[CANCER RESEARCH 42, 1967-1974, May 1982]

0008-5472/82/0042-0000$02.00

Estrone Receptor Formation During the Processing of Estradiol-Receptor

Complex in MCF-7 Cells1
E. R. Hansen and S. C. Brooks2
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201

ABSTRACT steroid hormones in neoplastic and normal target tissues be

Human breast cancer cells (MCF-7, maintained in long-term
culture) contain separate estrogen receptors specific for either
17/î-estradiol or estrone. Utilizing optimum conditions for the
protamine sulfate assay, it has been possible to demonstrate
both receptors in the 0.6 M KCI extract of nuclei and in the
cytosol. Similarly, in the exchange assay, high-affinity low-
capacity binding sites for 17/î-estradioland estrone have been
found in the salt-extracted nuclear residue. Dissociation con
stants and binding capacities were determined for either re
ceptor in the absence of the other [e.g., estrone receptor (E,R)
in the cytosol or nuclear residue from 17/J-estradiol-stimulated
cells] or, when both receptors were present, a saturating
amount of the other estrogen (unlabeled) was added to the
assay mixture (e.g., the salt-extractable nuclear receptors).
Specificity was demonstrated by the inability of estrone to
compete with 17/H2,4,6,7-3H]estradiol for the 17/î-estradiol
receptor (E2R) at molar excesses less than 10-fold. Likewise,
there was no inhibition of [6,7-3H]estrone binding to its receptor
by molar excesses of 17/î-estradiol below 100-fold. Other
steroid hormones were very weak competitors of [6,7-3H]es-
trone, even at 1000-fold molar excesses. The quantitative
relationships of these two estrogen receptors were shown to nuclear uptake and processing of E2R.
fluctuate in the various cellular compartments following incu
bation (37°) of MCF-7 cells with 10~8 M 17/î-estradiol. This
level of 17/î-estradiolelicited the translocation of all detectable
cytosolic E2R to the nucleus, where, after an incubation of 1 low capacity (4). Furthermore, estrone is formed within the
hr, the salt-resistant 17/î-estradioldisappeared and 40% of the nucleus from 17/î-estradiol, particularly in the salt-resistant
extractable 17/î-estradiol-binding capacity was lost (proc
essed). Simultaneously, the E,R which remained in the nuclear
residue appeared in the nuclear extract, and ultimately this
receptor accumulated in the cytosol. The estrone-binding ca
pacity (0.78 pmol/mg DNA) which appeared following the
processing of E2R nearly equalled the loss of 17/î-estradiol residual nuclear pellet displayed 2 separate high-affinity low-
binding sites per cell (0.85 pmol/mg DNA). Concentrations of capacity binding sites specific for either 17/8-estradiol or es
17/S-estradiol which elicited the greatest processing of E2R in
these incubations brought about the appearance of maximum
levels of E,R in MCF-7 cells.
Considering these results in the light of data previously
reported from this laboratory concerning the metabolic and
ligand fate of 17/8-[3H]estradiol in MCF-7 cells, processing
would appear to involve the formation of EiR in the salt-resist
ant nuclear compartment followed by the accumulation
in the cytosol.
INTRODUCTION
It is essential to the understanding of steroid effects on
breast cancer cells that the total fate of physiological levels of
' Supported in part by Grant CA 22828 from the National Cancer Institute.
2 To whom requests for reprints should be addressed.
Received June 8, 1981 ; accepted February 10. 1982.
known. For example, 17/î-estradiol is destined to be metabo
lized by specific enzymes with which it may come into contact
as well as being bound to its cytoplasmic receptor and subse
quently translocated to the nucleus (1, 5, 22, 25-27). Once
within the nucleus, 17/î-estradiol shares an association with
salt-extractable proteins and with certain salt-resistant com
ponents (4, 9, 23).
E2R3 complex has recently been shown to undergo process
ing within the nucleus of MCF-7 cells (18). Originally observed
by Horwitz and McGuire (18), processing was defined as the
disappearance of nuclear (as well as total cellular) E..R during
the induction of progesterone receptor. The fate of the lost E2R
complex has not been elucidated.
Previously reported studies with MCF-7 cells have estab
lished that within these cells 17/î-estradiol is oxidized to es
trone as well as being bound by its receptor followed by the
translocation of the complex to the nucleus (4). It has also been
demonstrated that, like uterine cells (6, 24, 29, 35-37), 17ß-
estradiol dehydrogenase activity exists within the nucleus of
these breast cancer cells (4). Since no other fate awaits 17/î-
estradiol in MCF-7 cells, this system is amenable to a study of
the relationship of 17/8-estradiol dehydrogenase activity to the
Utilizing the MCF-7 human breast cancer cell line, we have
demonstrated that estrone formed from 17/î-estradiolin situ is
bound to a cytoplasmic macromolecule with high affinity and
nuclear fraction. This unexpected discovery would predict the
presence of estrone-specific nuclear receptors in MCF-7 cells.
We have carried out studies designed to gain information
regarding the presence of a nuclear estrone-binding compo
nent. Whole nuclei, the salt-extracted nuclear proteins, and the
trone (3).
The data presented herein demonstrate that formation of the
E,R occurs concomitant with the nuclear processing of E2R
complex in MCF-7 cells.
MATERIALS AND METHODS
of E,R Materials. [3H]Estradiol (111 Ci/mmol and [3H]estrone (48.3 Ci/
mmol) were purchased from New England Nuclear (Boston, Mass.) and
purified on thin-layer chromatography before use (24). Nonradioactive
hormones were obtained from Research Plus Laboratories, Inc. (Den-
ville, N. J.).
