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[CANCER RESEARCH 42, 4394-4398, November 1982] 0008-5472/82/0042-OOOOS02.00 Epidermal Growth Factor Receptors and Effect of Epidermal Growth Factor on Growth of Human Breast Cancer Cells in Long-Term Tissue Culture1 Yasuo Imai,2 Clement K. H. Leung, Henry G. Friesen, and Robert P. C. Shiu Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada, R3E OW3 ABSTRACT Epidermal growth factor (EGF) may be important in regulating the proliferation of mammary epithelial cells. In the present study, we examined EGF binding and effect on growth in nine human mammary cell lines. The T-47D, MCF-7, SK-Br-3, AlAb 496, BT-20, and BT-474 tumor cell lines and a cell line (HBL- 100) derived from milk exhibited EGF binding; both high (Ka 1010 M~1)- and low (Ka 109 M-1)-affinity sites were detected. The total number of EGF receptors per cell of different cell lines varied from 1.6 x 103 sites/cell (for AlAb 496) to 1.5 x 106 sites/cell (for BT-20). The two floating cell lines, DU4475 and Lev III, had no detectable EGF binding. Effect of EGF on growth was studied by monitoring cell number and the incor poration of [3H]thymidine into DNA of cells maintained in Dul- becco's modified Eagle's medium supplemented with 0.1% fetal bovine serum. Using these procedures, only T-47D cells were stimulated by EGF at low concentrations (0.1 to 1 ng/ ml). At concentrations higher than 10 ng/ml, EGF was inhibi tory to varying degrees in most cell lines that contained EGF receptors. The growth of the two floating cell lines that had no detectable EGF binding was unaffected by EGF. Our results show that EGF receptors are not present in all human breast cancer cell lines. There is no apparent correlation between EGF binding and its mitogenic activity in cell lines with EGF receptors. EGF may have biological roles in human breast cancer other than growth regulation. INTRODUCTION Human breast cancers are known to be influenced by ste roids and polypeptide hormones (27). Recently, the use of in vitro long-term tissue culture of human breast cancer cells has provided an excellent model for studying the mechanisms of hormone actions in breast cancer (25). Receptors for insulin (30), steroid hormones (8, 25), and prolactin (37) in several established human breast cancer cell lines have been reported. In addition to the major steroid and peptide hormones, EGF3 has been shown to stimulate the growth of cultured normal mammary epithelium in both rodents (43, 45) and humans (39, 41, 42). The presence of high concentrations of EGF in human milk was recently reported (40). EGF was also reported to be mitogenic in mouse mammary carcinoma cells (44) and in one human breast cancer cell line (29). In addition, EGF receptors 1 Supported by the Medical Research Council of Canada and the National Cancer Institute of Canada. Portions of this work were reported at the Twenty- First Annual Meeting of the American Society for Cell Biology held in Anaheim, Calif., November 9 to 13. 1981 (14). 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: EGF, epidermal growth factor; DME, Dulbecco's modified Eagle's medium; HEPES, W-2-hydroxyethylpiperazine-/V'-ethanesul- fonic acid. Received February 4. 1982; accepted July 26. 