Proliferation of breast cancer cells: regulation, mediators, targets for therapy.

A majority of breast cancers (BC) display characteristics of epithelial cells and express estrogen receptors and/or HER-2 (a member of the epidermal growth factor receptor family). About one-fifth of BC is constituted of basal cells for which there is no specific category of proliferation regulators. Insulin-like growth factor (IGF) signaling is involved in most BC cells, irrespective of cell type. All inducers of cell proliferation employ transcriptional as well as non-transcriptional mechanisms to activate the cascade of cyclin-dependent kinases, which causes irreversible progression to the G1/S phase transition. We analyze the pathways of the different inducers that lead to this cascade. Several actors in the mitogenic signal transduction are required irrespective of the initial signal although their functions may differ: for example members of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) cascades. As some of these proteins are also involved in the cell survival mechanisms, they appear to be good targets for therapeutic intervention. In the case of the estrogen-dependent cells, complex interplay between the estrogen receptor (a conditional transcription factor), co-repressors and co-activators offers additional molecular targets for therapy. Besides, we have found that p21(WAF1), an inhibitor of cyclin-dependent kinases, can orient the cell to either proliferation or differentiation suggesting that at an early stage of BC development it may be possible to reverse the cellular changes associated with malignant transformation.

B-ind1, a novel mediator of Rac1 signaling cloned from sodium butyrate-treated fibroblasts.

Sodium butyrate is a multifunctional agent known to inhibit cell proliferation and to induce differentiation by modulating transcription. We have performed differential display analysis to identify transcriptional targets of sodium butyrate in Balb/c BP-A31 mouse fibroblasts. A novel butyrate-induced transcript B-ind1 has been cloned by this approach. The human homologue of this transcript contains an open reading frame that codes for a protein of 370 amino acids without known functional motifs. In transfected cells, the B-ind1 protein has been found to potentiate different effects of the small GTPase Rac1, such as c-Jun N-terminal kinase activation and transcriptional activity of nuclear factor kappaB (NF-kappaB). In addition, we have demonstrated that B-ind1 forms complexes with the constitutively activated Rac1 protein. To investigate the role of B-ind1 in Rac1 signaling, we have constructed several deletion mutants of B-ind1 and tested their ability to affect the activation of NF-kappaB by Rac1. Interestingly, the fragment encoding the median region of human B-ind1 acted as a dominant-negative variant to block Rac1-mediated NF-kappaB activity. These data define B-ind1 as a novel component of Rac1-signaling pathways leading to the modulation of gene expression.

Estrogen induction of the cyclin D1 promoter: involvement of a cAMP response-like element.

Estrogens induce cell proliferation in target tissues by stimulating progression through the G(1) phase of the cell cycle. Induction of cyclin D1 expression is a critical feature of the mitogenic action of estrogen. We have determined a region between -96 and -29 in the cyclin D1 promoter that confers regulation by estrogens in the human mammary carcinoma cells MCF-7. This region encompasses a unique known transcription factor binding site with a sequence of a potential cAMP response element (CRE-D1). The induction is strictly hormone dependent and requires the DNA binding domain as well as both AF-1 and AF-2 domains of the estrogen receptor (ER) alpha. Destruction of the CRE-D1 motif caused complete loss of estrogen responsiveness. Both c-Jun and ATF-2 transactivated the cyclin D1 promoter in transient transfection experiments, and a clear additional increase was detected when ER was cotransfected with either c-Jun or with c-Jun and ATF-2 but not with ATF-2 alone. Furthermore, the expression of a dominant negative variant of c-Jun, TAM67, completely abolished the induction of the cyclin D1 promoter both in the absence and presence of ER. We show that ATF-2 homodimers and ATF-2/c-Jun heterodimers, but not c-Jun homodimers, were able to bind the CRE of the cyclin D1 promoter. To interpret these results, we propose a mechanism in which ATF-2/c-Jun heterodimers bind to the CRE-D1 element and mediate the activation of cyclin D1 promoter by the ER. This mechanism represents a pathway by which estrogens control the proliferation of target cells.

Oestrogen receptor facilitates the formation of preinitiation complex assembly: involvement of the general transcription factor TFIIB.

