Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
0008-5472/82/0042-0000$02.00
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
E. R. Hansen and S. C. Brooks
and antibiotics and made 10% with respect to calf serum (33). Pas suspension was centrifuged at 800 x g for 10 min, and the supernatant
sages involved trypsin digestion (33) to obtain monocellular suspen was decanted. Resulting pellets were incubated in duplicate under
sions followed by the plating of 2 x 106 cells/flask. Cultures were conditions indicated in the legend to Chart 3 with 300 /il TrisrEDTA
confluent after 1 week and were harvested for experimentation after 2 containing increasing concentrations of [3H]estradiol (0.2 to 4.0 nM) or
weeks (approximately 30 x 10s cells/flask). [3H]estrone (0.3 to 6.0 nM) with or without 200-fold excess of respective
Following removal of medium, flasks were washed twice with NaCI steroid. After incubation, tubes were washed 3 times with Tris:EDTA
solution (9 mg/100 ml) and once with Tris:EDTA buffer [10 mM Tris- buffer and extracted with 1.5 ml ethanol, and 1 ml was counted as
HCI: 1.5 mM EDTA (pH 7.4 at 0°)]to remove residual medium. The cells described before. The data were analyzed by the method of Scatchard
were then removed from the flask using a rubber policeman with 2 ml (32).
of Tris:EDTA buffer made 8 mM with dithiothreitol and placed in a
Dounce tube.
RESULTS
Preparation of Cytosol, Microsomal, and Nuclear Extracts. The
suspension of harvested cells from 2 flasks was homogenized in the 4
Characteristics of the Protamine Assay of Various Estro
ml buffer made 0.1 % with saponin utilizing 15 strokes of a tightly fitting gen Receptors. The 0.6 M KCI extract of nuclei from MCF-7
pestle. The homogenate was centrifuged at 800 x g for 10 min (4°),
cells contained protamine-precipitable proteins which bind
and the supernatant was saved (crude cytosol). A more purified nuclear [3H]estradiol with high affinity and finite capacity. However, the
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 rate at which this association reached equilibrium and the
Tris-HCI:8 mw dithiothreitol (pH 7.6 at 0°)]3 times and centrifuging as maximum [3H]estradiol binding observed varied with the tem
before. The supernatants were added to the crude cytosol. The crude perature of the assay and the prior availability of 17/8-estradiol
cytosol was centrifuged at 105,000 x g (4°) for 60 min, yielding a
to the cells (Chart 1). When cultures were grown in media
supernatant (cytosol) and a high-speed pellet (microsomes). containing 10% calf serum (2 to 6 x 10~" M 17/S-estradiol;
The partially purified nuclear and microsomal pellets were extracted see Footnote 4 and Ref. 28), the extractable nuclear 17ß-
with Tris:EDTA:KCI buffer [10 mw Tris-HCI:1.5 mM EDTA:0.6 M KCI.1 estradiol receptor reaches equilibrium by 1 hr at 37°or 6 to 20
mM thioglycerol:10% glycerol (pH 8.5 at 0 °)] by triturating (with
hr at 0°.Regardless of which temperature is used, the maxi
Dounce pestle) every 10 min for 1 hr. Solubili/uri proteins were
mum binding is similar (approximately 0.52 pmol 17/î-estradiol
separated from insoluble nuclear or microsomal material by centrifu-
gation at 105,000 x g for 30 min. The concentrated nuclear or bound per mg DNA). Previously, this binding to (or exchange
with?) the protamine-precipitated nuclear receptor at 0°has
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 been defined as indicative of uncharged receptor (40). This
(39). DNA was determined from the residual nuclear and microsomal does not appear to be the case for receptor extracted from
pellet (105,000 x g pellet) by the diphenylamine method of Burton (7) substantially purified nuclei in these experiments since [3H]-
using Sigma type V DNA as a standard. estrone was not capable of occupying this site during cold
Protamine Sulfate Assays. The method used was that of Steggles equilibrations (Chart 2). Even at 37°, [3H]estrone occupied
and King (34) as modified by Zava et al. (39) and Chamness ef al. (8).
