THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 2-18, No. 17,Issue of September 10, PP. 6251~6253,1973

Printed in U.S.A.

Estrogen Receptor in a Human Cell Line receptor. The absence of estrogen binding protein in these
cells from a tumor of a nontarget tissue indicates that prolonged
(MCF -7) from Breast Carcinoma* maintenance in cell culture did not generate receptor. This was
(Received for publication, May 16, 1973) true in spite of constant exposure to the plasma hormones found
KI\MUEL C. BROOKS,~ ELIZABETH R. LOCKE, AND in calf serum (12).
HERBERT D. SOULE The 17fi-estradiol binding protein was somewhat labile to
extended storage of cells at -70”. For example, storage for 36
From the Michigan Cancer Foundation, and the Department
days reduced the number of binding sites in the experiment
of Biochemistry, Wayne State University School of Medi-
described in Fig. 2 to approximately onc-third (2.7 x 10W2
cine, Detroit, Michigan 48901
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-
SUMMARY
scribed in Fig. 1. Although this decrease in picomoles of 17/3-
A stable cell line (MCF-7) derived by pleural effusion estradiol bound per mg of protein occurred, the binding constant
from a breast cancer patient has been demonstrated to of the remaining receptor would be expected to be unchanged
contain significant amounts of 17fi-estradiol receptor. This (5). The Scatchard plot which resulted from analysis of nine

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binding protein is specific for 17@-estradiol and has a K, equal aliquots of the microsome-free supernatant fraction from frozen
to 2.5 nM, a sedimentation constant of 4.0 S (and 9.2 S), and cells is shown in Fig. 2. These binding determinations were
a mechanism capable of transporting the 17P-estradiol into carried out according to a procedure previously reported from
the nucleus. this laboratory which has taken into account the competitive

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 effusion 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 2.0 4.0 6.0 6.0 IO 12

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

to calf serum. Details of culturing and cell morphology will be [3H] Estradiol (nhl)
published elsewhere (8). FIG. 1. Saturation curve for 17kestradiol receptor in the microsome-
free supernatant of MCF-7 cells (0, radioactivity bound to protein from
For the experiments described herein, cells were inoculated MCF-7 cells; 0, radioactivity bound to protein from Det. 562 cells).
into closed plastic containers (Falcon T-75) and allowed to grow Cells were cultured for 19 days as described in the text, harvested, and
into a conlluent monolayer (approximately 20 X lo6 cells per 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,
bott’le, 15 to 21 days). Cells from passages 71 through 87 were 2 X 15 s at a setting of 8 with an interval of 60 s, in 2 ml of 10 nm Tris-
used in these investigations. The microsome-free supernatant 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
fraction prepared from these cells contained a significant number the resulting supernntnnt fluid was centrifuged in a Millipore filterfuge
of 17@-estradiol binding sites (6.3 X 1OW pmole per mg of pro- tube (top filter pore size 1.2 ,um, bottom filter 0.45 pm) at 600 X g for 15
tein, Fig. 1). Control cells (Det. 562), obtained in a similar 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
fashion from a patient with adenocarcinoma of the throat (11) NasEDTA, and dextran blue. All of the above operations were carried
and cultured as described for MCF-7, contained no 17@-estradiol out at 0”. Aliquots (0.4 ml) of the diluted supernatant fraction were
supplemented immediately after centrifugation with the appropriate
* This investigation was supported in part by United States Public amount of purified 17fi-[2,4,6, 7-3H]estradiol (110 Ci per mmole) dissolved
Health Service Research Grant CA-07177 and Contract NIH-71-2421 in 10 ~1 of ethanol. After incubating for 2 hours at 4’, each sample then
from the National Cancer Institute and by an institutional grant to the was passed through 3-g Sephadex G-25 columns (9). A void volume of
Michigan Cancer Foundation from the United Foundation of Greater approximately 5 ml (determined by dextrnn blue) was collected and ex-
Detroit. tracted with ethyl acetate. Radioactivity in the extract was counted in
$ To whom requests for reprints should be addressed at Michigan a liquid scintillation spectrometer. Protein was assayed by the method
Cancer Foundation, 110 East Warren St., Detroit, Mich. 48201. of Lowry et al. (10).