The action of oestrogen hormones is mediated through the oestrogen receptor (ER), a member of a large superfamily of nuclear receptors that function as ligand-activated transcription factors. Sequence-specific transcription factors, including the nuclear receptor superfamily, are thought to interact either directly or indirectly with general transcription factors to regulate transcription. Although numerous studies have focused on the identification of potential co-activators interacting with isolated trans-activation domains of ER, few have investigated the mechanisms by which ER transmits its signal to the basal transcription machinery. We show that ER does not stabilize the binding of the TATA-box binding protein (TBP) of the TFIID complex, or of TFIIB to the promoter, although a stable ER-TBP-TFIIB-promoter complex was detected, suggesting that ER, TBP and TFIIB might interact with each other to form a complex to the promoter. We also demonstrate that ER binds specifically to TFIIB, a key component of the preinitiation complex. Affinity chromatography with immobilized deletion mutants of ER maps a TFIIB interaction region that encompasses the DNA-binding domain. The addition of excess TFIIB to transcription reactions in vitro did not, however, affect the magnitude of transcriptional activation by ER. These results indicate that, in contrast with current models, ER does not activate transcription by increasing the rate of assembly of TFIIB into the transcription complex. An increased concentration of TFIIB was unable, by itself, to overcome the requirement for ER. By using an immobilized promoter-template assay employing nuclear extract from HeLa cells, recombinant human ER increased the stable association of subsequent components of the transcription machinery (TFIIE and TFIIF), in correlation with ER-induced transcription. Our results suggest that ER acts, in an early step, during or immediately after the formation of template-committed complexes containing TFIIB, favouring the recruitment of one or more components of the basic transcription machinery as well as co-activators.

Properties of overlapping EREs: synergistic activation of transcription and cooperative binding of ER.

We have designed a novel estrogen-responsive unit, overERE, which consists of two overlapping ERE separated by 5 bp (center-to-center). In gel retardation assays, this sequence forms a low-mobility complex that migrates like an estrogen receptor tetramer. The receptor-overERE complex was specific and was supershifted by anti-ER H222 antibodies. Dose response studies showed that the formation of the receptor tetramer-overERE complex was cooperative. Truncated receptors were used to assess the contribution of the receptor domains. Deletion of the E domain of the ER prevented the formation of an ER-tetramer complex, which reflects a novel function of this receptor domain. In transfection experiments, 17-beta-estradiol activated transcription from an overERE-containing promoter 4-6 times better than from an ERE-containing promoter. This synergistic effect was observed using either the natural hormone (17-beta-estradiol) or xenoestrogens (phenol red, chlordane). We conclude that two overlapping estrogen-responsive elements can elicit synergistic induction of transcription.

Direct inhibition of the expression of cyclin D1 gene by sodium butyrate.

In the mouse fibroblasts BP-A31 as well as in the human epidermoid carcinoma cells KB-3-1, both cyclin D1 mRNA and protein contents decreased rapidly during incubation with sodium butyrate. The decrease of cyclin D1 mRNA was not prevented by cycloheximide indicating that protein synthesis is not required for the inhibition of the expression of cyclin D1 gene by sodium butyrate. The 973 bp region upstream of the human cyclin D1 gene conferred inhibition of the expression of an indicator gene in transiently transfected cells. An 11 base-pair segment situated within this region, with a strong homology to the butyrate-response consensus element identified in butyrate-inducible promoters, also caused an inhibition of transcription under these conditions, indicating that cyclin D1 expression is inhibited by butyrate at the transcriptional level.

The 90 kDa heat-shock protein (hsp90) modulates the binding of the oestrogen receptor to its cognate DNA.

The role of heat-shock protein 90 (hsp90) in the regulation of the oestrogen receptor (ER) function is less well understood than for other steroid-hormone receptors because hsp90 is not involved in the stabilization or induction of a high-affinity ligand-binding state of ER nor in the inhibition of receptor dimerization. Electrophoretic mobility-shift assays, using purified ER and hsp90, were employed to investigate directly the effect of hsp90 on the ability of ER to bind to the oestrogen-response element (ERE) from the vitellogenin A2 gene. Contrary to models in which hsp90 binds to and passively inactivates steroid-hormone receptors, our studies show that the binding of ER to ERE is inversely dependent on the relative concentration of hsp90. Exposure of purified ER-hsp90 complexes to ERE led to the dissociation of hsp90 and concomitant specific binding of ER to ERE. We demonstrate that the amount of ER-ERE complex decreased with increasing concentrations of hsp90. Furthermore hsp90 dissociated preformed high-affinity ER-ERE complexes. Kinetic dissociation experiments indicate the hsp90 acts in a dynamic and specific process rather than by simple trapping of ER owing to its inherent off-rate. The receptor released from the ERE-bound state by hsp90 was recovered associated with hsp90 and was able to rebind to ERE. These results indicate that hsp90 does not suppress ER function merely by steric hindrance. On the basis of these results and others, we propose that, in vivo, hsp90 may play a dual role in ER function: (i) at a physiological temperature, hsp90 stabilizes an active form of the receptor in accordance with its general molecular chaperone role; (ii) at elevated temperatures or under other environmental stress, the increased cellular concentration of hsp90 negatively interferes with ER-dependent transcription, in accordance with the inhibition of gene transcription attributed to hsp90 after heat shock.

