estrogen receptor in MCF7

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Estrogen Receptor in a Human Cell Line

(MCF -7) from Breast Carcinoma*(Received for publication, May 16, 1973)

KI\MUEL C. BROOKS,~ ELIZABETH R. LOCKE, AND

HERBERT D. SOULE

From the Michigan Cancer Foundation, and the Department

of Bioche mistry, Wayne State University Sch ool of Medi-

cine, Detroit, Michigan 48901

SUMMARY

A stable cell line (MCF-7) derived by pleural effus ion

from a breast cancer patient has been demonstrated to

contain significant amounts of 17fi-estradiol receptor. This

binding protein is specif ic for 17@-estradiol and has a K, equal

to 2.5 nM, a sedimentation constant of 4.0 S (and 9.2 S), and

a mechanism capable of transporting the 17P-estradiol into

the nucleus.

Considerable work, carried out in recent years, has culminated

in elucidation of the receptor mechanism for the stimulation of

“target” tissues by steroid hormones (l-4). This knowledge

recently has given investigators greater insight into the molecular

events involved in hormonal control of normal and neoplastic

target tissues (5-7). Present understanding regarding the

estrogen receptor has been acquired through in vivo studies and

by short term in vitro incubations of tissue slices or cell fractions.

JIYc would like to report herein the first demonstration of 17/S

estradiol binding protein in a stable cell line.The primary culture of human breast carcinoma cells was

obtained originally by pleural effus ion from a female patient with

metastatic disease. A stable epithelioid cell line, MCF-7, was

derived from free-floating passages and had been maintained

through 71 weekly subcultivations. The cells were cultured in

Eagle’s minimal essential medium supplemented with nonessen-

tial amino acids and 20 pg per ml of insulin prepared in Hanks’

salt solution. All media contained 250 units of penicillin and

250 pg of streptomycin per ml and were made 10% with respect

to calf serum. Details of culturing and cell morphology will be

published elsewhere (8).

For the experiments described herein, cells were inoculated

into closed plastic containers (Falcon T-75) and allowed to grow

into a conlluent monolayer (approximately 20 X lo6 cells per

bott’le, 15 to 21 days) . Cells from passages 71 through 87 were

used in these investigations. The microsome-free supernatant

fraction prepared from these cells contained a significant number

of 17@-estradiol binding sites (6.3 X 1OW pmole per mg of pro-

tein, Fig. 1). Control cells (Det. 562), obtained in a similar

fashion from a patient with adenocarcinoma of the throat (11)

and cultured as described for MCF-7, contained no 17@-estradiol

* Thi s investigation was supported in part by United State s Public

Health Service Research Grant CA-07177 and Contract NIH-71-2421

fro m the National Cancer Inst itute and by an institutional gran t to the

Michigan Cancer Foundation fro m the United Foundation of Greater

Detroit.

$ To whom requests for reprints should be addressed at Michigan

Cancer Foundation, 110 East Warren St., Detroit, Mich. 48201.

THE JOURNAL OF BIOLOGICAL CHEM ISTRY

Vol. 2-18, No. 1 7,Issue of September 10, PP. 6251~6253,1973Printed in U.S.A.

receptor. The absence of estrogen binding protein in these

cells from a tumor of a nontarget tissue indicates that prolonged

maintenance in cell culture did not generate receptor. This wastrue in spite of constant exposure to the plasma hormones found

in cal f serum (12).

The 17fi-estradiol binding protein was somewhat labile to

extended storage of cells at -70”. For example, storage for 36

days reduced the number of binding sites in the experiment

described in Fig. 2 to approximately onc-third (2.7 x 10W2

pmole of 17,Kestradiol per mg of protein) that of an aliquot of

the same cells stored for 14 days and utilized in the studies de-

scribed in Fig. 1. Although this decrease in picomoles of 17/3-

estradiol bound per mg of protein occurred, the binding constant

of the remaining receptor would be expected to be unchanged

(5). The Scatchard plot which resulted from analysis of nine

aliquots of the microsome-free supernatant fraction from frozencells is shown in Fig. 2. These binding determinations were

carried out according to a procedure previously reported from

this laboratory which has taken into account the competitive

2.0 4.0 6.0 6.0 IO

[3H] Estradiol (nhl)

12

FIG. 1. Saturation curve for 17kestradiol receptor in the micro some-

free supernatant of MCF-7 cells (0, radioactivity bound to protein fro m

MCF-7 cells; 0, radioactivity bound to protein fro m Det. 562 cells).

