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Available estradiol receptors in nuclei from human endometrium
Jour~I of Steroid Biochemistry, Vol. 13, pp. 3 to I1 Pergamon Press Ltd 1980. Printed in GreatBritain
AVAILABLE ESTRADIOL FROM HUMAN HONOREE FLEMING
RECEPTORS IN NUCLEI ENDOMETRIUM and
ERLIO GURPIDE
Departments of Obstetrics and Gynecology (Division of Reproductive Biology) and of Biochemistry,
Mount Sinai School of Medicine, New York, N.Y. 10029,U.S.A. (Received 14 March 1979) SUMMARY
Unoccupied or available estradiol (E,) nuclear receptors were found in normal, as well as in cancerous, human endometrium. The concentrations of these receptors ranged from 0.2-1.7 pmol/mg DNA in proliferative endometrium to 0.1-0.6 pmol/mg DNA in secretory tissue, showing maximal average values at the mid-follicular phase. Available nuclear receptors were measured by incubating isolated nuclei with 10 nM [%I]-Ea f 1 pM DES for 60 min at 4°C. The available nuclear receptors could also be labeled in whoie tissue or in KC1 nuclear extracts. Receptors labeled at 4°C extracted from nuclei with 0.6 KC1 M and centrifuged in a sucrose density gradient appear in a DES competibie peak with a sedimentation constant slightly lower than that of BSA. A series of experiments demonstrated that E,-free estrogen receptors in endometr~al nuclei are not artifacts produced by cytoplasmic contamination, E2 exchange at 4°C. or loss of E, during preparation of the nuclei. A procedure to measure concentrations of available and occuplied nuclear receptors. as well as available cytoplasmic receptors for Er. is described.
to receptors from which endogenous Ez might have been stripped during isolation of the nuclei. We also show significant variations in the concentrations of available nuclear receptors during the menstrual cycle with maximal levels being found in mid-proliferative endometrium.
INTRODUCTION
It is currently believed that the physiologically important forms of the estradiol (E,) receptor are the available (not bound to the hormone) cytoplasmic receptor and the occupied (hormone bound) nuclear receptor, and that a major function of Ez is to convert the first into the second. However, accumulated evidence indicates the existence of available or unoccupied nuclear E2 receptor. This type of receptor has been found in uterine tissue from cows[l], immature [23 and mature rats [3], and pigs [4]. It has also been found in a rat pituitary cell line ES], in the human breast tumor cell line MCF-7 [6,7], and in the endometrial cancer cell line HEC 1B [8]. In rat uteri, the amount of available nuclear receptor is small compared to total receptor [3], but in MCF-7, the available nuclear receptor has been found under certain conditions to constitute as much as 75% of the total receptor [7]. The first human tissue shown to contain available nuclear receptors was breast tumor. Available nuclear receptors were measurable in 30-400/, of the tumors examined in two different studies [P. lo]. In this report, we demonstrate the existence in human endometrium of these available nuclear receptors, characterized by labeling with C3H]-E2 at 4°C. We find that available nuclear receptors exist in endometrial cancer. But we also find such receptors in normal endometrial tissues. Our results indicate that available receptors constitute a large fraction of total nuclear receptor. We show that labeling does not result from exchange of c3H]-E2 for nuclear receptorbound endogenous El, from binding of E3H]-E2 to cytoplasmic ~mponents which might contaminate the nuclear preparations, or from binding of E3H]-Ez
MATERIALS AND METHODS
Endometrium, scraped from the uterus by dilatation and curettage or following hysterectomy, was transferred from the operating room at 4”C, in EBSS (Earl’s balanced salt solution, GIBCO) containing antibiotics. The day of the cycle for each tissue was determined according to Hertig and Noyes[l 11. Experiments were done within two hours of obtaining the tissue. Labeling of available estradiol receptors in isolated nuclei
Approximately SO-l@) mg of endometrium were homogenized in 1 ml of SMTD (0.32 M sucrose, 1 mM MgCl, 10 mM Tris HCI pH 8.0 and 1 mM dithiothreitol, DTT) buffer in a glass homogenizer using a motor driven pestle, keeping the tissue in ice during this procedure. The nuclei were pelleted by centrifugation at 5OOg for 1-2 min, and resuspended for a second homogenization in the same buffer to which 0.1% Triton X-100 had been added. After 2-3 more washes with SMTD buffer, the nuclei were incubated with 10nM 6,7C3H]-E2 (48 Ci/mmol, New England Nuclear) with and without 1 CM diethystilbestrol (DES) at 4°C for 60min. Following incubation, the nuclei were washed once in TED buffer (1OmM Tris HCl pH 8.0, 1 mM EDTA, 1 mM DTT) containing 0.1% Triton X-100, and 4-5 times with TED buffer 3
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HONOREEFLEMINGand ERLIO GURPIDE
only. The C3H]-Ez bound to the chromatin was extracted with 1 ml EtOH. DNA was measured in the pellets from these extractions according to the Burton method [123. Labeling of occupied E2 receptors in isolated nuclei
Available and occupied nuclear receptors were measured by incubating nuclei in SMTD buffer containing 10nM C3H]-Et + 1 ,um DES at 4°C for 1 h and then at 30°C for 2 h. The preincubation was done to stabilize available nuclear receptors by binding them to E2 before the nuclei were exposed to the elevated temperatures necessary for exchange of t3H]-Ez for endogenous nuclear bound Ez. Labeling of avail~le nuclear receptors in whole tissue
Fragments of endometrial tissue were suspended in EBSS media with 1OOnM [3H]-E2 _t 10pM DES at 4°C for 2 h. Following incubation, the fragments were washed three times with EBSS and the tissue was homogenized and centrifuged as described above. The supernatant was used for measurement of cytosol receptors and the nuclear pellet was washed and extracted. In some experiments, the nuclear pehet from the tissue incubated at 4°C was divided after homogenization in SMTD plus 0.1% Triton. One portion was washed extensively and extracted as described. The remainder was centrifuged and resuspended in SMTD for a second incubation with 10nM C3H]-EZ, with or without 1 PM DES, at 30°C for 2 h to label receptors bound to endogenous E,. Labeling of total receptors in whole tissue
Tissue fragments were incubated with 1OOnM C3H]-E2 (48 Ci/mmol) + 10 PM DES at 30 or 37°C (we detect no difference in labeling at these two temperatures). Under these conditions, cytoplasmic receptors are translocated and exchange occurs between E3H]-E2 and endogenous nuclear bound E,. This procedure was used to measure total receptor in the ceil (available nuclear + occupied nuclear + translocated cytoplasmic receptor). Labeling of cytosol receptors in whole tissue
After separation of nuclei during the procedure described above, cytosol was prepared by centrifuging the supernatant at 105,OOOg for 45 min. Dextrancoated charcoal was added to the cytosol to obtain a final concentration of 0.25% and the mixture was incubated at 4°C for 30min. The charcoal was pelleted. and E3H]-E2 was measured in the sup~a~nt. Receptor concentrations are expressed as pmol specifically bound E2/mg DNA. Sedimentation analysis of extracted auailable. nuclear receptor
Available nuclear receptors, labeled by incubating tissue or isolated nuclei with r3H]-E2 at 4°C as previously described, were extracted by suspending the
nuclei in TED buffer containing 0.6 M KC1 for 1 h at 4°C. This extract was run on a 5-20x sucrose density gradient, prepared in TED buffer containing 0.5 M KCl. for 16 h, at 166,OOOg[13]. Two-tenth ml fractions were collected from a puncture in the bottom of the tube, mixed with Scintiverse (Fisher Scientific, Westford, Mass.). and counted in a liquid scintillation spectrometer (Isocap 300. Nuclear Chicago, Des Plaines, Ill.) to determine the distribution of radioactivity. A parallel gradient contained bovine serum albumin labeled by acetylation with 14C acetic anhydride.
