Effect of ligand binding and DNA binding on the structure of the mouse oestrogen receptor

We have investigated the effects of ligand and DNA binding on the structure of the oestrogen receptor by performing limited proteolysis and analysing DNA binding activity by gel shift analysis. The effects of oestradiol, 4-hydroxytamoxifen and ICI 164,384 have been examined and we have found that despite differences in the DNA binding activity or relative mobility of the receptor-DNA complex we were unable to detect differences in the cleavage pattern produced by trypsin, chymotrypsin, Staphylococcus aureus V8, papain or elastase. Inhibition of DNA binding by ICI 164,384 was lost in receptor fragments that lacked the hormone binding domain. In contrast to the full-length receptor, proteolytic fragments produced by chymotrypsin differed in their ability to bind to an oestrogen response element (ERE) vs a thyroid response element (TRE). Evidence is presented that this difference can be accounted for by the inability of fragments lacking the hormone binding domain to dimerise on a TRE.     

A comparative study of the interaction of oestradiol and the steroidal pure antioestrogen, ICI 164,384, with the molybdate-stabilized oestrogen receptor

The kinetics of binding of oestradiol and the steroidal pure antioestrogen ICI 164,384 to the molybdate-stabilized oestrogen receptor, partially purified from pig and human uterine tissue, were determined. ICI 164,384 bound directly to the oestrogen receptor protein and the kinetic parameters of this interaction were, in general, similar to those for the binding of oestradiol, regardless of the source of the receptor protein. However, the rate of association of oestradiol, regardless of the source of the receptor protein. However, the rate of association of the antagonist with the receptor protein was slower when compared to that of oestradiol. Furthermore, the concentration of binding sites for the two ligands was of the same order. The binding of oestradiol resulted in a steroid-receptor complex which could be transformed in vitro, to a form with increased affinity for DNA-cellulose. However, the complex formed between ICI 164,384 and the receptor protein did not show increased affinity for DNA-cellulose when exposed to conditions that transformed agonist-receptor complexes. Therefore, the binding of ICI 164,384 to the oestrogen receptor protein results in a suppression of the transformation process. A similar suppression in vivo may account for the pure antagonist properties of ICI 164,384.     

Differential DNA-binding abilities of estrogen receptor occupied with two classes of antiestrogens: studies using human estrogen receptor overexpressed in mammalian cells.

We have developed a transient transfection system using the Cytomegalovirus (CMV) promoter to express the human estrogen receptor (ER) at very high levels in COS-1 cells and have used it to study the interaction of agonist and antagonist receptor complexes with estrogen response element (ERE) DNA. ER can be expressed to levels of 20-40 pmol/mg or 0.2-0.3% of total soluble protein and all of the soluble receptor is capable of binding hormone. The ER binds estradiol with high affinity (Kd 0.2 nM), and is indistinguishable from native ER in that the receptor is capable of recognizing its cognate DNA response element with high affinity, and of transactivating a transgene in an estradiol-dependent manner. Gel mobility shift assays reveal interesting ligand-dependent differences in the binding of receptor complexes to ERE DNA. Receptors occupied by estradiol or the type I antiestrogen transhydroxytamoxifen bind to DNA response elements when exposed to the ligand in vitro or in vivo. Likewise, receptors exposed to the type II antiestrogen ICI 164,384 in vitro bind to ERE DNA. However, when receptor exposure to ICI 164,384 is carried out in vivo, the ER-ICI 164,384 complexes do not bind to ERE DNA, or do so only weakly. This effect is not reversed by subsequent incubation with estradiol in vitro, but is rapidly reversible by in vivo estradiol exposure of intact COS-1 cells. This suggests there may be some cellular process involved in the mechanism of antagonism by the pure antiestrogen ICI 164,384, which is not observed in cell-free extracts.     

Interaction of the antioestrogen ICI 164,384 with the oestrogen receptor

The use of partially purified preparations of the human uterine oestrogen receptor has enabled, for the first time, a study of the binding of the steroidal, pure antioestrogen ICI 164,384 [N-n-butyl-11-(3,17 beta-dihydroxy-oestra-1,3,5(10)-trien-7 alpha-yl)N-methyl-undecamide] to the oestrogen receptor. Scatchard analyses of the binding of [3H]oestradiol and [3H]ICI 164,384 to the receptor show that the equilibrium dissociation constants for the interactions of these ligands with the receptor at 0 degrees C are 0.44 and 0.69 nM respectively. The concentration of receptor binding sites for the agonist was 1986 fmol/mg protein whilst that for the antagonist was 1400 fmol/mg protein. The affinity of the antioestrogen-receptor complex for DNA-cellulose does not increase following exposure to conditions that transform the oestrogen-receptor complex.     