(exchanged with?) only 20% of these sites (Chart 2).
Fractions (500 /il) of cytosol and nuclear extracts were precipitated After exposure of the culture to 17/î-estradiol(10~8 M) during
with 300 jul of protamine sulfate (1 mg/ml; DSP injection, without a 1-hr incubation at 37°, there were dramatic changes in the
phenol preservative; Eli Lilly and Co.) and centrifuged at 800 x g for
10 min. amount and exchange characteristics of the extractable nu
For one-point assays, the protamine precipitates from aliquots of the clear 17/î-estradiolreceptor (Chart 1). A 3-fold increase (~1.5
pmol/mg DNA) was observed in the quantity of [3H]estradiol
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 which exchanged with receptor at 37°, probably representing
and temperatures. Nonspecific binding was determined by a parallel the translocation of cytoplasmic E2R complex to the nuclear
incubation with tritiated estrogen plus 200-fold excess nonradiolabeled compartment. On the other hand, little (0.2 pmol/mg DNA)
17/3-estradiol or estrone, respectively. After incubation, the precipitates [3H]estradiol was seen to exchange at 0°with nuclear receptor-
were washed 3 times with Tris:EDTA buffer and extracted with 1.5 ml bound 17/î-estradiol. Presenting these cells with the optimum
ethanol, and 1 ml was counted utilizing a scintillation spectropho-
level of 17/8-estradiol to bring about nuclear processing of the
tometer equipped with an absolute activity analyzer. The data were
analyzed after subtraction of nonspecific binding.
receptor complex (18) has significantly altered the exchange
properties of the ligand. At the same time, 17/S-estradiol stim
Saturation analysis on cytosol, microsomal extracts, or nuclear
extracts was carried out by incubating for indicated times and temper
ulation of MCF-7 cells brought about the appearance of an
appreciable amount of [3H]estrone binding to (0°)or exchange
atures the protamine sulfate precipitates in duplicate with 300 »Iof
Tris:EDTA containing increasing concentrations of [3H]estradiol (0.2 to (at 37°)with ligand(s) on the protamine precipitate of the 0.6
4.0 nw) or [3H]estrone (0.3 to 6.0 nw) with or without 200-fold excess M KCI extract of nuclei (Chart 2). The 0.12 pmol/mg DNA of
of respective estrogen. The protamine-precipitated proteins were then [3H]estrone binding at 0°is similar to the 0.2 pmol/mg DNA of
washed, extracted, and counted as described above. The binding [3H]estradiol bound in the cold (Chart 1), suggesting that both
capacities and KDs were determined using the method of Scatchard tritiated ligands were taken up by this small number of unoc
(32) after subtraction of nonspecific binding.
Nuclear Exchange Assay on 0.6 M KCI-extracted Nuclear Pellet. cupied sites in the nuclear extract, possibly from cytoplasmic
A modification of the exchange procedure of Anderson et al. (2) was
contamination (13). Under the assay conditions, the observed
[3H]estrone exchange at 37°would not be expected to occur
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 with charged classical E2R complex since the affinity of estrone
pellet) was transferred to a Dounce homogenizer, and 2 ml of buffered is considerably less than that of 17/î-estradiolfor this protein.
0.25 M sucrose solution were added. The pellet was homogenized into
Therefore, the 0.1-pmol/mg DNA [3H]estrone binding excess
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 4 Radioimmunoassay of total 17/i-estradiol (free plus hydrolyzed sulfates) in
analysis, and 500-/il samples were taken for an exchange assay. The media containing 10% calf serum.