Interplay between estrogens, progestins, retinoic acid and AP-1 on a single regulatory site in the progesterone receptor gene.

Transcriptional regulation of the progesterone receptor gene involves induction by estrogens and down-regulation by progestins, retinoic acid, and AP-1 proteins. We have previously identified an intragenic (+698/+723) estrogen-responsive element present in the progesterone receptor gene, which binds the estradiol receptor and mediates estrogen and 4-OH tamoxifen induction. Progesterone receptor gene expression was equally stimulated by estradiol and 4-OH tamoxifen in the presence of a NH2 terminally deleted estrogen receptor mutant lacking activation function 1, suggesting that activation function 2 was the predominant activation domain. This was confirmed by the lack of activity of an estrogen receptor mutant deleted of activation function 2. Repression by progestins, retinoic acid, and AP-1 was mediated by the same estrogen responsive element although retinoic and progesterone receptors as well as AP-1 proteins did not bind to this element. Repression by these proteins appears to involve different transactivating regions of the estrogen receptor. Repression by retinoic receptors involved only activation function 2 whereas repression by progesterone receptor and AP-1 necessitated both functional domains. Since these proteins act without directly contacting the DNA, it seems likely that repression may be achieved by protein-protein interactions among different domains of the estrogen receptor and/or the transcriptional machinery.

Estrogen receptor-induced bending of the Xenopus vitellogenin A2 gene hormone response element.

DNA bending is increasingly proposed as an essential step for the establishment of the multiprotein complexes required for transcription initiation. Polyamines and metallic cations, known to promote DNA-bending, enhance the binding of purified estrogen receptor (ER) to the estrogen response element (ERE) of the Xenopus vitellogenin A2 gene. Using both circular permutation electrophoretic mobility and cyclization assays, we provide evidence that ER bends the DNA at the estrogen response element. The same bending occurs as a result of estrogen receptor protein binding independently of its conformational changes induced by hormone or anti-hormone. We suggest a role of the observed DNA bending in estrogen-regulated transcription.

Characterization of the hormone responsive element involved in the regulation of the progesterone receptor gene.

The transcription of the progesterone receptor gene is induced by estrogens and decreased by progestins. Studies were performed to define the regions of the gene and the molecular mechanisms involved. No hormonal regulation could be observed using 5' flanking regions of the gene up to -2762 in front of a heterologous gene. Estrogen and progestin regulation could be observed only when using fragments of the gene extending down to +788. Progressive deletions from the 5' and 3' ends, site-directed mutagenesis and DNase protection experiments with purified estrogen receptor suggested that the biologically active estrogen responsive element (ERE) is present at +698/+723, overlapping the initiation of translation. An oligonucleotide was synthesized bearing this ERE and shown to impart estrogen inducibility to a heterologous gene. Its regulation by anti-estrogens corresponded to that of the in situ progesterone receptor gene since tamoxifen was a partial agonist whereas ICI 164384 was a full antagonist. This ERE also mediated down-regulation by progestins in the presence of the progesterone receptor, even though it has no progesterone receptor binding ability. DNase footprinting showed that this effect was not due to a decrease of estrogen receptor affinity for the ERE in the presence of progesterone receptor. Finally, use of deletion mutants of the progesterone receptor showed that the steroid binding and the DNA binding domains were necessary for down-regulation whereas deletions of various parts of the N-terminal domain were without effect.

Hormonal regulation of vitellogenin genes: an estrogen-responsive element in the Xenopus A2 gene and a multihormonal regulatory region in the chicken II gene.

