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Original Studies |
Service de Médecine, Laboratoire d’Investigation Clinique H. J. Tagnon, Laboratoire d’Endocrinologie, Institut Jules Bordet, Centre des Tumeurs de l’Université Libre de Bruxelles, B-1000 Brussels, Belgium
Address all correspondence and requests for reprints to: Abraham Borkowski, M.D., Service de Médecine, Institut Jules Bordet, 1 rue Héger-Bordet, B-1000 Bruxelles, Belgium.
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Abstract |
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We showed that: 1) IgGs Fab fragments (which contain only one antigen binding site) induced an estrogenic response in MCF-7 cells, producing estrogen receptor (ER) down-regulation and an increase in progesterone receptor concentration; 2) IgGs specifically inhibited MCF-7 cell surface labeling with fluorescent estradiol (E2)-BSA conjugates; 3) this inhibition of E2-BSA binding to membrane estrogen binding sites was largely caused by natural anti-E2-BSA antibodies (Ab) selectively associated with the natural anti-ER Ab within IgGs; 4) furthermore, these natural anti-E2-BSA Ab accounted for most of IgGs estrogenic activity in cell culture; 5) however, when incubated with cytosolic ER, they did not behave like estrogens, but they decreased ER hormone binding capacity; and 6) although IgGs stimulated cAMP production, their anti-E2-BSA Ab subpopulation did not.
In conclusion, the estrogenic activity of IgGs does not involve Ab mimicking E2 molecular configuration or ligand-independent cAMP mediated pathways, membrane Fc receptors, and membrane receptor cross-linking mechanisms. On the contrary, IgGs seem to function by neutralizing estrogen-like epitopes, associated with ER-related peptides, which might inhibit ER activation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The estrogen receptor is a 66-kDa ligand-inducible nuclear transcription factor (3). After estrogen binding, ER is thought to be phosphorylated and to undergo conformational changes that increase its affinity for a 13-nucleotide palindromic sequence, the estrogen response element, which controls the transcription of estrogen responsive genes (4). Recent reports also referred to the activation of steroid hormone receptors by ligand-independent pathways through cell surface receptors to neurotransmitters (5) and to growth factors (6). In this regard, data suggest the existence of cross-talks between steroid hormones and cAMP signaling pathways (7, 8), and there is evidence that phosphorylation mechanisms might activate steroid receptors, even in the absence of their steroid ligand (9, 10). On the other hand, estrogen binding sites identified in the plasma membrane of various cell types could be responsible for some rapid or nongenomic steroid actions (11, 12, 13).
The purpose of this study was to clarify IgGs’ biological mechanism of action and, therefore, to analyze: 1) the contribution of cell membrane receptor cross-linking and of Ab endocytosis through Fc receptors; 2) the interaction of IgGs with estrogen binding sites located at the cell surface; 3) the activation of ER through cAMP-mediated nonligand pathways; and 4) the possible presence and role of natural Ab recognizing estrogen-related molecules. Such receptor-like Ab, able to bind natural ligands, have been found for other hormones, and they do interfere with normal cellular functions (14, 15). With regard to estrogens, a monoclonal Ab being the internal image of E2 has been produced experimentally (16). Therefore, according to the idiotypic network theory (17) and to the unlimited diversity of the immune system (18), the antiidiotypic counterparts, i.e. Ab mimicking the estrogen binding domain of ER and thus recognizing estrogen-related structures, should also be synthesized by B lymphocytes. Such Ab might be found within the still very heterogeneous populations of Ab composing the IgGs and play a role in the biological properties of the latter.
Our data suggest that IgGs do not function like a ligand, by mimicking E2 molecular configuration, nor by stimulating a cAMP-mediated ligand-independent pathway. On the contrary, they seem to act mostly by neutralizing molecules that express estrogen-like epitopes and that might be inhibitors of ER activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unless otherwise specified, all reagents were purchased from Sigma-Aldrich (St. Louis, MO). Radioactive materials came from Amersham, Little Chalfont, UK.
Cell cultures
MCF-7 mammary carcinoma cells were cultured in MEM with 10% FCS, glutamine (4 mmol/L), and penicillin-streptomycin (100 IU/mL-100 µg/mL) (Life Technologies, Paisley, Scotland) and seeded at least twice in MEM with 10% dextran-coated charcoal (DCC)-treated FCS before experiments. ERC cells (derived from ER409: CHO cells transformed with the ER gene) were cultured as previously described (19) and were a kind gift of Dr. Kushner.
