Preparation of Growth Factors. TGF, from human plate-lets was purified by the method of Assoian et al. (17) withthe addition of a final purification step consisting of re-versed-phase C18 HPLC eluted with a 45-60% acetonitrilegradient in H20 with 0.1% trifluoroacetic acid. The plateletTGF,8 was shown to be homogenous on silver-stained 12.5%polyacrylamide/NaDodSO4 gels (32) under both reducingand nonreducing conditions. The protein content of theTGFO preparation was determined by weighing a thoroughlydried sample of the purified protein. EGF was purified fromadult male mouse submaxillary glands by the method of Sav-age and Cohen (33). Crystalline bovine insulin was pur-chased from Sigma. Purified human platelet-derived growthfactor (PDGF) was kindly provided by R. Ross (34). Fibro-blast growth factor (FGF) was partially purified from bovinepituitary glands in our laboratory. This FGF preparation,=50% pure, caused one-half maximal stimulation of [3H]thy-midine incorporation into AKR-2B cells in the presence ofinsulin at 150 pg/ml (13). Mouse embryo factor was partiallypurified from acid/ethanol-extracted 17-day mouse embryos(8) by using ion-exchange and gel exclusion chromatogra-phy. Serum-free conditioned medium from AKR-MCA cellswas chromatographed on a Bio-Gel P-60 column in 1 M ace-tic acid as described (9). The major colony-stimulating activ-ity (pool III) eluting at an apparent Mr of 13,000 ± 2000 waspooled.

Radioiodination of TGFf8. Purified TGF8 was radioiodi-nated with 125I-labeled Bolton and Hunter reagent (35) fromAmersham. Four micrograms of purified TGFP was dis-solved in 10 ,ul of 4mM HCl. To this was added 10 ,ul of 0.2M sodium borate (pH 8.5) and this mixture was transferredto a polypropylene tube containing 500 tiCi (1 Ci = 37 GBq)of dried 125I-labeled Bolton and Hunter reagent. The reac-tion was allowed to proceed for 30 min at 0°C with occasion-al agitation. To terminate the reaction, 500 ,ul of 0.2 M gly-cine in 0.1 M sodium borate buffer (pH 8.5) was added,mixed well, and incubated for 5 min at 0°C. Then 500,ug ofbovine serum albumin in 50,l of 0.1 M sodium borate buffer(pH 8.5) was added and this mixture was chromatographedon a 9-ml Sephadex G-25 column that had been pretreatedwith 10 mg of bovine serum albumin in 1 M acetic acid/0.1%Triton X-100. The column was eluted with 1 M acetic acidwith 0.1% Triton X-100 and the 125I-labeled TGFA (1251_TGFP) was collected in the void volume of the column. 13I'-labeled TGFO (131I-TGFI) was synthesized with 1311-labeledBolton and Hunter reagent (35) by using the above proce-dure described for 125I-TGF,3.Standard Binding Assay. In the standard binding assay the

AKR-2B (clone 84A) cells were plated in 6-well cultureplates (9.1 cm2 per well) at a density of 1 x 105 to 2 x 10cells per well using McCoy's Sa medium with 5% (vol/vol)fetal bovine serum. The cells were washed with binding buff-er [Dulbecco's phosphate-buffered saline (pH 7.4) contain-ing 0.1% bovine serum albumin and 5mM MgCl2] and then 1ml of binding buffer containing 0.2 ng of 125I-TGFJ3 (15,000cpm) with or Without various levels of competitors was add-ed to each well. Nonspecific binding of 125I-TGFI3 was deter-mined in the presence of 1,ug of 50% pure unlabeled TGFp.The incubation was continued for 2 hr at room temperatureon a rocker platform. The assay was terminated by aspira-tion of the binding solution and then the cells were washedthree times with binding buffer. The cells and their receptor-

bound TGFJ3 were removed from the plate by treatment with1 ml of collagenase solution (1 mg/ml in H20) for 5 min atroom temperature. The cell-bound 125I-TGFI3 in the colla-genase solution was then assayed for radioactivity in a gam-ma counter. The number of cells in replicate wells was deter-mined by counting in a hemocytometer.Comparative Binding Assay. The binding of 125I-TGF,8 to a

variety of cells was compared with its binding to AKR-2B(clone 84A) cells by using the conditions of the standardbinding assay. Some of the tumor cell lines required scrapingafter the collagenase treatment to more completely removethe cells. The relative binding was expressed as a ratio of thecpm of bound 125I-TGF3 per 106 cells for one cell type divid-ed by the cpm of bound 125I-TGFP per 106 cells for AKR-2B(clone 84A) cells.

