The effects of histone acetylation on estrogen responsiveness in MCF-7 cells.

Because histone acetylation is implicated in the facilitation of specific gene transcription, the effect of increasing histone acetylation on the expression of an endogenous gene was investigated. The ability of trichostatin A (TSA), a histone deacetylase inhibitor, to potentiate the estradiol (E2) induction of progesterone receptor (PR) levels in MCF-7 cells was studied. Although TSA alone had no effect on PR synthesis, measured by a whole-cell binding assay with [3H]R5020, TSA potentiated the effect of 10(-11) ME2 such that 10 ng of TSA/mL approximately doubled the hormone response. When TSA was removed from the cells after various incubation times (24 and 48 h) by successive washings with TSA-free medium, it was determined that TSA was required throughout the 96-h incubation period in order to achieve maximal potentiation for the E2 response. In addition, TSA potentiated E2 induction of pS2 mRNA. These results suggested that the estrogen receptor (ER) complex might alter histone acetylation as part of the gene activation process. To test this directly, MCF-7 cells were incubated for 48 h with E2 followed by incubation with sodium [3H]acetate for 1 h. From two-dimensional polyacrylamide gel electrophoresis, an increase in total acetate incorporation into histones in estrogen- treated cells compared to control was observed as well as a preferential increase in the mono- and diacetylated histone H4. Experiments with lysine-specific antiacetylated H4 antibodies suggest a preferential increase in acetylation at lysine 16, but not 5, 8, or 12. The results of this study support an important role for histone acetylation in the mechanism of action of the ER.

Effect of the histone deacetylase inhibitor trichostatin A on the responsiveness of rat hepatocytes to dioxin.

Since histone acetylation has been implicated in the facilitation of specific gene transcription, we investigated the effect of increasing histone acetylation through inhibition of histone deacetylase on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induction of P4501A activity in cultured rat hepatocytes. Inhibition of histone deacetylation was accomplished with addition of trichostatin A (TSA) to the incubation medium, and P4501A activity was measured spectrofluorometrically by determination of the rate of resorufin formation by ethoxyresorufin-O-deethylase (EROD). While TSA alone (5-200 ng/mL) had no effect on EROD activity, TSA potentiated the effect of various concentrations (10(-12) to 10(-10) M) of TCDD. Addition of 200 ng TSA/mL with TCDD resulted in an increased EROD activity of approximately 200% compared with TCDD alone. When TSA was removed from the cells after various incubation times (2, 6, 24 hr) by successive washings with TSA-free medium, it was determined that TSA was required for 24 hr in order to potentiate the effects of a 48-hr incubation with TCDD. In addition to measurement of EROD activity, P4501A1 and 1A2 microsomal protein were determined by western immunoblotting analysis. While neither P4501A1 nor 1A2 was detectable in the presence of TSA alone, P4501A1 was present after incubation of cells with TCDD in the presence or absence of TSA. TCDD plus TSA also resulted in the formation of P4501A2. The results of this study suggest an important role for histone acetylation in the action of TCDD on induction of P4501A enzymes.

Estrogen receptor interaction with specific histones. Binding to genomic DNA and an estrogen response element.

Chromosomal proteins that impart high affinity and specificity to the binding of the estrogen receptor (ER) to DNA are termed estrogen receptor binding factors (ERBFs). Certain partially purified chromosomal protein fractions obtained from rabbit uterine chromatin by extraction with various molarities of GdnHCl when reconstituted to double-stranded DNA demonstrated high affinity binding for the ER. We report the purification and characterization of ERBFs in the chromosomal protein fraction extracted with 4 M GdnHCl (CP4) after large scale purification. These protein fractions were further purified by CL-Sepharose 6B column chromatography which resolved fractions from CP4 that recognized the ER bound by estrogen only or antiestrogen only. Thus, these hydrophobic chromosomal proteins enhanced the binding of the ER to reconstituted chromatin. To further investigate the interaction of ERBFs with ER, gel mobility shift assays were performed. The highly purified CP4 fraction with ERBF activity in the binding assay with reconstituted chromatin caused an increase in the formation of the retarded ER-estrogen responsive element (ERE) band. Thus, chromatin contains specific ERBFs for ER bound by estrogen which enhance the binding of ER to genomic DNA and a target ERE sequence. Further purification of the CL-Sepharose fraction with ERBF activity was achieved by preparative SDS-PAGE. ERBF activity was attributed to proteins with approximate molecular weights of 16,000, 13,000, and 12,000 and a pl of > 9.0. Peptides were partially sequenced by Edman degradation and were found to have identity with histones H2B and H4. A 17 kDa protein without ERBF activity was identified as H3. Since these histones were not readily extracted from chromatin with 3 M NaCl or 1-3 M GdnHCl, we postulate that some ERBFs may be histone variants or modified histones that display a very high affinity for DNA and ER.