Purification of anti-ER Ab (IgGs)
Purifications were performed as previously described (1, 2). Briefly, total IgG (IgGt) from normal blood donor plasmas was purified on a DEAE-Sephacel column (Pharmacia, Uppsala, Sweden). An IgG subpopulation with enhanced reactivity for ER (IgGs) was isolated by adsorption of IgGt on a calf uterine cytosol-Blue B Matrex gel (Amicon, Danvers, MA) column that selectively binds the ER. Flow through Ab was designated as IgGe.
Preparation of Ab Fab fragments
Immobilized papain (Pierce, Rockford, IL) was used to prepare Fab fragments from IgGs, following the manufacturer’s protocol. Separation of Fab fragments from the intact Ig and Fc fragments was carried out by adsorption of the digested mixture on a protein A-sepharose column (Pharmacia).
Measurement of ER down-regulation and of increase of PR concentration in MCF-7 cell monolayer cultures (whole cell assays)
Experiments were carried out as previously described (1, 2).
Labeling of MCF-7 cells with E2-BSA-fluorescein isothiocyanate conjugates (E2-BSA-FITC); inhibition by IgGs
MCF-7 cells were removed from flasks by incubation with 2.5 mmol/L ethylenediamine tertra-acetate-phosphate-buffered saline (EDTA-PBS) (10 mmol/L NaHPO4, 150 mmol/L NaCl, pH 7.4), centrifuged 10 min at 1,000 x g, and washed twice with 0.01% BSA-10-2 mol/L NaN3-PBS. The cells (106) were suspended in 200 µL of the same buffer and 1–10 µg of E2-BSA-FITC (E2 17ß-hemisuccinate:BSA-fluorescein isothiocyanate containing 4–9 mol steroid and 2–5 mol FITC per mol BSA) were added for 30 min at 4 C. The cells were finally washed three times with BSA-NaN3-PBS, suspended in 200–500 µL of this buffer and analyzed by fluorescent activated cell sorter. For competition experiments, the cells were preincubated during 20 min with competitors before labeling with E2-BSA-FITC.
Enzyme-linked immunosorbent assay (ELISA) on E2-BSA-coated plates
Briefly, 96-well plates were coated with 0.5 µg per well (50 µL) of E2-BSA (17 ß-E2 17-hemisuccinate:BSA containing 40 mol E2 per mol of BSA) in PBS. After washing, the plates were saturated with 1% BSA-PBS, and dilutions of the Ab to be tested were added overnight in 0.1% BSA-0.01% Tween80-PBS. After washing, the plates were incubated with goat antihuman biotinylated Ab (Amersham) for 2 h and with streptavidin-peroxidase (Amersham) for 20 min. O-phenylenediamine was used as chromogen. For inhibition assays, the Ab to be tested and potential competitors (E2 or E2-hemisuccinate = 17 ß-E2 17-hemisuccinate) were preincubated overnight together and put on E2-BSA-coated plates for 4 h.
Preparation of E2-BSA columns
E2-BSA-sepharose columns were prepared by coupling E2-BSA to AH-sepharose 4B beads (Pharmacia) using the carbodiimide method described in: Affinity Chromatography, Principles and Methods, edited by Pharmacia. Coupling was realized with N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride with a ratio of 5 mg E2-BSA per mL of gel.
Purification of natural anti-E2-BSA Ab
Either total plasma IgG (IgGt) or the IgGs subpopulation were incubated overnight with E2-BSA columns in 0.2 mol/L Tris, 0.5 mol/L NaCl (pH 7.8). After extensive washing with the same buffer, bound Ab (anti-E2-BSA Ab) were eluted with 0.2 mol/L glycine, 0.5 mol/L NaCl (pH 3.6), and the pH was immediately neutralized with 1 mol/L Tris. Controls corresponding to bound and flow through Ab were, respectively: 0.2 mol/L glycine, 0.5 mol/L NaCl (pH3.6) buffer passed through E2-BSA columns and 0.2 mol/L Tris, 0.5 mol/L NaCl (pH 7.8) incubated 18 h with the columns. Both were supplemented thereafter with IgGt. Bound and flow through Ab (FT-E2-BSA Ab), as well as controls, were then treated with DCC (final concentration: 0.5%), dialyzed in 10 mmol/L Tris, 10 mmol/L MgCl2 (pH 7.5), and purified on DEAE-Sephacel columns to remove any potential free E2 or E2-BSA.