RESULTS
Concentration of available E2 receptors (not occupied by E,) in nuclei of human endometrium throughout the menstrual cycle
Figure 1 shows the concentrations (pmol Ez bound at 4”C/mg DNA) of available estrogen receptor in endometrial nuclei during the cycle. As described in the preceding section, these values were obtained from experiments in which either isolated nuclei or tissue fragments were labeled with C3H]-E, at 4°C and measurements of radioactivity and DNA were carried out in nuclear pellets thoroughly washed with Triton X-100 and buffer. Con~ntrations of nuclear estrogen receptors available for binding at low temperature were estimated after subtraction of results from parallel incubations carried out under identical conditions with mixtures of C3H]-E2 and DES in a molar ratio 1: 100. Available nuclear receptors were detected in almost all specimens of normal endometrium tested. Their concentrations were highest during the mid-follicular phase of the cycle, reaching values of about 1.7 pmol/mg DNA, a si~i~cant proportion of the expected total concentration of estrogen receptors in the tissue. The concentrations appeared to decrease during the follicular phase. Lower values persisted during the luteal phase, when the concentrations of total endometrial E, receptors were also low [14]. The presence of available (possibly unoccupied) receptors in nuclei of human endometrium, both normal and cancerous, represents a novel finding. Therefore, detailed studies were conducted to define optimal conditions for measuring this type of binding site and to ascertain that they are not artifactual. Some of the results of these studies are shown below. EJkt of concen~ation of [3H]-E2 and time of incubation on labeling of nuclear receptors at 4°C
Figure 2 shows representative binding curves for nuclei isolated from mid-proliferative (low and high receptor level), late proliferative and secretory endometrial tissue incubated with increasing amounts of [3H]-E2 in buffer at 4°C for periods of up to 2 h. The data indicate saturation of available nuclear sites at concentrations of E, between 8 and 12 nM.
Available estradiol receptors in nuclei
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Fig. 1. Concentration of available nuclear receptors in normal endometrium during the menstrual cycle and in aden~~~noma= Available nuckar receptors were measured in isolated nuclei incubated with 10 nM C3H]-Ea f 1 FM DES at 4°C for 60 min or in nuclei isokted from tissue fragments ~cubat~ with lOOnM [aH]-F& f 1OpM DES at 4°C for 9N20min. Each point represents the amount of avaitaMe nnciear receptors in each endometrial specimen dated according to the system of Hertig and
Noyes[lI]. Figure 3 shows results from a study in which the time of incubation of nuclei with f3H]-Ez (10 nM) at 4°C was varied from 15 to 180 min. No significant difference in the values obtained at 60, 120 or 180 min were noted. About 80% of the saturation level was reached at about 30 min of incubation. The labeled receptor is relatively stable at 4°C; only about 20% decline in binding was noted to occur after overnight incubations.
Figure 4 shows a binding profife of a 0,6M KC1 extract from nuclei labeled with [3N]-E, at 4°C and run on a high salt (0.5 M KCI) sucrose gradient. The
bound C3H}-Ez moves more slowly than “C-labeted BSA, indicating an S value below 4.6. The available receptors can be extracted prior to labeling and then labeled at 4”C, or the receptor can be first labeled and then extracted from the nuclei with 0.6 KCl. These two procedures yield equivalent results but the recovery of avatiabie receptors is not complete (8@900/,). For that reason, we decided to perform the measurements described in this paper in whole nuctei rather than in the salt extract.
Several tests were conducted to determine whether the rapid and specific binding of C3H]-E2 at 4°C in
Fig. 2 maturation of available nuclear receptors with increasing concentrations of r3H]-E2 + DES at 4°C for 6tImin. Specific binding is plotted for four different endometriai specimens. The amounts of availabIe receptor in mu-proliferative endometrium are higher than those found in late proliferative and secretory tissues.
HONOR& FLEMING and ERLIO GUKPIDI:
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Fig. 3. Binding of [3H]-E2 to available nuclear receptors with increasing time of incubation. Nuclei from a proliferative endometrium were incubated with 10 nM [3H]-Ez f 1 PM DES at 4°C for increasing periods of time. Maximum specific binding was attained after 1 h of incubation.
lated from the same tissue. The results are shown in Table 1. In some of the preparations, the concentrations in nuclei are higher than the concentrations in cytosol, an unlikely distribution if the available nuclear receptors were actually cytoplasmic. Furthermore, the nucle~/cytoplasmic ratios show a dispersion of values larger than expected from contamination. b. Estradiol exchange at 4°C. Although results obtained from labeling experiments with 3H-E2 in rat uteri have shown no exchange at 4°C [15], the possibility that such an exchange may occur in human endometrium was investigated. Isolated nuclei were labeled for 1.5min with C3HJ-E2 (10 nM), washed, and resuspended for another 15 min either in buffer alone or in buffer containing unlabeled E2 (1 ,uM) or unlabeled DES (1 FM). If the original labeling occurred by exchange
isolated nuclei could be explained by trivial methodological artifacts, such as contamination of the nuclear preparation with cytoplasmic estrogen receptors, exchange at 4°C of receptor-bound endogenous E2 for 3H-Ez, or generation of “unoccupied” binding sites by removal of E2 from receptors during the preparation of the nuclear pellet. a. Cytopfu~mic co~tami~ution, The endometrial cytosol contains ligand-free estrogen receptors which might contaminate the nuclear pellet. However, thorough washing of the nuclei with 0.1% Triton and buffer before labeling is expected to remove the outer nuclear membrane with adsorbed cytoplasmic material. The nuclei were again washed with Triton and buffer after labeling. In some experiments, the concentration of available receptors in cytosol was measured and compared with the con~n~ation of available receptors in nuclei iso-
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Fig. 4. Sedimentation profile of a 0.6 KC1 extract of nuclei labeled with 10 nM E2 at 4°C for 60 min. The nuclei were washed once with 0.1% Triton in TED buffer and with TED buffer 3-4 times more. An 0.6KCI in TED buffer extract of the nuclei was run on a 10-307; discontinuous sucrose gradient prepared in TED buffer containing 0.5 M KCI. The [‘4C]-BSA was run in a parallel gradient.
Available estradiol receptors in nuclei Table 1. A comparison of concentrations of available nuclear and available cytoplasmic receptors in endometrial specimens in different phases of the menstrual cycle Concentration c3H]-E2 bound (4°C) (pmol/mg DNA) Endometrium Early proliferative Early proliferative Mid-proliferative Mid-proliferative Secretory (d 16) Secretory (d 19)
Nuclei
Cytosol
0.38 0.76 0.63 1.7 0.39 0.21
0.21 0.22 1.7 12 0.28 0.14
of available Receptor Ratio of available receptor Nuclei/cytosol 1.8 3.7 0.36 0.14 1.4 1.5
Fragments of endometrium were incubated with 1OOnM c3H]-E2 f 10pM DES for 9(r120min. Cytosol and nuclei were separated and analyzed for specific 3H-E, binding as described in Materials and Methods.
of C3H]-Es for endogenous
Es, loss of bound C3H]-E2 would also be expected to occur in the presence of excess unlabeled competitor during the second incubation period. The results of this experiment are shown in Table 2. The specific labeling of the nuclei (total minus competed with lOO-fold excess DES) was 0.36 pmol/mg DNA. Incubation of labeled nuclei with excess Es or DES (lOO-fold the concentration of Es
used to label the nuclei) at 4°C resulted in less than 8% reduction in the concentration of specifically bound C3H]-Ea. Therefore, at least 90% of the specific nuclear labeling that occurred during the fir& 15 min of incubation could not be accounted for by exchange and corresponded to binding of c3H]-E2 to sites not occupied by Ea. c. Generation of available binding sites during preparation of nuclei. It is possible that endogenous E2 could be lost from some fraction of the nuclear receptors during the isolation of nuclei, leaving binding sites available. To test this possibility, tissue fragments were pre-incubated with E2 to fill the available sites before isolating and attempting to label the nuclei at 4°C. Secretory endometrium was incubated with Ea (10 nM) for 90min at 30°C. To serve as control, a portion of the tissue was maintained in buffer at 4°C. Nuclei were then isolated, washed and labeled with C3H]-E2 (1OnM) at 4°C for 60min. As shown in
Table 3, the nuclei from the control tissue were found to contain 0.14 pmol/mg DNA of available nuclear receptor, whereas no specific C3H]-E2 uptake was detected in the nuclei from the tissue previously exposed to unlabeled E2. This result indicates that available receptor sites originally present in the nuclei have been filled with unlabeled E2 during the preincubation period and that no binding sites were made available during preparation of nuclei. It might be questioned, however, whether some of the unlabeled Ea used in the preincubation might not remain in the nuclei and interfere with the binding of C3H]-E2 at 4°C. This concern is dispelled by the results from labeling at 30°C which indicates that the amount of total nuclear receptors measured under exchange conditions (available and exchangeable) is not diminished by the preincubation with Ea. A similar experiment was carried out with a specimen of mid-proliferative endometrium, expected to contain higher levels of estrogen receptor than the secretory tissue. As before, ahquots of the tissue were preincubated in buffer at 4°C (control) or with E2 (10 nM) at 30°C for 90 min. Nuclei were isolated and labeled with C3H]-E, (10nM) at different temperatures (4 and 30°C) and for two different periods of time (15 and 180 min). In every case, labeling was also conducted with mixtures of C3H]-E2 and unlabeled DES (1: 100) to correct for non-specific binding. Table 4
Table 2. Nuclear exchange of E2 does not occur at 4°C under conditions adequate for labeling available nuclear receptors First incubation 15 min/4”C 10 nM [aH]-E2
Second incubation 15 min/4”C Buffer InME 1 HIMDES
C3H]-Ez bound (pmol/mg DNA)
Type of binding
0.41 0.34 0.34
Total Not exchangeable Not exchangeable
Available receptors labeled in isolated nuclei incubated at 4°C for 15 min with C3H]-E, alone or in the presence of unlabeled E2 or DES. Only negligible amounts of the nuclear bound C3H]-E2 could be exchanged during a subsequent, identical incubation of nuclei with excess unlabeled estrogens.