Cell Culture and Harvesting. MCF cells of human breast tumor
origin were cultured in T75 flasks utilizing 20 ml Eagle's minimal
essential medium supplemented with nonessential amino acids, insulin,
3 The abbreviations used are: E2R, 17/?-estradiol receptor; E,R, estrone re
ceptor; [3H]estradiol, 17/3-{2,4,6,7-3H]estradiol; [3H]estrone, [6.7-3HJestrone.

MAY 1982 1967

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E. R. Hansen and S. C. Brooks
and antibiotics and made 10% with respect to calf serum (33). Pas
sages involved trypsin digestion (33) to obtain monocellular suspen
sions followed by the plating of 2 x 106 cells/flask. Cultures were
confluent after 1 week and were harvested for experimentation after 2
weeks (approximately 30 x 10s cells/flask).
Following removal of medium, flasks were washed twice with NaCI
solution (9 mg/100 ml) and once with Tris:EDTA buffer [10 mM Tris-
HCI: 1.5 mM EDTA (pH 7.4 at 0°)]to remove residual medium. The cells
were then removed from the flask using a rubber policeman with 2 ml
of Tris:EDTA buffer made 8 mM with dithiothreitol and placed in a
Dounce tube.
Preparation of Cytosol, Microsomal, and Nuclear Extracts. The
suspension of harvested cells from 2 flasks was homogenized in the 4
ml buffer made 0.1 % with saponin utilizing 15 strokes of a tightly fitting
pestle. The homogenate was centrifuged at 800 x g for 10 min (4°),
and the supernatant was saved (crude cytosol). A more purified nuclear
pellet was prepared by washing the crude pellet obtained with 2 ml of
buffered 0.25 M sucrose solution [0.25 M sucrose:3 mM MgCI:10 mw
Tris-HCI:8 mw dithiothreitol (pH 7.6 at 0°)]3 times and centrifuging as
before. The supernatants were added to the crude cytosol. The crude
cytosol was centrifuged at 105,000 x g (4°) for 60 min, yielding a
supernatant (cytosol) and a high-speed pellet (microsomes).
The partially purified nuclear and microsomal pellets were extracted
with Tris:EDTA:KCI buffer [10 mw Tris-HCI:1.5 mM EDTA:0.6 M KCI.1
mM thioglycerol:10% glycerol (pH 8.5 at 0 °)] by triturating (with
Dounce pestle) every 10 min for 1 hr. Solubili/uri proteins were
separated from insoluble nuclear or microsomal material by centrifu-
gation at 105,000 x g for 30 min. The concentrated nuclear or
microsomal extracts were diluted with Tris:EDTA buffer to a concentra
tion of less than 0.1 M KCI to enable precipitation by protamine sulfate
(39). DNA was determined from the residual nuclear and microsomal
pellet (105,000 x g pellet) by the diphenylamine method of Burton (7)
using Sigma type V DNA as a standard.
Protamine Sulfate Assays. The method used was that of Steggles
and King (34) as modified by Zava et al. (39) and Chamness ef al. (8).
Fractions (500 /il) of cytosol and nuclear extracts were precipitated
with 300 jul of protamine sulfate (1 mg/ml; DSP injection, without
phenol preservative; Eli Lilly and Co.) and centrifuged at 800 x g for
10 min.
For one-point assays, the protamine precipitates from aliquots of the
cytosol or nuclear extracts were incubated with a single concentration
(3.6 to 5.0 nM) of [3H]estradiol or [3H]estrone for the designated times
and temperatures. Nonspecific binding was determined by a parallel
incubation with tritiated estrogen plus 200-fold excess nonradiolabeled
17/3-estradiol or estrone, respectively. After incubation, the precipitates
were washed 3 times with Tris:EDTA buffer and extracted with 1.5 ml
ethanol, and 1 ml was counted utilizing a scintillation spectropho-
tometer equipped with an absolute activity analyzer. The data were
analyzed after subtraction of nonspecific binding.
Saturation analysis on cytosol, microsomal extracts, or nuclear
extracts was carried out by incubating for indicated times and temper
atures the protamine sulfate precipitates in duplicate with 300 »Iof
Tris:EDTA containing increasing concentrations of [3H]estradiol (0.2 to
4.0 nw) or [3H]estrone (0.3 to 6.0 nw) with or without 200-fold excess
of respective estrogen. The protamine-precipitated proteins were then
washed, extracted, and counted as described above. The binding
capacities and KDs were determined using the method of Scatchard
(32) after subtraction of nonspecific binding.
Nuclear Exchange Assay on 0.6 M KCI-extracted Nuclear Pellet.
A modification of the exchange procedure of Anderson et al. (2) was
utilized. Nuclei were purified and extracted with Tris:EDTA:KCI buffer
as described above. The 0.6 M KCI-extracted pellet (105,000 x g
pellet) was transferred to a Dounce homogenizer, and 2 ml of buffered
0.25 M sucrose solution were added. The pellet was homogenized into
an even suspension with 15 strokes of a tightly fitting pestle. This
suspension was diluted to 15 ml, 1-ml samples were taken for DNA
analysis, and 500-/il samples were taken for an exchange assay. The
1968
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
suspension was centrifuged at 800 x g for 10 min, and the supernatant
was decanted. Resulting pellets were incubated in duplicate under
conditions indicated in the legend to Chart 3 with 300 /il TrisrEDTA
containing increasing concentrations of [3H]estradiol (0.2 to 4.0 nM) or
[3H]estrone (0.3 to 6.0 nM) with or without 200-fold excess of respective
steroid. After incubation, tubes were washed 3 times with Tris:EDTA
buffer and extracted with 1.5 ml ethanol, and 1 ml was counted as
described before. The data were analyzed by the method of Scatchard
(32).