Expression of the vitellogenin genes in avian and amphibian liver is regulated by estrogens. The DNA elements mediating estrogen induction of the various vitellogenin genes of chicken and Xenopus encompass one or more copies of a 13-mer palindromic sequence called the estrogen-responsive element (ERE). Here we show that upon incubation with the purified estrogen receptor (ER) from calf uterus the Xenopus vitellogenin A2 gene yields a DNase-I footprint over the ERE between -331 and -319. This element does not mediate the response to glucocorticoids or progestins in T47D cells. The three guanine residues in each half of the palindrome are protected against methylation by dimethylsulfate after incubation with ER, but not with glucocorticoid (GR) or progesterone (PR) receptors. In contrast, the chicken vitellogenin II gene exhibits multihormonal regulation by estrogens, progestins, and glucocorticoids in T47D and MCF7 cells. Regulation is mediated by the DNA region between -721 and -591 that contains four binding sites for hormone receptors, as demonstrated by DNase-I footprints and methylation protection experiments. The two distal and most proximal binding sites are recognized by ER, GR, and PR, whereas the central binding site is only bound by ER and GR. At suboptimal concentrations, estrogens and progestins or glucocorticoids act synergistically. In experiments using a DNA fragment containing an ERE adjacent to a glucocorticoid-responsive element/progesterone-responsive element, ER and PR bind synergistically to their corresponding sites, perhaps explaining the functional synergism of both hormones. Thus, two very different regulatory elements are used to mediate estrogen induction of related genes in chickens and amphibians.

Structural differences between the hormone and antihormone estrogen receptor complexes bound to the hormone response element.

To investigate the molecular mechanism(s) by which the estrogen receptor (ER) modulates transcription, we compared the structures of receptor complexes containing estradiol, the agonist diethylstilbestrol, and the antagonist ICI164,384. The binding of ICI164,384 to nontransformed 8-9S ER does not preclude the salt-induced dissociation of the 90-kDa heat shock protein and releases the transformed homodimeric 5S ER as classically observed in the presence of agonist. We report that calf ER binds to the estrogen response element of the Xenopus vitellogenin A2 gene in either the absence or presence of hormone, agonist, or antagonist. These binding interactions were highly sequence- and receptor-specific. These findings indicate that ligand binding in vitro is not absolutely required for dimerization or specific DNA binding of the ER. As demonstrated by gel retardation assays, the ICI164,384-ER complex bound to the response element displays a slower mobility than complexes formed in the presence of estradiol or agonist. This difference in electrophoretic mobility is suggestive of a conformational change in the complex induced by the ligand. An exchange experiment demonstrated that this alteration of the structure is reversible. We suggest that ICI164,384 induces conformational modifications within the ligand-binding domain of the receptor that do not prevent binding to the response element but could fail to promote subsequent events required for gene transcription.

Several regions of human estrogen receptor are involved in the formation of receptor-heat shock protein 90 complexes.

Several mutants of the human estrogen receptor (ER) were transiently expressed in Cos 7 cells in order to determine the regions involved in the formation of complexes with the heat shock protein Mr approximately 90,000 (hsp 90). The formation of the cytosol non-DNA binding 8-9 S complexes (8-9 S ER) was monitored by glycerol gradient ultracentrifugation. It was established that the N-terminal region of the receptor, including the two zinc fingers of the DNA binding domain (DBD), is not required for the formation of the 8-9 S ER complexes. Conversely, deletion of the entire ligand binding domain (LBD) produced truncated receptor mutants that are constitutive transcriptional activators and did not form 8-9 S ER complexes, confirming results obtained previously with the glucocorticosteroid receptor (Pratt, W.B., Jolly, D.J., Pratt, D.V., Hollenberg, S.M., Giguerre, V., Cadepond, F., Schweizer-Groyer, G., Catelli, M.G., Evans, R.M., and Baulieu, E.E. (1988) J. Biol. Chem. 263, 267-273). However, no limited subregion of the LBD was found to be uniquely involved in hsp 90 binding. A highly positively charged region situated at the C-terminal extremity of the DBD (between amino acids 251 and 271) also appeared to be implicated. Although not sufficient, this sequence is necessary for the formation of the 8-9 S ER; it also corresponds to the NL1 nuclear localization domain of steroid receptors. Taken together, these results suggest that the formation of complexes with hsp 90 involves several receptor regions, and they are consistent with the proposal that hsp 90 inhibits receptor function and can be released by hormone binding to the LBD.

Role of chromatin structure in transcriptional regulation of MMTV LTR hormone-dependent promoter.