HONOR& FLEMINGand ERLIOGURPIDE
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Table 3. Filling of available nuclear receptors in secretory endometrium by preincubation with EZ at 30°C Labeling cf isolated nuclei (pmol/mg DNA) Tissue preincubation (90 min)
Available receptors (4°C) 1OnM C3H]-Ez f 1 ,uM DES (60 min)
Total receptors (30°C) 10nM c3H]-E2 + 1 PM DES (60 min)
0.14 -0
No steroid (4°C) 10 nM E2 (30°C)
0.29 0.32
Nuclei, isolated from fragments of control or preincubated secretory endometrium, were incubated with r3H]-Ez for 120 min at 4°C or at 30°C. Nuclei from the tissue preincubated with Ez were not labeled at 4°C. This loss of binding must be due to occupation of available nuclear receptors by E2 during preincubation since the concentration of total nuclear receptor (available + occupied), when measured under exchange conditions, is not decreased in the treated tissue.
presents the results from these ex~riments. The control tissue showed a ievei of available nuclear receptors of 1.4pmol/mg DNA. The same value was obtained whether the labeling was done at 0 or 30°C indicating that in this tissue practically all of the nuclear receptor was available. The tissue preincubated with E2 at 30°C showed the expected reduction in the level of available (unfilled) receptors and an elevation in the concentration of total binding sites, measured by exchange at 30°C. The increase in tota binding sites in the nuclei is likely due to translocation of cytoplasmic receptors during the preincubation period. In the preincubated tissue, approx. 10% of the total nuclear sites are still available. These receptors may be a fraction of the original population of available receptors not filled during the preincubation period, or they may have been created during nuclear isolation. Another experiment was done to determine whether available nuclear receptors could also be filled by preincubating tissue fragments at 4°C. It was found that it was necessary to increase the concentration of E2 from the 10 nM used at 30°C to 100 nM. As table 5 shows, 85% of the available nuclear receptors in this secretory tissue could be filled during preincubation for 90min with IOOnM E, at either 4 or 30°C.
L~eljng receptors in whole tissue
A concentration of 0.76 pmol/mg DNA of available receptor was found in nuclei when fragments of a specimen of secretory endometrium were incubated at 4°C with either C3H]-Ez (100 nM) or [3H]-E2 (100nM) + DES (10 FM). When nuclei from the same tissue were first isolated and then incubated with a saturating concentration of C3H]-EZ, 0.55 pmol/mg DNA of available receptor was found. The similarity of the results obtained with these two methods of labeling demonstrated that available nuclear receptors were present as such in the tissue, prior to isotation of the nuclei. The lower value found when labeling isolated nuclei may be due to the action on the receptors of proteolytic enzymes released during homogenization of the tissue. Available or unoccupied receptors are known to be more susceptible to destruction than occupied receptors. Table 6 presents results which illustrate two methods used to measure available and total estrogen receptors in human endometrium. Method I involves incubation of tissue fragments at 4°C with [jH]-E2 and t3H]-E2 + DES as described above. After 2 h of incubation, cytosols are prepared and treated with dextr~~oated charcoal to remove unbound C3H]-EZ, After ~ntrifugation, aliquots of the supernatants are counted. Available cytosol receptor concentrations are expressed as pmol E2 specifi-
Table 4. Filling of available nuclear receptors in proliferative endometrium by preincubation with E2 at 30°C Labeling of isolated nuclei (pmol/mg DNA) Total receptors (30°C) Available receptors (4°C) 1OnM E3H]-Ez + 1 PM DES 1OnM E3H]-Ez + 1 PM DES Tissue preincubation (90 min)
(15 mm)
(120min)
(IS min)
(I 20 min)
1.3 0.31
i.4 0.31
1.4 2.0
1.4 3.6
No steroid (4°C) 10 nM E2 (30C)
Nuclei, isolated from fragments of control or preincubated proliferative endometrium, were incubated with [jH]-E2 for 120 min at 4°C or at 30°C. Approximately 80% of the available nuclear receptors were filled during preincubation. A substantial increase in total nuclear receptor in the preincubation tissue can be explained by translocation of cytoplasmic receptor into the nucleus. Almost all of the nuclear recep_ tors were available in this tissue prior to preincubation, a situation observed in some, but not all, proliferative
tissue.
Available estradiol receptors in nuclei Table 5. Filling of available nuclear receptors by preincubation either 4°C or at 30°C
of tissue fragments at
Labeling of isolated nuclei (pmol/mg DNA)
Tissue preincubation (90 min) No steroid (4°C) 100 nM E2 (4°C) 100 nM Ez (30°C)
Total receptors (30°C) Available receptors (4°C) 10 nM C3H]-Ez f 1 PM DES 10 nM c3H]-EI f 1 PM DES (60 min) (60 min) 0.34 0.29 0.49
0.28 0.04 0.03
Secretory tissue was preincubated with E2 at 4°C or at 30°C prior to isolation of the nuclei and their subsequent incubation with C3H]-E2at 4°C or 30°C. The substantial
decrease in binding to nuclei from both preincubated tissues demonstrates that available nuclear receptors can be filled in tissue fragments at either 3O”C,as previously shown, or at 4°C. The concentration of total nuclear receptors, similar in control tissue or tissue preincubated at 4”C, increases in tissue preincubated at 3o”C, dut to translocational of cytoplasmic receptors.
tally bound/mg DNA. Available nuclear receptor concentrations are estimated from measurements of bound C3H]-Ez in isolated nuclei (Materials and Methods). Occupied nuclear receptors are measured by incubating portions of the isolated nuclei with additional c3H]-E2 (+ DES) for 2 h at 3O”C, conditions appropriate for exchange of endogenous receptorbound E2 for C3H]-Ez. The concentration of occupied
Table 6. A comparison
nuclear receptors is calculated as the difference between the concentration of total nuclear receptors labeled under these conditions and available nuclear receptors labeled at 4°C. This labeling scheme provides estimates of the concentration of total receptors in the cell (total nuclear + available cytosol). Method II involves parallel incubations of tissue fragments with C3H]-E2 (1OOnM) or C3H]-E,
of two methods for measuring Ez receptor distribution human endometrial tissue Exp No. 1 (pmol/mg DNA)
Exp No. 2 (pmol/mg DNA)
0.42 0.48 0.60 1.0
1.6 0.32 0.49 2.0
0.42 0.42
1.6 1.0
0.48 0.57 1.0
0.32 0.46 2.0
in
Method I (preferred)
Available cytosolic Available nuclear Total nuclear *Total in cell Method II
Cytosolic Available (original) TTranslocated at 37°C Nuclear Available ITotal Total in cell
* Total in cell = Available Cytosolic + Total Nuclear. t Translocated at 37°C = Available Cytosolic (4°C) - Available Cytosol(37”C). 1 Total Nuclear = Total in cell - Available Cytosol. Method I: Endometrial fragments are incubated with 1OOnM C3H]-E, f 10 PM DES for 120 min at 4”C, the tissue is homogenized, and specific binding in the cytosol and a portion of the nuclei is measured. Some of the isolated nuclei, with available receptor already labeled, are then incubated with 10nM “H-E2 k 1 PM DES for 120 min at 30°C to label occupied nuclear receptors by exchange. The concentrations of available cytosolic, available nuclear, and total nuclear (available + occupied) can be measured in the single specimen of tissue and the concentration of total receptors in the cell can be computed from these values. Method II: Endometrial fragments are incubated with 100 nM [aH]-Ez k 10 PM DES for 120 min at 4°C and for 60 min at 37°C the tissue is homogenized, and specific binding in the cytosol and nuclei is measured. The translocated cytosol receptor is computed as the difference between the concentration of available cytosol receptor after incubation at 4°C and receptor still present in cytosol after incubation at 37°C. The concentration of total nuclear receptor is determined as the difference between total receptor measured in the cell at 37°C and the concentration of available cytoplasmic receptor measured at 4°C.
