Tamoxifen and metabolites in MCF7 cells: correlation between binding to estrogen receptor and inhibition of cell growth.

The binding of [3H]tamoxifen ([3H]Tam), a nonsteroidal antiestrogen, and of 4-[3H]hydroxytamoxifen ([3H]OH-Tam), a metabolite accumulated in vivo in target cell nuclei, was characterized in soluble extracts of human breast cancer MCF7 cells growing in a medium depleted in estrogens. Saturation analysis indicated a much higher affinity for OH-Tam (Kd = 0.15 nM) than for Tam (Kd = 4.8 nM). The binding of [3H]Tam and [3H]estradiol was competitive and mutually exclusive, and the binding site concentration (0.16 to 0.47 pmol/mg total protein) was similar for both ligands, strongly suggesting that antiestrogens were binding to the estrogen receptor (ER) in these cells. The ability of Tam and of some of its metabolites or derivatives to prevent the MCF7 cell growth was found to be correlated with their affinity for ER as determined by direct interaction or by binding competition with [3H]estradiol on the uterine and MCF7 cytosol ER. OH-Tam was the highest-affinity compound and was 100-fold more active than Tam. The inhibitions observed were actually due to Tam and OH-Tam, respectively, since we did not detect any significant metabolism of these two labeled compounds by the MCF7 cells. N-desmethyltamoxifen, the other Tam metabolites found in high concentration in human plasma, was as effective as Tam while cis-tamoxifen appeared less effective. Compound E, which has no lateral chain, was the only tested compound having a good affinity for ER and a poor efficiency in preventing cell growth. These results support the hypothesis that antiestrogens control the growth of breast cancer by acting directly on the ER located in cancer cells.

Effect of ligand and DNA binding on the interaction between human transcription intermediary factor 1alpha and estrogen receptors.

Hormonal regulation of gene activity is mediated by nuclear receptors acting as ligand-activated transcription factors. To achieve efficient regulation of gene expression, these receptors must interact with different type of molecules: 1) the steroid hormone, 2) the DNA response element, and 3) various proteins acting as transcriptional cofactors. In the present study, we have investigated how ligand and DNA binding influence the in vitro interaction between estrogen receptors (ERs) and the transcription intermediary factor hTIF1alpha (human transcriptional intermediary factor 1alpha). We first optimized conditions for the coactivator-dependent receptor ligand assay to lower ED50, and we then analyzed the ability of various natural and synthetic estrogens to allow the binding of the two types of proteins. Results were compared with the respective affinities of these ligands for the receptor. We then developed a protein-protein-DNA assay allowing the quantification of cofactor-ER-estrogen response element (ERE) complex formation in the presence of ligand and used measurements of fluorescence anisotropy to define the equilibrium binding parameters of the interaction. We demonstrated that the leucine-charged domain of hTIF1alpha is sufficient to interact with ERE-bound ERalpha in a ligand-dependent manner and showed that binding of ERalpha onto DNA does not significantly affect its hormone-dependent association with TIF1alpha. Finally, we show that, mainly in the absence of hormone, hTIF1alpha interacts better with ERbeta than with ERalpha independently of the presence of ERE.

Cordycepin and early effects of estradiol on the immature rat uterus.

Estradiol induces the synthesis of a specific protein fraction (IP) in the uterus of the immature rat. The injection of cordycepin (3' deoxyadenosine), an inhibitor of poly A synthesis, inhibits the synthesis of IP. This fact suggests that one of the earliest effects of estrogen is the production of Hn-RNA poly-A relative to IP. Moreover, using electron microscopy, the stimulation by estradiol of the nucleolus of the immature rat uterine epithelium has been shown. Cordycepin does not affect this stimulation to any appreciable extent. Biochemical studies (incorporation of radioactive stracers into NRA, affinity chromatography on poly U-Sepharose) carried out in parallel with and under conditions comparable to those used in electron microscopy show that cordycepin does not greatly affect the increase in ribosomal RNA observed under the effect of estradiol. The blocking of IP by cordycepin and the lack of inhibition at the nucleolus level under the same conditions, show that the two early effects of the action of estrogen on the immature rat uterus are not directly correlated.