RESULTSTime Course of 125I-TGFP Binding. The time course of

125I-TGFJ3 binding to 2.88 x 105 AKR-2B (clone 84A) cellswas determined at room temperature, 220C ± MC (Fig. 1). Ateach time point the specific binding of 125I-TGFp (total bind-ing minus nonspecific binding) was determined. Of the15,000 cpm (0.2 ng) of 251I-TGF,3 added per well, -1.2% (175cpm) was nonspecifically bound. The level of nonspecificbinding changed very little during the time course of thebinding assay. At the point of maximal binding, 2 hr, -9%(1310 cpm) of the added 125I-TGFP3 was specifically bound tothe AKR-2B (clone 84A) cells. About 7-12% (1000-1800cpm) of the added 125I-TGFI3 was bound to the surfaces ofthe tissue culture plate as determined by elution with 0.2 MNaOH after removal of the cell receptor-bound 125I-TGFI3with collagenase. The collagenase treatment, therefore, al-lowed removal of the cell receptor-bound 125I-TGFf3, leavingthe surface-bound 125I-TGF/3 on the culture plate. The possi-ble degradation of the 1251-TGFf3 during the 2-hr incubationat room temperature was tested by comparing the trichloro-acetic acid-precipitable cpm in the binding buffer between 0and 2 hr. At the start of the assay, 0 hr, 95% of the added125I-TGFP was precipitable and this decreased only slightlyto 94% precipitable cpm after a 2-hr incubation under stan-dard assay conditions, indicating no significant degradationof the ligand during this time period.

Equilibrium Binding of 1251-TGFI3 to AKR-2B (Clone 84A)Cells. The equilibrium binding of 125I-TGF/3 to 2.49 x 105AKR-2B (clone 84A) cells at room temperature was ana-lyzed by the method of Scatchard (36) (Fig. 2). An apparentKd of 33 ± 3 pM with 10,500 binding sites per cell was calcu-lated by using the ligand-binding curve-fitting program de-

5

4

3-

6 .3

~'xX1

O0 1 2 3 4 5 6

Time, hr

FIG. 1. Time course of binding of 0.2 ng (15,000 cpm) of 125i-TGFP to 2.88 x 105 AKR-2B (clone 84A) cells at 220C. At varioustimes the cell-associated 125I-TGFP was determined after collagen-ase treatment. At each time point the binding of125I-TGFP3 was cor-rected for nonspecific binding and the specifically bound125I-TGF,8was plotted.

6758 Cell Biology: Tucker et aL

Proc. NatL Acad Sci. USA 81 (1984) 6759

0.08

0.04.

00 1 2 3 4 5

Bound TGFP, pM

FIG. 2. Scatchard plot of 125I-TGF/3 binding to 2.49 x 10i AKR-2B (clone 84A) cells at 22TC. Various concentrations of unlabeledTGFB were incubated with a tracer level (15,000 cpm; 0.2 ng) of 1251TGFB for 2 hr with the AKR-2B (clone 84A) cells. After washing,the cell-associated 125I-TGFP was determined.

veloped by Munson and Rodbard (37). Of the 15,000 cpm(0.2 ng) of 125I-TGFP added per well, about 3% (425 cpm)was nonspecifically bound and -11% (1615 cpm) was specif-ically bound to the AKR-2B (clone 84A) cells. Scatchardanalysis of 125I-TGFB3 binding to NRK (49F) cells gave verysimilar results (data not shown). The ED50 for TGF, stimu-lation of AKR-2B (clone 84A) colony formation in soft agarwas 40 pM, which agrees well with the apparent Kd forTGFP binding to these cells. Mg2+ ions (5 mM) were re-quired for the maximal binding of I251-TGF,3 to AKR-2B(clone 84A) cells. Another divalent cation, Ca2+, was notmaximally active as a replacement for Mg2+ ions (data notshown).Comparison of the Properties of 125I-TGFf3 and Unlabeled