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HONORBEFLEMINGand
(1OOnM) + DES (IO FM) at 4°C and at 37°C. Following incubation, cytosol and nuclear binding are measured. Specific nuclear binding at 37°C includes total nuclear receptor (available + occupied) in the originai tissue and translocated cytoplasmic receptors. The latter parameter can be estimated by subtracting the concentration of cytosol receptors remaining after incubation at 37°C from the concentration of total cytosol receptors measured at 4°C. If it is assumed that all of the receptor lost from the cytoplasm appears in the nucleus, then the total nuclear receptor concentration prior to incubation, and the level of total estrogen receptor in the cell, can be calculated. Receptor concentrations determined by the two methods on two different specimens are shown in Table 6. The concentrations of total nuclear receptors or of total receptor in the cell, measured by either method were in close agreement. Method I may be preferable since it can be applied to less tissue and does not require the assumption that all of the receptor lost from the cytoplasm at 37°C is found in the nucleus.
ERLIOGURPIDE
mature cow endometrium by Jackson and Chatkey[l]. These Ez binding sites were characterized as having high affinity and low capacity and were observed to vary in quantity during the cycle: peaking at diestrus, almost disappearing at proestrus. and peaking again at ovulation. As opposed to the receptors from human uteri and breast tissue, available nuclear receptors in cow uterus could not be extracted with KU. Available nuclear receptors (0.08 pmol/uterus) have been documented for the immature rat by Carlson and Gorski[Z]. We also looked at immature rats using techniques described in this paper and found similar amounts of available nuclear receptors. Zava and McGuire found between 0.2 and 0.4 pmoljmg DNA of ‘available receptors in uterine nuclei of immature rats [3]. More recently Jungblut et ai.[4] reported that available nuclear receptors could be found in uteri from ovariectomized, adrenalectomized pigs. The discovery of available nuclear receptors in a significant number of breast tumors and in a breast cell line led to speculation that their existence may account for the category of hormone-independent DISCUSS1ON breast tumor which contains receptor, yet is able to The results reported in this paper demonstrate the grow under conditions of steroid deprivation (e.g. endocrine gland ablation). If receptor in certain tumors existence in human endometrium of nuclear estrogen is already present in the nucleus and does not require receptors which are not bound to E,. The evidence presented indicates that these available nuclear recap- steroid to translocate, the tumor might not require tors are not artifactual. It was shown that exchange steroid to proliferate. Our results demonstrate that does not account for the rapid labeling in nuclei at available nuclear receptors are not unique to malig4°C. Since available nuclear sites could be labeled in nant tissues. They are, in fact, a prominent feature of normal human endometrium. Available receptor conwhole tissue fragments, it is clear that the receptors stitutes almost all of the nuclear receptor in the proliare not “made available” by stripping of endogenous Ez during nuclear isolation. Our procedures for isola- ferative tissues shown in Tables 3 and 4. In secretory tion of nuclei and the results obtained make it ex- tissues, available receptor is usually between 40 and tremely unlikely that the available receptors result 507; of total nuclear receptor. Available or unoccufrom cytoplasmic contamination, One possibility not pied nuclear receptors are quantitatively as important yet explored is that some nuclear receptors may be as the occupied receptors. As can be seen in Fig. 1, there is a wide variation in occupied by a steroid that E2, with a higher affinity for the receptor, is able to displace. Since this is a the amounts of available nuclear receptor within each possibility, we use the term “available” rather than of the three proliferative periods. This is not surprising since each tissue was obtained from a different “unoccupied” to describe the nuclear receptors subject. Nevertheless, there is a definite peak in conmeasured in our experiments. centration of available receptor in the mid-follicular Brooks et aI.[6] and Zava and McGuire[7] observed that a large fraction of the E2 receptor in the phase. Available receptor concentrations are low in early proliferative and appear to be declining in late breast tumor cell line MCF-7 was in the nucleus and proliferative tissues, perhaps due to the high levels of could be labeled at 4°C. In human breast tumors, Garola and McGuire found available nuclear recep- endogenous E2 present at the time. During the luteal tors in 40% of th.e 28 breast tumors examined 193. phase, the concentrations remain low. The period in which avaifabfe receptor is most Panko and MacLeod examined 139 breast tumors and found that nuclear extracts from 300/, of these abundant coincides with the period of rapid cellular tissues could be labeled at 4”C, indicating the pres- proliferation. Chalkey and Jackson showed in cows ence of available receptors [lo]. Sonnenschein et that the peak in unoccupied receptors coincided with the peak in endometrial growth. They also showed aI.[5] measured available receptor in nuclear extracts from two cell lines cloned from rat pituitary ‘tumors. that available receptors are present in endometrium. a tissue characterized by active cellular proliferation. Even after 40 generations of growth in E,-depleted medium, 30% of the total receptor was found to be in and not in myometrium Cl]. Even in cell culture, more available receptor is found in nuclei when the the nucleus, unbound to Ez. Available nuclear receptors were also measured in cells are actively growing [7].
Available estradiol receptors in nuclei What is the significance of the fact that peak amounts of available nuclear receptor appear to correlate with active cell proliferation? An explanation consistent with current dogma of steroid hormone action is that the relatively larger amounts of estrogen-receptor complex accumulating in the nucleus during a period of proliferation, such as the follicular phase, dissociate rapidly, leaving intact available receptor in the nucleus. The elevated levels of available receptor thus generated would gradually decline as the rate of entry of ER complex into the nucleus decreased and as available receptor was destroyed or returned to the cytoplasm. When the period of rapid growth has passed, it might be expected that a steady state would be established between entry of ER into the nucleus, dissociation of E from R and disposition of R. Contradicting this interpretation of the data is the fact that available receptors are found in nuclei of systems from which E, has been excluded. Several cell lines have been shown to contain available nuclear Ez receptors even after they have been grown in E,-depleted medium [S]. Available receptors are also found in the uterine nuclei of immature rats [2], as well as in pigs which have been ovariectomized and adrenalectomized [4]. These results suggest that the receptor may translocate into the nucleus and bind chromatin without itself being bound to E,, a situation not expected on the basis of the current accepted mechanisms of steroid receptor function.
Acknowledgrments-We are grateful to Drs. S. Kaplan, H. May, A. Crosdale and other physicians of the Department of Obstetrics and Gynecology for their collaboration in making specimens of endometrium available to us. also to Dr. L. Deglidisch. of the Department of Pathology for the histologic dating of the tissue. This work was supported by Grants HD-07197 and CA 15648. awarded by the National Institutes of Health (DHEW).