1982. have been studied in several breast cancer cell lines (28).4 Furthermore, EGF was shown to act as a tumor promoter in an in vivo study involving application of methylcholanthrene to mouse skin (34). On the basis of these observations, it seemed worthwhile to determine the biological effect of EGF on human breast cancer cells. In the present study, we examined the binding of EGF to 9 human mammary cell lines established from various sources and compared the receptors and mito genic activity of EGF in these cells. MATERIALS AND METHODS Cell Lines and Growth Conditions. MCF-7 was generously provided by Dr. M. Rich, Michigan Cancer Foundation. BT-474 was a gift of Dr. E. Y. Lasfargues, Institute for Medical Research, Camden, N. J. T-47D and HBL-100 were obtained from EG&G/Mason Research Institute, Rockville, Md. AlAb 496, DU4475, Lev III, BT-20, and SK-Br-3 were provided by J. F. Weaver, Naval Biosciences Laboratory, Oakland, Calif. All of these cell lines are characterized with respect to human origin (8). The source and growth pattern of these cell lines are summarized in Table 1. All cell lines were routinely maintained in DME supplemented with L- glutamine (4 mw), glucose (4.5 g/liter), penicillin (100 lU/ml), strep tomycin (100 /ig/ml), bovine insulin (10 |ug/ml), and 10% (v/v) fetal bovine serum. Trypsin-EDTA in Hanks' balanced salt solution was used for cell passages. Cells were maintained in a humidified atmosphere of 95% air-5% CO2 at 37°. Culture flasks and dishes were obtained from Corning (Fisher Scientific Co., Winnipeg, Manitoba, Canada); culture medium, trypsin, antibiotics, and fetal bovine serum were purchased from Grand Island Biological Co. (Burlington, Ontario, Canada). Preparation and lodination of EGF. EGF was purified from male mouse submaxillary glands according to the method of Savage and Cohen (35). EGF was iodinated by a modification of the chloramine-T method (12), using Na'25l (Amersham). The specific activity of ¡he126I- labeled EGF was 200 to 250 fiCi/fig. Determination of Specific Binding of 125l-Labeled EGF to Cultured Cells. Four dishes (35 mm) of subconfluent monolayer cells were washed once with 25 mw HEPES-buffered (pH 7.4) Hanks' balanced salt solution containing 0.1% (w/v) bovine serum albumin. After aspi ration of the washing medium, each dish received 1.5 ml of the same buffer containing about 0.05 ng of 125l-labeled EGF with or without various doses of unlabeled EGF (0.01 to 100 ng). The dishes were incubated at 24° (room temperature) for 4 hr when equilibrium was achieved. At the end of this incubation period, the radioactive medium was aspirated, and the dishes were washed twice with 2-ml portions of ice-cold buffer. The cells were then dissolved with 2 ml of 0.1 N NaOH containing 0.5% Triton X-100. The solutions were transferred to dis posable glass tubes, and radioactivity was determined in a LKB Wallac gamma counter with a counting efficiency of 70% for 125I. Specific binding was taken as the total radioactivity bound (mean of duplicates) in the absence of unlabeled hormone minus that bound (mean of ' S. Fitzpatrick, M. Chu, W. Dean, and G. Schulte. Detection of epidermal growth factor receptors in human breast tumors and cell lines. Proceedings of 63rd Annual Meeting of the Endocrine Society. Abstract 110, 1981. 4394 CANCER RESEARCH VOL. 42 Association for Cancer Research. by guest on September 2, 2020. 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Page 1: Epidermal Growth Factor Receptors and Effect of Epidermal ... · Epidermal growth factor (EGF) may be important in regulating the proliferation of mammary epithelial cells. In the