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
Eif? Formation during Processing
1.5
tu
04bnM
5 O.O7
o Kd
130 nM
? 0.06
10 •o
S3 0.05
I 004
0.5
Û 0.03
Si
«0.02
o
2 4 6 8 10 12 14 16 18 20
0.01
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 2S-0* 23-0° ZyO«23'0* ZyO" 23-0«
the 0.6 M KCI nuclear extract. Nuclear extracts were prepared, and one-point MEM »CS »IO~8ME2 CDM
protamine sulfate assays were performed at the indicated times and temperatures Chart 3. Exchange characteristics of the 0.6 M KCI-insoluble nuclear residue
under incubation with 5 nM [3H]estradiol. Details of the procedures are described
in "Materials and Methods." Points, mean of duplicate samples. Assays carried 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
out on the nuclear extracts from untreated cells in 3 T75 flasks: •,0°:O. 37°.
at 0°for 18 hr as indicated. Cells (15 T75 flasks) were grown in regular media
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- (minimal essential medium plus 10% calf serum (MEM + CS) as described in
"Materials and Methods." Fourteen additional flasks were incubated for 1 hr
estradiol: A. 0°;A, 37°.
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
03 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.
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
E. R. Hansen and S. C. Brooks
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
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
••"TÃ-*,
?'"'.'
It was also possible to demonstrate the different affinities of ter concentrations over a period of 5 hr revealed the relation
[3H]estrone for both receptors in the cytosol of cells exposed ship of E2R loss to the gain in cellular Et R (Chart 7). In these
to a minimal level of 17/î-estradiol (1CT11 M, media with 10% experiments, the level of each receptor was determined by
calf serum). A concentration of 4 X 10~9 M 17/î-estradiol one-point analyses (carried out in duplicate), and therefore the
completely displaced [3H]estrone from the cytosolic E2R. The results cannot be construed as quantitative but merely repre
remaining [3H]estrone binding (to the E,R) was not totally sentative of each receptor's temporal fluctuations. The disap
competed out until a concentration of 4 x 10~6 M 17/8-estradiol 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
was reached (Chart 66). The presence of some cytosolic EiR
in control cells is also indicated by the initial slope of the of EiR was stabilized (Chart 7A). Interestingly, the nuclear E2R
unlabeled estrone competition line in Chart 6A. continued to decline for the entire 5 hr; however, this decrease
Dynamics and Cellular Distribution of 17/i-Estradiol and 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
Estrone Receptors. Data presented above demonstrate that
there are 2 estrogen-binding components in MCF-7 cells; one (Chart 78). Nuclear E,R underwent a transient peak at 30 min
preferentially bound 17/î-estradiol in the presence of excess but diminished thereafter maintaining a very low level. Most of
estrone, and another displayed higher affinity for estrone than the EiR in these cells could be found in the cytosol where its
for 17/î-estradiol. The experimental results indicated that the concentration rose rapidly for 1 hr to a significant level which
quantitative relationships of these individual receptor sites var then increased only slightly through 5 hr. While E2R was lost to
ied with cellular compartment and estrogen stimulation of the the salt-extractable nuclear compartment, E,R, and much later
culture. In order to demonstrate these relationships, a quanti E2R, accumulated in the cytoplasm.
tative analysis (Scatchard plot) of each receptor was carried The appearance of estrone-binding protein in the cytoplasm
out on cell fractions before and after a 1-hr exposure of the displayed a dependence on the concentration of 17/î-estradiol
culture to 10~8 M 17/î-estradiol(Table 2).