RESULTS
Characteristics of the Protamine Assay of Various Estro
gen Receptors. The 0.6 M KCI extract of nuclei from MCF-7
cells contained protamine-precipitable proteins which bind
[3H]estradiol with high affinity and finite capacity. However, the
rate at which this association reached equilibrium and the
maximum [3H]estradiol binding observed varied with the tem
perature of the assay and the prior availability of 17/8-estradiol
to the cells (Chart 1). When cultures were grown in media
containing 10% calf serum (2 to 6 x 10~" M 17/S-estradiol;
see Footnote 4 and Ref. 28), the extractable nuclear 17ß-
estradiol receptor reaches equilibrium by 1 hr at 37°or 6 to 20
hr at 0°.Regardless of which temperature is used, the maxi
mum binding is similar (approximately 0.52 pmol 17/î-estradiol
bound per mg DNA). Previously, this binding to (or exchange
with?) the protamine-precipitated nuclear receptor at 0°has
been defined as indicative of uncharged receptor (40). This
does not appear to be the case for receptor extracted from
substantially purified nuclei in these experiments since [3H]-
estrone was not capable of occupying this site during cold
equilibrations (Chart 2). Even at 37°, [3H]estrone occupied
(exchanged with?) only 20% of these sites (Chart 2).
After exposure of the culture to 17/î-estradiol(10~8 M) during
a 1-hr incubation at 37°, there were dramatic changes in the
amount and exchange characteristics of the extractable nu
clear 17/î-estradiolreceptor (Chart 1). A 3-fold increase (~1.5
pmol/mg DNA) was observed in the quantity of [3H]estradiol
which exchanged with receptor at 37°, probably representing
the translocation of cytoplasmic E2R complex to the nuclear
compartment. On the other hand, little (0.2 pmol/mg DNA)
[3H]estradiol was seen to exchange at 0°with nuclear receptor-
bound 17/î-estradiol. Presenting these cells with the optimum
level of 17/8-estradiol to bring about nuclear processing of the
receptor complex (18) has significantly altered the exchange
properties of the ligand. At the same time, 17/S-estradiol stim
ulation of MCF-7 cells brought about the appearance of an
appreciable amount of [3H]estrone binding to (0°)or exchange
(at 37°)with ligand(s) on the protamine precipitate of the 0.6
M KCI extract of nuclei (Chart 2). The 0.12 pmol/mg DNA of
[3H]estrone binding at 0°is similar to the 0.2 pmol/mg DNA of
[3H]estradiol bound in the cold (Chart 1), suggesting that both
tritiated ligands were taken up by this small number of unoc
cupied sites in the nuclear extract, possibly from cytoplasmic
contamination (13). Under the assay conditions, the observed
[3H]estrone exchange at 37°would not be expected to occur
with charged classical E2R complex since the affinity of estrone
is considerably less than that of 17/î-estradiolfor this protein.
Therefore, the 0.1-pmol/mg DNA [3H]estrone binding excess
4 Radioimmunoassay of total 17/i-estradiol (free plus hydrolyzed sulfates) in
media containing 10% calf serum.
CANCER RESEARCH VOL. 42

1.5
10
0.5
2 4 6 8 10 12 14 16 18 20
Hours
Chart 1. Effect of time, temperature, and the exposure of MCF-7 cells to 1CT8
M 17/?-estradiol on the exchange of [3H]estradiol ([3H]EZ) with the receptors in
the 0.6 M KCI nuclear extract. Nuclear extracts were prepared, and one-point
protamine sulfate assays were performed at the indicated times and temperatures
under incubation with 5 nM [3H]estradiol. Details of the procedures are described
in "Materials and Methods." Points, mean of duplicate samples. Assays carried
out on the nuclear extracts from untreated cells in 3 T75 flasks: •,0°:O. 37°.
Assays carried out on the nuclear extracts from cells in 3 T flasks incubated
prior to harvest for 1 hr at 37° with regular media containing 10~8 M ^^ß-
estradiol: A. 0°;A, 37°.
03
0.2
f--..
Ul
f O.I
0.0
IO 14 16 18 20
Hours
Chart 2. Effect of time, temperature, and the exposure of MCF-7 cells to 10~8
M 17/S-estradiol on the exchange of [3H]estrone ([3H]Ei) with receptors in the 0.6
M KCI nuclear extract. Nuclear extracts were prepared, and one-point protamine
sulfate assays were performed at the indicated times and temperatures using
incubations with 5 nM pHJestrone. Details of the procedure are described in
"Materials and Methods." Points, means of duplicate samples. Assays carried
out on the nuclear extracts from untreated cells in 2 T75 flasks: •,0°;O, 37°.
Assays carried out on the nuclear extracts from cells in 2 T75 flasks incubated
prior to harvest for 1 hr at 37° with regular media containing 10~8 M 17/3-
estradiol: A, 0°:A, 37°.
in the 37° exchange assay may represent specific estrone
nuclear receptor.
The binding curves depicted in Charts 1 and 2 clearly dem
onstrate that optimum binding assays for [3H]estradiol and
[3H]estrone may be carried out on the nuclear extract by
equilibrating the protamine precipitate with either tritiated ste
roid for 1 hr at 37°. These conditions allow quantitation of
nuclear estrogen receptors whether the culture has been pre
viously exposed to 17/S-estradiol or not.