Steroidal affinity labels of the estrogen receptor. 1. 17 alpha-(Bromoacetoxy)alkyl/alkynylestradiols.

To develop steroidal affinity labels for the estrogen receptor, we prepared five electrophilic estradiol derivatives bearing the 17 alpha-propyl, 17 alpha-(1'-butynyl), or 17 alpha-(1'octynyl) chain, with either a terminal epoxy function (for the 17 alpha-propyl substituent) or a terminal bromoacetoxy function (for all three 17 alpha-substituent types). These compounds displayed low affinity for the lamb uterine estrogen receptor; with apparent relative affinity constants ranging from 0.02% to 0.24% that of estradiol. They were also rapidly transformed in cytosol, probably to the corresponding vicinal diols (epoxy compounds) or primary alcohols (bromoacetoxy compounds). Nevertheless, bromoacetates induced irreversible inactivation of the hormone-binding site but only with ligand-free binding sites. The effect of bromoacetates was prevented by treatment of the cytosol with the thiol-specific reagent methyl methanethiosulfonate. Inactivation of the receptor at 0 degrees C was rapid (< 1 h) and strongly dependent on both compound concentration and pH, with significant effects obtained at either > 150 nM (at pH 9) or pH > 7.5 (at 5 microM). Regardless of the conditions used, the order of efficiency for bromoacetates was always: 17 alpha-propyl derivative < 17 alpha-butynyl derivative < 17 alpha-octynyl derivative, with maximal inactivation of approximately 30% and approximately 70% of the hormone-binding sites obtained for the less active and more active compounds, respectively. Characteristics of the receptor inactivation suggest that (i) prepared bromoacetates are highly reactive affinity labels for the estrogen receptor, (ii) they react with similar (or even a single) nucleophilic amino acid residues located within or near the hormone-binding site of the receptor; these residues are probably the -SH of cysteines, and (iii) position 17 alpha of steroidal ligands is suitable for introducing electrophilic substituents to develop efficient affinity labels for the receptor.

Steroidal affinity labels of the estrogen receptor. 2. 17 alpha-[(Haloacetamido)alkyl]estradiols.

In a previous study, we described affinity labeling of the lamb uterine estrogen receptor by 17 alpha-[(bromoacetoxy)alkyl/alkynyl]estradiols. However, the intrinsic receptor-alkylating activities of these compounds were probably very hampered by their poor hydrolytic stability in estrogen receptor-containing tissue extracts. Therefore, (i) to develop affinity labels of the receptor not susceptible to hydrolysis and (ii) to specify the structural requirements for 17 alpha-electrophilic estradiol derivatives to be potent affinity labels of the receptor, we prepared four 17 alpha-[(haloacetamido)alkyl]estradiols. Three were bromoacetamides differing at the alkyl substituent (methyl, ethyl, or propyl), and the last was an [(iodoacetamido)propyl]estradiol prepared under both nonradioactive and 3H-labeled forms. Although their affinities for the estrogen receptor were very low (from 0.008% to 0.02% that of estradiol), they appeared to be efficient affinity labels of the receptor due to their irreversible inhibition of [3H]estradiol specific binding in lamb uterine cytosol. The effect of the compounds was time-, pH-, and concentration-dependent, with > 50% and > 80% estrogen-binding sites inactivated at 0 degrees C and pH 8.5, for the less active and more active compounds, respectively; the corresponding IC50 values varied from approximately 20 nM to approximately 10 microM. The order of efficiency was [(bromoacetamido)methyl]estradiol < [(bromoacetamido)ethyl]estradiol << [(bromoacetamido)propyl]estradiol < [(iodoacetamido)propyl]estradiol. Affinity labeling was directly demonstrated by ethanol-resistant binding of [3H][(iodoacetamido)propyl]estradiol to the receptor. The irreversible inactivation of the hormone-binding site by the four haloacetamides was prevented by treatment of the cytosol with the thiol-specific reagent methyl methanethiosulfonate, suggesting that the target of these compounds was probably the -SH of cysteines. Negative results obtained with other 17 alpha-electrophilic estradiol derivatives suggested that affinity labeling of the receptor by such derivatives required a minimal distance, including at least four C-C or C-N bonds, between the steroid and the electrophilic carbon. We therefore concluded that target cysteines in the hormone-binding site were not in direct contact with the steroid but probably in the immediate neighborhood of the D ring of the bound steroid.