by which the culture was stimulated. Chart 8 presents the
Cells maintained in 10% calf serum (10~11 M 17/î-estradiol)
levels of cytosolic E2R, extractable nuclear E2R, and Et R
had most of their E2R in the cytoplasm (cytosol plus micro- (mostly found in the cytosol; see Chart 7) which were present
somes, 2.04 pmol/mg DNA) with a lesser amount of salt- 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
extractable nuclear E2R (0.44 pmol/mg DNA). These same
cells also contained 0.75 pmol/mg DNA of cytosolic EtR (Table 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
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 the maximum 17/î-estradiol-stimulatory level (10~9 M), the nu
previously described loss (processing) of total cellular E2R was clear E2R reached a capacity (0.70 pmol/mg DNA) in these
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 .°00-o-
I5
ments from a total of 0.75 to 1.53 pmol/mg DNA. Therefore,
v—-0-—*
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 IO
DNA. Total cellular binding capacity for both estrogens had
remained essentially unchanged during 17/î-estradiolstimula 0 -^ ^ ^
tion. 05
Examination of the time course of these alterations in recep- •o
C
3
O
Å“
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
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
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,
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
EiR Formation during Processing
a possible relationship between E2R and EtR is suggested. The estrone for sites on EtR until its concentration is at least 100-
quantitative data in Table 2 clearly show that both these recep fold greater. On the other hand, 17/8-estradiol and estrone are
tors could be found in the cytoplasm and nucleus. Furthermore, equally effective in the inhibition of [3H]estradiol binding to type
as the 17/8-estradiol exposure of this culture was increased to II sites (10). Finally, the number of binding sites on EiR does
optimum concentrations that bring about processing, the spe not approach that reported for type II [greater than 4 times type
cific E2R accumulated within the nucleus as the E(R reached I in uterus, (10) and in MCF-7 cells5].
its highest level in the cytosol. Most importantly, the total The data derived from these investigations do not allow for
estrogen (17/?-estradiol plus estrone)-binding capacity re a determination of the origins of EtR nor do they permit con
mained virtually unaltered after a 1-hr exposure of MCF-7 cells clusions as to the relationship between E2R and E,R in MCF-7
to the processing level of 17/8-estradiol. The 0.85-pmol/mg cells. It is, however, pertinent to discuss receptor alterations
DMA value of "lost" E2R had been accompanied by a nearly
which might account for the observations reported herein.
equal increase in cellular EtR (0.78 pmol/mg DMA). Information presently available indicates that the D-ring of
This quantitative similarity between the disappearance of estrogens is linked to the receptor protein through a hydrogen-
E2R and the appearance of additional E,R in MCF-7 cells was bonding system involving the 17-oxygen and some point on the
also reflected in their temporal relationship (Chart 7). As cellular protein which lies above Ring D (15). The 17/2-hydroxyl of 17ß-
E2R was processed, the level of E,R increased. For the most estradiol would be expected to contribute to higher affinity if
part, the E2R was lost from the nuclear extract and the EtR, the ligand were the hydrogen donor to such bonding. However,
while present in the same extract, was shown to accumulate in small alterations to this site on the receptor resulting in a
the cytosol. Over the entire 5-hr period, the total detected protein which served as the hydrogen donor above Ring D
cellular estrogen-binding capacity (17/8-estradiol plus estrone) might produce a receptor with greater binding to estrone with
remained unchanged in these 6 timed assays which were its 17-keto group acting now as a hydrogen acceptor. A similar
carried out under identical saturation conditions for each li- situation has been elucidated for the hydrogen bonding above
gand. Ring D of the estrogen-specific bovine adrenal estrogen sulfo-
Not only did the formation of E,R occur simultaneously with transferase (30).
the processing of E2R, but also the maximum appearance of More important is the contribution which the aromatic Ring
this novel receptor depends on the exposure of MCF-7 cells to A makes to the affinity of estrogens to their receptor(s). X-ray
optimum levels of E2 (10~9 to 10~8 M) for the promotion of
crystallographic studies have shown discrete but important
processing (Chart 8). 17/8-Estradiol concentrations which were differences to exist between the A ring-binding properties of
too low or too high for optimum processing of E2R resulted in estradici and estrone (11, 12). Principally, these differences
decreased formation of EiR. are a lower pK for the phenol group of estrone and the unique
There are several possibilities which must be considered as requirement of a hemihydrate of the C(3)-hydroxyl of 17/8-
possible explanations for the observed estrone binding. For estradiol for receptor interaction. This latter condition results
example, 17/S-estradiol dehydrogenase has been shown to be in the capacity of 17/8-estradiol to act as a hydrogen bond
distributed throughout target tissues (29). Although it has been donor and acceptor at the C(3)-hydroxyl, while estrone (which
demonstrated in these investigations that there was no inter- does not hydrate) acts as a donor only at this position. Subtle
conversion of 17/8-estradiol and estrone during the incubation changes in the estrogen receptor site near C(3) of the ligand
of tritiated estrogen and the protamine sulfate precipitate, it may also produce a protein with greater affinity for estrone.