Following salt extraction of the nuclear pellet, the nuclear
residue has been shown to contain components with classical
E2R properties (3, 9, 23). Utilizing an exchange assay for
Scatchard analyses at 23°or 0°,it is possible to determine the
binding properties of both tritiated estrogen ligands to these
salt-resistant sites (Chart 3). Chart 3, left, depicts approxi-
MAY 1982
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
Eif? Formation during Processing
tu
04bnM
5 O.O7
o Kd
130 nM
? 0.06
•o
S3 0.05
I 004
Û 0.03
Si
«0.02
o
0.01
2S-0* 23-0° ZyO«23'0* ZyO" 23-0«
MEM »CS »IO~8ME2 CDM
Chart 3. Exchange characteristics of the 0.6 M KCI-insoluble nuclear residue
from MCF-7 cells. Nuclear exchange assays were performed as described under
"Materials and Methods." Incubations were carried out either at 23°for 1 hr or
at 0°for 18 hr as indicated. Cells (15 T75 flasks) were grown in regular media
(minimal essential medium plus 10% calf serum (MEM + CS) as described in
"Materials and Methods." Fourteen additional flasks were incubated for 1 hr
prior to harvest with 10~6 M 17/ì-estradiol (£2)in minimal essential medium plus
10% dextran-coated charcoal-stripped calf serum (17) + 10~8 M 17/S-estradiol.
Twelve flasks were grown in minimal essential medium plus 10% calf serum for
9 days prior to transfer to hormone-free chemically defined medium (CDM) (16)
for 3 days prior to harvest. D, [3H)estrone exchange; •[3H]estradiol exchange.
K0s indicated above each bar.
mately 0.06 pmol of both [3H]estradiol and [3H]estrone ex
change per mg DNA. In the salt-extracted nuclei from cells
grown in regular media (10% calf serum containing 2 to 6 x
10~11 M 17/J-estradiol), [3H]estrone required elevated temper
ature for exchange while [3H]estradiol exchanged at both tem
peratures (23°and 0°).At this time, it is not possible to explain
these temperature-dependent exchange variations as they per
tain to receptors bound to the nuclear residue. It is, however,
interesting to note that nuclei from cells grown with stimulatory
levels of 17/8-estradiol (10~8 M) contained only [3H]estrone
exchangeable sites (23°) in the salt-extracted residue (Chart
3, middle). Furthermore, cells maintained in a hormone- and
serum-free medium [chemically defined medium (16)] showed
no salt-resistant estrogen-binding sites (Chart 3, right).
It is also possible to demonstrate significant cytosolic estrone
binding in the absence of a detectable 17/8-estradiol receptor.
This is accomplished by carrying out binding studies on the
protamine precipitate obtained from the 105,000 x g super
natant from cells exposed to 10~8 M 17^-estradiol for 1 hr.
Assay methods which utilize an adsorbant for the free ligand
[e.g., dextran-coated charcoal (1:10)] will strip the [3H]estrone
from its receptor. It is also important that the protamine sulfate
assay be carried out at 0°since both the E2R and EiR are
destroyed if cytosol precipitates are incubated at 37°.Utilizing
the described conditions, a minimum of 4 hr is required for
[3H]estrone to equilibrate with its receptor, while there is no
loss of binding for at least 22 hr at 0°(Chart 4). This cytosol
was devoid of E2R due to its translocation to the nucleus during
the previous incubation of cells with 10~8 M 17/8-estradiol
(Chart 4). Cytosolic E,R displays classical saturation kinetics,
and the data are amenable to Scatchard analyses (Chart 5)
yielding results typical of receptors with high affinity (KD 4.6
x 10~9 M) and limited capacity (1.22 pmol/mg DNA). The
[3H]estrone binding found in MCF-7 cells is not prevented by a
1000-fold excess of nonestrogenic steroid hormones in the
assay (Table 1).
1969
E. R. Hansen and S. C. Brooks
1.0
0.5
IO 12 14 16 20
Hours
Chart 4. Time curve ot estrone-specific binding in the cytosol from cells
treated with 10*' M 17/ì-estradiol. Three T75 flasks were incubated with regular
media containing an additional 10"' M 17/ì-estradiol for 1 hr at 37°.The cytosol
was prepared, and duplicate one-point protamine sulfate assays were performed
under incubations with 5 nw | !H|estradiol (O) or ( H [estrone (•)for the indicated
times at 0°.Points, means of triplicate incubations.
I.O 20 3.0 4.0 5.0 6.0 7.0
nMpH]E,
Chart 5. Saturation curve and Scatchard plot of estrone (L } cytosol receptor
observed after exposure of MCF-7 cells to 10"8 M 17/3-estradiol. Four T75 flasks
were incubated prior to harvest for 1 hr at 37°with regular medium containing an
additional 10~8 M 17/3-estradiol. Details of the procedure are described in
"Materials and Methods." . total binding; O. nonspecific binding; ©,specific
binding. The Scatchard polt (inset) indicates 0.055 pmol bound (B) per sample
containing 0.045 mg DMA. Therefore, the binding capacity was 1.22 pmol
estrone per mg DNA. The K, indicated was 4.6 nM. No cytosolic ER was
observed in these cells.