Effects of the antioestrogen, ICI 164,384, on oestrogen induced RNAs in MCF-7 cells

The effects of ICI 164,384 on the expression of six oestrogen-regulated RNAs (pNR-1, pNR-2, pNR-13, pNR-17, pNR-25 and pNR-100) and the 46 kDa secreted protein were measured in MCF-7 cells. In marked contrast to tamoxifen, an antioestrogen commonly used in the treatment of breast cancer, ICI 164,384 administered alone had little or no effect on the RNAs or protein. ICI 164,384 completely inhibited the induction of the RNAs and 46 kDa protein by oestradiol. Although ICI 164,384 has an affinity for the human oestrogen receptor only slightly less than that of oestradiol, half maximal inhibition of oestradiol action was attained with between a 50 and 150-fold molar excess of ICI 164,384. The pNR-1 RNA is induced by tamoxifen but this induction was abolished by ICI 164,384. Thus, ICI 164,384 acts as a potent antioestrogen for the regulation of the expression of specific oestrogen-responsive genes in human breast cancer cells.     

ICI 182,780, a new antioestrogen with clinical potential.

Previous studies in this laboratory identified a series of 7 alpha-alkylamide analogues of 17 beta-oestradiol which are pure antioestrogens. Among this initial lead series of compounds, exemplified by ICI 164,384, none was of sufficient in vivo potency to merit serious consideration as a candidate for clinical evaluation. Further structure-activity studies identified a new compound, ICI 182,780, 7 alpha-[9-(4,4,5,5,5-pentafluoro-pentylsulphinyl)nonyl]oestra-1,3,5(10)- triene-3,17 beta-diol, with significantly increased antioestrogenic potency. The antiuterotrophic potency of ICI 182,780 is more than 10-fold greater than that of ICI 164,384. ICI 182,780 has no oestrogen-like trophic activity and, like ICI 164,384 is peripherally selective in its antioestrogenic effects. The increased in vivo potency of ICI 182,780 was also reflected, in part, by intrinsic activity at the oestrogen receptor and in the growth inhibitory potency of ICI 182,780 in MCF-7 human breast cancer cells. ICI 182,780 was a more effective inhibitor of MCF-7 growth than 4'-hydroxytamoxifen, producing an 80% reduction of cell number under conditions where 4'-hydroxytamoxifen achieved a maximum of 50% inhibition. Sustained antioestrogenic effects of ICI 182,780, following a single parenteral dose of ICI 182,780 in oil suspension, were apparent in both rats and pigtail monkeys. In vivo, the antitumour activity of ICI 182,780 was demonstrated with xenografts of MCF-7 and Br10 human breast cancers in athymic mice where, over a 1 month period, a single injection of ICI 182,780 in oil suspension achieved effects comparable with those of daily tamoxifen treatment. Thus, ICI 182,780 provides the opportunity to evaluate clinically the potential therapeutic benefits of complete blockade of oestrogen effects in endocrine-responsive human breast cancer.     

Therapeutic potential of pure antioestrogens in the treatment of breast cancer

Novel 7-analogues of 17β-oestradiol like ICI 164,384, differ from all antioestrogens described previously in being entirely free of partial agonist activity. In adult rats, ICI 164,384 blocks completely the stimulatory effects of endogenous or exogenous oestrogens and produces a castration-like involution of the uterus without affecting the hypothalamic-pituitary-ovarian axis. If analogues effects were achived in patients, peripherally-selective complete oestrogen withdrawal would occur, which presents a novel pharmacological option not achieved by any current treatment. Studies with human breast cancer cells showed that ICI 164,384 reduced to a greater extent than did tamoxifen, the mitotic fraction. This difference may reflect a synergistic stimulatory interaction between serum growth factors like insulin, and the partial agonist effect of tamoxifen which is not seen with ICI 164,384. In long-term culture in the presence of ICI 164,384 no resistant cell lines developed, as has been observed previously in studies with tamoxifen. Pure antioestrogens might thus have a further therapeutic advantage over partial agonists like tamoxifen in reducing the probability of treatment failure due to the regrowth of tumours from resistant cells.     

Biology and mode of action of pure antioestrogens

The properties of a series of 7 alpha-alkyl analogues of oestradiol are described. Studies of chemical structure and activity in the immature rat uterotrophic/antiuterotrophic assay revealed that molecules containing a terminal functional group (acid, alcohol, amine, amide) linked to the steroid by a decamethylene bridge possess both oestradiol agonist and antagonist activity. However, certain amides, exemplified by the compound ICI 164,384 [N-n-butyl-11-(3,17 beta-dihydroxyoestra-1,3,5(10)-trien-7 alpha-yl)-N-methylundecanamide], were devoid of oestrogenic activity but possessed potent antioestrogenic activity. Comparison of receptor binding and biological potency of steroid 7 alpha- and 7 beta-isomers showed that activity is confined largely to the 7 alpha-isomer. Comparison of the effects of tamoxifen and ICI 164,384 on progesterone receptor (PR) concentration in the rat uterus showed that, unlike tamoxifen, ICI 164,384 did not induce PR and blocked induction of PR by oestradiol. Chronic treatment of mature female rats with ICI 164,384 led to an ovariectomy-like regression of the uterus without affecting LH secretion or the rate of growth. ICI 164,384 was also an effective antitumour agent in rats bearing carcinogen-induced mammary tumours.     