TGF,8. Two methods were used for determining the proteincontent and specific activity of the 125I-TGF,6 preparation.The ability of 125I-TGF/3 and unlabeled TGFA to stimulatecolony formation of AKR-2B (clone 84A) cells in soft agarand compete for the binding of 1311-TGF/3 to the receptor onAKR-2B (clone 84A) cells were compared. Both methodsshowed that the 125I-TGFf3 preparation contained the equiv-alent of 1 ,g of TGFB with a specific activity of 60 ,uCi/,tg(data not shown). The loss of TGFB protein mass during thelabeling procedure was expected because of the adherenceof TGFB to surfaces especially during lyophilization. Thereseemed to be no gross modification of TGFl during its ra-

dioiodination since both 125I-TGFf3 and unlabeled TGFp mi-grate the same on 12.5% polyacrylamide/NaDodSO4 gels(32) under both reducing and nonreducing conditions (datanot shown).

Specificity of the TGF,8 Binding Sites on AKR-2B (Clone84A) Cells. Four purified growth factors were tested for theirability to inhibit 125I-TGFP binding to AKR-2B (clone 84A)cells (Fig. 3). FGF, EGF, and insulin had no significant ef-fect on the binding of TGF/3 at a concentration of the growthfactors at least 100 times that required to give a half-maximalresponse in their appropriate biological assay. PDGF causeda 20% inhibition of 25I-TGF,8 binding at a concentration 100times that needed to half-maximally stimulate [3H]thymidineincorporation into AKR-2B cells. This inhibition of TGFPbinding by PDGF may be an intrinsic property of PDGFsince this PDGF preparation stimulated colony formation ofAKR-2B (clone 84A) cells in soft agar at similar concentra-tions. However, the possibility of TGFP contamination ofthe PDGF preparation cannot be excluded.Two partially purified preparations of TGFP from mouse

embryos and AKR-MCA cell conditioned medium, P-60(pool III), caused a 50% inhibition of 125I-TGF/3 binding at 4pug/ml and 9 ,ug/ml, respectively (Fig. 3). Both of these TGFpreparations had an ED50 of 5 Ag/ml for the stimulation ofcolony formation of AKR-2B cells in soft agar. PurifiedTGFP caused 50% inhibition of I251-TGFj3 binding to AKR-2B (clone 84A) cells at 0.8 ng/ml (Fig. 3) and had an ED50 of1 ng/ml for stimulation of colony formation of these samecells.

Relative Binding of 12SI-TGFI3 to Various Cultured Cells.Three pairs of nontransformed:chemically transformed mousecell lines-the AKR-2B:AKR-MCA, C3H/10T1/2:MCA-58,and BALB/3T3:BP-3T3 cells-showed decreased 125I1TGFB binding on the chemically transformed cell line whencompared to their nontransformed parent cell line (Table 1).The AKR-MCA cells showed only 11% reduction in bindingrelative to the nontransformed AKR-2B parents, and thiswas reproducible in two repeat experiments. The chemicallytransformed C3H and BALB cells showed a greater reduc-tion in binding relative to their nontransformed parents, 41%and 45%, respectively. This reduction in 125I-TGFI3 bindingis likely the result of higher levels of production of TGFBcompeting material by these chemically transformed celllines, resulting in down-regulation of the receptor. Down-regulation of TGFP binding sites on AKR-2B (clone 84A)

go x80~~~~~~~~.00

la U

C to..\060£0U.0U~ ~

40

20~~~~~~~~~~~~~> 0

0

Competitor, g/ml

FIG. 3. Specificity of 1251I-TGF/3 binding to AKR-2B (clone 84A) cells at 220C. Cultures of AKR-2B (clone 84A) cells were incubated for 2 hrwith a tracer level (15,000 cpm; 0.2 ng) of 1151I-TGFp8 and various amounts of unlabeled TGF/3 (in), PDGF (i4), FGF (e), EGF (x), insulin (n),mouse embryo factor (0), or AKR-MCA cell conditioned medium, P-60 (pool III) (A&). Binding was terminated by washing and the cell-bound1251I-TGFp8 was determined.