[CANCER RESEARCH 42, 4394-4398, November 1982]0008-5472/82/0042-OOOOS02.00

Epidermal Growth Factor Receptors and Effect of Epidermal Growth Factoron Growth of Human Breast Cancer Cells in Long-Term Tissue Culture1

Yasuo Imai,2 Clement K. H. Leung, Henry G. Friesen, and Robert P. C. Shiu

Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada, R3E OW3

ABSTRACT

Epidermal growth factor (EGF) may be important in regulatingthe proliferation of mammary epithelial cells. In the presentstudy, we examined EGF binding and effect on growth in ninehuman mammary cell lines. The T-47D, MCF-7, SK-Br-3, AlAb496, BT-20, and BT-474 tumor cell lines and a cell line (HBL-

100) derived from milk exhibited EGF binding; both high (Ka1010 M~1)- and low (Ka 109 M-1)-affinity sites were detected.

The total number of EGF receptors per cell of different celllines varied from 1.6 x 103 sites/cell (for AlAb 496) to 1.5 x106 sites/cell (for BT-20). The two floating cell lines, DU4475

and Lev III, had no detectable EGF binding. Effect of EGF ongrowth was studied by monitoring cell number and the incorporation of [3H]thymidine into DNA of cells maintained in Dul-becco's modified Eagle's medium supplemented with 0.1%

fetal bovine serum. Using these procedures, only T-47D cells

were stimulated by EGF at low concentrations (0.1 to 1 ng/ml). At concentrations higher than 10 ng/ml, EGF was inhibitory to varying degrees in most cell lines that contained EGFreceptors. The growth of the two floating cell lines that had nodetectable EGF binding was unaffected by EGF. Our resultsshow that EGF receptors are not present in all human breastcancer cell lines. There is no apparent correlation betweenEGF binding and its mitogenic activity in cell lines with EGFreceptors. EGF may have biological roles in human breastcancer other than growth regulation.

INTRODUCTION

Human breast cancers are known to be influenced by steroids and polypeptide hormones (27). Recently, the use of invitro long-term tissue culture of human breast cancer cells hasprovided an excellent model for studying the mechanisms ofhormone actions in breast cancer (25). Receptors for insulin(30), steroid hormones (8, 25), and prolactin (37) in severalestablished human breast cancer cell lines have been reported.

In addition to the major steroid and peptide hormones, EGF3

has been shown to stimulate the growth of cultured normalmammary epithelium in both rodents (43, 45) and humans (39,41, 42). The presence of high concentrations of EGF in humanmilk was recently reported (40). EGF was also reported to bemitogenic in mouse mammary carcinoma cells (44) and in onehuman breast cancer cell line (29). In addition, EGF receptors

1 Supported by the Medical Research Council of Canada and the NationalCancer Institute of Canada. Portions of this work were reported at the Twenty-First Annual Meeting of the American Society for Cell Biology held in Anaheim,Calif., November 9 to 13. 1981 (14).

2 To whom requests for reprints should be addressed.3 The abbreviations used are: EGF, epidermal growth factor; DME, Dulbecco's

modified Eagle's medium; HEPES, W-2-hydroxyethylpiperazine-/V'-ethanesul-

fonic acid.Received February 4. 1982; accepted July 26. 1982.

have been studied in several breast cancer cell lines (28).4

Furthermore, EGF was shown to act as a tumor promoter in anin vivo study involving application of methylcholanthrene tomouse skin (34). On the basis of these observations, it seemedworthwhile to determine the biological effect of EGF on humanbreast cancer cells. In the present study, we examined thebinding of EGF to 9 human mammary cell lines establishedfrom various sources and compared the receptors and mitogenic activity of EGF in these cells.

MATERIALS AND METHODS

Cell Lines and Growth Conditions. MCF-7 was generously providedby Dr. M. Rich, Michigan Cancer Foundation. BT-474 was a gift of Dr.E. Y. Lasfargues, Institute for Medical Research, Camden, N. J. T-47Dand HBL-100 were obtained from EG&G/Mason Research Institute,Rockville, Md. AlAb 496, DU4475, Lev III, BT-20, and SK-Br-3 were

provided by J. F. Weaver, Naval Biosciences Laboratory, Oakland,Calif. All of these cell lines are characterized with respect to humanorigin (8). The source and growth pattern of these cell lines aresummarized in Table 1.

All cell lines were routinely maintained in DME supplemented with L-

glutamine (4 mw), glucose (4.5 g/liter), penicillin (100 lU/ml), streptomycin (100 /ig/ml), bovine insulin (10 |ug/ml), and 10% (v/v) fetalbovine serum.

Trypsin-EDTA in Hanks' balanced salt solution was used for cell

passages. Cells were maintained in a humidified atmosphere of 95%air-5% CO2 at 37°. Culture flasks and dishes were obtained from

Corning (Fisher Scientific Co., Winnipeg, Manitoba, Canada); culturemedium, trypsin, antibiotics, and fetal bovine serum were purchasedfrom Grand Island Biological Co. (Burlington, Ontario, Canada).