11 REFERENCES
1. Jackson V. and Chalkey R.: The binding of estradial-178 to bovine endometrial nuclear membrane. J. BioI. Chem. 249 (1974) 1615-1626. 2. Carlson R. A. and Gorski J.: Estrogen-soecific binding sites in the nuclear myofibrillar fr&tion of immature rat uteri. Fed. Proc. 36 (1977) 386, Abstract NO. 617. 3. Zava D. T.. Harrington N. Y. and McGuire E. L.: Nuclear E2 receptors in the adult rat uterus: A new exchange assay. Biochemistry 15 (1976) 4292-4297. 4. Jungblut P. W., Kallweit E.. Sierralta W.. Truitt A. J. and Wagner R. K.: Estradiol and receptor cantent of uterine nuclei from ovariectomized and adrenalectomized pigs. Acta Endocr., Copenh. 87 (1978) Suppl. 215, 136. 5. Sonnenschein C.. Soto A. M., Colofiore J. and Farookhi R.: Estrogen target cells. Establishment of a cell line derived from the rat pituitary tumor MtT/F,. Exp. cell. Res. 101 (1976) 15-22. 6. Brooks S. C.. Locke E. R. and Soule H. D.: Estrogen receptors in a human cell line (MCF-7) from breast carcinoma. J. biol. Chem. 248 (1973) 6251-6253. 7. Zava D. T. and McGuire W. L.: Estrogen receptor. Unoccupied sites in nuclei of a breast tumor cell-line. J. biol. C’hem. 252 (1977) 3703-3708. 8. Satyaswaroop P. &.. Fleming H., Bressler R. S. and Gurpide E.: Human endometrial cell cultures for hormonal studies. Cancer Res. 38 (1978) 4367-4375. 9. Garola R. E. and McGuire W. L.: An improved assay for nuclear estrogen receptor in experimental and human breast cancer. Cancer Res. 37 (1977) 3333-3337. 10. Panko W. B. and MacLeod R. M. : Unchanged nuclear receptors for estrogen in breast cancers. Cancer Res. 38 (1978) 1948-1951. 11. Noyes R. W.. Hertig A. T. and Rock J.: Dating the endometrial biopsy. .ert. Steril. 1 (1950) 3-25. 12. Burton K. A.: A study of the conditions and mechanisms of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 65 (1956) 315-323. 13. Greene G. L., Closs L. E., Fleming H., Desombre E. R. and Jensen E. V.: Antibodies to estrogen receptor: Immunochemical similarly of estrophilin from various mammalian species. PNAS 74 (1977) 3681-3685. 14. Tseng L. and Gurpide E.: Nuclear concentration of estradiol in superfused slices of human endometrium. Am. J. Obstet. Gynec. 114 (1972) 995%1004. 15. Anderson J., Clark J. H. and Peck E. J. Jr: Oestrogen and nuclear binding sites. Biochem. J. 126 (1972) 561-567.
AVAILABLE ESTRADIOL FROM HUMAN HONOREE FLEMING
RECEPTORS IN NUCLEI ENDOMETRIUM and
ERLIO GURPIDE
Departments of Obstetrics and Gynecology (Division of Reproductive Biology) and of Biochemistry,
Mount Sinai School of Medicine, New York, N.Y. 10029,U.S.A. (Received 14 March 1979) SUMMARY
Unoccupied or available estradiol (E,) nuclear receptors were found in normal, as well as in cancerous, human endometrium. The concentrations of these receptors ranged from 0.2-1.7 pmol/mg DNA in proliferative endometrium to 0.1-0.6 pmol/mg DNA in secretory tissue, showing maximal average values at the mid-follicular phase. Available nuclear receptors were measured by incubating isolated nuclei with 10 nM [%I]-Ea f 1 pM DES for 60 min at 4°C. The available nuclear receptors could also be labeled in whoie tissue or in KC1 nuclear extracts. Receptors labeled at 4°C extracted from nuclei with 0.6 KC1 M and centrifuged in a sucrose density gradient appear in a DES competibie peak with a sedimentation constant slightly lower than that of BSA. A series of experiments demonstrated that E,-free estrogen receptors in endometr~al nuclei are not artifacts produced by cytoplasmic contamination, E2 exchange at 4°C. or loss of E, during preparation of the nuclei. A procedure to measure concentrations of available and occuplied nuclear receptors. as well as available cytoplasmic receptors for Er. is described.
to receptors from which endogenous Ez might have been stripped during isolation of the nuclei. We also show significant variations in the concentrations of available nuclear receptors during the menstrual cycle with maximal levels being found in mid-proliferative endometrium.
INTRODUCTION
It is currently believed that the physiologically important forms of the estradiol (E,) receptor are the available (not bound to the hormone) cytoplasmic receptor and the occupied (hormone bound) nuclear receptor, and that a major function of Ez is to convert the first into the second. However, accumulated evidence indicates the existence of available or unoccupied nuclear E2 receptor. This type of receptor has been found in uterine tissue from cows[l], immature [23 and mature rats [3], and pigs [4]. It has also been found in a rat pituitary cell line ES], in the human breast tumor cell line MCF-7 [6,7], and in the endometrial cancer cell line HEC 1B [8]. In rat uteri, the amount of available nuclear receptor is small compared to total receptor [3], but in MCF-7, the available nuclear receptor has been found under certain conditions to constitute as much as 75% of the total receptor [7]. The first human tissue shown to contain available nuclear receptors was breast tumor. Available nuclear receptors were measurable in 30-400/, of the tumors examined in two different studies [P. lo]. In this report, we demonstrate the existence in human endometrium of these available nuclear receptors, characterized by labeling with C3H]-E2 at 4°C. We find that available nuclear receptors exist in endometrial cancer. But we also find such receptors in normal endometrial tissues. Our results indicate that available receptors constitute a large fraction of total nuclear receptor. We show that labeling does not result from exchange of c3H]-E2 for nuclear receptorbound endogenous El, from binding of E3H]-E2 to cytoplasmic ~mponents which might contaminate the nuclear preparations, or from binding of E3H]-Ez
MATERIALS AND METHODS
Endometrium, scraped from the uterus by dilatation and curettage or following hysterectomy, was transferred from the operating room at 4”C, in EBSS (Earl’s balanced salt solution, GIBCO) containing antibiotics. The day of the cycle for each tissue was determined according to Hertig and Noyes[l 11. Experiments were done within two hours of obtaining the tissue. Labeling of available estradiol receptors in isolated nuclei
Approximately SO-l@) mg of endometrium were homogenized in 1 ml of SMTD (0.32 M sucrose, 1 mM MgCl, 10 mM Tris HCI pH 8.0 and 1 mM dithiothreitol, DTT) buffer in a glass homogenizer using a motor driven pestle, keeping the tissue in ice during this procedure. The nuclei were pelleted by centrifugation at 5OOg for 1-2 min, and resuspended for a second homogenization in the same buffer to which 0.1% Triton X-100 had been added. After 2-3 more washes with SMTD buffer, the nuclei were incubated with 10nM 6,7C3H]-E2 (48 Ci/mmol, New England Nuclear) with and without 1 CM diethystilbestrol (DES) at 4°C for 60min. Following incubation, the nuclei were washed once in TED buffer (1OmM Tris HCl pH 8.0, 1 mM EDTA, 1 mM DTT) containing 0.1% Triton X-100, and 4-5 times with TED buffer 3
4
HONOREEFLEMINGand ERLIO GURPIDE
only. The C3H]-Ez bound to the chromatin was extracted with 1 ml EtOH. DNA was measured in the pellets from these extractions according to the Burton method [123. Labeling of occupied E2 receptors in isolated nuclei
Available and occupied nuclear receptors were measured by incubating nuclei in SMTD buffer containing 10nM C3H]-Et + 1 ,um DES at 4°C for 1 h and then at 30°C for 2 h. The preincubation was done to stabilize available nuclear receptors by binding them to E2 before the nuclei were exposed to the elevated temperatures necessary for exchange of t3H]-Ez for endogenous nuclear bound Ez. Labeling of avail~le nuclear receptors in whole tissue
Fragments of endometrial tissue were suspended in EBSS media with 1OOnM [3H]-E2 _t 10pM DES at 4°C for 2 h. Following incubation, the fragments were washed three times with EBSS and the tissue was homogenized and centrifuged as described above. The supernatant was used for measurement of cytosol receptors and the nuclear pellet was washed and extracted. In some experiments, the nuclear pehet from the tissue incubated at 4°C was divided after homogenization in SMTD plus 0.1% Triton. One portion was washed extensively and extracted as described. The remainder was centrifuged and resuspended in SMTD for a second incubation with 10nM C3H]-EZ, with or without 1 PM DES, at 30°C for 2 h to label receptors bound to endogenous E,. Labeling of total receptors in whole tissue
Tissue fragments were incubated with 1OOnM C3H]-E2 (48 Ci/mmol) + 10 PM DES at 30 or 37°C (we detect no difference in labeling at these two temperatures). Under these conditions, cytoplasmic receptors are translocated and exchange occurs between E3H]-E2 and endogenous nuclear bound E,. This procedure was used to measure total receptor in the ceil (available nuclear + occupied nuclear + translocated cytoplasmic receptor). Labeling of cytosol receptors in whole tissue
After separation of nuclei during the procedure described above, cytosol was prepared by centrifuging the supernatant at 105,OOOg for 45 min. Dextrancoated charcoal was added to the cytosol to obtain a final concentration of 0.25% and the mixture was incubated at 4°C for 30min. The charcoal was pelleted. and E3H]-E2 was measured in the sup~a~nt. Receptor concentrations are expressed as pmol specifically bound E2/mg DNA. Sedimentation analysis of extracted auailable. nuclear receptor
Available nuclear receptors, labeled by incubating tissue or isolated nuclei with r3H]-E2 at 4°C as previously described, were extracted by suspending the
nuclei in TED buffer containing 0.6 M KC1 for 1 h at 4°C. This extract was run on a 5-20x sucrose density gradient, prepared in TED buffer containing 0.5 M KCl. for 16 h, at 166,OOOg[13]. Two-tenth ml fractions were collected from a puncture in the bottom of the tube, mixed with Scintiverse (Fisher Scientific, Westford, Mass.). and counted in a liquid scintillation spectrometer (Isocap 300. Nuclear Chicago, Des Plaines, Ill.) to determine the distribution of radioactivity. A parallel gradient contained bovine serum albumin labeled by acetylation with 14C acetic anhydride.