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 REFERENCES
(37) indicating significantly less ligand affinity compared to that
of estrone (or 17/3-estradiol) found in these investigations (KD 1. Adams, J. B., and Chandra, D. P. Dehydroepiandrosterone sulfotransferase
as a possible shunt for the control of steroid metabolism In human mammary
s nM). In addition, the widely different binding characteristics carcinoma. Cancer Res., 37. 278-284,1977.
of the protein described herein for 17/3-estradiol and estrone 2. Anderson, J., Clark, J. H., and Peck, E. J., Jr. Oestrogen and nuclear
binding sites; determination of specific sites by 'H-oestradiol exchange.
(100-fold 17/8-estradiol required to displace estrone) are unlike Biochem. J., 126: 561-567, 1972.
those of the target tissue dehydrogenase (KM for 17/8-estradiol 3. Brooks, S. C., Horn, L., Pack, B. A., Rozhin, J., Hansen, E., and Goldberg,
is 20 times that of estrone). Finally, the apparent level of this R. Estrogen metabolism and function in vivo and in vitro. In: J. A. McLachland
(ed.), Estrogens in the Environment, pp. 147-167. Amsterdam: Elsevier/
dehydrogenase in target tissues is increased by progesterone North-Holland Biomedicai Press, 1980.
(36), not by 17/8-estradiol as demonstrated in these studies. 4. Brooks, S. C., Lock, E. R., and Horn, L. Metabolism of 17/3-estradiol during
receptor mediated nuclear migration in breast cancer cells. Cancer Res.,
Numerous recent publications have reported the existence 38. 4238-4242. 1978.
of a type II estrogen receptor in uterine and breast tissue (10, 5. Brooks, S. C., Rozhin, J., Pack. B. A., Horn, L., Godefroi, V. C., Locke, E.
14, 38). This binding has been characterized by a high KD (30 R., Zemlicka, J., and Singh, D. V. Role of sulfate conjugation in estrogen
metabolism and activity. J. Toxicol. Environ. Health, 4: 283-300, 1978.
nM) for 17/î-estradiol and the lack of nuclear migration during 6. Buirchell, B. J., and Hähnel, R. Metabolism of estradiol-17/3 in human
17/8-estradiol stimulation, both properties of the EiR docu endometrium during the menstrual cycle. J. Steroid Biochem., 6. 1489-
mented above. There are, however, several discrepancies be 1494, 1975.
7. Burton, K. A study of the conditions and mechansim of the diphenylamine
tween the properties of the type II 17/S-estradiol binding and reaction for the colorimetrie estimation of deoxyribonucleic acid. Biochem.
the E,R described herein. First, the cytoplasmic type II E2R J., 62. 315-322, 1956.
concentration remains unchanged during 17/S-estradiol stimu 8. Chamness, G. C., Huff, K., and McGuire. W. L. Protamine-precipitated
estrogen receptor: a solid-phase ligand exchange assay. Steroids, 25. 627-
lation (14). Conversely, the level of cytoplasmic EiR increases 635, 1975.
significantly following the exposure of MCF-7 cells to 17ß-
estradiol. Furthermore, 17/8-estradiol does not compete with ' Unpublished data from these laboratories.
