Roles of antiestrogen binding sites in human endometrial cancer cells

Estrogen-noncompatible antiestrogen binding sites (AEBS) as well as estrogen receptors (ER), and the growth-inhibitory effect of tamoxifen were investigated in two human endometrial cancer cell lines, IK-90 and HEC-IA cells. IK-90 cells contained specific AEBS, but no ER was found in these cells. Scatchard plot analysis of AEBS in 12,000 g supernatant from IK-90 cells showed a high affinity binding site for tamoxifen (Kd:5.6 +/- 1.0 nM) with the maximum binding site of 457 +/- 47 fmol/mg protein. However, no measurable ER or AEBS was found in HEC-IA cells. The effect of tamoxifen on the growth of cells was found to be identical in both cell lines; the addition of 10 microM tamoxifen to culture medium was cytocidal whereas tamoxifen at lower concentrations (1 nM-1 microM) did not significantly affect the growth of both IK-90 and HEC-IA cells. These results demonstrate for the first time the presence of AEBS in human endometrial cancer cells. The present results also suggest that AEBS does not play a fundamental role in mediating the growth-inhibitory effect of tamoxifen in endometrial cancer cells.     

Characterization of estrogen and antiestrogen binding to the cytosol and microsomes of breast tumors

The binding of [3H]estradiol and [3H]hydroxytamoxifen to the cytosol and microsomal fractions of several human breast tumors was investigated. By washing microsomal membranes with a KCl-free or a KCl-containing medium we could distinguish between intrinsic, extrinsic and contaminant estradiol binding sites in these membranes. We observed that treatment of the microsomes with low salt medium removes about 80% of the total estradiol binding sites, whereas 20% are not extractable. The concentration of unextractable [3H]estradiol binding sites in the microsomes varies in proportion to the level of cytosolic estrogen receptors (ER). About 10% of the total extranuclear specific estrogen binding sites was consistently found tightly associated to the microsomal fraction, which displays an affinity for estradiol (Kd = 0.1-0.6 nM) similar to that of the cytosolic ER. The displacement of [3H]estradiol with unlabeled hormone or with the antiestrogens, nafoxidine, enclomiphene and tamoxifen (TAM) exhibits identical IC50 values either in the cytosol or in the microsomal membranes. On the other hand, the microsomal fraction of breast tumors also binds [3H]hydroxyTAM, but with higher capacity and lower affinity than those of the cytosolic fraction. Furthermore, we did not observe correlation between the concentrations of ER and of antiestrogen binding sites (AEBS) in the tumors. These results indicate that microsomal membranes of human breast tumors contain estrogen binding sites which may be related to the cytosol ER recycling and that specific AEBS are predominantly localized in this membrane system. Furthermore, it is shown that the magnitude of estradiol binding to microsomes depends on the ER positive degree of the tumors, whereas the magnitude of the antiestrogen binding to the microsomes is independent of the ER status of the tumors.     

Role of estrogen receptors and antiestrogen binding sites in an early effect of antiestrogens, the inhibition of cholesterol biosynthesis

The effects of estradiol (E2), 4-hydroxy-tamoxifen (OH-Tam), and LY117018 on cholesterogenesis were investigated in two human breast cancer cell lines (MCF-7 and BT20), and in rat hepatoma (HTC) and fibroblastic (NRK-49F) cell lines. It was found that 10(-10) M E2 stimulated and 10(-8) M OH-Tam inhibited cholesterol synthesis in the estrogen-sensitive MCF-7 cell line. The OH-Tam effect occurred in less than 15 min whereas E2 only stimulated after 8 h. The inhibition of cholesterol synthesis was not reversed by E2. E2 was without effect in the HTC and estrogen-resistant BT20 cell lines whereas OH-Tam was as effective as in the MCF-7 cells. LY117018 had nearly as much effect on cholesterol synthesis as OH-Tam, in both MCF-7 and BT20 cells. Neither E2 nor OH-Tam had any effect on the NRK-49F cell line, even at micromolar concentrations. The three lines (MCF-7, BT20, HTC), whose cholesterol synthesis has been shown to be OH-Tam sensitive, appeared to contain high-affinity antiestrogen binding sites (AEBS); since the OH-Tam-resistant line (NRK) only contained low-affinity AEBS, there appears to be some relationship between OH-Tam sensitivity and high-affinity AEBS content. This suggests that the cholesterogenesis inhibition induced by antiestrogens is ER-independent and may involve AEBS. The cholesterogenesis stimulation induced by E2 occurred via a different pathway that appears to be related to the presence of ER in the cells.     