RESULTS
Concentration of available E2 receptors (not occupied by E,) in nuclei of human endometrium throughout the menstrual cycle
Figure 1 shows the concentrations (pmol Ez bound at 4”C/mg DNA) of available estrogen receptor in endometrial nuclei during the cycle. As described in the preceding section, these values were obtained from experiments in which either isolated nuclei or tissue fragments were labeled with C3H]-E, at 4°C and measurements of radioactivity and DNA were carried out in nuclear pellets thoroughly washed with Triton X-100 and buffer. Con~ntrations of nuclear estrogen receptors available for binding at low temperature were estimated after subtraction of results from parallel incubations carried out under identical conditions with mixtures of C3H]-E2 and DES in a molar ratio 1: 100. Available nuclear receptors were detected in almost all specimens of normal endometrium tested. Their concentrations were highest during the mid-follicular phase of the cycle, reaching values of about 1.7 pmol/mg DNA, a si~i~cant proportion of the expected total concentration of estrogen receptors in the tissue. The concentrations appeared to decrease during the follicular phase. Lower values persisted during the luteal phase, when the concentrations of total endometrial E, receptors were also low [14]. The presence of available (possibly unoccupied) receptors in nuclei of human endometrium, both normal and cancerous, represents a novel finding. Therefore, detailed studies were conducted to define optimal conditions for measuring this type of binding site and to ascertain that they are not artifactual. Some of the results of these studies are shown below. EJkt of concen~ation of [3H]-E2 and time of incubation on labeling of nuclear receptors at 4°C
Figure 2 shows representative binding curves for nuclei isolated from mid-proliferative (low and high receptor level), late proliferative and secretory endometrial tissue incubated with increasing amounts of [3H]-E2 in buffer at 4°C for periods of up to 2 h. The data indicate saturation of available nuclear sites at concentrations of E, between 8 and 12 nM.
Available estradiol receptors in nuclei
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Fig. 1. Concentration of available nuclear receptors in normal endometrium during the menstrual cycle and in aden~~~noma= Available nuckar receptors were measured in isolated nuclei incubated with 10 nM C3H]-Ea f 1 FM DES at 4°C for 60 min or in nuclei isokted from tissue fragments ~cubat~ with lOOnM [aH]-F& f 1OpM DES at 4°C for 9N20min. Each point represents the amount of avaitaMe nnciear receptors in each endometrial specimen dated according to the system of Hertig and
Noyes[lI]. Figure 3 shows results from a study in which the time of incubation of nuclei with f3H]-Ez (10 nM) at 4°C was varied from 15 to 180 min. No significant difference in the values obtained at 60, 120 or 180 min were noted. About 80% of the saturation level was reached at about 30 min of incubation. The labeled receptor is relatively stable at 4°C; only about 20% decline in binding was noted to occur after overnight incubations.
Figure 4 shows a binding profife of a 0,6M KC1 extract from nuclei labeled with [3N]-E, at 4°C and run on a high salt (0.5 M KCI) sucrose gradient. The
bound C3H}-Ez moves more slowly than “C-labeted BSA, indicating an S value below 4.6. The available receptors can be extracted prior to labeling and then labeled at 4”C, or the receptor can be first labeled and then extracted from the nuclei with 0.6 KCl. These two procedures yield equivalent results but the recovery of avatiabie receptors is not complete (8@900/,). For that reason, we decided to perform the measurements described in this paper in whole nuctei rather than in the salt extract.
Several tests were conducted to determine whether the rapid and specific binding of C3H]-E2 at 4°C in
Fig. 2 maturation of available nuclear receptors with increasing concentrations of r3H]-E2 + DES at 4°C for 6tImin. Specific binding is plotted for four different endometriai specimens. The amounts of availabIe receptor in mu-proliferative endometrium are higher than those found in late proliferative and secretory tissues.
HONOR& FLEMING and ERLIO GUKPIDI:
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Fig. 3. Binding of [3H]-E2 to available nuclear receptors with increasing time of incubation. Nuclei from a proliferative endometrium were incubated with 10 nM [3H]-Ez f 1 PM DES at 4°C for increasing periods of time. Maximum specific binding was attained after 1 h of incubation.
lated from the same tissue. The results are shown in Table 1. In some of the preparations, the concentrations in nuclei are higher than the concentrations in cytosol, an unlikely distribution if the available nuclear receptors were actually cytoplasmic. Furthermore, the nucle~/cytoplasmic ratios show a dispersion of values larger than expected from contamination. b. Estradiol exchange at 4°C. Although results obtained from labeling experiments with 3H-E2 in rat uteri have shown no exchange at 4°C [15], the possibility that such an exchange may occur in human endometrium was investigated. Isolated nuclei were labeled for 1.5min with C3HJ-E2 (10 nM), washed, and resuspended for another 15 min either in buffer alone or in buffer containing unlabeled E2 (1 ,uM) or unlabeled DES (1 FM). If the original labeling occurred by exchange
isolated nuclei could be explained by trivial methodological artifacts, such as contamination of the nuclear preparation with cytoplasmic estrogen receptors, exchange at 4°C of receptor-bound endogenous E2 for 3H-Ez, or generation of “unoccupied” binding sites by removal of E2 from receptors during the preparation of the nuclear pellet. a. Cytopfu~mic co~tami~ution, The endometrial cytosol contains ligand-free estrogen receptors which might contaminate the nuclear pellet. However, thorough washing of the nuclei with 0.1% Triton and buffer before labeling is expected to remove the outer nuclear membrane with adsorbed cytoplasmic material. The nuclei were again washed with Triton and buffer after labeling. In some experiments, the concentration of available receptors in cytosol was measured and compared with the con~n~ation of available receptors in nuclei iso-
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Fig. 4. Sedimentation profile of a 0.6 KC1 extract of nuclei labeled with 10 nM E2 at 4°C for 60 min. The nuclei were washed once with 0.1% Triton in TED buffer and with TED buffer 3-4 times more. An 0.6KCI in TED buffer extract of the nuclei was run on a 10-307; discontinuous sucrose gradient prepared in TED buffer containing 0.5 M KCI. The [‘4C]-BSA was run in a parallel gradient.
Available estradiol receptors in nuclei Table 1. A comparison of concentrations of available nuclear and available cytoplasmic receptors in endometrial specimens in different phases of the menstrual cycle Concentration c3H]-E2 bound (4°C) (pmol/mg DNA) Endometrium Early proliferative Early proliferative Mid-proliferative Mid-proliferative Secretory (d 16) Secretory (d 19)
Nuclei
Cytosol
0.38 0.76 0.63 1.7 0.39 0.21
0.21 0.22 1.7 12 0.28 0.14
of available Receptor Ratio of available receptor Nuclei/cytosol 1.8 3.7 0.36 0.14 1.4 1.5
Fragments of endometrium were incubated with 1OOnM c3H]-E2 f 10pM DES for 9(r120min. Cytosol and nuclei were separated and analyzed for specific 3H-E, binding as described in Materials and Methods.