Preparation and lodination of EGF. EGF was purified from malemouse submaxillary glands according to the method of Savage andCohen (35). EGF was iodinated by a modification of the chloramine-Tmethod (12), using Na'25l (Amersham). The specific activity of ¡he126I-

labeled EGF was 200 to 250 fiCi/fig.Determination of Specific Binding of 125l-Labeled EGF to Cultured

Cells. Four dishes (35 mm) of subconfluent monolayer cells werewashed once with 25 mw HEPES-buffered (pH 7.4) Hanks' balanced

salt solution containing 0.1% (w/v) bovine serum albumin. After aspiration of the washing medium, each dish received 1.5 ml of the samebuffer containing about 0.05 ng of 125l-labeled EGF with or without

various doses of unlabeled EGF (0.01 to 100 ng). The dishes wereincubated at 24° (room temperature) for 4 hr when equilibrium was

achieved. At the end of this incubation period, the radioactive mediumwas aspirated, and the dishes were washed twice with 2-ml portions ofice-cold buffer. The cells were then dissolved with 2 ml of 0.1 N NaOHcontaining 0.5% Triton X-100. The solutions were transferred to dis

posable glass tubes, and radioactivity was determined in a LKB Wallacgamma counter with a counting efficiency of 70% for 125I. Specific

binding was taken as the total radioactivity bound (mean of duplicates)in the absence of unlabeled hormone minus that bound (mean of

' S. Fitzpatrick, M. Chu, W. Dean, and G. Schulte. Detection of epidermal

growth factor receptors in human breast tumors and cell lines. Proceedings of63rd Annual Meeting of the Endocrine Society. Abstract 110, 1981.

4394 CANCER RESEARCH VOL. 42

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EGF and Human Breast Cancer Cell Lines

Table 1

Tissue source and growth pattern of human mammary cell lines

CelllinesT-47DMCF-7BT-20BT-474SK-Br-3AlAb

496HBL-100DU4475Lev

IIITissue

sourcePleural

effusionPleuraleffusionOriginaltumorOriginaltumorPleuraleffusionLungmetastasisNormalmilkCutaneousmetastasisPleural

effusionGrowth

patternMonolayerMonolayerMonolayerMonolayerMonolayerMonolayerMonolayerSuspensionSuspensionRef.17382221103231209

duplicates) in the presence of excess unlabeled hormone (100 ng).The latter represents nonspecific binding to cells and culture dish.

In the case of cells cultured in suspension (DU4475 and Lev III), thecells were incubated in plastic tubes. Free and bound hormones wereseparated by centrifugation of the cell suspension at 250 x g for 5min. The pellets were resuspended and washed twice with cold buffer.The cell pellets were counted in the gamma counter.

Determination of Effect of EGF on Growth of Cultured Cells.Human breast cancer cells (5 to 8 x 104 cells/dish) were plated in

DME supplemented with 10% fetal bovine serum and incubated for 24hr. The cells were then washed with fresh medium without serum andincubated in 2 ml of DME containing 0.1% fetal bovine serum for 24hr. Twenty jul of EGF dissolved in the above culture medium wereadded. Twenty hr later, the cells were labeled with [3H]thymidine (1

^Ci/dish) for 4 hr. The medium was aspirated, and the cells werewashed once with phosphate-buffered saline, followed by one washing

with 3 ml of cold 10% trichloroacetic acid and 2 washings with cold5% trichloroacetic acid. Acid-insoluble radioactivity was dissolved in 1ml of 0.1 N NaOH containing 0.5% Triton X-100. The solution was

mixed with Aguasol 2 (New England Nuclear) and counted in a liquidscintillation counter. In the case of floating cells, cells were cultured inplastic culture tubes (12 x 75 mm) in the same volume as monolayercells. Trichloroacetic acid-insoluble materials were collected by cen

trifugation (1500 x g, 30 min).Cell number was also determined 4 days after addition to EGF. Cells

were plated and cultured in the same manner as that of the [3H]-

thymidine incorporation study. Cells were trypsinized and counted byCoulter Counter.