Studies of [3H]-E2 binding to isolated ER in the presence of Ab
We studied [3H]-E2 binding to ER from
MCF-7 and from ERC cell cytosols. Cells were removed from the culture
dishes with 2.5 mmol/L EDTA-PBS, disrupted in a potter tube (Weaton,)
at 240 rpm (3 cycles of 20 strokes each) and centrifuged 30 min at
30,000 x g. Hundred microliters of cytosol, 100 µL
Ab or buffer, and 100 µL 3H-E2 (final
concentrations: 0.75–6.7 x 10-9 mol/L) were
incubated together for 18 h at 4 C. Unbound
3H-E2 was removed by addition of 300 µL 0.5%
DCC solution and centrifugation; the supernatant was added to
scintillation solution (National Diagnostics, Atlanta, GA) and counted.
Nonspecific binding was obtained by adding 10-5 mol/L
unlabeled E2 to the tubes. The buffer used was 10 mmol/L
KHPO4, 10% glycerol, 1 mmol/L 
-monothioglycerol (pH
7.4) containing protease inhibitors (1 µg/mL leupeptin, 2 µg/mL
aprotinin, and 100 µg/mL phenyl methane sulfonyl fluoride)
(Boehringer, Mannheim, Germany), 0.2% gelatin, and 1 mmol/L EDTA.
LIGAND program (20) was used for Scatchard plot analyses of the data
(21).
Measurement of cAMP level in MCF-7 cell cultures
MCF-7 cells cultured in 10% DCC-treated FCS-MEM were seeded in 24-well plates. When cells were three fourths confluent, the medium was replaced by RPMI 1640 without phenol red and with 2 µmol/L isobuthylmethylxanthine (IBMX). After 20 min, E2 and the Ab to be tested were added to the plates for 30 to 60 min in RPMI plus IBMX. The medium was then discarded, plates were washed once with PBS, and cAMP was extracted with 0.5 mL 95% isopropanol for 1 h, on ice. Isopropanol was evaporated in a Buchler vortex-evaporator and the dried material suspended in 0.05 mol/L acetate (pH 6.2). The concentrations of cAMP were measured by ELISA kits from Amersham.
Statistical analyses
Data are expressed as the means ± SD. The statistical significance was analyzed by Student’s t test using Statworks Macintosh program. Differences between groups were considered significant at P < 0.05. For ELISA, Scatchard plot analyses, and whole-cell assays, we show representative experiments to illustrate the Ab variability among individuals. However, whenever feasible, means ± SD are specified in the text.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
Because Ab could function through their constant or through their
variable regions, we first investigated whether the mechanism of IgGs
estrogenic activity resulted from the interaction of their antigen
binding site (IgGs Fab part) with specific molecules or from the
interaction of their Fc part with putative Fc receptors. For this
purpose, we measured the estrogenic activity of isolated IgGs Fab
fragments in MCF-7 cell cultures. As shown in Fig. 1, the IgGs Fab
fragments were as active as intact IgGs, or as E2, in
producing a dose-dependent increase in PR concentration and a
down-regulation of ER content. In three different experiments, maximal
PR induction was 167 ± 25% for IgGs Fab fragments, 165 ± 20% for
IgGs, and 232 ± 55% for E2 (P < 0.05 in
unpaired t tests between E2 and IgGs or IgGs Fab
fragments). The concentrations of Fab fragments and IgGs required to
achieve these increases were, respectively: 20 ± 26 µg/mL and 25 ±
20 µg/mL. Although IgGs data for ER down-regulation do not appear in
Fig. 1, these IgGs were tested apart for this parameter, and they do
down-regulate the ER. Comparatively, IgGs Fab fragments and IgGs
induced 50% ER down-regulation, respectively, at 10 ± 9 µg/mL and
12 ± 10 µg/mL (n = 3).