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
£.R. Hansen and S. C. Brooks
9. Clark, J. H., Eriksson, H. A., and Hardin, J. W. Uterine receptor-estradiol rat uterine minces. Endocrinology, 87. 924-933, 1970.
complexes and their interaction with nuclear binding sites. J. Steroid Bio- 25. Pack, B. A., Brooks, C. L., Dukelow, W. R.. and Brooks, S. C. The metabolism
chem., 7. 1039-1043, 1977. and nuclear migration of estrogen in porcine uterus throughout the implan
10. Clark, J. H., Hardin, J. W., Upchurch, S., and Eriksson, H. Heterogeneity of tation process. Biol. Reprod.. 20: 545-551, 1979.
estrogen binding sites in the cytosol of the rat uterus. J. Biol. Chem., 253. 26. Pack, B. A., Christensen, C., Douraghy, M., and Brooks. S. C. Nuclear and
7630-7634, 1978. cytosolic estrogen receptor in gilt endometrium throughout the estrous
11. Duax, W. L., Smith. G. D., Swenson, D. C., Strong, P. D., Weeks, C. M., cycle. Endocrinology, »03.2129-2136, 1978.
Anancenko, S. N., and Egorova, V. V. Steroid structure and function. IX. 27. Pack, B. A., Tovar, R., Booth, E., and Brooks, S. C. The cyclic relationship
Molecular conformation and receptor binding of isomerie analogs of o-homo- of estrogen sulfurylation to the nuclear receptor level in human endometrial
estradiol. J. Steroid Biochem.. 14:1-7, 1981. curertings. J. Clin. Endocrinol. Metab., 48: 420-424, 1979.
12. Duax, W. L., and Weeks, C. M. Molecular basis of estrogenicity: x-ray 28. Pavlik, E. S., and Katzenellenbogen, B. S. Human endometrial cells in
crystallographic studies. In: J. A. McLachlan (ed.), Estrogens in the Environ primary tissue culture: estrogen interactions and modulation of cell prolif
ment, pp. 11-32. Amsterdam: Elsevier/North-Holland Biomedicai Press, eration. J. Clin. Endocrinol. Metab., 47: 333-343, 1978.
1980. 29. Follow. K.. Schmidt-Gollwitzer, M., and Nevinny-Stickel, J. Progesterone
13. Edwards. D. P., Martin, P. M., Horwitz, K. B.. Chamness. G. C., and McGuire. receptors in normal human endometrium and endometrial carcinoma. In: W.
W. L. Subcellular compartmentalization of estrogen receptors in human L. McGuire (ed.). Progesterone Receptors in Normal and Neoplastic Tissues,
breast cancer cells. Exp. Cell Res., »27.197-213, 1980. pp. 313-338. New York: Raven Press. 1977.
14. Eriksson, H., Upchurch, S., Hardin, J. W., Peck, E. J., and Clark, J. H. 30. Rozhin, J., Soderstrom, R. L., and Brooks, S. C. Specificity studies on
Heterogeneity of estrogen receptors in the cytosol and nuclear fractions of bovine adrenal estrogen sulfotransferase. J. Biol. Chem., 249. 2079-2087,
the rat uterus. Biochem. Biophys. Res. Commun., 8). 1-7, 1978. 1974.
15. Hähnel, R., Twaddle, E., and Ratajczak, T. The specificity of the estrogen 31. Ruh, T. S., Katzenellenbogen, B. S., Katzenellenbogen, J. A., and Gorski. J.
receptor of human uterus. J. Steroid Biochem., 4: 21 -31, 1973. Estrone interaction with the rat uterus: in vitro response and nuclear uptake.
16. Higuchi, K.. and Robinson, R. C. Studies on the cultivation of mammalian Endocrinology, 92. 125-134, 1973.
cell lines in a serum-free chemically defined medium. In Vitro (Rockville), 9 32. Scatchard, G. The attraction of proteins for small molecules and ions. Ann.
114-121. 1973. N. Y. Acad. Sci., 5): 660-672, 1949.