6251
6252

binding of 17P-estradiol by Scphadex G-25 column material (9). receptor complex are typical of those described for incubat,ed
The lco was found to be 2.5 nM which is comparable to the dis- tissues (15).
sociation constants determined by others for estrogen receptor A recognized property of the cytoplasmic estrogen receptor is
in human breast tumors (5). its migration into the nucleus of target tissue (16). Nuclear
1 he specificity of the binding for 1 Tfi-estradiol is shown in migration has been thought to be temperature-dependent; how-
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 significantly decreased radioactivity in the receptor
complex. 1 his inhibition was observed also in experiments
with high concentration of a specific 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- 75

Ringer bicarbonate buffer with 20 no tritiated 17P-estradiol IIll I I I I I I I I I

, 2 3 4 6 8 IO 12 14 I6 I8 20 22
showed peaks at 4.0 and 9.2 S (Fig. 3). Although incubated in
Bottom FRACTION NUMBER TOP
bicarbonate buffer salts, these cells were homogenized and cen-

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trifuged through a gradient in Tris-EDTA buffer containing no FIG. 3. Sucrose gradient sedimentation of cytoplasmic receptor pre-
pared from MCF-7 cells. A total of 32 X lo6 viable cells were incubated
KCl. The two sedimentation constants found for the estrogen with 20 nM tritiated 17P-estradiol in 2 ml of Krebs-Ringer bicarbonate
buffer, pH 7.4, for 60 min at 0”. After 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 buffer, 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
2.4 strokes of a Dounce homogenizer in 2 ml of Tris-EDTA containing 5 rnM
dithiothreitol. After centrifugation, 0.5 ml of dextran (0.05%.coated
2.1 charcoal (0.570) was added to 0.6 ml of the 105,000 X g supernatant and
I.8 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
1.5 sucrose gradient (5 to 20%) in Tris-EDTA. The proteins were sedi-
mented in a 50.1 SW rotor at 41 X lo3 rpm for 15 hours at 4”. The tube
1.2 was punctured and 3-drop fractions were collected for measurement of
radioactivity. The marker protein, 10 mg per ml of bovine serum albu-
0.9 min (arrow), was treated similarly and the fractions were assayed at
260 nm.
0.6

0.3 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
Bound [3~] Estradial x Protein mg-’ x pM Tris-EDTA containing 2 rnM unlabeled 17&estradiol. The other aliquot
was washed three times with 6 ml of Krebs-Ringer bicarbonate buffer,
FIG. 2. Scatchard plot derived from the 17fi-estradiol receptor in pH 7.4, and then incubated in 2 ml of this buffer for 30 min at 37” under
frozen MCF-7 cells (20 X 106). Homogenization and incubations with 95% 0~5% COz. At the end of the warm incubation these cells were
tritiated 170.e&radio1 were carried out as described in Fig. 1. As out- also washed twice with Tris-EDTA containing a 102-fold excess of un-
lined in a previous publication (9), each point was determined from three labeled 17@-estradiol. Homogenization of both aliquots of cells was car-
analyses and represents the radioactivity of bound tritiated 17&estradiol ried out in 2 ml of Tris-EDTA containing 5 rnM dithiothreitol. Cen-
which has been corrected for competitive binding to column material. 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-EDTA containing a 102-fold
TABLE I excess of unlabeled steroid, both nuclear pellets were extracted for 40
min with 1 ml of 0.4 M KC1 in Tris-EDTA. These nuclear extracts were
Inhibition of 17/3-[3H]estradiol binding to cytoplasmic receptor
cleared by centrifugation at 1000 X D prior to passing over 3-g Sephadex
Homogenization (20 X lOa frozen cells) and incubations were carried G-25 columns. The cytosol supernatants were also chromatographed
out as described in Fig. 1 except that in these experiments the tritiated through 3-g Sephadex G-25 columns. Radioactivity in the void volume
17p-estradiol (in 10 ~1 of ethanol) was added 10 min after the unlabeled was measured.
compound (in 5 ~1 of ethanol). The bound radioactivity was determined Porcine endometrium was obtained by scraping one horn of an imma-
in the effluent from 3-g Sephadex columns. ture pig uterus. The removed tissue was washed with Krebs-Ringer
bicarbonate buffer, and a O&ml packed volume of endometrium was
Per cent of 17p- utilized in the experiments carried out exactly as described for the
Compound added Concentration [JHlestradiol bound MCF-7 cells.

%?4 Tissue Temperature Time Cytosol Nuclei

17fi-[sHIEstradio 1.8 100
+ 17P-Estradiol 180 41 nin % dfim % dh
1800 12 MCF-7 cells O0 60 24 76
+ Progesterone 1600 86 37 30 7 93
+ Ull, 1OOA 210 49 Porcine endometrium 0 60 40
2100 14 37 30 12
ever, many of the experiments carried out to demonstrate uptake constants, transport mechanisms, and the mode of nuclear up-
of the receptor complex by nuclei are presently in doubt due to take.
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