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)
We first incubated 106 MCF-7 cells with various
concentrations of E2-BSA-FITC (5–50 µg/mL) and observed
a similar cell surface staining, which ranged from 50–80% of the
cells. Thereafter, we used a constant concentration of
E2-BSA-FITC (20 µg/mL, which corresponds to approximately
3.6 x 10-6 mol/L E2) to investigate the
inhibition of cell surface labeling caused by various reagents. As
shown in Table 1, MCF-7 labeling was inhibited after incubation with
IgGs, whereas IgGe (although used at 10-fold higher concentrations)
were ineffective.
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Binding of IgGs, IgGe, and IgGt on E2-BSA-coated plates
(Fig. 2)
Because of the specific inhibition of MCF-7 cell surface labeling
with E2-BSA-FITC in the presence of IgGs, we investigated
the binding of IgGs, IgGe, and IgGt to E2-BSA. ELISA in
Fig. 2A shows that IgGs are much enriched in anti-E2-BSA
Ab, as compared with IgGt or IgGe. The relative concentration of
anti-E2-BSA Ab within IgGs varied greatly from preparation
to preparation, IgGs concentrations required to reach 50% of maximum
binding in ELISA ranging from 0.2–60 µg/mL. Of 10 plasmas, IgGs
contained, in mean, 20x more anti-E2-BSA Ab than the
corresponding IgGt or IgGe (range: 5–100x).
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shows that
even 5 x 10-5 mol/L in E2 and
10-4 mol/L in E2-hemisuccinate did not inhibit
the binding of the two different IgGs preparations. On the contrary,
binding to E2-BSA of a standard anti-E2 Ab
obtained from immune rabbit serum was efficiently inhibited by both
free E2 and free E2-hemisuccinate. On the other
hand, free E2-BSA used as inhibitor did prevent IgGs
binding to E2-BSA, and IgGs were not found to bind to BSA
or phosphotyrosine-BSA-coated plates (data not shown). This indicates
that IgGs recognize a distinct epitope on the E2-BSA
molecule, resulting from the association of both E2 and
BSA, but neither free E2, nor BSA, nor the linker
(hemisuccinate), nor E2-unrelated molecules coupled to
BSA. Similar results were obtained with the natural anti-E2-BSA Ab, purified by adsorption of IgGt on E2-BSA columns (data not shown).
Estrogen-like activity of the natural anti-E2-BSA Ab in
MCF-7 cell culture (Fig. 3)
To assess the contribution of the anti-E2-BSA Ab
subpopulation in IgGs biological activity, we studied ER
down-regulation in MCF-7 cell culture, comparing IgGs, IgGs adsorbed on
E2-BSA column (IgGs-anti-E2-BSA Ab), and IgGs
not retained by the column (IgGs-FT-E2-BSA Ab). As shown in
Fig. 3A, IgGs-anti-E2-BSA Ab were much more active than
IgGs, because at least 10x higher concentrations of the latter were
needed to achieve the same ER down-regulation.
IgGs-FT-E2-BSA Ab were even less efficient. All
purifications and cell experiments were run in parallel with buffers
previously incubated with E2-BSA columns, supplemented with
IgG, and finally treated like the various IgGs subpopulations (see
Materials and Methods). No estrogenic effects were observed
with these nonspecific IgG (NS IgG), thus ruling out E2
contamination.
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shows that total plasma IgG, similarly purified on
E2-BSA column (anti-E2-BSA Ab), also have
estrogenic effects on MCF-7 cells (induction of PR and down-regulation
of ER) and are 100x more active than the Ab not retained by the column
(FT-E2-BSA Ab). In the three experiments carried out, we
observed that natural anti-E2-BSA Ab were, in mean, 40x
more active than FT-E2-BSA Ab. For example, a 50% ER
down-regulation was obtained, respectively, with 31 ± 14 µg/mL of
anti-E2-BSA Ab and 1250 ± 600 µg/mL of
FT-E2-BSA Ab. IgGt purified on BSA columns had no
estrogenic activity (data not shown).