Tamoxifen and AEBS ligands induced apoptosis and autophagy in breast cancer cells through the stimulation of sterol accumulation

Tamoxifen (Tx) interacts with high affinity to the microsomal antiestrogen binding site (AEBS) which is a hetero-oligomeric complex involved in cholesterol metabolism. We established that Tx and other AEBS ligands induce breast cancer cell differentiation, apoptosis and autophagy through the induction of sterol accumulation. We determined that cell death is sterol- and ROS-dependent and is prevented by the antioxidant vitamin E. Macroautophagy is characterized by the accumulation of autophagic vacuoles, an increase in the expression of Beclin 1 and the stimulation of autophagic flux. We established that macroautophagy is sterol-dependent and is associated with cell survival rather than cytotoxicity, since blockage of macroautophagy sensitizes cells to AEBS ligands. These results show that the accumulation of sterols by AEBS ligands in MCF-7 cells induces both apoptosis and macroautophagy. Collectively, these data support a therapeutic potential for selective AEBS ligands in breast cancer management and reveal a mechanism that explains the induction of autophagy in MCF-7 cells by Tx and other selective estrogen receptor modulators. Moreover these data give pharmacological clues to improve the apoptotic efficacy of AEBS ligands.     

Interactions of tamoxifen in the chicken

The triphenylethylene antiestrogens are very potent antagonists of estrogen action in the chicken and manifest little agonist activity compared to their action in other species. The estrogen antagonism is most probably mediated by the estrogen receptor, to which tamoxifen binds with a Ki of 2.6 nM. Tamoxifen is readily metabolized by liver to 4-hydroxytamoxifen, which binds the liver nuclear estrogen receptor with a Ki of 0.1 nM. The Kd of the receptor is 0.7 nM. Estrogen receptor concentrations in liver from immature chickens are relatively low both in nuclear and cytosol fractions. Treatment with estradiol results in 10-fold up-regulation of the nuclear levels to give a total receptor concentration of about 2 pmol/g tissue. Tamoxifen can promote this up-regulation to a limited extent, but interpretation of experimental results is compromised by difficulties with exchange assays in the face of the very high binding affinity of 4-hydroxytamoxifen. Tamoxifen also binds with high affinity (Kd 2-4 nM) and distinctive specificity to antiestrogen binding sites (AEBS) present in a wide variety of chicken tissues and in the highest concentration in the liver (800 pmol/g tissue). Liver and serum contain ether-soluble components which can compete for binding of [3H]tamoxifen to the AEBS. The serum AEBS inhibitory activity is chromatographically heterogeneous and is associated with a sterol-like fraction as well as with a fatty-acid-containing fraction. Tamoxifen treatment of cockerels results in dose- and time-dependent decreases in serum free and esterified cholesterol, and in phospholipids and triglycerides. These changes may reflect estrogen-receptor-independent interactions of tamoxifen.     

Comparative analysis of estrogen receptors covalently labeled with an estrogen and an antiestrogen in several estrogen target cells as studied by limited proteolysis

Estrogen receptors covalently labeled with the estrogen affinity label [3H]ketononestrol aziridine (KNA) or with the antiestrogen affinity label [3H]tamoxifen aziridine (TAZ) were subjected to limited proteolysis with trypsin, alpha-chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on 10-20% sodium dodecyl sulfate-polyacrylamide gradient gels followed by fluorography. The similar molecular weights of intact receptors (Mr 66,000 daltons) and the proteolytic digest patterns indicate extensive homology among estrogen receptors from MCF-7 human breast cancer cells, GH4 rat pituitary cells and rat uterus when liganded with estrogen or antiestrogen. Each protease generated a distinctive ladder of estrogen receptor fragments, and the fragmentation patterns were virtually identical for estrogen receptors labeled with estrogen (KNA) or antiestrogen (TAZ). Each protease yielded a relatively "resistant" receptor fragment of about 28,000-35,000 daltons. Trypsin and chymotrypsin at higher concentrations generated a much smaller 6,000-8,000 dalton digest product that still contained the [3H]KNA- or [3H]TAZ-labeled receptor binding site. Moreover, the receptor digest patterns were similar for estrogen receptors from the three different target cells. Our studies suggest considerable structural relatedness among these three estrogen receptors and also indicate that these two affinity labels bind to a similar, perhaps identical, region of the receptor molecule.     