Cell Biology: Tucker et aL

CXP-.co..-X

Proc. Natl. Acad Sci. USA 81 (1984)

Table 1. Binding of 125I-TGFf3 to cultured cells

Relative bindingCell line Cell origin Description of l25I-TGFP*

AKR-2B (clone 84A) Mouse embryo Fibroblast-like 1.00AKR-MCA AKR-2B Chemically transformed 0.89C3H/10T1/2 Mouse embryo Fibroblast-like 0.97MCA-58 C3H/10T'/2 Chemically transformed 0.57BALB/3T3 Mouse embryo Fibroblast-like 1.18BP-3T3 BALB/3T3 Chemically transformed 0.65NR-6 Swiss/3T3 EGF receptorless 1.79NRK (49F) Rat kidney Fibroblast 0.96WI38, PDL47t Human embryonic lung Fibroblast 1.31Flow 2000, PDL13t Human embryonic lung Fibroblast 2.38HFF, PDL8t Human neonatal foreskin Fibroblast 1.00NF272, PDL20t Human neonatal foreskin Keratinocyte 0.25BSC-1 Green monkey kidney Epithelial 0.30

*Ratio of 125I-TGFI3 bound compared to that bound by an equivalent number of AKR-2B (clone 84A)cells.

tPopulation doubling level number.

cells was demonstrated by incubation of AKR-2B (clone84A) cells with TGFB (100 ng/ml), PDGF (50 ng/ml), FGF(20 ng/ml), EGF (1 ,ug/ml), or insulin (1 ,ug/ml) at 37°C for 3hr and 18 hr. After the initial incubation period, the cellswere rinsed, and the binding capacity for 123I-TGF,8 was de-termined. Only TGFB pretreatment resulted in >10% reduc-tion in the TGF/3 binding capacity, with a 64% and 76% de-crease in binding after 3 hr and 18 hr, respectively (data notshown).The level of TGFB binding to NRK (49F) cells was very

similar to the level of TGFf3 binding to the AKR-2B (clone84A) cells. The NR-6 cells, an EGF receptorless derivativeof Swiss/3T3 cells, responded to TGFP by forming coloniesin soft agar (data not shown) and showed a higher level ofTGF/3 binding than the AKR-2B (clone 84A) cells (Table 1).Two fibroblast cell types derived from human embryonic

lung, WI38 and Flow 2000 cells, showed higher levels ofTGFP3 binding than the AKR-2B (clone 84A) cells (Table 1).Another fibroblast cell type derived from human neonatalforeskin, the HFF cells, had similar TGFB binding levels tothe AKR-2B (clone 84A) cells. Two cell types of epithelialorigin, secondary cultures of human neonatal foreskin kera-tinocytes (NF272) and green monkey kidney epithelial cells(BSC-1), demonstrated binding of 125I-TGFf3. The level ofbinding to these epithelial cell types was lower than that ob-tained with the secondary cultures of foreskin fibroblasts(HFF cells) and the other mesenchymal cell types (Table 1).

Binding of 1251-TGFf3 to Cultured Human Tumor Cells. Sixhuman tumor cell lines were compared for their ability toform colonies in soft agar and their relative binding of 125i-TGFP. There was a good inverse correlation between thelevel of TGFB binding and the number of soft colonies

Table 2. Binding of 1251I-TGFA to cultured tumor cells

Soft agarRelative colonies,

Cell binding of no. perline Origin l2SI-TGFp* 75,000 cellst

A204 Human rhabdomyosarcoma 0.00 18,500A549 Human lung carcinoma 0.38 9,6001072-F Human kidney carcinoma 0.23 2,400A498 Human kidney carcinoma 0.79 370A431 Human carcinoma of vulva 0.81 300CAKI-2 Human kidney carcinoma 0.87 300

*Ratio of '251-TFG,3 bound corhpared to that bound by an equivalentnumber of AKR-2B (clone 84A) cells.tNumber of colonies formed per 75,000 cells per ml seeded by usingconditions of standard soft agar assay.

formed per 75,000 cells per ml (Table 2). The tumor cell linesthat grow well in soft agar (A204, A549, 1072-F) had lowerTGF,8 binding capacities, whereas the tumor cell lines thatdo not grow well in soft agar had higher levels ofTGFp bind-ing, more comparable to that of the nontransformed AKR-2B (clone 84A) cells.