Steroidal affinity labels of the estrogen receptor alpha. 4. Electrophilic 11beta-aryl derivatives of estradiol.

Ten electrophilic estradiol 11beta-aryl derivatives were synthesized, with three different types of 11beta-substituent: (i) pOO(CH(2))(2)X (compounds: 6, X = OSO(2)CH(3); 7, X = I; 13, X = NHCOCH(2)Cl; 15, X = N(CH(3))COCH(2)Br; and 16, X = N(CH(3))COCH(2)Cl); (ii) pOO(CH(2))(5)X (compounds: 17, X = I; 20, X = NHCOCH(2)Br; and 22, X = N(CH(3))COCH(2)Br); and (iii) pOC(triple bond)CCH(2)X (compounds: 27, X = NHCOCH(2)Cl; and 29, X = N(CH(3))COCH(2)Cl). The range of their apparent affinity constants for binding the lamb uterine estrogen receptor alpha (ERalpha) was 3-40% that of estradiol. Six electrophiles, chloroacetamides 13, 16, 27, and 29, iodide 17, and bromoacetamide 20 (whose arm linking the electrophilic carbon to the 11beta-phenyl group includes at least six bonds), were able to irreversibly inhibit the binding of [(3)H]estradiol to ER (25-60% decrease in binding sites), with the following compound effectiveness order: 17 < 13 < 16 approximately 20 approximately 27 approximately 29. Mesylate 6, iodide 7 (whose linking arm includes only three bonds), and bromoacetamides 15 and 22 (which differ from 16 by the Cl to Br change and from 20 by the NH to NCH(3) change, respectively) were much less effective (<10% decrease in binding sites, if any). The fact that the inactivation of estradiol-binding sites by the six electrophiles was totally prevented by estradiol indicated that they were ER affinity labeling agents. When ER was modified by methyl methanethiosulfonate, an SH-specific reagent, the different compounds led to very contrasting results in ER affinity labeling. With modified ER, iodide 17 and chloroacetamides 27 and 29 were practically inactive, chloroacetamides 13 and 16 and bromoacetamide 20 were still active but less effective than on the native ER, whereas tertiary bromoacetamides 15 and 22, found to be practically inactive on native ER, became the most effective electrophiles ( approximately 45% and approximately 65% binding sites inactivated, respectively). The results indicate that in the steroid-filled hormone-binding pocket: (i) nucleophilic residues are localized on the beta-side but relatively remote from the steroid nucleus (distance from C-11 > "seven bonds"); (ii) relatively discrete changes in the electrophilic functionality, such as Cl to Br or NH to NCH(3) of haloacetamido compounds, can markedly modify the positioning of the electrophilic center which could no longer react with the nucleophilic residues; and (iii) cysteine residues (probably homologues of human ERalpha cysteine 381 and/or cysteine 530) are, at least partly, the covalent attachment sites of the electrophiles. Moreover, modification of cysteine residues by methyl methanethiosulfonate changes the structure of the hormone-binding pocket, whose labeling by the various electrophiles is profoundly altered.

Steroidal affinity labels of the estrogen receptor. 3. Estradiol 11 beta-n-alkyl derivatives bearing a terminal electrophilic group: antiestrogenic and cytotoxic properties.