Specificity of 2 Estrogen Receptors for 17/?-Estradiol and
Estrone. The steroid receptor theory does not prohibit the
binding of similar steroids to the same site. Specificity is
established by the extreme molar excess of a competitor re
quired to displace the labeled steroid ligand. It is well estab
lished that estrone will not only bind to the classical estrogen
receptor but the bound estrone will activate the complex and
promote nuclear translocations (21, 31 ). Although triggered by
nonphysiological levels of estrone, the nuclear estrone com
plex will promote the induction of specific proteins (21). The
data in Chart 6A clearly demonstrate the competition of estrone
for the cytosolic and extractable nuclear E2R. Labeled 17ß-
estradiol was displaced from its cytosolic receptor by estrone
with increasing effectiveness as the molar excess of estrone
1970
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
increased. However, it was not possible to displace all the
bound [3H]estradiol until the assay was performed in the pres
ence of a 1000-fold molar excess of estrone. Greater resist
ance to exchange with estrone was displayed by the nuclear
E2R complex. In this case, there was no displacement of the
ligand until the complex was exposed to a 100-fold excess of
estrone (Chart 6A).
Similarly, the EtR in MCF-7 cells was very resistant to ex
change with 17/8-estradiol. When examined in the absence of
the 17/8-estradiol cytoplasmic receptor [cells previously incu
bated with 10~8 M 17/8-estradiol (Chart 60)], 17/î-estradiol
22
would not displace [3H]estrone on its receptor unless the un-
labeled 17/S-estradiol was present in the incubation at molar
excesses of 100-fold or greater (Chart 66). Similar results were
obtained with salt-extractable nuclear estrone-binding compo
nent (data not shown). Clearly, at nM concentrations, [Destra-
dio! does not bind to or exchange with the EiR (Chart 4).
Table 1
Specificity of pHjestrone binding
MCF-7 cells in 3 T75 flasks were incubated with regular media containing an
additional 10~8 M 17/S-estradiol for 1 hr at 37° prior to harvest. Cytosol was
prepared, and one-point protamine sulfate assays were performed in triplicate.
The assay incubations contained 5 nM [3H]estrone in all tubes, with the experi
mental tubes being supplemented with a 1000-fold concentration of the indicated
steroids. Assay incubations were carried out at 0°for 18 hr.
[3H]Estrone
bound (pmol/mg
Steroids added DNA) % of inhibition
[3H]Estrone (5 nM) 0.66
4 f>MMprogesterone 0.38 42
4- 5 JIM Sa-dihydrotestosterone 0.43 35
+ 5 /IM cortisol 0.64 3
4000
4 40 400 4000
itMEg
Chart 6. Competition of 17/3-estradiol (E2) and estrone (E,) for various recep
tors in MCF-7 cells before and after optimum processing. In A, cytosol and
nuclear extract were prepared, and one-point protamine sulfate assays were
performed in duplicate. Cytosol assays used 18-hr incubations at 0°,and nuclear
extracts were incubated for 1 hr at 37°. Both fractions were assayed utilizing 4
nM | H|estmdiol In the presence of increasing concentrations of unlabeled
estrone. O. cytosol from control cells (4 flasks) cultured only in regular media;
A, nuclear extract from cells exposed to 10~" M 17/3-estradiol for 1 hr at 37°
prior to harvesting (4 flasks). In B, cytosol protamine sulfate assays were
performed in duplicate on preparations from control cells (•)and from cells
exposed to 10~8 M 17/3-estradiol for 1 hr at 37° prior to harvesting (A). These
assays were carried out with 4 nM [3H]estrone being incubated in the presence
of increasing concentration of unlabeled 17/8-estradiol for 18 hr at 0°.
CANCER RESEARCH VOL. 42
••"TÃ-*,
?'"'.'
It was also possible to demonstrate the different affinities of
[3H]estrone for both receptors in the cytosol of cells exposed
to a minimal level of 17/î-estradiol (1CT11 M, media with 10%
calf serum). A concentration of 4 X 10~9 M 17/î-estradiol
completely displaced [3H]estrone from the cytosolic E2R. The
remaining [3H]estrone binding (to the E,R) was not totally
competed out until a concentration of 4 x 10~6 M 17/8-estradiol
was reached (Chart 66). The presence of some cytosolic EiR
in control cells is also indicated by the initial slope of the
unlabeled estrone competition line in Chart 6A.
Dynamics and Cellular Distribution of 17/i-Estradiol and
Estrone Receptors. Data presented above demonstrate that
there are 2 estrogen-binding components in MCF-7 cells; one
preferentially bound 17/î-estradiol in the presence of excess
estrone, and another displayed higher affinity for estrone than
for 17/î-estradiol. The experimental results indicated that the
quantitative relationships of these individual receptor sites var
ied with cellular compartment and estrogen stimulation of the
culture. In order to demonstrate these relationships, a quanti
tative analysis (Scatchard plot) of each receptor was carried
out on cell fractions before and after a 1-hr exposure of the
culture to 10~8 M 17/î-estradiol(Table 2).
Cells maintained in 10% calf serum (10~11 M 17/î-estradiol)
had most of their E2R in the cytoplasm (cytosol plus micro-
somes, 2.04 pmol/mg DNA) with a lesser amount of salt-
extractable nuclear E2R (0.44 pmol/mg DNA). These same
cells also contained 0.75 pmol/mg DNA of cytosolic EtR (Table
2). EtR could not be detected elsewhere in these cells. One hr
after incubating MCF-7 cells with 10~8 M 17/î-estradiol, the
previously described loss (processing) of total cellular E2R was
seen [E2R decreased from 2.68 to 1.63 pmol/mg DNA (Table
2)]. During this incubation, the receptor with highest affinity for
estrone had increased in the cytosolic and nuclear compart
ments from a total of 0.75 to 1.53 pmol/mg DNA. Therefore,
incubation with 10"8 M 17/î-estradiolhad diminished total E2R
by 0.85 pmol/mg DNA while increasing EiR by 0.78 pmol/mg
DNA. Total cellular binding capacity for both estrogens had
remained essentially unchanged during 17/î-estradiolstimula
tion.