DISCUSSIONScatchard analysis of the equilibrium binding of TGFB toAKR-2B (clone 84A) cells demonstrates saturable bindingwith only one class of high-affinity binding sites (Fig. 2). TheKd for this 125I-TGF,8 binding is 33 pM. This agrees well withthe ED50 of 40 pM for the stimulation of colony formation ofAKR-2B (clone 84A) cells in soft agar, indicating that TGFJbinding to its receptor is responsible for the biological activi-ty of the molecule. There are 10,500 TGFJ3 binding sites onAKR-2B (clone 84A) cells. Very similar results were ob-tained with NRK (49F) cells, the other cell type commonlyused as indicators for detecting TGF,8 activity (6). For com-parison, PDGF binds to Swiss/3T3 cells with a Kd of 10 pMand there are -200,000 binding sites per cell (38). EGF bindsto human fibroblasts with a Kd of 300-500 pM and with-100,000 binding sites per cell (39) and to AKR-2B cells witha Kd of 1 nM and with 100,000 binding sites per cell (40).Thus, there appear to be significantly fewer TGFf3 bindingsites on fibroblasts and fibroblast-like cells (i.e., AKR-2B,C3H/10T1/2, and BALB/3T3) than PDGF or EGF bindingsites.The specificity of the TGF8 binding sites on AKR-2B

(clone 84A) cells was shown by the absence of competitionby FGF, EGF, and insulin (Fig. 3). The significance of thelow level of competition shown by high concentrations ofPDGF is not known. This may represent contamination ofthe PDGF preparation with small amounts of TGF/3, sincethese two growth factors from platelets tend to copurify dur-ing the early steps of the PDGF purification scheme (unpub-lished observations). Alternatively, PDGF may be able tobind with low affinity to the TGF/3 receptor in a manneranalogous to insulin binding to the somatomedin C receptor(41).The binding of TGFI to NR6 cells, an EGF-receptorless

Swiss 3T3-derived cell line, and the ability ofTGF/3 to stimu-late colony formation of these cells in soft agar demonstratethat EGF is not required for the expression of the biologicactivity of TGFO3. Added EGF is also not required for TGF/3stimulation of growth of AKR-2B (clone 84A) cells in softagar (9). This is in contrast to the NRK (49F) cells that dorequire added EGF for the full expression of TGFB biologi-

6760 Cell Biology: Tucker et aL

Proc. Natl. Acad. Sci. USA 81 (1984) 6761

cal activity (12). It has been shown recently that TGFJ3 in-creases the levels of EGF receptors on NRK (49F) cells (42).This may be one mechanism that allows EGF to modulatethe colony-stimulating activity of TGFIB on NRK (49F) cells.Our results demonstrate the binding of TGFIB to a variety

of cultured cells of both mesenchymal and epithelial origin(Table 1). This is in contrast to PDGF (43), which only bindsto cells of mesenchymal origin, but is similar to EGF (39,44), which binds to both mesenchymal and epithelial cells. Afactor that may contribute to the low level of TGFB bindingon certain human tumor cell lines and chemically trans-formed cell lines (see Tables 1 and 2) is the down-regulationof TGFI3 binding sites caused by the production of TGFP bythese cells. It has been shown previously that both nontrans-formed and transformed cells in culture (9) and both normaland tumor tissues in vivo (6, 8, 10, 11) produce TGFO-likemolecules. We have now demonstrated that two partially pu-