of C3H]-Es for endogenous
Es, loss of bound C3H]-E2 would also be expected to occur in the presence of excess unlabeled competitor during the second incubation period. The results of this experiment are shown in Table 2. The specific labeling of the nuclei (total minus competed with lOO-fold excess DES) was 0.36 pmol/mg DNA. Incubation of labeled nuclei with excess Es or DES (lOO-fold the concentration of Es
used to label the nuclei) at 4°C resulted in less than 8% reduction in the concentration of specifically bound C3H]-Ea. Therefore, at least 90% of the specific nuclear labeling that occurred during the fir& 15 min of incubation could not be accounted for by exchange and corresponded to binding of c3H]-E2 to sites not occupied by Ea. c. Generation of available binding sites during preparation of nuclei. It is possible that endogenous E2 could be lost from some fraction of the nuclear receptors during the isolation of nuclei, leaving binding sites available. To test this possibility, tissue fragments were pre-incubated with E2 to fill the available sites before isolating and attempting to label the nuclei at 4°C. Secretory endometrium was incubated with Ea (10 nM) for 90min at 30°C. To serve as control, a portion of the tissue was maintained in buffer at 4°C. Nuclei were then isolated, washed and labeled with C3H]-E2 (1OnM) at 4°C for 60min. As shown in
Table 3, the nuclei from the control tissue were found to contain 0.14 pmol/mg DNA of available nuclear receptor, whereas no specific C3H]-E2 uptake was detected in the nuclei from the tissue previously exposed to unlabeled E2. This result indicates that available receptor sites originally present in the nuclei have been filled with unlabeled E2 during the preincubation period and that no binding sites were made available during preparation of nuclei. It might be questioned, however, whether some of the unlabeled Ea used in the preincubation might not remain in the nuclei and interfere with the binding of C3H]-E2 at 4°C. This concern is dispelled by the results from labeling at 30°C which indicates that the amount of total nuclear receptors measured under exchange conditions (available and exchangeable) is not diminished by the preincubation with Ea. A similar experiment was carried out with a specimen of mid-proliferative endometrium, expected to contain higher levels of estrogen receptor than the secretory tissue. As before, ahquots of the tissue were preincubated in buffer at 4°C (control) or with E2 (10 nM) at 30°C for 90 min. Nuclei were isolated and labeled with C3H]-E, (10nM) at different temperatures (4 and 30°C) and for two different periods of time (15 and 180 min). In every case, labeling was also conducted with mixtures of C3H]-E2 and unlabeled DES (1: 100) to correct for non-specific binding. Table 4
Table 2. Nuclear exchange of E2 does not occur at 4°C under conditions adequate for labeling available nuclear receptors First incubation 15 min/4”C 10 nM [aH]-E2
Second incubation 15 min/4”C Buffer InME 1 HIMDES
C3H]-Ez bound (pmol/mg DNA)
Type of binding
0.41 0.34 0.34
Total Not exchangeable Not exchangeable
Available receptors labeled in isolated nuclei incubated at 4°C for 15 min with C3H]-E, alone or in the presence of unlabeled E2 or DES. Only negligible amounts of the nuclear bound C3H]-E2 could be exchanged during a subsequent, identical incubation of nuclei with excess unlabeled estrogens.
HONOR& FLEMINGand ERLIOGURPIDE
8
Table 3. Filling of available nuclear receptors in secretory endometrium by preincubation with EZ at 30°C Labeling cf isolated nuclei (pmol/mg DNA) Tissue preincubation (90 min)
Available receptors (4°C) 1OnM C3H]-Ez f 1 ,uM DES (60 min)
Total receptors (30°C) 10nM c3H]-E2 + 1 PM DES (60 min)
0.14 -0
No steroid (4°C) 10 nM E2 (30°C)
0.29 0.32
Nuclei, isolated from fragments of control or preincubated secretory endometrium, were incubated with r3H]-Ez for 120 min at 4°C or at 30°C. Nuclei from the tissue preincubated with Ez were not labeled at 4°C. This loss of binding must be due to occupation of available nuclear receptors by E2 during preincubation since the concentration of total nuclear receptor (available + occupied), when measured under exchange conditions, is not decreased in the treated tissue.
presents the results from these ex~riments. The control tissue showed a ievei of available nuclear receptors of 1.4pmol/mg DNA. The same value was obtained whether the labeling was done at 0 or 30°C indicating that in this tissue practically all of the nuclear receptor was available. The tissue preincubated with E2 at 30°C showed the expected reduction in the level of available (unfilled) receptors and an elevation in the concentration of total binding sites, measured by exchange at 30°C. The increase in tota binding sites in the nuclei is likely due to translocation of cytoplasmic receptors during the preincubation period. In the preincubated tissue, approx. 10% of the total nuclear sites are still available. These receptors may be a fraction of the original population of available receptors not filled during the preincubation period, or they may have been created during nuclear isolation. Another experiment was done to determine whether available nuclear receptors could also be filled by preincubating tissue fragments at 4°C. It was found that it was necessary to increase the concentration of E2 from the 10 nM used at 30°C to 100 nM. As table 5 shows, 85% of the available nuclear receptors in this secretory tissue could be filled during preincubation for 90min with IOOnM E, at either 4 or 30°C.
L~eljng receptors in whole tissue
A concentration of 0.76 pmol/mg DNA of available receptor was found in nuclei when fragments of a specimen of secretory endometrium were incubated at 4°C with either C3H]-Ez (100 nM) or [3H]-E2 (100nM) + DES (10 FM). When nuclei from the same tissue were first isolated and then incubated with a saturating concentration of C3H]-EZ, 0.55 pmol/mg DNA of available receptor was found. The similarity of the results obtained with these two methods of labeling demonstrated that available nuclear receptors were present as such in the tissue, prior to isotation of the nuclei. The lower value found when labeling isolated nuclei may be due to the action on the receptors of proteolytic enzymes released during homogenization of the tissue. Available or unoccupied receptors are known to be more susceptible to destruction than occupied receptors. Table 6 presents results which illustrate two methods used to measure available and total estrogen receptors in human endometrium. Method I involves incubation of tissue fragments at 4°C with [jH]-E2 and t3H]-E2 + DES as described above. After 2 h of incubation, cytosols are prepared and treated with dextr~~oated charcoal to remove unbound C3H]-EZ, After ~ntrifugation, aliquots of the supernatants are counted. Available cytosol receptor concentrations are expressed as pmol E2 specifi-
Table 4. Filling of available nuclear receptors in proliferative endometrium by preincubation with E2 at 30°C Labeling of isolated nuclei (pmol/mg DNA) Total receptors (30°C) Available receptors (4°C) 1OnM E3H]-Ez + 1 PM DES 1OnM E3H]-Ez + 1 PM DES Tissue preincubation (90 min)
(15 mm)
(120min)
(IS min)
(I 20 min)
1.3 0.31
i.4 0.31
1.4 2.0
1.4 3.6
No steroid (4°C) 10 nM E2 (30C)
Nuclei, isolated from fragments of control or preincubated proliferative endometrium, were incubated with [jH]-E2 for 120 min at 4°C or at 30°C. Approximately 80% of the available nuclear receptors were filled during preincubation. A substantial increase in total nuclear receptor in the preincubation tissue can be explained by translocation of cytoplasmic receptor into the nucleus. Almost all of the nuclear recep_ tors were available in this tissue prior to preincubation, a situation observed in some, but not all, proliferative
tissue.
Available estradiol receptors in nuclei Table 5. Filling of available nuclear receptors by preincubation either 4°C or at 30°C
of tissue fragments at
Labeling of isolated nuclei (pmol/mg DNA)
Tissue preincubation (90 min) No steroid (4°C) 100 nM E2 (4°C) 100 nM Ez (30°C)
Total receptors (30°C) Available receptors (4°C) 10 nM C3H]-Ez f 1 PM DES 10 nM c3H]-EI f 1 PM DES (60 min) (60 min) 0.34 0.29 0.49
0.28 0.04 0.03
Secretory tissue was preincubated with E2 at 4°C or at 30°C prior to isolation of the nuclei and their subsequent incubation with C3H]-E2at 4°C or 30°C. The substantial
decrease in binding to nuclei from both preincubated tissues demonstrates that available nuclear receptors can be filled in tissue fragments at either 3O”C,as previously shown, or at 4°C. The concentration of total nuclear receptors, similar in control tissue or tissue preincubated at 4”C, increases in tissue preincubated at 3o”C, dut to translocational of cytoplasmic receptors.