Cells were cultured for 19 days as described in the tex t, harvested, and

stored frozen at -70” for 14 days. The cells (40 X lo6 MCF-7 and

50 X lo6 Det. 562) were homogenized with a Willems Polytron 10 ST,

2 X 15 s at a setting of 8 with an interval of 60 s, in 2 ml of 10 nm Tris-

HCl, pH 7.4, containing 1.5 nm MgC12, 10 rn~ KCI, and 5 mu dithio-

threitol. The homogenate was centrifuged at 15,000 X 0 for 15 min and

the resulting supernntnnt fluid was centrifug ed in a Millipore filterf uge

tube (top filter pore size 1.2 ,um, bottom filter 0.45 pm) at 600 X g for 15

min. This microsome-free filtrate was diluted 1:l (v/v) with 10 rn~

Tris- I-ICI , pH 8.5, containing 10 rn~ KCI, 5 nm dithiothreitol, 1 nm

NasE DTA, and dextran blue. All of the above operations were carried

out at 0”. Aliquots (0.4 ml) of the diluted supernatant fraction were

supplemented immediately after centrifugation with the appropriate

amou nt of purified 17fi-[2 ,4,6, 7-3H]estradiol (110 Ci per mm ole) dissolved

in 10 ~1 of ethanol. Afte r incubating for 2 hours at 4’, each sample then

was passed through 3-g Sephadex G-25 columns (9). A void volume of

approxima tely 5 ml (determine d by dextrnn blue) was collected and ex-

tracted with ethyl acetate. Radioactivity in the extract was counted in

a liquid scintillation spect rometer. Protein was assayed by the metho d

of Lowry et al. (10).

6251

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6252

binding of 17P-estradiol by Scphadex G-25 column material (9).

The lco was found to be 2.5 nM which is comparable to the dis-

sociation constants determined by others for estrogen receptor

in human breast tumors (5).

1 he specific ity of the binding for 1 Tfi-estradiol is shown in

Table I. Preincubation of cytosol from cells with a 103-fold

excess of progesterone did not depress the binding by tritiated

17P-estradiol (1.8 nM). However, prior exposure to unlabeled

17fi-estradiol at lo2 and lo3 times the concentration of tritiated

estrogen signif icantly decreased radioactivity in the receptor

complex. 1 his inhibition was observed also in experiments

with high concentration of a speci fic estrogen blocking agent,

UllJOOA (13).

While sediment.ation constants reported for the complex vary

according to conditions of the experiment, most of the values

obtained for tissues homogenized in Tris buffer with high or low

KC1 concentrations are near 4 and 9, respectively (14). The

density gradient pattern of cytoplasmic l’ifi-estradiol binding

protein derived from the incubation of MCF-7 cells in Krebs-

Ringer bicarbonate buffer with 20 no tritiated 17P-estradiol

showed peaks at 4.0 and 9.2 S (Fig. 3). Although incubated in

bicarbonate buffer salts, these cells were homogenized and cen-

trifuged through a gradient in Tris-EDTA buffer containing noKCl. The two sedimentation constants found for the estrogen

2.4

2.1

I.8

1.5

1.2

0.9

0.6

0.3

Bound [3~] Estrad ial x Protein mg-’ x pM

FIG . 2. Scatchard plot derived from the 17fi-estradiol receptor in

frozen MCF-7 cells (20 X 106). Homoge nization and incubations with

tritiated 170.e&radio1 were carried out as described in Fig . 1. As out-

lned in a previous publication (9), each point was determined fro m three

analyses and represents the radioactivity of bound tritiated 17&estradiol

which has been corrected for competitive binding to column material.

TABLE I

Inhibition of 17/3-[3H]es tradiol binding to cytoplas mic receptor

Homogenization (20 X lOa frozen cells) and incubations were carried

out as described in Fig . 1 except that in these experiments the tritiated

17p-estradiol (in 10 ~1 of ethanol) was added 10 min afte r the unlabeled

compound (in 5 ~1 of ethanol). The bound radioactivity was determined

in the effluent from 3-g Sephadex columns.