17. Horwitz. K. B., Costlow, M. E., and McGuire, W. L. MCF-7: a human breast 33. Soûle,H. D., Vasquez, J., Long, A., Albert, S., and Brennan, M. A human
cancer cell line with estrogen, androgen, progesterone, and glucocorticoid cell line from a pleural effusion derived from a breast carcinoma. J. Nati.
receptors. Steroids, 26. 785-795, 1975. Cancer Inst., 5».1409-1416, 1973.
18. Horwitz, K. B., and McGuire, W. L. Estrogen control of progesterone receptor 34. Steggles, A. W., and King, R. J. B. The use of protamine to study 6,7-3H
in human breast cancer: correlation with nuclear processing of estrogen oestradiol-17ß binding in rat uterus. Biochem. J., Ã-»8.695-701, 1970.
receptor. J. Biol. Chem., 253. 2223-2228, 1978. 35. Tseng, L., and Gurpide, E. Changes in the in vitro metabolism of estradici by
19. Kreitmann, B.. Faye, J. C., Derach, B., and Bayard, F. Unequal binding of human endometrium during the menstrual cycle. Am. J. Obstet. Gynecol.,
estrogens in human endometrium. J. Steroid Biochem., 11: 1253-1258, 114: 1002-1011, 1972.
1979. 36. Tseng, L., and Gurpide, E. Stimulation of various 17ß-and 20a-hydroxyste-
20. Kreitmann-Gimbel. B., Bayard. F.. and Hodgen, G. D. Changing ratios of roid dehydrogenase activities by progestins in human endometrium. Endo
nuclear estrone to estradici binding in endometrium at implantation: regu crinology, »04.1745-1748, 1979.
lation by chorionic gonadotropin and progesterone during rescue of the 37. Tseng. L., Mazella, J., and Tseng, L. Kinetic studies of human endometrial
primate corpus luteum. J. Clin. Endocrino!. Metab., 52. 133-137, 1981. hydroxysteroid dehydrogenase. J. Steroid Biochem., »4.437-442, 1981.
21. Lippman, M., Monaco. M. E., and Bolán, G. Effects of estrone, estradici and 38. Watson, C. S., Medina, D., and Clark, J. H. Characterization of progesterone
estriol on hormone responsive human breast cancer in long-term tissue receptors, estrogen receptors and estrogen (Type ID-binding sites in the
culture. Cancer Res.. 37. 1901-1907, 1977. hormone-independent variant of the MXT-3590 mouse mammary tumor.
22. Liu, H. C., and Tseng. L. Estradici metabolism in isolated human endometrial Endocrinology, »07.1432-1437, 1980.
epithelial glands and stremai cells. Endocrinology, 104: 1674-1681, 1979. 39. Zava, D. T.. Harrington, N. Y., and McGuire, W. L. Nuclear estradici recep
23. Mester, J., and Baulieu, E. E. Dynamics of oestrogen-receptor distribution tors in the adult rat uterus: a new exchange assay. Biochemistry »5:4292-
between the cytosol and nuclear fractions of immature rat uterus after 4297, 1976.
oestradiol administration. Biochem. J., »46.617-623. 1975. 40. Zava, D. T., and McGuire, W. L. Estrogen receptor: unoccupied sites in
24. Pack, B. A., and Brooks, S. C. Metabolism of estrogens and their sulfates in nuclei of a breast tumor cell line. J. Biol. Chem., 252: 3703-3708. 1977.
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.
Estrone Receptor Formation During the Processing of
Estradiol-Receptor Complex in MCF-7 Cells
E. R. Hansen and S. C. Brooks
Updated version Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/42/5/1967
E-mail alerts Sign up to receive free email-alerts related to this article or journal.
Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Subscriptions Department at pubs@aacr.org.
Permissions To request permission to re-use all or part of this article, use this link
http://cancerres.aacrjournals.org/content/42/5/1967.
Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
Rightslink site.
Downloaded from cancerres.aacrjournals.org on July 24, 2019. © 1982 American Association for Cancer Research.