tally bound/mg DNA. Available nuclear receptor concentrations are estimated from measurements of bound C3H]-Ez in isolated nuclei (Materials and Methods). Occupied nuclear receptors are measured by incubating portions of the isolated nuclei with additional c3H]-E2 (+ DES) for 2 h at 3O”C, conditions appropriate for exchange of endogenous receptorbound E2 for C3H]-Ez. The concentration of occupied
Table 6. A comparison
nuclear receptors is calculated as the difference between the concentration of total nuclear receptors labeled under these conditions and available nuclear receptors labeled at 4°C. This labeling scheme provides estimates of the concentration of total receptors in the cell (total nuclear + available cytosol). Method II involves parallel incubations of tissue fragments with C3H]-E2 (1OOnM) or C3H]-E,
of two methods for measuring Ez receptor distribution human endometrial tissue Exp No. 1 (pmol/mg DNA)
Exp No. 2 (pmol/mg DNA)
0.42 0.48 0.60 1.0
1.6 0.32 0.49 2.0
0.42 0.42
1.6 1.0
0.48 0.57 1.0
0.32 0.46 2.0
in
Method I (preferred)
Available cytosolic Available nuclear Total nuclear *Total in cell Method II
Cytosolic Available (original) TTranslocated at 37°C Nuclear Available ITotal Total in cell
* Total in cell = Available Cytosolic + Total Nuclear. t Translocated at 37°C = Available Cytosolic (4°C) - Available Cytosol(37”C). 1 Total Nuclear = Total in cell - Available Cytosol. Method I: Endometrial fragments are incubated with 1OOnM C3H]-E, f 10 PM DES for 120 min at 4”C, the tissue is homogenized, and specific binding in the cytosol and a portion of the nuclei is measured. Some of the isolated nuclei, with available receptor already labeled, are then incubated with 10nM “H-E2 k 1 PM DES for 120 min at 30°C to label occupied nuclear receptors by exchange. The concentrations of available cytosolic, available nuclear, and total nuclear (available + occupied) can be measured in the single specimen of tissue and the concentration of total receptors in the cell can be computed from these values. Method II: Endometrial fragments are incubated with 100 nM [aH]-Ez k 10 PM DES for 120 min at 4°C and for 60 min at 37°C the tissue is homogenized, and specific binding in the cytosol and nuclei is measured. The translocated cytosol receptor is computed as the difference between the concentration of available cytosol receptor after incubation at 4°C and receptor still present in cytosol after incubation at 37°C. The concentration of total nuclear receptor is determined as the difference between total receptor measured in the cell at 37°C and the concentration of available cytoplasmic receptor measured at 4°C.
10
HONORBEFLEMINGand
(1OOnM) + DES (IO FM) at 4°C and at 37°C. Following incubation, cytosol and nuclear binding are measured. Specific nuclear binding at 37°C includes total nuclear receptor (available + occupied) in the originai tissue and translocated cytoplasmic receptors. The latter parameter can be estimated by subtracting the concentration of cytosol receptors remaining after incubation at 37°C from the concentration of total cytosol receptors measured at 4°C. If it is assumed that all of the receptor lost from the cytoplasm appears in the nucleus, then the total nuclear receptor concentration prior to incubation, and the level of total estrogen receptor in the cell, can be calculated. Receptor concentrations determined by the two methods on two different specimens are shown in Table 6. The concentrations of total nuclear receptors or of total receptor in the cell, measured by either method were in close agreement. Method I may be preferable since it can be applied to less tissue and does not require the assumption that all of the receptor lost from the cytoplasm at 37°C is found in the nucleus.
ERLIOGURPIDE
mature cow endometrium by Jackson and Chatkey[l]. These Ez binding sites were characterized as having high affinity and low capacity and were observed to vary in quantity during the cycle: peaking at diestrus, almost disappearing at proestrus. and peaking again at ovulation. As opposed to the receptors from human uteri and breast tissue, available nuclear receptors in cow uterus could not be extracted with KU. Available nuclear receptors (0.08 pmol/uterus) have been documented for the immature rat by Carlson and Gorski[Z]. We also looked at immature rats using techniques described in this paper and found similar amounts of available nuclear receptors. Zava and McGuire found between 0.2 and 0.4 pmoljmg DNA of ‘available receptors in uterine nuclei of immature rats [3]. More recently Jungblut et ai.[4] reported that available nuclear receptors could be found in uteri from ovariectomized, adrenalectomized pigs. The discovery of available nuclear receptors in a significant number of breast tumors and in a breast cell line led to speculation that their existence may account for the category of hormone-independent DISCUSS1ON breast tumor which contains receptor, yet is able to The results reported in this paper demonstrate the grow under conditions of steroid deprivation (e.g. endocrine gland ablation). If receptor in certain tumors existence in human endometrium of nuclear estrogen is already present in the nucleus and does not require receptors which are not bound to E,. The evidence presented indicates that these available nuclear recap- steroid to translocate, the tumor might not require tors are not artifactual. It was shown that exchange steroid to proliferate. Our results demonstrate that does not account for the rapid labeling in nuclei at available nuclear receptors are not unique to malig4°C. Since available nuclear sites could be labeled in nant tissues. They are, in fact, a prominent feature of normal human endometrium. Available receptor conwhole tissue fragments, it is clear that the receptors stitutes almost all of the nuclear receptor in the proliare not “made available” by stripping of endogenous Ez during nuclear isolation. Our procedures for isola- ferative tissues shown in Tables 3 and 4. In secretory tion of nuclei and the results obtained make it ex- tissues, available receptor is usually between 40 and tremely unlikely that the available receptors result 507; of total nuclear receptor. Available or unoccufrom cytoplasmic contamination, One possibility not pied nuclear receptors are quantitatively as important yet explored is that some nuclear receptors may be as the occupied receptors. As can be seen in Fig. 1, there is a wide variation in occupied by a steroid that E2, with a higher affinity for the receptor, is able to displace. Since this is a the amounts of available nuclear receptor within each possibility, we use the term “available” rather than of the three proliferative periods. This is not surprising since each tissue was obtained from a different “unoccupied” to describe the nuclear receptors subject. Nevertheless, there is a definite peak in conmeasured in our experiments. centration of available receptor in the mid-follicular Brooks et aI.[6] and Zava and McGuire[7] observed that a large fraction of the E2 receptor in the phase. Available receptor concentrations are low in early proliferative and appear to be declining in late breast tumor cell line MCF-7 was in the nucleus and proliferative tissues, perhaps due to the high levels of could be labeled at 4°C. In human breast tumors, Garola and McGuire found available nuclear recep- endogenous E2 present at the time. During the luteal tors in 40% of th.e 28 breast tumors examined 193. phase, the concentrations remain low. The period in which avaifabfe receptor is most Panko and MacLeod examined 139 breast tumors and found that nuclear extracts from 300/, of these abundant coincides with the period of rapid cellular tissues could be labeled at 4”C, indicating the pres- proliferation. Chalkey and Jackson showed in cows ence of available receptors [lo]. Sonnenschein et that the peak in unoccupied receptors coincided with the peak in endometrial growth. They also showed aI.[5] measured available receptor in nuclear extracts from two cell lines cloned from rat pituitary ‘tumors. that available receptors are present in endometrium. a tissue characterized by active cellular proliferation. Even after 40 generations of growth in E,-depleted medium, 30% of the total receptor was found to be in and not in myometrium Cl]. Even in cell culture, more available receptor is found in nuclei when the the nucleus, unbound to Ez. Available nuclear receptors were also measured in cells are actively growing [7].
Available estradiol receptors in nuclei What is the significance of the fact that peak amounts of available nuclear receptor appear to correlate with active cell proliferation? An explanation consistent with current dogma of steroid hormone action is that the relatively larger amounts of estrogen-receptor complex accumulating in the nucleus during a period of proliferation, such as the follicular phase, dissociate rapidly, leaving intact available receptor in the nucleus. The elevated levels of available receptor thus generated would gradually decline as the rate of entry of ER complex into the nucleus decreased and as available receptor was destroyed or returned to the cytoplasm. When the period of rapid growth has passed, it might be expected that a steady state would be established between entry of ER into the nucleus, dissociation of E from R and disposition of R. Contradicting this interpretation of the data is the fact that available receptors are found in nuclei of systems from which E, has been excluded. Several cell lines have been shown to contain available nuclear Ez receptors even after they have been grown in E,-depleted medium [S]. Available receptors are also found in the uterine nuclei of immature rats [2], as well as in pigs which have been ovariectomized and adrenalectomized [4]. These results suggest that the receptor may translocate into the nucleus and bind chromatin without itself being bound to E,, a situation not expected on the basis of the current accepted mechanisms of steroid receptor function.
Acknowledgrments-We are grateful to Drs. S. Kaplan, H. May, A. Crosdale and other physicians of the Department of Obstetrics and Gynecology for their collaboration in making specimens of endometrium available to us. also to Dr. L. Deglidisch. of the Department of Pathology for the histologic dating of the tissue. This work was supported by Grants HD-07197 and CA 15648. awarded by the National Institutes of Health (DHEW).
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