Examination of the time course of these alterations in recep-
Table 2
Cellular distribution of E2R and E,R before and after optimum processing
Scatchard analysis was carried out on each receptor utilizing the protamine
sulfate assay procedure: 0°incubation for 18 hr for cytosol receptors; and 37°
for 1 hr for nuclear and microsomal extracted receptors. Estrone binding was
determined utilizing a range of [3H]estrone (see "Materials and Methods")
incubated in the presence of 5 nw unlabeled 17/ì-estradiol, a level capable of
saturating the unchanged E2R (see Chart 68).
17/8-Estradiol
bindingCapacity(pmol/
bindingCellular
X1Q-*M0.220.350.410.280.76Estrone
xmg Ko
10~9M0.75
DNA)
compartmentControlCytosolMicrosomesNuclearTotal+
DNA)1.870.170.442.480.2001.431.63KD
3.83000.751.30
70-estradiolCytosolMicrosomesNuclearTotalCapacity(pmol/mg
1Q-8M 1
4.5900.23
1.761.53E,R(pmol/mgDNA)2.620.170.443.231.5001.663.16
nuclear EiR; A, total cytosolic and nuclear E2R;O, total receptors(E2R plus E,R).
MAY 1982
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
EiR Formation during Processing
ter concentrations over a period of 5 hr revealed the relation
ship of E2R loss to the gain in cellular Et R (Chart 7). In these
experiments, the level of each receptor was determined by
one-point analyses (carried out in duplicate), and therefore the
results cannot be construed as quantitative but merely repre
sentative of each receptor's temporal fluctuations. The disap
pearance of E2R in MCF-7 cells had plateaued by 1 hr of
incubation with 10~8 M 17/î-estradiolat which time the elevation
of EiR was stabilized (Chart 7A). Interestingly, the nuclear E2R
continued to decline for the entire 5 hr; however, this decrease
was offset to some degree by the appearance of a small amount
of cytosolic E2R after 3 hr of exposure to 10~8 M 17/î-estradiol
(Chart 78). Nuclear E,R underwent a transient peak at 30 min
but diminished thereafter maintaining a very low level. Most of
the EiR in these cells could be found in the cytosol where its
concentration rose rapidly for 1 hr to a significant level which
then increased only slightly through 5 hr. While E2R was lost to
the salt-extractable nuclear compartment, E,R, and much later
E2R, accumulated in the cytoplasm.
The appearance of estrone-binding protein in the cytoplasm
displayed a dependence on the concentration of 17/î-estradiol
by which the culture was stimulated. Chart 8 presents the
levels of cytosolic E2R, extractable nuclear E2R, and Et R
(mostly found in the cytosol; see Chart 7) which were present
in MCF-7 cells incubated for 1 hr with increasing levels (10~11
to 10~6 M) of 17/î-estradiol. Cytosolic E2R was seen to disap
pear and accumulate in the nucleus as the 17/î-estradiol con
centration in the incubation increased from 10~11 to 10~9 M. At
the maximum 17/î-estradiol-stimulatory level (10~9 M), the nu
clear E2R reached a capacity (0.70 pmol/mg DNA) in these
.°00-o-
I5
v—-0-—*
IO
0 -^ ^ ^
05
•o
C
3
O
Å“
Chart 7. Time curve of formation of EiR concomitant with processing of E2R
in MCF-7 cells. Prior to harvest, 2 T75 flasks were incubated at 37° for the
indicated times with regular media containing 10" M 17/i-estmdiol. Cytosol and
nuclear extracts were prepared as described in "Materials and Methods." One-
point receptor assays were performed with 4.2 nw [3H)estrone plus 5 nM 17/î-
estradiol for the cytosolic and nuclear EiR determinations and with 3.8 nw
[3H]estradiol for cytosolic and nuclear E. R assays. At these concentrations, the
EiR is approximately 50% saturated (see Chart 5). and the E2R is approximately
80% saturated.' Points, means of duplicate assays. A O, total cytosolic and
B: 0, cytosolic E,R; •,cytosolic E2R; A, nuclear E,R; •nuclear E;,R.
1971
E. R. Hansen and S. C. Brooks
[3H]estradiol of the detection system.
l The first of these assumptions would involve the loss of total
Õ tritiated estrogen bound with high affinity in intact cells exposed
to physiological levels of [3H]estradiol, while the latter proposal
would possibly exhibit a consistency of protein-bound label in
i such experiments. An earlier publication from this laboratory
(4) has shown the sum of cytosolic and nuclear tritiated estro
gen not to decline during a 1-hr, 37°incubation of MCF-7 cells
containing receptor previously complexed with [3H]estradiol in
l
IO'" IO'1 icre io-r the cold. It would appear then that there is no loss of bound
labeled estrogen in cells exposed to physiological levels of
M E, [3H]estradiol.