Compound added ConcentrationPer cent of 17p-

[JHlestradiol bound

%?4

17fi-[sHIEstradio 1.8 100

+ 17P-Estradiol 180 41

1800 12

+ Progestero ne 1600 86+ Ull, 1OOA 210 49

2100 14

receptor complex are typical of those described for incubat,ed

tissues (15).

A recognized property of the cytoplasmic estrogen receptor is

its migration into the nucleus of target tissue (16). Nuclear

migration has been thought to be temperature-dependent; how-

75

IIl l I I I I I I I I I

, 2 3 4 6 8 IO 12 14 I6 I8 20 22

Bottom FRACTION NUMBER TO P

FIG . 3. Sucrose gradient sedimentation of cytoplasmic receptor pre-pared from MCF-7 cells. A total of 32 X lo6 viable cells were incubated

with 20 nM tritiated 17P-estradiol in 2 ml of Krebs-Ringer bicarbonate

buffer, pH 7.4, for 60 min at 0”. Afte r incubation the cells were cen-

trifuged at 600 X g for 10 min at O”, the supernatant was discarded, and

the cells were washed in 6 ml of Krebs-Ringer phosphate buff er, pH 7.4,

followed by two washes with 2 ml of 10 rnM Tris , pH 7.4, containing 1.5

rnM NazEDTA (Tris- EDTA ). Then the cells were ruptured with 10

strokes of a Dounce homogenizer in 2 ml of Tris-ED TA containing 5 rnM

dithiothreitol. After centrifugation, 0.5 ml of dextran (0.05%.coated

charcoal (0.570) was added to 0.6 ml of the 105,000 X g supernatant and

the mixture was allowed to stand 60 min at 0”. The charcoal was sedi-

mented and 0.4 ml of the supernatant was layered on top of 4.6 ml of a

sucrose gradient (5 to 20%) in Tris-E DTA . The proteins were sedi-

mented in a 50.1 SW rotor at 41 X lo3 rpm for 15 hours at 4”. The tube

was punctured and 3-drop fractions were collected for measurement of

radioactivity. The marker protein, 10 m g per ml of bovine serum albu-

min (arrow), was treated similarly and the fractions were assayed at

260 nm.

TABLE II

Migration of bound radioactive 17p-e&radio1 into nuclei of target tissues

Two equal aliquots of viable MCF-7 cells (20 X 106, 0.3-ml packed

cell volume) were incubated at 0’ for 60 min as described in Fig . 3. Fol-

lowing the cold incubation, one aliquot was washed with 2 ml of cold

Tris -ED TA containing 2 rnM unlabeled 17&estradiol. The other aliquot

was washed three times with 6 ml of Krebs-Ringer bicarbonate buffer,

pH 7.4, and then incubated in 2 ml of this buffer for 30 min at 37” under

95% 0~5% COz. At the end of the warm incubation these cells were

also washed twice with Tris-E DTA containing a 102-fold excess of un-

labeled 17@-estradiol. Homoge nization of both aliquots of cells was car-ried out in 2 ml of Tris-E DTA containing 5 rnM dithiothreitol. Cen-

trifugation at 1000 X Q for 10 min yielded each nuclear pellet and a super-

natant which was subsequently centrifuged at 105,000 X Q for 60 min.

Following two washes with 2 ml of Tris-E DTA containing a 102-fold

excess of unlabeled steroid, both nuclear pellets were extracte d for 40

min with 1 ml of 0.4 M KC1 in Tris-E DTA . These nuclear extracts werecleared by centrifugation at 1000 X D prior to passing over 3-g Sephadex

G-25 columns. The cytosol supernatants were also chromatographed

through 3-g Sephadex G-25 column s. Radioactivity in the void volume

was measured.

Porcine endom etrium was obtained by scraping one horn of an imm a-

ture pig uterus. The removed tissue was washed with Krebs-Ringer

bicarbonate buffer, and a O&m l packed volume of endometrium was

utilized in the experiments carried out exactly as described for the

MCF-7 cells.