With the aim of developing a new series of steroidal affinity labels of the estrogen receptor, six electrophilic 11 beta-ethyl (C2), 11 beta-butyl (C4), or 11 beta-decyl (C10) derivatives of estradiol bearing an 11 beta-terminal electrophilic functionality, i.e. bromine (C4), (methylsulfonyl)oxy (C2 and C4), bromoacetamido (C2 and C4), and (p-tolylsulfonyl)oxy (C10), were synthesized. The range of their affinity constants for binding the estrogen receptor was 0.4-37% that of estradiol; the order of increasing affinity (i) relative to the 11 beta-alkyl arm was ethyl < butyl and (ii) relative to the electrophilic functionality was bromoacetamido < bromine < (methylsulfonyl)oxy. Regardless of the conditions used, including prolonged exposure of the receptor to various pH levels (7-9) and temperatures (0-25 degrees C), the extent of receptor affinity labeling by the 11 beta-ethyl and 11 beta-butyl compounds, if any, was under 10%. This was in sharp contrast to results obtained using 11 beta-((tosyloxy)decyl)estradiol which labeled from 60% to 90% of the receptor hormone-binding sites with an EC50 of approximately 10 nM. Estrogenic and antiestrogenic activities of the compounds were determined using the MVLN cell line, which was established from the estrogen-responsive mammary tumor MCF-7 cells by stable transfection of a recombinant estrogen-responsive luciferase gene. The two 11 beta-ethyl compounds were mainly estrogenic, whereas the three 11 beta-butyl and the 11 beta-decyl compounds essentially showed antiestrogenic activity. The fact that the chemical reactivities of 11 beta-ethyl and 11 beta-butyl compounds were not compromised by interaction with the estrogen receptor made the synthesized high-affinity compounds potential cytotoxic agents which might be able to exert either (i) a specific action on estrogen-regulated genes or (ii) a more general action in estrogen-target cells. Therefore the ability of the compounds (1) to irreversibly abolish estrogen-dependent expression of the luciferase gene and (2) to affect the proliferation of MVLN cells were determined. All electrophiles were able to irreversibly suppress expression of the luciferase gene; the antiestrogenic electrophiles were more potent than the estrogenic ones but less efficient than 4-hydroxytamoxifen, a classical and chemically inert triphenylethylene antiestrogen. Only the antiestrogenic electrophiles decreased cell proliferation; however, they were less potent than 4-hydroxytamoxifen. In conclusion, the synthesized electrophilic estradiol 11 beta-ethyl and 11 beta-butyl derivatives (i) were not efficient affinity labels of the estrogen receptor and (ii) did not display significant cytotoxicity in estrogen-sensitive mammary tumor cells. However, since these derivatives displayed high affinity for the estrogen receptor, they could be used to prepare potential cytotoxic agents which might be selective for tumors affecting estrogen-target tissues, by coupling them with a toxic moiety.

Ellipticine derivatives with an affinity to the estrogen receptor, an approach to develop intercalating drugs with a specific effect on the hormone-dependent breast cancer.

In order to obtain breast tumor directed agents, we have prepared mixed compounds using estradiol or (E)-clomiphene as specific vectors of the breast tissue and a DNA intercalator from the ellipticine series as the cytotoxic agent. Among the newly synthesized ellipticine derivatives, only the 2-[3-aza-5-(3,17 beta-dihydroxy-1,3,5-estratrien-17 alpha-yl)-4-oxopentamethylene]ellipticinium bromide shows the desired properties, DNA intercalation and affinity for estrogen receptor. Competition experiments with estradiol on the hormone-dependent human MCF-7 breast cancer cell line demonstrate that a transport by the estrogen receptor system is not involved in the antitumor activity of derivative 24.

Mode of action of LN 1643 (a triphenylbromoethylene antiestrogen): probable mediation by the estrogen receptor and high affinity metabolite.

After in vivo administration of [3H]LN 1643, a triphenylbromoethylene antiestrogen, to immature female rats, polar metabolites were selectively accumulated in uterine nuclear fractions which contained most of the estrogen receptor. One metabolite comigrated with the 4-hydroxylated derivative (LN 2839) of LN 1643. LN 1643 and LN 2839 inhibited competitively and reversibly the binding of estradiol to the estrogen receptor, and the affinity of LN 2839 for the estrogen receptor was about 150-fold higher than that of LN 1643. Both compounds prevented the growth of the MCF7 human breast cancer cells and LN 2839 was about 10-fold more efficient than LN 1643. These results and previous data obtained with tamoxifen (a parent triphenylethylene antiestrogen) and its 4-hydroxylated metabolite, suggest that the antiestrogenic action of LN 1643 is mediated by the estrogen receptor as for the other synthetic antiestrogens, and that LN 1643 acts at least partly via its 4-hydroxy metabolite.