Chart 8. Effect of exposure of MCF-7 cells to increasing concentrations of
17/3-estradiol (£2)on their content of E2R and EiR. Before harvest, cells were These initial studies also demonstrated that the labeled li
incubated for 1 hr at 37°with regular media plus the indicated concentrations of gand was composed of both 17/8-estradiol and estrone. In fact,
17/S-estradiol (2 T75 flasks of cells for each level of 17/S-estradiol). Cytosol and
nuclear extracts were prepared as described in "Materials and Methods." One- the portion of bound tritium found in estrone increased with the
point receptor assays were performed with 4.0 nM [3H]estrone plus 5 nw 17/3- length of incubation of MCF-7 cells containing pulse-labeled
estradiolforcytosolic(0°. 18 hr) and nuclear (37°. 1 hr)Ei R assays. Utilizing the [3H]E2R.Furthermore, the [3H]estrone increased at the expense
same incubation conditions, the E2R was determined in separate aliquots of the of [3H]estradiol in the salt-resistant nuclear component while a
cytosol and nuclear extract utilizing 3.5 nw | 'H Jestradiol. Each column represents
the mean of duplicate assays. Eä, cytosolic E2R; 0, salt-extractable nuclear E2R; [3H]estrone-protein complex accumulated in the cytosol in the
•total of cytosolic and nuclear E,R. presence of a 100-fold excess of unbound cytoplasmic [3H]-
estradiol. These initial experiments suggested the existence of
one-point assays which this receptor maintained throughout an EiR in these cells, a fact documented in later work (3).
the incubations with elevated 17/8-estradiol concentrations. Although unusual, our earlier reports of a specific E,R in MCF-
The total cellular EiR rose with increasing 17/3-estradiol in the 7 human breast tumor cells have been supported by the pub
incubations until the stimulatory 17/S-estradiolreached 10~9to
lished investigations of Kreitmann ef al. showing the presence
10"8 M. Thereafter, at the highest concentration of media 17ß- of both 17/î-estradiol and estrone receptors in human uterine
estradiol, the E,R per mg DNA diminished, accompanied by an cytosol (19) and in monkey endometrial nuclei (20).
increase in cytosolic E2R (Chart 8). Although the binding ca It has been the purpose of the studies contained herein to
pacity of the salt-extractable nuclear E2R after maximal proc establish the optimum conditions for the assay of E2Rand EiR
essing (brought about by 10~9 to 10~8 M 17/8-estradiol) was
in the various cellular compartments, to document the specific
independent of the concentration of stimulatory 17/8-estradiol ity of E!R, and to show the dynamics and cellular distribution
in the incubation, the cellular E,R (mostly cytosolic) fluctuated of E2Rand EiR, particularly following 17/8-estradiol stimulation
in a manner opposite to that of the variations in cytosolic E2R of MCF-7 cells.
and dependent on the concentration of 17/3-estradiol in the Utilizing optimum conditions, it was possible to determine
media. dissociation constants and binding capacities for either recep
tor in the absence of the other (e.g., E,R in the cytosol from
DISCUSSION
17/3-estradiol-stimulated cells) or, in the case where both re
ceptors were present, saturating amounts of the other estrogen
The processing (loss) of E2R has been related to the induc (unlabeled) may be added to the assay mixture (e.g., salt-
tion of progesterone receptor in MCF-7 cells (18). Experimen extractable nuclear receptors). These procedures have shown
tally, a portion of the detectable cellular E2R is seen to disap both E2R and E,R to exist in the cytosol and in the salt-
pear within 1 hr of stimulation of this culture by 10~9 to 10~8 extractable and the salt-resistant nuclear compartments. The
M 17/8-estradiol. Since the cytosolic receptor has been trans binding of [3H]estradiol to the extractable nuclear E2Rwas not
located to the nucleus immediately following administration of competed by estrone at molar excesses below 10-fold (Chart
17/S-estradiol, it is the diminishing level of salt-extractable 6A). Below this competitive excess, there was also very little
nuclear E2R which initially reveals the effects of processing. displacement of [3H]estradiol from its cytosolic receptor by
Horwitz and McGuire (18) have reported that the lost receptor estrone. At the same time, the data in Chart 6 show little or no
was not detected in the salt-resistant fraction, nor have the binding of 17/S-estradiol to the E,R (particularly in the cytosol)
experiments reported herein shown additional E2Rin any other since there was no displacement of [3H]estrone by 17/8-estra
cellular compartment. diol until a molar excess of 1000-fold was reached (Chart 6ß).
Detection of the estrogen receptor, or its complex with The exchange of 17/8-estradiol for [3H]estrone on the classical
endogenous 17/î-estradiol,has heretofore relied on the spe cytosolic E2R is seen to occur in control cells which contain
cific binding of, or ligand exchange with, tritiated 17/8-estradiol. both receptors in their cytoplasm (half-displacement occurring
Without this high-affinity adsorption of the [3H]estradiol ligand, at 5 X 10~10M or near the KDfor cytosolic E2R). Aside from
the presence of receptor would be undetected. The observed the indicated specificity, 17/S-estradiol binds more tightly to its
cytosolic (K0 = 0.3 x 10~9 M) and nuclear (K0 = 0.8 x 10~9
loss of E2Rcould therefore be the result of any alteration to the
receptor which results in a significant decrease in its affinity M) receptors than estrone is bound by its receptors (cytosolic
for the labeled 17/8-estradiol in the assay. On the other hand, E,R, KO= 4 x 10"9M; nuclear E,R, KD = 1.7 x 10~9M).
a circumstance could exist in which the nuclear estrogen- The fact that MCF-7 cells contain separate proteins which
receptor complex was changed in a manner which no longer preferentially bind either 17/8-estradiol or estrone is intriguing
allowed the endogenous ligand to be exchanged with the and, since estrone is derived endogenously from 17/8-estradiol,

1972 CANCER RESEARCH VOL. 42

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a possible relationship between E2R and EtR is suggested. The
quantitative data in Table 2 clearly show that both these recep
tors could be found in the cytoplasm and nucleus. Furthermore,
as the 17/8-estradiol exposure of this culture was increased to
optimum concentrations that bring about processing, the spe
cific E2R accumulated within the nucleus as the E(R reached
its highest level in the cytosol. Most importantly, the total
estrogen (17/?-estradiol plus estrone)-binding capacity re
mained virtually unaltered after a 1-hr exposure of MCF-7 cells
to the processing level of 17/8-estradiol. The 0.85-pmol/mg
DMA value of "lost" E2R had been accompanied by a nearly
equal increase in cellular EtR (0.78 pmol/mg DMA).