Factors affecting anti-estrogen binding site concentration in rat liver

It is known that synthetic anti-estrogens such as tamoxifen bind to specific high affinity anti-estrogen binding sites (AEBS), which are distinct from estrogen receptors. These binding sites are widely distributed in animal and human tissues, the highest concentrations being found in the liver. The physiological role of these intracellular binding sites, which are located predominantly in the microsomal fraction, is currently unknown, as is the nature and identity of their endogenous ligands. In an attempt to gain information which may provide clues to the possible physiological role of these binding sites, studies were carried out to determine whether the concentration of these binding sites in rat liver was affected by a number of physiological variables. The results of these studies indicated that in the rat (i) liver AEBS increased progressively with age; (ii) liver AEBS concentration tended to be higher among females than males after 100 days of age; (iii) there was no significant variation in liver AEBS level with different phases of the estrous cycle; (iv) liver AEBS level was not significantly affected by castration in both males and females or by estradiol replacement in castrated females; (v) liver AEBS concentration increased significantly with increases in ambient temperature; (vi) there was no clearly detectable alteration in liver AEBS levels with changes in the light:dark cycle; (vii) starvation for 24, 48, and 72 hr increased liver AEBS by approximately 1.5-, 3-, and 2-fold, respectively, while refeeding decreased its level; and (viii) liver AEBS was not affected by increasing dietary fat content from 0.5% to 20% (w/w), but was increased modestly by the addition of cholesterol (2% w/w) to the diet. These observations identify several physiological variables which are associated with changes in liver AEBS concentration and suggest possible avenues for future studies to define the physiological role of these binding sites.     

Binding of oxygenated cholesterol metabolites to antiestrogen binding sites from chicken liver

High affinity (KD 2-4 nM) binding sites (AEBS) for the triphenylethylene antiestrogen, tamoxifen, are found in chicken liver cytosol preparations. These sites exhibit affinity for triphenylethylene derivatives typical of those reported for AEBS in other systems. Although steroids and cholesterol do not compete for the sites, certain oxygenated cholesterol metabolites, particularly 7-ketocholesterol, do so. Hot ethanol extracts of chicken and human serum contain AEBS inhibitory activity. This activity is chromatographically heterogeneous, but a sterol-containing fraction has been isolated in which the 7-ketocholesterol concentration can account for the AEBS inhibitory activity of the fraction.     

High affinity specific antiestrogen binding sites are concentrated in rough microsomal membranes of rat liver

Saturation and competitive binding analyses demonstrated the presence of a high affinity (K_D = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver.     

Molecular characterization of the microsomal tamoxifen binding site

Tamoxifen is a selective estrogen receptor modulator widely used for the prophylactic treatment of breast cancer. In addition to the estrogen receptor (ER), tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS), which is involved in ER-independent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands. As a consequence of the treatment with the antitumoral drugs tamoxifen or PBPE, a selective AEBS ligand, we show that tumor cells produced a significant concentration- and time-dependent accumulation of cholesterol precursors. Sterols have been purified by HPLC and gas chromatography, and their chemical structures determined by mass spectrometric analysis. The major metabolites identified were 5alpha-cholest-8-en-3beta-ol for tamoxifen treatment and 5alpha-cholest-8-en-3beta-ol and cholesta-5,7-dien-3beta-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase. Steroidal antiestrogens such as ICI 182,780 and RU 58,668 did not affect these enzymatic steps, because they do not bind to the AEBS. Transient co-expression of human 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-reductase and immunoprecipitation experiments showed that both enzymes were required to reconstitute the AEBS in mammalian cells. Altogether, these data provide strong evidence that the AEBS is a hetero-oligomeric complex including 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase as subunits that are necessary and sufficient for tamoxifen binding in mammary cells. Furthermore, because selective AEBS ligands are antitumoral compounds, these data suggest a link between cholesterol metabolism at a post-lanosterol step and tumor growth control. These data afford both the identification of the AEBS and give new insight into a novel molecular mechanism of action for drugs of clinical value.     