Studies of chromatin structure were performed in mouse fibroblast cell lines containing Bovine Papilloma Virus (BPV) based artificial minichromosomes containing Mouse Mammary Tumor Virus (MMTV) Long Terminal Repeat (LTR), a retroviral promoter regulated by glucocorticoids, driving the transcription of v-Ha-ras. These minichromosomes fractionate with the "active chromatin", indicating an association of the minichromosomes with components of the "nuclear matrix". Two regions of the minichromosomes upstream and downstream of v-Ha-ras are involved in this interaction. MMTV LTR promoter is associated with nucleosomes precisely positioned on the DNA sequences. Hormonal activation is accompanied by a structural change of the nucleosome associated with the hormone response elements (HREs). This structural change can be visualized by the appearance of a hormono-dependent DNaseI hypersensitive site. Anti-hormones, even when able to promote a strong binding of the receptor to the nucleus, are unable to induce the chromatin structural change. The strong association of the hormone-receptor complex with the nucleus is necessary to induce the DNaseI hypersensitive site and to maintain the transcription, but is not necessary for DNaseI hypersensitivity maintenance. This suggests a double role for the hormone-receptor complex: 1) induction of a chromatin rearrangement and 2) transcriptional transactivation.

Subunit composition of the estrogen receptor. Involvement of the hormone-binding domain in the dimeric state.

The purified estrogen receptor (ER) whether in 9 or 5 S molecular form, binds more than one molecule of the monoclonal antibody JS 34/32 (Redeuilh, G., Moncharmont, B., Secco, C., and Baulieu, E.-E. (1987) J. Biol. Chem. 262, 6969-6975). We now have investigated the effects of controlled trypsin proteolysis and of a dissociating chaotropic salt (NaSCN) on the structure of the estrogen receptor covalently labeled with radioactive tamoxifen aziridine. When the DNA-binding transformed 5S ER was dialyzed against a buffer containing 0.5 M NaSCN, it was converted into a form sedimenting at 3.7 S +/- 0.1 (n = 3). It reverted to the 5 S molecular form when NaSCN was dialyzed away. Fluorographic analysis of both the 5 and 3.7 S ER following SDS-gel electrophoresis revealed one main band corresponding to Mr congruent to 66,000. After limited trypsin treatment of the 5 S ER, tamoxifen aziridine-binding protein sedimented at 4.3 S +/- 0.1 (n = 5), had a Stokes radius of 3.6 nm (calculated Mr = 65,000), and did not bind DNA. The same form was obtained after limited trypsin digestion of the ER bound to DNA-cellulose or to hsp 90 (the nontransformed 8-9 S ER molecular form). This 4.3 S trypsinized ER was reversibly dissociated by NaSCN into a congruent to 3 S +/- 0.1 (n = 3) molecular form. Fluorographic analysis of both the 4.3 and 3 S ER after SDS-gel electrophoresis showed one main radioactive band of Mr congruent to 30,000. Taken together our results suggest that 1) the 5 S ER is a homodimer of two Mr congruent to 66,000 hormone binding subunits which may be released as such from the nontransformed 8-9 S ER, 2) the trypsin digestion products yield two carboxyl-terminal fragments of Mr congruent to 30,000 that remain in the form of a dimer having lost their DNA-binding region, and 3) the trypsin cleavage would occur within the region located between the hormone-binding domain and the DNA-binding domain. These data indicate that the dimerization of the receptor occurs through hydrophobic interaction of its hormone-binding domain but cannot exclude that other part(s) of the receptor may also contribute to the dimer formation. The dimerization may be critically involved in the mechanism by which estradiol-receptor complexes promote change of gene transcription.

The binding activity of estrogen receptor to DNA and heat shock protein (Mr 90,000) is dependent on receptor-bound metal.

1,10-Phenanthroline inhibited the DNA-cellulose binding of the transformed calf uterus estrogen receptor (homodimer of 66-kDa molecules: 5 S estrogen receptor) in a temperature- and concentration-dependent manner. This result appears related to the metal-chelating property of 1,10-phenanthroline, since the inhibition was decreased by addition of Zn2+ and Cd2+, but not by Ca2+, Ba2+, or Mg2+ for which the affinity of the chelator is low. Only a slight inhibition was observed in the presence of the 1,7-phenanthroline, a nonchelating analogue. After dialysis or filtration to remove free 1,10-phenanthroline, DNA binding of the 5 S estrogen receptor was still inhibited. Conversely, the chelator was unable to release prebound 5 S estrogen receptor from DNA-cellulose. The 5 S estrogen receptor DNA binding was inhibited when 1,10-phenanthroline was present during the transformation to activated receptor of the hetero-oligomeric nontransformed 9 S estrogen receptor, in which the hormone binding subunits are associated with heat shock protein, Mr 90,000 (hsp 90) molecules. In contrast, if 1,10-phenanthroline was removed before the transformation took place, only a slight inhibition was observed. Other experiments with EDTA indicated a similar inhibition of DNA-cellulose binding by the 5 S estradiol receptor, and all metal ions chelated by this agent prevented its inhibitory effect. The results indicate that 1,10-phenanthroline inhibited the DNA binding of the transformed 5 S estradiol receptor by chelating metal ion tightly bound to the receptor, which is not accessible to the chelator when the receptor is bound to DNA or to hsp 90. Therefore, they suggest that the metal ion may play a critical role in the interaction with DNA and hsp 90 by maintaining the structural integrity of the implicated receptor domain.