High-affinity binding to the estrogen receptor of [3H]4-hydroxytamoxifen, an active antiestrogen metabolite.

The tritiated 4-hydroxytamoxifen (OHT), an active metabolite of tamoxifen found in estrogen target nuclei, was prepared in vitro and its interaction with the cytosol estrogen receptor (R) of uterus and chick oviduct was specified. OHT bound to the 8S R with the same affinity and the same kinetic parameters as estradiol (E2). Its ability to protect the E2-binding sites and to stabilize the R--DNA interaction was also similar to that of E2. We conclude that the binding characteristics in vitro of ligands on the cytosol R do not presently allow us to discriminate between agonist and antagonist ligands.

The dissociation rate of estrogen receptor-ligand complexes is increased by high concentrations of steroids and antiestrogens.

The first-order dissociation rate constant, k-, of estradiol from uterine estrogen receptor, measured kin the presence of micromolar concentrations of diethylstilbestrol, increased linearly over a large concentration range (0-300 microM) of diethylstilbestrol. The experimental K- measured appears to be the sum of a basal dissociation rate constant corresponding to the spontaneous dissociation in the absence of diethylstilbestrol, and a diethylstilbestrol-induced dissociation rate constant, which is proportional to both the diethylstilbestrol concentration and the inverse of the cytosol concentration. Diethylstilbestrol induced the dissociation of estradiol in all species studied (lamb, calf and rat) and of estrone and 2 antiestrogens in lamb uterus. Various steroids and triphenylethylene antiestrogens also efficiently induced the dissociation of estradiol from the estrogen receptor. However, the potency of these inducers, which varied greatly, was not correlated with the binding affinity for the estrogen receptor. Structural characteristics and the hydrophobicity of the inducers, however, did appear to be important parameters. The relative efficiency of inducers varied depending on the ligand that was bound to the receptor. This induced dissociation allows the complete dissociation of estrogen receptor-[3H]-ligand complexes in a short time (less than 24 h) at low temperatures without alteration of the level of [3H] ligand bound non-specifically and can therefore be used to measure the [3H] ligand bound to the receptor by exchange at 0-4 degree C. From the specificity and the high doses of inducers required to make possible the observation of a significant effect, we conclude that the induced dissociation probably does not have a biological role.

New steroidal nitrosoureas.

Reformatsky reaction of 3beta, 21-diacyloxy-and 3beta-methoxy-21-acyloxy-5-pregnen-20-one with ethyl bromoacetate yields, through an intramolecular 1,2-acyl migration, 38, 20 XI-diacyloxy- and 3 beta-methoxy-20 XI-acyloxy-14alpha-card-5-enolide respectively. The 20 XI-acyloxy-14, alpha-card-5-enolides were converted into the respective 20 XI-hydroxy-14alpha-card-5-enolides and the 14 alpha card-5,20(22)-dienolides. Experimental support to the proposed intramolecular 1,2-acyl migration is provided by the use of labelled compounds.

[The elucidation of the antiestrogen and antitumoral mechanisms of tamoxifen]. / Contribution à l'élucidation du mécanisme d'action antioestrogénique et antitumoral du tamoxifène.