Other Reagents. Human growth hormone (2.6 lU/mg) was obtainedfrom the Endocrine Study Section, National Institute of Arthritis, Metabolism, and Digestive Diseases, NIH. Human prolactin (>25 lU/mg)was kindly provided by Dr. I. Worsley, University of Manitoba. Fibro-blast growth factor, nerve growth factor, multiplication-stimulating activity, and anti-EGF antiserum were obtained from Collaborative Research, Inc. (Waltham, Mass.). Bio-Gel P-6 (200 to 400 mesh) waspurchased from Bio-Rad Laboratories. All other reagents were pur

chased from Sigma Chemical Co. (St. Louis, Mo.).

binding experiments, cells were incubated at 24°for 4 hr in anequilibrium condition. Under this condition, 125l-labeled EGF in

the incubation media was not significantly degraded as compared with the 125l-labeled EGF which has not been exposed to

the cells (Table 2). Furthermore, methylamine (10 mivi), ammonium chloride (10 mM), and sodium azide, which are knownto inhibit the internalization or degradation of bound hormonesto cells (5, 19), did not alter significantly the amount of 125I-

labeled EGF bound to the cells (data not shown). The specificbinding of 125l-labeled EGF achieved with HEPES-bufferedHanks' balanced salt solution was similar to that obtained by

using HEPES-buffered DME without sodium bicarbonate. In

addition, there was no apparent change in the morphology ofthe cells as judged by phase-contrast microscopy after 4 hr ofincubation at any of the above temperatures.

Specificity of EGF Receptor. To assess the specificity ofEGF binding to the human breast cancer cells, excess amountsof unlabeled polypeptide hormones and growth factors wereadded in the incubation media of T-47D cells (Table 3). Noneof the hormones added altered the binding of 125l-labeled EGFto the cells. 12-O-Tetradecanoylphorbol-13-acetate, which is

known to interact with EGF receptor (23), inhibited the bindingof EGF to the T-47D cells when preincubated at 37° for 40

min. This inhibition was also observed to varying degrees inother breast cancer cell lines with EGF receptors.

Equilibrium Analysis of Binding of 125l-Labeled EGF to

Human Breast Cancer Cell Lines. For all cell lines tested,

01234Time (hour)

Chart 1. Effect of time and temperature on the binding of 125l-labeled EGF tointact T-47D monolayer cells. Each dish contained 1.2 x 106 cells and 12,500cpm of 125l-labeled EGF. Each point represents the specific binding of 125l-labeledEGF to the T-47D cells. Specific binding was determined by subtracting the meanvalue of duplicates for nonspecific binding from the mean value of duplicates fortotal binding.

RESULTS

Optimization of Conditions for the Binding of 125l-Labeled

EGF by Intact Human Breast Cancer Cells. In order to determine the optimal conditions for studying the binding of 125I-

labeled EGF to the human mammary cell lines, the effects oftime and temperature on the binding of 125l-labeled EGF to

several cell lines were tested. Chart 1 shows the results of EGFbinding to T-47D cells. At 37°,maximum binding of 125l-labeled

EGF was observed at about 90 min, and the binding decreasedgradually with time. At 4°,the rate of association of 125l-labeledEGF was slow. At 24°, the binding increased gradually and

reached a plateau after 4 hr without a decrease thereafter. Thetime course of BT-20 and HBL-100 showed a pattern similar tothat of the T-47D cells (data not shown). In all subsequent

Table 2Integrity of 125/-/abe/ed EGF after incubation with human breast cancer cells

125l-labeled EGF (24,500 cpm/dish) was incubated at 24° for 4 hr in thepresence of T-47D (2.1 x 106 cells/dish) and BT-20 (0.26 x 106 cells/dish) orin the absence of cells. Under this condition, about 30% of added 125l-labeledEGF bound to the cells of both cell lines. The integrity of '25l-labeled EGF in the

incubation media was examined by immunoreactivlty to an excess anti-EGFantiserum or by gel filtration on Blol-Gel P-6 (1 x 25 cm). Incubation medium(500 fil) applied on the column and eluted with phosphate-buffered saline containing 0.1% bovine serum albumin. The radioactivity eluted in the fractionswhere intact '"l-labeled EGF appeared was collected and counted. The values

were expressed as percentages of total radioactivity.