Cooperation of proto-signals for nuclear accumulation of estrogen and progesterone receptors.

Multiple proto-signals (p-NLSs) for nuclear targeting, none of which suffices on its own, cooperate in the estrogen (ER) and progesterone (PR) receptors. In the ER, an estrogen-inducible p-NLS was found in the hormone binding domain (HBD), in addition to three lysine/arginine-rich motifs resembling prototype constitutive nuclear localization signals (NLSs). The inducible and the constitutive ER p-NLSs cooperate in the presence of estrogen and hydroxy-tamoxifen, but not in the presence of ICI 164,384. In the PR, three p-NLSs, two of which are located within and directly adjacent to the second zinc finger, cooperate with each other and a weak hormone-inducible p-NLS in the PR HBD. No 'masking' of p-NLSs by the HBD was observed for ER and PR, while the ligand-free glucocorticoid receptor HBD inhibited the activity of both homologous and heterologous NLSs. Nuclear co-translocation experiments indicated that in vivo the stability of ER and PR dimers is hormonally controlled, but that, in the absence of the cognate ligand, ER dimers are more stable than PR dimers. This is likely to account for the differential hormone requirement of ER and PR DNA binding in vitro.     

Hormone-regulated v-rel estrogen receptor fusion protein: reversible induction of cell transformation and cellular gene expression.

We describe the construction of a v-rel estrogen receptor fusion protein (v-relER) which allows the regulation of v-rel oncoprotein activity by hormone. In the presence of estrogen, v-relER readily transformed primary chicken fibroblasts and bone marrow cells in vitro. In both cell types, v-rel-specific transformation was critically dependent on the presence of estrogen or the estrogen agonist 4-hydroxytamoxifen (OHT). Withdrawal of estrogen or application of an estrogen antagonist, ICI164,384 (ICI) caused a reversal of the transformed phenotype. We also demonstrate that the v-relER protein binds to NF-kappa B sites in an estrogen-dependent manner, thereby showing that sequence-specific DNA binding of v-relER is critical for the activation of its transforming capacity. In transient transfection experiments, we failed to demonstrate a clear repressor or activator function of the v-rel moiety in v-relER. However, in v-relER-transformed bone marrow cells, estrogen and OHT induced elevated mRNA levels of two cellular genes whose expression is constitutive and high in v-rel-transformed cells. These results suggest that v-rel might exert part of its activity as an activator of rel-responsive genes.     

Comparative studies on the effects of steroidal and nonsteroidal oestrogen antagonists on the proliferation of human breast cancer cells

Pure and partial agonist antioestrogens, exemplified by ICI 164,384 and tamoxifen or 4-hydroxytamoxifen (4'-OHT) respectively, differ in their capacity to inhibit the growth of MCF-7 human breast cancer cells. Under basal conditions which maintain but do not permit proliferation of MCF-7 cells, growth rate was enhanced by oestradiol, Phenol Red, insulin and 4'-OHT but not by ICI 164,384. Oestradiol and insulin together enhanced cell growth rate synergistically. 4'-OHT, but not ICI 164,384, similarly increased insulin-stimulated cell growth in the absence of oestradiol. ICI 164,384 blocked the stimulatory action of 4'-OHT. The stimulatory effect of the peptide growth factors TGF-alpha and IGFI on MCF-7 cells were attenuated by ICI 164,384 and 4'-OHT; ICI 164,384 was more effective than 4'-OHT. The antiproliferative action of ICI 164,384 on oestradiol or growth factor stimulated MCF-7 cells was only weakly inhibited by TGF-beta antibodies.     

Altered phenotypic characteristics of T47d human breast cancer cells after prolonged growth in estrogen-deficient medium.