Scatchard plot analysis of [3H]-E2
binding to ER in the presence of anti-E2-BSA Ab (Fig. 4)
As shown in Fig. 4, Ab not adsorbed on the
E2-BSA-sepharose column (FT-E2-BSA Ab) did not
modify [3H]-E2 binding to ER, whereas Ab that
were retained by the resin (anti-E2-BSA Ab) decreased the
number of ER hormone binding sites and might also reduce the ER
affinity for E2, but only at highest Ab concentrations and
for 10% of plasmas. The decrease in the number of estrogen binding
sites was an anti-E2-BSA Ab dose-dependent response (data
not shown) but varied from plasma to plasma, as illustrated in the
figure. Similar decreases in the number of ER hormone binding sites
were observed in the presence of IgGs, whereas IgGe did not modify the
ER ligand binding properties (data not shown). On the contrary, as
expected, increasing E2 concentrations always progressively
decreased ER affinity but never decreased its number of hormone binding
sites.
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For practical reasons, i.e. higher amounts of ER per cell, we mainly used ER from ERC cell cytosols; however, the same patterns of Scatchard plot analysis were observed with ER from MCF-7 cells.
cAMP production in MCF-7 cell culture (Fig. 5)
We measured cAMP accumulation in MCF-7 cells after 30–60 min
incubation with E2 and various Ab in the presence of IBMX
as phosphodiesterase inhibitor. As shown in Fig. 5, only IgGs
significantly (P < 0.001) increased cAMP production;
E2 (0.5 nmol/L) was inactive, as were the other Ab tested.
Forskolin (10-6 mol/L), used as positive control, always
produced at least a 10-fold increase of cAMP content (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We then decided to investigate cell surface receptors that might be recognized by the Fab part of the natural Ab and be a source of estrogenic activity. Estrogen binding sites have been identified in the outer membrane of various cells, especially by methods using fluorescent E2 conjugates (12, 13). We confirm these observations in MCF-7 mammary carcinoma cells and show a specific membrane staining by E2-BSA-FITC molecules. However, as previously reported by Berthois et al (12), E2-BSA was much more efficient than E2 in inhibiting cell labeling, thus suggesting some distinct properties for the membrane sites. In addition, we show that IgGs also did specifically and efficiently inhibit cell surface labeling by E2-BSA-FITC. Such inhibition is compatible with a competition between IgGs and E2-BSA-FITC for the same cell membrane receptors. However, it might also be caused by Ab recognizing the E2-BSA-FITC molecules and, therefore, behaving themselves like soluble estrogen binding sites. The IgGs would then compete with the cell membrane receptors for the same ligand.
Natural anti-E2-BSA Ab were found, in ELISA, to be present in all normal human plasmas. Furthermore, these Ab were selectively concentrated within the IgGs population. Indeed, they were copurified with the anti-ER Ab and thus might be directed towards uterine proteins that are bound to ER-related peptides or that, like ER, bind to the Blue B Matrex column. These natural anti-E2-BSA Ab seem to recognize a distinct epitope resulting from the association between E2 and BSA. In addition, Scatchard plot analyses indicate that, although the anti-E2-BSA Ab do modify the ligand binding properties of cytosolic ER, they do not behave like estrogens, because they reduce the total number of detected ER estrogen binding sites. This decrease may result from the steric hindrance of [3H]-E2 binding to ER or from the modification of ER molecular configuration and may occur either through the direct recognition of the ER molecule itself by the Ab or indirectly through the interaction of the Ab with ER-associated proteins (22). The last hypothesis is more probable because, using eight different Ab preparations, we never detected any direct anti-E2-BSA Ab recognition of ER, both in Western blots and in immunoprecipitation studies carried out with labeled ER (personal observations).
By acting as antiligand Ab, which neutralize the E2-BSA-FITC molecules, the natural anti-E2-BSA Ab may thus account, to a significant extent, for the inhibition of E2-BSA-FITC cell surface labeling caused by the IgGs.
The anti-E2-BSA Ab also were found (and this was totally unexpected) to account for a major part of the estrogenic properties of IgGs in cell experiments, producing ER down-regulation and an increase in PR concentration. Therefore, it seems again that IgGs do not behave like the internal image of an estrogen but, on the contrary, like a soluble hormone binding site that recognizes estrogen-like molecules looking like E2-BSA.