Differences in the form of the salt-transformed estrogen receptor when bound by estrogen versus antiestrogen

Our laboratory has previously reported that antiestrogen binding to molybdate-stabilized non-transformed estrogen receptor results in a larger form of the receptor in 0.3 M KCl when compared with estrogen bound receptor. Estradiol promoted the formation of monomers in the presence of 0.3 M KCl whereas antiestrogen appeared to promote dimer formation. We have extended these studies examining the rabbit uterine salt-transformed estrogen receptor partially purified by DEAE-cellulose chromatography. We previously demonstrated that estrogen receptor prepared in this way bound to different sites on partially deproteinized chromatin subfractions or reconstituted chromosomal protein/DNA fractions when the receptor was complexed with estrogen vs antiestrogen. Analysis of these receptor preparations indicated that DEAE-cellulose step-elution resulted in a peak fraction which sedimented as a single 5.9S peak in 5-20% sucrose density gradients containing 0.3 M KCl for receptor bound by the antiestrogens H1285 and trans-hydroxytamoxifen. However, receptor bound by estradiol sedimented as 4.5S. These receptor complexes bound DNA-cellulose indicating that these partially purified receptors were transformed. DEAE rechromatography or agarose gel filtration of the partially purified antiestrogen-receptor complexes resulted in significant dissociation of the larger complex into monomers. Incubations of 5.9S antiestrogen-receptor complexes with antibodies against nontransformed steroid receptor-associated proteins (the 59 and 90 kDa proteins) did not result in the interaction of this larger antiestrogen-receptor complex with these antibodies (obtained from L. E. Faber and D. O. Toft, respectively). Our results support the concept that antiestrogen binding induces a different receptor conformation which could affect monomer-dimer equilibrium, thus rendering the antiestrogen-receptor complex incapable of inducing complete estrogenic responses in target tissues.     

The estrogenic and antiestrogenic properties of tamoxifen in GH4C1 pituitary tumor cells are gene specific.

We have examined the effects of the antiestrogen tamoxifen (TAM) and the estrogen 17 beta-estradiol (E2) on several estrogen-regulated responses in GH4C1 pituitary tumor cells. After 5 days of treatment with either TAM (1.0 microM) or E2 (1.0 nM), the level of PRL mRNA was markedly increased when measured by the cytosolic dot blot procedure. In contrast, only E2 was able to increase the levels of beta-actin mRNA and cytosolic protein, suggesting that this estrogen may stimulate cell proliferation over the course of treatment. This apparent difference in the abilities of TAM and E2 to stimulate GH4C1 cell proliferation was examined directly. TAM had no effect on cell proliferation as evidenced by its inability to increase cellular DNA or deoxythymidine triphosphate incorporation by nuclei isolated from treated cells. In contrast, E2 stimulated cell proliferation as evidenced by increases in cellular DNA and deoxythymidine triphosphate incorporation by isolated nuclei. The abilities of TAM and E2 to induce progesterone receptor (PR) and PR mRNA were also examined. TAM was unable to increase the levels of PR or PR mRNA, whereas E2 was effective in both of these regards. When added in combination with E2, TAM acted as a classical antiestrogen, partially blocking the induction of PR by E2. To determine whether the inabilities of TAM to stimulate cell proliferation and induce PR were a function of TAM concentration, dose-response experiments were performed. TAM at concentrations ranging from 10(-8)-10(-6) M was effective in inducing PRL mRNA, but at none of the tested concentrations was TAM effective in stimulating cell proliferation or inducing PR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of cholesterol and oxysterols metabolism in the pharmacology of tamoxifen and other AEBS ligands