This quantitative similarity between the disappearance of
E2R and the appearance of additional E,R in MCF-7 cells was
also reflected in their temporal relationship (Chart 7). As cellular
E2R was processed, the level of E,R increased. For the most
part, the E2R was lost from the nuclear extract and the EtR,
while present in the same extract, was shown to accumulate in
the cytosol. Over the entire 5-hr period, the total detected
cellular estrogen-binding capacity (17/8-estradiol plus estrone)
remained unchanged in these 6 timed assays which were
carried out under identical saturation conditions for each li-
gand.
Not only did the formation of E,R occur simultaneously with
the processing of E2R, but also the maximum appearance of
this novel receptor depends on the exposure of MCF-7 cells to
optimum levels of E2 (10~9 to 10~8 M) for the promotion of
processing (Chart 8). 17/8-Estradiol concentrations which were
too low or too high for optimum processing of E2R resulted in
decreased formation of EiR.
There are several possibilities which must be considered as
possible explanations for the observed estrone binding. For
example, 17/S-estradiol dehydrogenase has been shown to be
distributed throughout target tissues (29). Although it has been
demonstrated in these investigations that there was no inter-
conversion of 17/8-estradiol and estrone during the incubation
of tritiated estrogen and the protamine sulfate precipitate, it
remains conceivable that a precipitated estrogen dehydrogen
ase might bind the estrogen. This does not appear to be the
case, however, since the target tissue enzyme has a JUMKM
(37) indicating significantly less ligand affinity compared to that
of estrone (or 17/3-estradiol) found in these investigations (KD
s nM). In addition, the widely different binding characteristics
of the protein described herein for 17/3-estradiol and estrone
(100-fold 17/8-estradiol required to displace estrone) are unlike
those of the target tissue dehydrogenase (KM for 17/8-estradiol
is 20 times that of estrone). Finally, the apparent level of this
dehydrogenase in target tissues is increased by progesterone
(36), not by 17/8-estradiol as demonstrated in these studies.
Numerous recent publications have reported the existence
of a type II estrogen receptor in uterine and breast tissue (10,
14, 38). This binding has been characterized by a high KD (30
nM) for 17/î-estradiol and the lack of nuclear migration during
17/8-estradiol stimulation, both properties of the EiR docu
mented above. There are, however, several discrepancies be
tween the properties of the type II 17/S-estradiol binding and
the E,R described herein. First, the cytoplasmic type II E2R
concentration remains unchanged during 17/S-estradiol stimu
lation (14). Conversely, the level of cytoplasmic EiR increases
significantly following the exposure of MCF-7 cells to 17ß-
estradiol. Furthermore, 17/8-estradiol does not compete with
MAY 1982
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
EiR Formation during Processing
estrone for sites on EtR until its concentration is at least 100-
fold greater. On the other hand, 17/8-estradiol and estrone are
equally effective in the inhibition of [3H]estradiol binding to type
II sites (10). Finally, the number of binding sites on EiR does
not approach that reported for type II [greater than 4 times type
I in uterus, (10) and in MCF-7 cells5].
The data derived from these investigations do not allow for
a determination of the origins of EtR nor do they permit con
clusions as to the relationship between E2R and E,R in MCF-7
cells. It is, however, pertinent to discuss receptor alterations
which might account for the observations reported herein.
Information presently available indicates that the D-ring of
estrogens is linked to the receptor protein through a hydrogen-
bonding system involving the 17-oxygen and some point on the
protein which lies above Ring D (15). The 17/2-hydroxyl of 17ß-
estradiol would be expected to contribute to higher affinity if
the ligand were the hydrogen donor to such bonding. However,
small alterations to this site on the receptor resulting in a
protein which served as the hydrogen donor above Ring D
might produce a receptor with greater binding to estrone with
its 17-keto group acting now as a hydrogen acceptor. A similar
situation has been elucidated for the hydrogen bonding above
Ring D of the estrogen-specific bovine adrenal estrogen sulfo-
transferase (30).
More important is the contribution which the aromatic Ring
A makes to the affinity of estrogens to their receptor(s). X-ray
crystallographic studies have shown discrete but important
differences to exist between the A ring-binding properties of
estradici and estrone (11, 12). Principally, these differences
are a lower pK for the phenol group of estrone and the unique
requirement of a hemihydrate of the C(3)-hydroxyl of 17/8-
estradiol for receptor interaction. This latter condition results
in the capacity of 17/8-estradiol to act as a hydrogen bond
donor and acceptor at the C(3)-hydroxyl, while estrone (which
does not hydrate) acts as a donor only at this position. Subtle
changes in the estrogen receptor site near C(3) of the ligand
may also produce a protein with greater affinity for estrone.
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CANCER RESEARCH VOL. 42
Estrone Receptor Formation During the Processing of
Estradiol-Receptor Complex in MCF-7 Cells
E. R. Hansen and S. C. Brooks

Cancer Res 1982;42:1967-1974.

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