T47D human breast cancer cells were cultured in estrogen-deficient media for up to 32 months and the resulting cell line (L(hE(-))) exhibited unique phenotypic and genotypic characteristics. Compared to low passage (L) cells, the L(hE(-)) cells exhibited a significantly higher rate of proliferation, unique morphological features, advanced ploidy status and 5- to 10-fold higher levels of the estrogen receptor (ER) as determined by ligand binding and Western blot analysis. Sequence analysis of the DNA binding domain of the ER revealed a C-->A transversion which resulted in a H513N amino acid change. Treatment of L cells with 10 n m 17beta-estradiol (E2) resulted in a greater than two-fold increase in cell proliferation which was inhibited by tamoxifen, 4'-hydroxytamoxifen, ICI 164,384 and ICI 182,780. In contrast, 10 n m E2 caused a 70% decrease in growth of L(hE(-)) cells and this antimitogenic activity was blocked by ICI 164,384 and ICI 182,780 but not by tamoxifen or 4'-hydroxytamoxifen. L(hE(-)) cells were E2-responsive in transient transfection studies using a plasmid containing an estrogen-responsive element derived from the vitellogenin A2 gene promoter. These data show that the phenotypic and genotypic characteristics of L(hE(-)) T47D cells resemble those described for ER-negative cell lines stably transfected with the ER.     

Unregulated expression of c-Jun or c-Fos proteins but not Jun D inhibits oestrogen receptor activity in human breast cancer derived cells.

We present evidence that oestrogen receptor activity in human MCF-7 breast cancer cells is reduced by over-expression of c-Jun or c-Fos proteins and to a lesser extent by Jun B overexpression. In contrast, overexpression of Jun D protein does not affect the activity of the oestrogen receptor. A region of c-Jun found to be required for repression of oestrogen receptor activity is located outside the DNA binding domain and is not conserved among the three Jun proteins. Finally, we suggest that c-Jun and c-Fos act independently to inactivate the oestrogen receptor.     

Regulation of progesterone receptor mRNA by oestradiol and antioestrogens in breast cancer cell lines

The induction of progesterone receptor mRNA by oestradiol and antioestrogens has been characterised in the MCF-7 breast cancer cell line. Progesterone receptor mRNA was induced more than 100-fold by oestradiol. The induction was half-maximal in the presence of 10(-10) M oestradiol and maximum levels were reached after 24 h treatment. Progesterone receptor mRNA was induced to 10% of the oestrogen-induced level by tamoxifen and its metabolite 4'-hydroxytamoxifen. The increase was half-maximal in the presence of 5 X 10(-8) M tamoxifen or 5 X 10(-10) M 4'-hydroxytamoxifen. In contrast, neither the benzothiophene antioestrogen LY117018 nor the 7 alpha-alkyl steroidal antioestrogen ICI 164,384 had any effect on progesterone receptor mRNA. The progesterone receptor mRNA was also induced by oestrogen in a T47D subline and in two other oestrogen-responsive breast cancer cell lines (ZR-75, EFM-19). Tamoxifen was a partial oestrogen for progesterone receptor mRNA induction in each of these cell lines. The large induction of the progesterone receptor mRNA by oestrogen in all 4 breast cancer cell lines supports the contention that the progesterone receptor may be a good predictive marker of hormonal response in human breast cancer.     

Oestrogenic activity of parabens in MCF7 human breast cancer cells

Parabens (4-hydroxybenzoic acid esters) have been recently reported to have oestrogenic activity in yeast cells and animal models. Since the human population is exposed to parabens through their widespread use as preservatives in foods, pharmaceuticals and cosmetics, we have investigated here whether oestrogenic activity of these compounds can also be detected in oestrogen-sensitive human cells. We report on the oestrogenic effects of four parabens (methylparaben, ethylparaben, n-propylparaben, n-butylparaben) in oestrogen-dependent MCF7 human breast cancer cells. Competitive inhibition of [3H]oestradiol binding to MCF7 cell oestrogen receptors could be detected at 1,000,000-fold molar excess of n-butylparaben (86%), n-propylparaben (77%), ethyl-paraben (54%) and methylparaben (21%). At concentrations of 10(-6)M and above, parabens were are able to increase expression of both transfected (ERE-CAT reporter gene) and endogenous (pS2) oestrogen-regulated genes in these cells. They could also increase proliferation of the cells in monolayer culture, which could be inhibited by the antiestrogen ICI 182,780, indicating that the effects were mediated through the oestrogen receptor. However, no antagonist activity of parabens could be detected on regulation of cell proliferation by 17 beta-oestradiol at 10(-10)M. Molecular modelling has indicated the mode by which paraben molecules can bind into the ligand binding pocket of the crystal structure of the ligand binding domain (LBD) of the oestrogen receptor alpha (ERalpha) in place of 17beta-oestradiol; it has furthermore shown that two paraben molecules can bind simultaneously in a mode in which their phenolic hydroxyl groups bind similarly to those of the meso-hexoestrol molecule. Future work will need to address the extent to which parabens can accumulate in hormonally sensitive tissues and also the extent to which their weak oestrogenic activity can add to the more general environmental oestrogen problem.