Tamoxifen is one of the major drugs used for the hormonotherapy of estrogen receptor positive breast cancers. However, its therapeutic efficacy can be limited by acquired resistance and tumor recurrence can occur after several years of treatment. Tamoxifen is known as the prototypical modulator of estrogen receptors, but other targets have been identified that could account for its pharmacology. In particular, tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS) and inhibits cholesterol esterification at therapeutic doses. We have recently shown that the AEBS was a hetero-oligomeric complex composed of 3β-hydroxysterol-Δ(8)-Δ(7)-isomerase and 3β-hydroxysterol-Δ(7)-reductase, that binds different structural classes of ligands, including selective estrogen receptor modulators, several sigma receptor ligands, poly-unsaturated fatty acids and ring B oxysterols. We established a link between the modulation of cholesterol metabolism by tamoxifen and other AEBS ligands and their capacity to induce breast cancer cell differentiation, apoptosis and autophagy. Moreover, we showed that the AEBS carries out cholesterol-5,6-epoxide hydrolase activity and established that cholesterol-5,6-epoxide hydrolase is a new target for tamoxifen and other AEBS ligands. Finally in this review, we report on recent data from the literature showing how the modulation of cholesterol and oxysterol metabolism can be linked to the antitumor and chemopreventive properties of tamoxifen, and give new perspectives to improve the clinical outcome of the hormonotherapy of breast cancers.     

Binding of estradiol and 5 alpha-androstane-3 beta-17 beta-diol by the male rat enriched gonadotrope cells

Dispersed pituitary cells from 42-day old male rats were separated using centrifugal elutriation. Based on LH and PRL cellular contents, fractionated cells were pooled into two fractions: "Lactotrope++ population" and "gonadotrope++ population". Estradiol and 5 alpha-androstane-3 beta, 17 beta-diol binding was measured in these fractions. Results revealed that: (1) The steroid receptors are not destroyed by cell dispersion and elutriation. (2) The estradiol receptor content is higher in gonadotrope++ cells than in lactotrope++ cells. (3) The number of binding sites for the two steroids changes in the different fractions: whereas it is exactly similar in "lactotrope++ population", it is much higher for estradiol than for 5 alpha-androstane-3 beta, 17 beta-diol in "gonadotrope++ population". These results suggest two different species--or conformations--of receptor binding sites for estradiol in the male rat pituitary; the first one could link both steroids, the second one would be specific for estradiol.     

New evidence that the antiestrogen binding site may be a novel growth-promoting histamine receptor (?H3) which mediates the antiestrogenic and antiproliferative effects of tamoxifen

Using as a probe [3H]-DPPE (N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine HCl), a novel compound selective for the antiestrogen binding site (AEBS), new evidence is presented that this site could be a growth-promoting histamine receptor of a type not previously described (?H3). In the rat uterus, DPPE alone at a concentration of 4 mg/kg acts as an estrogen antagonist, unlike TAM alone which is a partial estrogen agonist. In the presence of exogenous estradiol, both TAM and DPPE are partial antagonists. This suggests that the "antiestrogenic" effects of tamoxifen are mediated through AEBS/?H3 while the estrogenic effects are mediated through ER.     

Effects of clomiphene citrate on the pituitary gland in chronically estrogenized rat

Effects of clomiphene citrate (clomiphene) on the pituitary gland of chronically estrogenized ovariectomized rats were investigated. Estradiol-17 beta (E2) pellet implanted subcutaneously in castrated rats for 7 days caused significant increases in pituitary weight and serum prolactin (PRL) level but suppressed serum luteinizing hormone (LH) level. In the estrogenized rats about 40% of estrogen receptor (ER) found in whole pituitary cells (65 +/- 7 fmol/10 mg tissue) was observed in the nucleus, while 60% of ER was present in the cytosol fraction. A single injection of 5 micrograms E2 translocated cytosol ER immediately to nuclear compartment; amounts of ER found in cytosol and nuclear fractions were 16 +/- 1 and 37 +/- 4 fmol/10 mg tissue, respectively, at 1 h. However, the distribution of ER returned to the pre-injection level within 4 h. In the non-estrogenized castrated rats, the nuclear retention of ER was significantly longer than that in the estrogenized rats. A single administration of 200 micrograms clomiphene in the estrogenized rats, on the other hand, increased nuclear ER gradually. Nuclear ER reached the peak level at 4 h (62 +/- 5 fmol/10 mg tissue) and the level remained almost unchanged for 24 h. Cytosol ER decreased and reached a nadir at 4 h (4.3 +/- 0.3 fmol), and the replenishment of cytosol ER could not be detected for 24 h. Similar patterns of cytosol and nuclear ER following the clomiphene injection were also found in the castrated rats. The clomiphene administration in the estrogenized rats resulted in a significant reduction of the pituitary weight 48 h after the administration. The present results seem to show the antiestrogenic action of clomiphene in the pituitary gland.     