Incubation medium

% of radioactivity precipitated by excess anti

body

% of radioactivity elutedas intact I26l-labeled

EGF

With T-47DWith BT-20Without cells

878690

95.594.8

96

NOVEMBER 1982 4395

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Y. Imai et al.

Table 3Specificity of binding of '"/-/abe/ed EOF to T-4 7D cells

The indicated unlabeled polypeptides were added simultaneously with 125I-labeled EGF (22.700 cpm/dish) to T-47D cells (1.3 x 10e cells/dish) andincubated at 24°for 4 hr. 12-O-Tetradecanoylphorbol-13-acetate was preincu-bated with cells at 37° for 40 min. The medium was then changed to binding

buffer, and binding assay was performed in the same manner as above. Eachvalue represents the mean of duplicates.

Specific bindingConcentra- of ' "(-labeled

Addition tion (jig/ml) EGF (cpm/dish)

NonePorcineInsulinHuman

growthhormoneHumanprolactinMultiplication-stimulatingactivityFibroblast

growthfactorNervegrowthfactor1

2-O-tetradecanoylphorbol- 13-acetate1.01.01.01.01.01.00.0010.010.15,2765,5245,6675,5015,2115,4315,2062,5251,7751,512

specific binding of 125l-labeled EGF was a direct function of the

concentration of hormone in the medium. For clarity, the binding of 125l-labeled EGF to T-47D and HBL-100 cells as a

function of hormone concentration is illustrated in Chart 2.The amount of hormone bound gradually increased as the

hormone concentrations increased, until a plateau wasreached. Scatchard analysis of the binding data (Chart 2, inset)exhibited a curvilinear pattern. Scatchard plots of the other celllines also showed a curvilinear pattern. The apparent affinityconstants and total binding sites were determined from thesedata by the method of Kahn et al. (16).

Summary of EGF Receptor Contents in Human BreastCancer Cells. The optimal conditions illustrated in Chart 1were used to quantitate EGF receptors by Scatchard analysis.Table 4 shows that 6 human breast cancer cell lines (T-47D,MCF-7, SK-Br-3, AlAb 496, BT-20, BT-474) and HBL-100

which grew in monolayer had EGF receptors with both highand low affinities. The association constant for the high-affinitysites was approximately 1010 M~1, and that of low-affinity siteswas approximately 109 M"1.

The 2 solid tumor-derived cell lines, BT-20 and BT-474,showed very high specific binding of EGF (1.5 x 106 and 3.0X 10" sites/cell, respectively). The MCF-7 and T-47D lines,

both of which were derived from pleural effusions, had lowernumbers of EGF receptors (2.8 x 103 and 7.6 x 103 sites/

cell, respectively). The AlAb 496 cells, which were derivedfrom lung métastases,have 1.6 x 103 sites/cell. The HBL-100

cells, which were derived from normal human milk, had 1.9 x104 EGF receptors/cell. Interestingly, the 2 floating cell lines,

DU4475 and Lev III, did not exhibit any detectable binding of125I-EGF.

Effect of EGF on Growth of Human Breast Cancer Cells.The effects of EGF on incorporation of [3H]thymidine into DNA

of 9 human mammary cell lines are shown in Chart 3. The DNAsynthesis of T-47D cell lines was stimulated by EGF at 0.1 ng/

ml, and maximal stimulation occurred at 1 ng/ml. The DNAsynthesis of T-47D cells was inhibited slightly at higher EGF

concentrations (10 and 100 ng, respectively). In contrast, EGFdid not stimulate DNA synthesis in MCF-7, SK-Br-3, BT-20,and BT-474. High concentrations of EGF inhibited DNA syn

thesis in these cells. The AlAb 496 cells did not respond to anyof the EGF concentrations tested. Inhibition by higher concentrations of EGF was also observed in the HBL-100. The 2

floating cell lines DU4475 and Lev III, which had no detectableEGF receptors, did not show any response to EGF.