Natural autoantibodies can behave like hormones by recognizing and activating receptors (23). This biological mimicry of antireceptor Ab is attributed to their molecular resemblance to the hormones (24). Conversely, considering the diversity potential of the Ig-variable regions (18) and the idiotypic network theory (17), Ab mimicking the hormone binding sites of receptors can also be found. They are then internal images of receptors and, by neutralizing normal ligands, such receptor-like Ab can be a source of hormone resistance, as shown in man by autoantibodies to erythropoietin (14) or insulin (15). In our experiments, the Ab with estrogenic activity seem to function like hormone binding sites and bind estrogen-related molecules. To explain the paradoxical hormonal activity of these receptor-like Ab, we suggest that they neutralize a ligand that functions like a natural inhibitor of the ER activation. Although our arguments supporting this hypothesis are indirect, the latter would account for all our experimental observations, including the inhibition by IgGs of E2-BSA-FITC binding to cell membrane. Indeed, as this neutralization occurs at cell surface, it might then somehow reflect a physiological situation where a natural antiestrogenic molecule is prevented, by natural Ab, from binding to its receptor.
However, Ab might also be endocytosed and activate ER within the cell. Indeed, Ab have been shown to penetrate into living cells (16, 25, 26). One cannot exclude, therefore, an intracellular activity of the anti-E2-BSA Ab, but as discussed above, inside the cells again, they should similarly neutralize ER repressors bearing estrogen-like determinants and/or modify ER molecular configuration by interacting with ER-associated proteins.
We also considered the possibility of a ligand-independent stimulation of membrane receptors by the natural Ab. Cross-talks between the ER and membrane signal transduction pathways, involving adenylate cyclase and protein kinases activation, have been documented and may play an important role in modulating ER-dependent transcription (5, 10, 27). We undertook, therefore, the study of cAMP production in MCF-7 cell cultures in the presence of various Ab. Our results show that IgGs produce a specific increase of intracellular cAMP concentration, whereas IgGe, anti-E2-BSA Ab, and 0.5 nmol/L E2 (a concentration that, in our experience, produces maximal estrogenic effects on MCF-7 cells) are ineffective. This suggests that some Ab within IgGs, but not their anti-E2-BSA component, could activate the ER through nonligand cAMP-dependent pathways. Because the anti-E2-BSA Ab are responsible for most of the estrogenic properties of IgGs on MCF-7 cells, a significant contribution of cAMP-dependent nonligand pathways in IgGs estrogenic activity is improbable. Our results and others (28), showing no cAMP induction by E2 in MCF-7 cells, do not correspond with those reported by Aronica et al (8). This discrepancy might be caused by differences in cell lines and/or culture conditions. Nevertheless, our results suggest that although cAMP cascade may participate in ER activation by E2, it is not an absolutely necessary pathway.
We cannot exclude, however, ER activation through the stimulation of membrane receptors that are not associated with the cAMP pathways (29, 30, 31) but that share cross-reactive epitopes with E2-BSA.
In conclusion, neither Ab mimicking the E2 molecular configuration nor cAMP-mediated nonligand pathways, membrane Fc receptors, or membrane receptor cross-linking mechanisms play a significant role in the estrogenic properties of natural autoantibodies in MCF-7 cell culture. On the contrary, Ab with estrogenic activity seem to function, to a major extent, through their Fab regions and to recognize estrogen-like epitopes that can associate with ER-related peptides. They might function by neutralizing some natural inhibitors of ER activation expressing estrogen-like epitopes at the cell surface and/or inside the cells.
Our observations could be particularly helpful in understanding the hormonal control of normal mammary cells and breast cancer in man.
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Acknowledgments |
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Footnotes |
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Received December 2, 1996.
Revised April 17, 1997.
Accepted July 1, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), IgGs (
), and IgGs
Fab fragments (•). The binding of [3H]-E2
(5 x 10-10 mol/L) or [3H]-ORG 2058
(6 x 10-10 mol/L) to the cells was measured to
estimate ER and PR concentrations, respectively. Results are expressed
in percent, considering the control (medium alone) as 100%. Results
above and below the 100%
line correspond, respectively, to the PR induction and to the
ER down-regulation. This figure illustrates one of three similar
experiments.
, IgGs2; O, IgGe2; +, IgGt2. This
represents one of five similar experiments. B, Inhibition studies: a
constant amount of various Ab was incubated on
E2-BSA-coated plates in the presence of increasing
concentrations of E2 or E2-hemisuccinate as
potential competitors. Symbols: 