Tissue Temperature Time Cytosol Nuclei

nin % dfim % dh

MCF-7 cells O0 60 24 76

37 30 7 93

Porcine endom etrium 0 60 40

37 30 12

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ever, many of the experiments carried out to demonstrate uptake

of the receptor complex by nuclei are presently in doubt due to

evidence of posthomogenization binding of extracellular tritiated

l’ifi-estradiol by released cytoplasmic receptor (17). In Table

II experiments are presented which utilized a wash with a 102-fold

excess of nonlabeled 17,B-estradiol prior to homogenization of the

tissue, cells, or nuclei. The results clearly show migration of

cytoplasmic 17P-estradiol receptor complex into the nuclei during

incubation at 37”. Both porcine uterine nuclei and nuclei fromRICF-7 cells exhibited appreciable nuclear uptake after 1 hour

at 0”; a similar observation has been reported in the recent publi-

cation of Williams and Gorski (17).

These experiments demonstrated the presence of significant

amounts of 17fi-estradiol binding protein in a stable cell line

derived from a human breast tumor. As previously reported

for the receptor in human tumors, the estrogen binding protein

from MCF-7 has a Ku equal to 2.5 no, a sedimentation constant

of 4.0 S (and 9.2 S), and a mechanism capable of transporting

the 17P-estradiol complex into the nucleus.

Utilizing in viva experimentation and short term incubations,

it previously has not been possible to investigate the induction

of steroid receptor protein with strictly controlled environmentalexposure to hormones and nutrients. With cell culture tech-

niques these investigations are now feasible; and, in addition, a

stable cell line will permit experiments to be carried out which

will add to the present knowledge regarding intracellular binding

constants, transport mechanisms, and the mode of nuclear up-

take.REFERENCES

1. JENSEN, E. V., AND JACOBSON, H. I. (1962) Recent Proor. Hormone

Res. 18, 3872. JENSEN, E. V., AND DESOMBRE, E. R. (1972) in Biochemical Actior~s

of Hokmones (LITWACK, F., ed) p. 215, Academic Press, New York

3. FANG, S., AND LIAO, S. (1971) J. Biol. Chem. 246, 16

4. O’MALLEY, B. W., MEANS, A. R., AND SHERMAN, M. R. (1971) in

The Sex Steroids (MCKERXS, K. W., ed) p. 315, Appleton-Century-

Crofts, New York

5. H~HNEL, It. , AND TWADDLE, E. (1973) Cancer Res. 33, 559

6. MCGUIRE. W. L., Huw, K., JENNINGS, A., AND CRAMNESS, G. C.

(1972) Science 175, 335

7. MCGUIRE, W. L., AND JULIIN, J. A. (1971) Cancer Res. 31, 1440

8. SOULE, H D., VAZQUEZ, J., ALBERT, S., AND LONG, A. (1972) J.

Nat. Cancer Inst ., in press

9. GODEFROI, V. C., AND BROOKS, S. C. (1973) Anal. B&hem. 51, 335

10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, It. J.

(1951) J. Biol. Chem. 193, 265

11. PETERSON, W. D., STULBERG, C. S., AND SIMPSON, W. F. (1971) Proc.

Sot. Esp. Biol. M ed. 136, 1187

12. ESBER, H. J., PAYNE, I. J., AND BOGDEN, A. E., J. Nut. Cancer Inst.

50, 55913. JENSEN, E. V., JACOBSON, H. I., FLESHER, J. W., SAHA, N. N.,

GUPTA, G. N., SMITH, S., COLUCCI, J., SHIPLACOFF, D., NEUMANN,

H. G.. DESOMBRE. E. R.. AND JUNGBLUT, P. W. (1966) in Steroid

Dgnarks (PINCUB, G., N.~KAo, T. , AND TA IT , i., ehs) p. 133,

Academic Press, New York

14. CHAMNESS, G. C., AND MCGUIRE, W. L. (1972) Biochemistry l&246615. STEGG LES, A. W., AND KING, R. J. B. (1970) B&hem. J. 118, 69516. JENSEN, E. V., SUZUKI, T. , KAWASHIMA, T. , STUMPF, W. E., JUNG-

BLUT , P. W., AND DESOMBRE, E. R. (1968) Proc. Nat. Acad. Sci.

U. S. A. 59, 632

17. WILLIAMS, D., *ND GORSKI, J. (1971) Biochem. Biophus. Res. Com-

mun. 45, 258

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