Biochemical evidence that human placental lactogen and human chorionic gonadotropin are not stored in cytoplasmic secretion granules

The intracellular storage sites for the human placental hormones placental lactogen (hPL) and chorionic gonadotropin (hCG) are unknown. To determine whether hPL and hCG are stored in cytoplasmic secretion granules, we have compared the localization of hPL and hCG in placental homogenates following differential and density-gradient centrifugations to those of prolactin (PRL) and luteinizing hormone (LH) in human and rat pituitary homogenates. In the differential centrifugation studies, 93.1 +/- 4.1% (mean +/- SE) of the hPL and 79.4 +/- 6.0% of the hCG were detected in the postmicrosomal supernatant of placental homogenates. In contrast, 95-98% of the hPRL and hLH in the pituitary homogenates were detected in particulate fractions. Following centrifugation on sucrose-density gradients, particulate hPL and hCG were distributed diffusely throughout the gradients, while greater than 90% of the pituitary hormones sedimented as single peaks with densities of 1.22 g/cm3. When human placental and rat pituitary tissues were homogenized together prior to differential and density-gradient centrifugations, similar marked differences were observed between the distribution of the placental and pituitary hormones. These results strongly suggest that the placental hormones hPL and hCG, unlike pituitary PRL and LH, are not stored in large secretory granules. Differences in the intracellular storage sites of the hormones may explain, in part, differences in the regulation of peptide hormone secretion by placental and pituitary tissues.     

Estrogen-like effects of 7,12-dimethylbenz(a)anthracene on the female rat hypothalamo-pituitary axis

We have recently demonstrated that 7,12-dimethylbenz(a)anthracene (DMBA), a potent inducer of mammary tumors in rodents, can in vitro decrease the number of membrane dopamine D2 receptors and stimulate prolactin (PRL) release, by direct estrogen-like actions on anterior pituitary. In the present study, we tested the ability of DMBA to mimic the in vivo estradiol (17 beta E2) effects on pituitary D2 receptors and on PRL as well as LH release. We have found that DMBA, like 17 beta E2, when injected to ovariectomized rats, induced a decrease in the number of anterior pituitary D2 receptors, a release of PRL and exerted a biphasic (acute negative and longer term positive) action on LH secretion. We thus examined the ability of DMBA to interact with 17 beta E2 receptors in the hypothalamo-pituitary axis: DMBA binds to the pituitary cytosolic estrogen receptors with an affinity 0.001% that of 17 beta E2. Finally [3H]DMBA binds to hypothalamus-containing brain sections. This binding was displaced partially by RU 2858 a pure estrogen agonist and totally by tamoxifen, a purported estrogen antagonist. No competition for [3H]DMBA binding was observed with an androgen (RU 1881) or a glucocorticoid (RU 26988) agonist. From these data, it may be concluded that DMBA can act as a partial estrogen in pituitary and hypothalamic tissues.     

The inhibition of prolactin synthesis in GH3 rat pituitary tumor cells by monohydroxytamoxifen is associated with changes in the properties of the estrogen receptor

E2 (1 nM) stimulated the synthesis of PRL in GH3 cells. OH TAM (100 nM) did not affect basal PRL synthesis, but completely inhibited the increase produced by 1 nM E2. [3H]E2 and [3H]OH TAM both bound to the cytosolic 8S ER and these were split into 4S subunits on sucrose gradients containing 0.4 M KCl. By comparison, ER complexes extracted from nuclei of GH3 cells cultured in media containing [3H]E2 or [3H]OH TAM both sedimented at 5S on sucrose gradients containing 0.4 M KCl. Both 4S and 5S ER complexes were recognized by the monoclonal antibody D547 which increased their sedimentation coefficients to 8-9S. In contrast, a polyclonal antibody raised to calf uterine ER in the goat, interacted with the cytosolic ER so that the binding of [3H]E2 was inhibited but the binding of [3]OH TAM was only slightly reduced. A molecular model is proposed to describe the binding of E2 and OH TAM to the ER that might contribute to an understanding of estrogen and antiestrogen action.