Our contribution to elucidation of the mechanism of action of tamoxifen can be summarized as follows: (i) hydroxylated metabolites of tamoxifen (especially 4-hydroxytamoxifen), with high affinity for the oestrogen receptor are more potent antioestrogen and antitumoral agents than tamoxifen; they might play an important role in the in vivo antioestrogenic and antitumoral activities of tamoxifen; (ii) the activity of tamoxifen and derivatives in vitro is closely related to their affinity for the receptor; (iii) certain oestrogen receptor properties (dissociation kinetics, immunoreactivity, sensitivity to specific reagents) vary according to whether oestrogen or antioestrogen is bound to the receptor. These variations probably result from different "positioning" of oestrogen and antioestrogen at the receptor hormone-binding site. They indicate that antioestrogens may induce altered conformation and probably defective activation of the oestrogen receptor. This could be the cause of the antioestrogenic and antitumoral effects of these compounds. It is now widely accepted that the oestrogen receptor is the major if not the only mediator of antioestrogenic and antitumoral effects of triphenylethylene antioestrogens in mammary tumor cells. These compounds efficiently promote dimerization and binding of the receptor to target DNA. Their relative lack of oestrogenic activity could result from (i) their inability to activate the transactivating function of the receptor involved in transcription regulation of certain oestrogen-target genes; (ii) the accumulation of anomalous receptor forms; and (iii) deficient phosphorylation state of the receptor. Besides their conventional antioestrogenic activity, antioestrogens could also exert action which antagonizes the "oestrogenic" effects of various growth factors in cells expressing the oestrogen receptor. This action could cause antitumoral effects in the absence of oestrogens. Two main mechanisms could account for the resistance to antioestrogens in sensitive mammary tumor cells: (i) loss of oestrogen-dependent expression of growth factors, and (ii) change in the dominant activity of antioestrogens (antagonist-->agonist) in the cells, whose proliferation would be then stimulated by antioestrogens. New steroidal "pure" antioestrogens have been developed. These compounds seem capable of promoting dimerization and then receptor binding to target DNA. However, they could induce rapid and marked decreases in the oestrogen receptor concentration in cells, which might account for their antioestrogenic activity. These compounds are able to antagonize the oestrogenic effects of triphenylethylene antioestrogens and especially the growth of resistant mammary tumors stimulated by the latter compounds. They are of great interest for the future treatment of mammary breast tumors expressing the oestrogen receptor.

Differential interactions of estrogens and antiestrogens at the 17 beta-hydroxy or counterpart function with the estrogen receptor.

The action of diethylpyrocarbonate on lamb uterine estrogen receptor produced an homogeneous population of the receptor (approximately 55%) which still bound triarylethylene antiestrogens such as 4-hydroxytamoxifen with a high affinity but bound classical potent estrogens such as estradiol or diethylstilbestrol with a very low affinity. To specify the structural features of the ligands involved in the decrease of ligand affinity upon modification of the estrogen receptor, we determined the relative affinity constants of 17 steroidal estrogens or antiestrogens (deriving from estradiol by a 7 alpha- or 11 beta-substitution) and 14 nonsteroidal estrogens or antiestrogens (all including the 1,2-trans-diphenylethylene structure of diethylstilbestrol) for native and diethylpyrocarbonate-modified estrogen receptors. Then the ratio of the relative affinity constant for the native receptor to that for the modified receptor (rho) was calculated for each ligand, to compare the variation in the affinity of the ligand upon modification of the receptor to that of 4-hydroxytamoxifen (rho = 1). The results showed that the strong decrease of ligand affinity upon modification of the receptor displayed by classical estrogens (rho greater than or equal to 200) is strictly dependent on the presence of the 17 beta-hydroxyl group in steroidal compounds or its alpha-4- and beta-4-counterparts in diethylstilbestrol-related compounds. However, for the 7 alpha- or 11 beta-derivatives of estradiol displaying potent antiestrogenic properties, the relative decrease in affinity was much more limited (rho less than or equal to 19). For 11 beta-derivatives displaying a relative estrogenic activity weaker than that of estradiol itself, an average decrease in affinity was observed (23 less than or equal to rho less than or equal to 62). With the diethylstilbestrol-related compounds, bearing or not the alpha-4-hydroxyl and/or the beta-4-hydroxy functions and showing either weak relative estrogenic or antiestrogenic properties, the relative variation in affinity was weak (0.6 less than or equal to rho less than or equal to 24). These results indicate that the interaction of 7 alpha- or 11 beta-substituted steroidal antiestrogens and of 1,2-trans-diphenylethylene or triphenylethylene derivatives, displaying either weak relative estrogenic or antiestrogenic properties with the receptor, differs at the 17 beta-hydroxy or at the alpha-4-/beta-4-hydroxy functions from that of potent estrogens. They suggest that the strong decrease in the relative affinity of ligands upon receptor modification may reflect the high efficiency of the ligands to activate the receptor properly.(ABSTRACT TRUNCATED AT 400 WORDS)

[In vivo occupation of estrogen receptors by hydroxylated metabolites of tamoxifen]. / Occupation in vivo des récepteurs oestrogènes par des métabolites hydroxylés du tamoxifène.