To confirm our results from the [3H]thymidine incorporation

studies, the effect of the EGF on cell number was examined.The results of 4 cell lines are shown in Chart 4. The T-47D

cells were maximally stimulated by EGF at 1 ng/ml. In contrast,the growth of both MCF-7 and AIAb-496 was not stimulated by

500 1000 1500EGF (fmoD/dish (1.8 x 10 cells)6

oE 20

OLU

O01

.1 10

aCO

B HBL-100

10 20EGF Bound (tmol)

1000 2000 3000EGF (fmoO/dish (0.9 x 106 cells)

Chart 2. Analysis of EGF binding in T-47D and HBL-100 cells. Cultures of T-47D (A) and HBL-100 (8) cells were grown to a density of 1.8 x 10e and 0.9 x106 cells/dish (35 mm), respectively. After the cells were washed with bindingbuffer, 0.05 ng of 125l-labeled EGF (1.8 x 10" cpm) mixed with various known

amounts (0.01 to 100 ng) of unlabeled EGF was added to the dishes in 1.5 ml ofbinding buffer. After a 4-hr incubation at room temperature (24°), the dishes

were washed, and the cells were lysed as described. Inset, Scatchard analysisof the binding data.

Table 4

EGF receptors in human mammary cell linesThe binding assays were performed as described in "Materials and Methods."

Affinity constant (Ka) and total binding sites were determined by Scatchardanalyses.

CelllinesAlAb

496MCF-7T-47DHBL-100BT-474SK-Br-3BT-20DU4475Lev

IIIK"High6.2

x10'°8.4x10'°6.7x10103.2X10'°I2.0X10108.0X10'°1.2X 10'°(M-')Low8.2

X10"9.7X1093.1X10'°2.8X10a1.2x10'°1.6X10a0.2X10"NondetectableNondetectable-

Total binding sites/

cell1.6x1032.8x1037.6x1031

.9 x10"3.0x10*9.0X10*1.5X 10e

4396 CANCER RESEARCH VOL. 42

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EGF and Human Breast Cancer Cell Lines

3

e& 2

§g 2

ll

Ã5̈

4

T-47D

AI Ab 496

. HBL-100

10

9

8

0.8

0.6

0.4

MCF-7

BT-20

DU 4475

3

2

1.0

0.8

0.6

0.1

005-

SK-Br-3

BT-474

Lev ID

0 0.1 1 10 100 0 0.1 1 10 100 0 0.1 1 10 100

EGF Concentration (ng/ml)Chart 3. Effect of EGF on incorporation of [3H]thymidine in 9 human mammary

cell lines. Triplicate dishes (5 x 10" cells/dish) were plated in DME with 10%fetal bovine serum. Twenty-four hr later, the media were changed to DME with0.1 % fetal bovine serum and incubated for 24 hr. Then EGF was added, and 20hr later the cells were pulse labeled with [3H]thymidine (1 fiCi/dish) for 4 hr. Bars,

S.D.

12 - T-47D

10 -

OX 8

z= 120)" 11

10

I I 1 1 1

AlAb 496

MCF-7

1 1 I 1 1

BT-20

0 0.1 1 10 100 0 0.1 1 10 100

EGF Concentration (ng/ml)Chart 4. Effect of EGF on growth of human breast cancer cells. The cells

were plated and incubated as described in legend of Chart 3. Cell number wasdetermined 4 days after addition of EGF. Each point represents mean of triplicates. Bars, S.D.

EGF at concentrations of 0.1 to 100 ng/ml. The cell number ofBT-20 decreased at concentrations of EGF higher than 1 ng/ml. Thus, the results from the cell number study are in agreement with the results from [3H]thymidine incorporation studies.

DISCUSSION

Our results show that the mammary cell lines maintained asmonolayers have EGF receptors while 2 cell lines grown in

suspension had no detectable EGF receptors. Scatchard analysis of the binding data revealed a curvilinear pattern whichmay be interpreted as negative cooperativity between bindingsites (7). In our studies, the Scatchard plots were analyzed by2 components with high and low affinity in view of recentevidence suggesting heterogeneity of EGF receptors (18, 26).High-affinity constants determined from the analysis were onthe order of 1010M~' which were similar to those reported for