rified preparations of TGF from mouse embryos and AKR-MCA cell conditioned medium bind to the same cell mem-brane receptor on AKR-2B (clone 84A) cells as purified hu-man TGFP3 (Fig. 3). We have also demonstrated in thepresent study that incubation of the AKR-2B (clone 84A)cells with TGFP3 at 370C for 18 hr caused a 76% reduction inthe level of '25I-TGF3 binding, probably through the process

of receptor down-regulation. The relatively higher level ofTGFf binding on the normal human W138, Flow 2000, andneonatal foreskin fibroblasts may reflect either a higher in-trinsic number of TGFP cell membrane receptors or a lowerlevel of production of TGFB competing material or both.The specific binding assay for TGFf3 described in the pres-

ent study will be valuable in determining TGFB3 levels in tis-sues, cell extracts, and cell conditioned media. This assay

may be very important in elucidating the role of TGF,3 in cellproliferation, differentiation, and neoplastic transformation.The ubiquitous presence of TGFf3-like molecules in normaltissues and cultured cells and the broad spectrum of celltypes that exhibit TGF,8 binding sites implies that thisgrowth factor may have important roles in normal cellularprocesses. The induction by TGF3 of phenotypic changescharacteristic of neoplastic cells (i.e., morphologic transfor-mation in monolayer and anchorage-independent growth insoft agar) in nontransformed, anchorage-dependent culturedcells indicates that TGF, may also be important in the proc-

ess of neoplastic transformation.

We thank Dr. Edward B. Leof for helpful discussions, Mary E.Aakre for technical assistance, and Patricia Hart and Mary Johnsonfor typing the manuscript. This work was supported by Grant CA27217 awarded by the National Cancer Institute. G.D.S. was sup-

ported by National Cancer Institute training Grant CA 09441.

1. Marquardt, H., Hunkapiller, M. W., Hood, L. E., Twardzik,D. R., DeLarco, J. E., Stephenson, J. R. & Todaro, G. J.(1983) Proc. Natl. Acad. Sci. USA 80, 4684-4688.

2. Marquardt, H., Hunkapiller, M. W., Hood, L. E. & Todaro,G. J. (1984) Science 223, 1079-1082.

3. Stromberg, K., Pigott, D. A., Ranchalis, J. E. & Twardzik,D. R. (1982) Biochem. Biophys. Res. Commun. 106, 354-361.

4. Twardzik, D. R., Ranchalis, J. E. & Todaro, G. J. (1982) Can-cer Res. 42, 590-593.

5. DeLarco, J. E. & Todaro, G. J. (1978) Proc. Natl. Acad. Sci.USA 75, 4001-4005.

6. Roberts, A. B., Anzano, M. A., Lamb, L. C., Smith, J. M. &Sporn, M. B. (1981) Proc. Natl. Acad. Sci. USA 78, 5339-5343.

7. Moses, H. L., Branum, E. L., Proper, J. A. & Robinson,

R. A. (1981) Cancer Res. 41, 2842-2848.

8. Proper, J. A., Bjornson, C. L. & Moses, H. L. (1982) J. Cell.Physiol. 110, 169-174.

9. Tucker, R. F., Volkenant, M. E., Branum, E. L. & Moses.H. L. (1983) Cancer Res. 43, 1581-1586.

10. Nickell, K. A., Halper, J. & Moses, H. L. (1983) Cancer Res.43, 1966-1971.

11. Halper, J. & Moses, H. L. (1983) Cancer Res. 43, 1972-1979.12. Anzano, M. A., Roberts, A. B., Smith, J. M., Sporn, M. B. &

DeLarco, J. E. (1983) Proc. Natl. Acad. Sci. USA 80, 6264-6268.

13. Shipley, G. D., Childs, C. B., Volkenant, M. E. & Moses,H. L. (1984) Cancer Res. 44, 710-716.

14. Roberts, A. B., Anzano, M. A., Meyers, C. A., Wideman, J.,Blacher, R., Pan, Y.-C. E., Stein, S., Lehrman, S. R., Smith,J. M., Lamb, L. C. & Sporn, M. B. (1983) Biochemistry 22,5692-5698.

15. Frolik, C. A., Dart, L. L., Meyers, C. A., Smith, D. M. &Sporn, M. B. (1983) Proc. Natl. Acad. Sci. USA 80, 3676-3680.