We report that after in vivo administration of (3H) tamoxifen, the cytosol and nuclear estrogen receptor sites of Rat uterus and Chicken oviduct are mostly occupied by polar metabolites. One of the major metabolites is 4-hydroxy-tamoxifen which we have identified by cocrystallisation withe non radioactive compound and which is known to display a high affinity for the estrogen receptor. In the Rat uterus, the proportion of the metabolites versus tamoxifen, increases with time with a maximum at 8 hrs. for the 4-hydroxy-tamoxifen. Other hydroxylated metabolites (M2) became predominant after 24 hrs. We propose that in vivo, the synthetic antiestrogens act mostly via their transformation into hydroxylated metabolites.

Differences between oestrogen receptor activation by oestrogen and antioestrogen.

Triphenylethylene antioestrogens such as tamoxifen, nafoxidine and Ci 628 specifically inhibit oestrogen action at the target cell level, probably by interacting with the oestrogen receptor (ER) and competitively displacing oestrogens from their binding sites. It is not clear, however, why these ligands are less biologically active than oestrogens when the bind to the ER, as no reliable difference has been found either in the binding affinity of these two series of ligands to the ER or in their ability to translocate the ER to the nucleus. In fact, these antioestrogens are transformed in vivo into hydroxylated metabolites which display a better antioestrogenic activity than the injected compound and at least the same high affinity as oestradiol for the ER. With the aim of finding an in vitro criterion to predict the agonistic or antagonistic properties of ER ligands, we have stabilized the ER in its 'native' or non-activated form by the use of molybdate and have compared the binding of oestradiol (E2) and of 4-hydroxytamoxifen (OHT), an active metabolite of tamoxifen, to the molybdate-treated and to the activated ER. We report here that molybdate prevented the DNA binding and the 4S to 5S transformation of the ER bound to both ligands, and that it increased the dissociation rate of oestrogens but not that of antioestrogens. Moreover, in the absence of molybdate, receptor activation by heating decreased the dissociation rate of E2 but not that of OHT. We conclude that a difference exists between the ER activation triggered by oestrogens and antioestrogens and propose that antioestrogens are acting as allosteric ligands of the ER.

Hydroxylated metabolites of tamoxifen are formed in vivo and bound to estrogen receptor in target tissues.

After in vivo administration of [3H]tamoxifen to immature female rats and chickens, polar metabolites of tamoxifen were found in plasma, liver, uterus, and oviduct. 4-Hydroxytamoxifen and M2, another hydroxylated metabolite of tamoxifen, were the major tritiated compounds of the cytosol and the KCl-nuclear extract of target tissues and appeared to occupy the estrogen receptor sites since their accumulation in these fractions was saturable, resistant to charcoal, and prevented by estradiol. In the rat uterus, 4-hydroxytamoxifen was predominant during the 24 h following the [3H]tamoxifen injection, then its concentration declined while M2 became predominant. 4-Hydroxytamoxifen was also found in chicken oviduct where tamoxifen is acting as a full estrogen antagonist. Moreover, liver, chicken oviduct, and lamb uterus were able to convert tamoxifen into 4-hydroxytamoxifen in vitro. Other estrogen target tissues, such as the rat uterus, dimethylbenz (a) anthracene-induced rat mammary tumors, and MCF7 cells, did not transform tamoxifen significantly. 4-Hydroxytamoxifen formed in vitro was able to bind selectively to estrogen receptor with a high affinity and with a low dissociation rate similar to estradiol. These results demonstrate that 4-hydroxytamoxifen is formed in vivo and retained on estrogen receptor in target tissues, due to its high affinity. Since we have separately shown that 4-hydroxytamoxifen is a full anti-estrogen, more potent than tamoxifen itself in MCF7 cells, we conclude that tamoxifen is mostly acting in vivo indirectly via hydroxylated metabolite(s).