the other cells (1). As for total binding sites on cells, cell linesestablished from metastatic sites (AlAb 496, MCF-7, T-47D,DU4475, and Lev III) tend to have fewer EGF receptors thando cell lines established from human milk (HBL-100) andprimary breast tumor (BT-474, BT-20). The significance of thisfinding is not clear. Yuhas ef al. (47) suggested that breastcancer cells derived from pleural effusions were geneticallydifferent from the bulk of the tumor cells in solid breast cancersamples. Cell lines from effusion were unable to grow asorganized spheroid aggregates, while cell lines derived fromprimary cancers could. It is possible that metastatic mammarycancer cells, reflecting such a difference, may possess fewerEGF receptors than do primary tumors. Alternatively, the differences in EGF binding of these cell lines may be due to theproduction of endogenous EGF-like substances, which maybind to the EGF receptors, thus limiting the binding of exoge-nously applied EGF. We have tested this possibility by lookingfor EGF-like activity in serum-free conditioned medium of thesecell lines using a radioreceptor assay described previously(15). No EGF-like activity was detected in all the cell linestested.5

It is interesting to observe that the 2 floating cell lines,DU4475 and Lev III, had no detectable binding of EGF. It hasbeen shown that the induction of increased adhesion of 3T3cells to culture dishes is mediated by specific EGF receptors(3). EGF also stimulates the production of extracellular matrixwhich is necessary for cell adhesion (6, 24). Thus, a correlationbetween the capability of these cell lines to grow in suspensioncultures and loss of EGF receptor may exist.

Since all the cell lines grown in monolayer cultures had EGFreceptors with high affinity, we then tested whether EGF hadgrowth-promoting activity in these cells. Under our experimental conditions, EGF stimulates the incorporation of [3H]thymi-dine into DNA only in the T-47D cells. The stimulatory effect ofEGFon T-47D cells were confirmed by monitoring the increasein cell number. EGF inhibited the DNA synthesis of all the othermonolayer cell lines except AlAb 496. Recent reports indicatethat EGF also inhibits the growth of the human epidermoidA431 cells (11) and a pituitary tumor cell line GH4C, (36) atconcentrations which are stimulatory in other cells.

Recently, EGF was reported to stimulate the incorporation of[3H]thymidine into MCF-7 cells (29). However, our results are

at variance with these findings. These authors studied theeffect of EGF (obtained from Collaborative Research) on theincorporation of [3H]thymidine into MCF-7 cells which wereplated to subconfluent density and cultured in serum-free medium. We have also tested the effect of commercially availableEGF on MCF-7 cells using a similar protocol. However, wecould not observe any mitogenic activity of EGF on MCF-7cells. The discrepancy could be due to difference in the maintenance of this cell line. Also, serial passages of the cells may

5 Y. Imai and C. K. H. Leung, unpublished observations.

NOVEMBER 1982 4397

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Page 5: Epidermal Growth Factor Receptors and Effect of Epidermal ... · Epidermal growth factor (EGF) may be important in regulating the proliferation of mammary epithelial cells. In the

Y. Imai ef al.

affect responsiveness of MCF-7 cells to EGF, and the passage

number of the cells used in our study may be different fromthat of cells used by Osborne ef al. (29).

It has been shown that EGF-binding sites and responsiveness

to EGF change during the differentiation of granulosa cells(46), pheochromocytoma cells (13), and mouse teratocarci-noma cells (33). Therefore, the variation in EGF-binding sitesand responses to EGF observed in 9 human mammary celllines may reflect the diverse origins and different stages ofdifferentiation of these cells.

It is possible that EGF may have biological roles in mammarycells other than regulation of cell proliferation. For instance,EGF stimulates human chorionic gonadotropin secretion bycultured choriocarcinoma cells (2) and inhibits histamine-me-

dlated gastric acid secretion from gastric chief cells (4). Further, EGF modulates the production of growth hormone andprolactin from GH4Ci pituitary tumor cells (36). Whether or notEGF plays a role in influencing the differentiated functions ofhuman breast cancer cells remained to be elucidated.

ACKNOWLEDGMENTS

We thank Marilyn Lavallee for typing this manuscript.

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