Subunit composition of the molybdate-stabilized "8-9 S" nontransformed estradiol receptor purified from calf uterus.

The structure of the calf uterus nontransformed molybdate-stabilized estradiol receptor (ER) has been investigated using affinity labeling with tamoxifen aziridine and several monoclonal antibodies directed either against the steroid binding protein (Mr approximately 65,000) or against the heat shock protein of Mr approximately 90,000 (hsp 90). The purification was performed using affinity chromatography and a DEAE-Sephacel column. The [3H] estradiol-ER complex was obtained as a well-defined radioactive peak, the specific activity varying between 1,600 and 3,400 pmol/mg of protein. The purified ER sediments in glycerol gradients at 9.4 S +/- 0.2 (n = 5) and at 8.1 S +/- 0.2 (n = 15) in a 0.15 M KCl containing gradient ("8-9 S" ER). From a measured Stokes radius of 7.4 +/- 0.2 nm (n = 12), an Mr of approximately 300,000 has been calculated. Studies of the purified 8-9 S ER by glycerol gradient centrifugation and by "twin antibody" assay with the JS34/32 anti-ER monoclonal antibody suggest the presence of two binding subunits in the nontransformed molecular complex. Results of immunological analysis with polyclonal and several monoclonal antibodies against hsp 90 suggest the association of two molecules of this protein to the two steroid binding subunits. In high salt medium (0.4 M KCl), the purified ER sediments at 5.2 +/- 0.3 (n = 8), has a Stokes radius of 5.7 nm +/- 0.1 (n = 2) and the Mr is approximately 129,000, values expected for a homodimer consisting of two hormone-binding subunits (Mr approximately 65,000), a result confirmed by glycerol gradient centrifugation experiments, using the monoclonal antibody JS34/32. The relationship between the nontransformed 8-9 S ER and the transformed 5 S-ER forms are discussed, the simplest possibility being the release of the already formed homodimeric ER from 8-9 S ER during transformation.

Transformation of the 8-9 S molybdate-stabilized estrogen receptor from low-affinity to high-affinity state without dissociation into subunits.

The rate of dissociation of labeled estradiol from [3H] estradiol-8-9 S receptor complexes ([3H]E2-8-9 S ER) molybdate-stabilized was determined in the presence of either an excess of unlabeled hormone ("chase") or of charcoal/dextran suspension ("stripping"). Biphasic dissociation of the hormone was observed in both cases, but the fraction of the fast-dissociating component was dramatically reduced (5% instead of 60%) when stripping was used. As the dissociation patterns were independent of the degree of saturation of the receptor, the results do not favor the possibility of cooperative effects between binding sites in the 8-9 S ER. After pretreatment of cytosol by charcoal at 28 degrees C for 15 min, the dissociation studied by chase displayed only the slowly dissociating component (t1/2 approximately 65 min). This effect was dependent on temperature and influenced by the ligand bound to 8-9 S ER, being pronounced with estradiol (E2) and absent with [3H]4-hydroxytamoxifen. The slow-dissociating component obtained after charcoal treatment was reconverted to fast-dissociating state by adding dithiothreitol or by incubation with cytosol at 20 degrees C. The charcoal treatment did not change the sedimentation coefficient (approximately 9 S) and the Stokes radius (approximately 7 nm) of the [3H]E2-8-9 S ER, and the slow-dissociating form obtained did not bind to DNA-cellulose either in the presence or absence of molybdate ions. Thus there are likely small but functionally significant changes of structure in the 8-9 S ER which remain in a non-DNA-binding form, whereas the rate of estradiol dissociation is modified.

The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography.

Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.