16. Childs, C. B., Proper, J. A., Tucker, R. F. & Moses, H. L.(1982) Proc. Natl. Acad. Sci. USA 79, 5312-5316.

17. Assoian, R. K., Komoriya, A., Meyers, C. A., Miller, D. M.& Sporn, M. B. (1983) J. Biol. Chem. 258, 7155-7160.

18. Getz, M. J., Reiman, H. M., Jr., Siegal, G. P., Quinlan, T. J.,Proper, J., Elder, P. K. & Moses, H. L. (1977) Cell 11, 909-921.

19. Moses, H. L., Proper, J. A., Volkenant, M. E., Wells, D. J. &Getz, M. J. (1978) Cancer Res. 38, 2807-2812.

20. Reznikoff, C. A., Bertram, J. S., Bronkow, D. W. & Heidel-berger, C. (1973) Cancer Res. 33, 3239-3249.

21. Brown, K. D. & Holley, R. W. (1979) J. Cell. Physiol. 100,139-146.

22. Pledger, W. J., Stiles, C. D., Antoniades, H. N. & Scher,C. D. (1978) Proc. Natl. Acad. Sci. USA 75, 2839-2843.

23. Pruss, R. M. & Herschman, H. R. (1977) Proc. Nati. Acad.Sci. USA 74, 3918-3921.

24. DeLarco, J. E. & Todaro, G. J. (1978) J. Cell. Physiol. 94,335-342.

25. Gierd, D. J., Aaronson, S. A., Todaro, G. J., Arnstein, P.,Kersey, J. H., Dosik, H. & Parks, W. P. (1973) J. Natl. Can-cer Inst. 51, 1417-1423.

26. Fogh, J. & Trempe, G. (1975) in Human Tumor Cells In Vitro,ed. Fogh, J. (Plenum, New York), pp. 115-159.

27. Williams, R. D. (1980) Invest. Urol. 17, 359-363.28. Hopps, H. E., Bernheim, B. C., Nisalak, A., Tijo, J. H. &

Smadel, J. E. (1963) J. Immunol. 91, 416-424.29. Hayflick, L. (1965) Exp. Cell Res. 37, 614-636.30. Boyce, S. T. & Ham, R. G. (1983) J. Invest. Dermatol. 81,

Suppl., 33S-40S.31. Chen, T. R. (1977) Exp. Cell Res. 104, 255-262.32. Laemmli, U. K. (1970) Nature (London) 227, 680-685.33. Savage, C. R., Jr., & Cohen, S. (1972) J. Biol. Chem. 247,

7609-7611.34. Raines, E. W. & Ross, R. (1982) J. Biol. Chem. 257, 5154-

5160.35. Bolton, A. E. & Hunter, W. M. (1973) Biochem. J. 133, 529-

538.36. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672.37. Munson, P. J. & Rodbard, D. (1980) Anal. Biochem. 107, 220-

239.38. Bowen-Pope, D. F. & Ross, R. (1982) J. Biol. Chem. 257,

5161-5171.39. Carpenter, G., Lembach, K. J., Morrison, M. M. & Cohen, S.

(1975) J. Biol. Chem. 250, 4297-4304.40. Robinson, R. A., Volkenant, M. E., Ryan, R. J. & Moses,

H. L. (1981) J. Cell. Physiol. 109, 517-524.41. Van Wyk, J. J., Underwood, L. E., Baseman, J. B., Hintz,

R. L., Clemmons, D. R. & Marshall, R. N. (1975) Adv. Me-tab. Disorders 8, 127-150.

42. Assoian, R. K., Frolik, C. A., Roberts, A. B., Miller, D. M. &Sporn, M. B. (1984) Cell 36, 35-41.

43. Heldin, C.-H., Westermark, B. & Wasteson, A. (1981) Proc.Natl. Acad. Sci. USA 78, 3664-3668.

44. Fabricant, R. N., DeLarco, J. E. & Todaro, G. J. (1977) Proc.Natl. Acad. Sci. USA 74, 565-569.

Cell Biology: Tucker et aL