Purification and characterization of an Ah receptor binding factor in chromatin.

Dioxin induces biological responses through interaction with a specific intracellular receptor, the Ah receptor, and the subsequent interaction of the Ah receptor with chromatin. We previously reported the binding of the Ah receptor, partially purified form rabbit liver, to receptor binding factors (termed AhRBFs) in chromatin. Rabbit liver chromatin proteins (CP) were isolated by absorption of chromatin to hydroxylapatite followed by sequential extraction with 3 M NaCl and 1-8 M guanidine hydrochloride (GdnHCl). In the present study, we continued the purification of the CP5 fraction, which exhibited AhRBF activity. The proteins in CP5 were separated by CL-Sepharose 6B column chromatography resolving lower molecular weight fractions. To assay for receptor binding, a portion of each Cl-Sepharose 6B fraction was reconstituted to rabbit double-stranded DNA (dsDNA) using a reverse gradient dialysis of 7.5 to 0.0 M GdnHCl. These reconstituted chromatins were then examined for binding to [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD)-receptor complexes by the streptomycin filter binding assay. Two protein fractions with a molecular weight in the range of 10,000-14,000 demonstrated high affinity binding to the Ah receptor. The binding of AhRBFs reconstituted to dsDNA was shown, by competition experiments with Ah receptor bound by unlabeled TCDD (TCDD-R), to be > 90% specific for [3H]TCDD-R. Further purification was achieved by preparative ADS-PAGE, and AhRBF activity was attributed to two fractions with molecular weights between 12,000 and 10,000. A kDa protein with AhRBF activity was found to have an isoelectric point (pI) of > or = 10. The 12 kDa AhRBF was sequenced by Edman degradation after cyanogen bromide cleavage and identified as histone H4. Although histone H4 has been postulated to interact with transcription factors in a variety of systems, this is the first report of a specific interaction of AhR with histone H4.

Interrelationships between nuclear structure and ligand-activated intracellular receptors.

The role of ligand-activated intracellular receptors in activation of gene expression most probably involves a multistep process that requires alteration in chromatin structure. Results of studies with members of the steroid hormone receptor superfamily as well as the ligand-activated member of the basic region/helix-loop-helix family of transcriptional activators suggests that two different nuclear structures and their associated nuclear proteins are important in the initial stages of gene activation: the nuclear matrix and nucleosomes. Cell- and tissue-specific nuclear matrix proteins and the variant and modified histones appear to be important for tissue and species specificity of ligand-induced responses. Because the function of a receptor may be limited in vivo by promoter and transcription factor accessibility, the various roles of nuclear ligand-receptor complexes may involve interaction with nuclear matrix proteins and/or nucleosomes. Tissue-specific structural nuclear proteins could control the conformation (looping through matrix attachment regions) of the DNA and unwinding or rearrangement of nucleosomes, thus providing specificity to the expression of certain genes. Modulation of cooperative elements required for gene activation may involve association of the gene promoter with the nuclear matrix together with the presence of nucleosomes. Thus, the series of events involved in ligand-receptor activation of genes requires alterations in chromatin structure, which allow access of the receptor complex to elements within the gene.

Binding of the Ah receptor to receptor binding factors in chromatin.

Dioxin induces biological responses through interaction with a specific intracellular receptor, the Ah receptor, and the subsequent interaction of the Ah receptor with chromatin. We report the binding of the Ah receptor, partially purified from rabbit liver, to receptor binding factors in chromatin. Rabbit liver chromatin proteins (CP) were isolated by adsorption of chromatin to hydroxylapatite followed by sequential extraction with 1-8 M GdnHCl. To assay for receptor binding a portion of each CP fraction was reconstituted to rabbit double-stranded DNA using a reverse gradient dialysis of 7.5 to 0 M GdnHCl. These reconstituted nucleoacidic proteins were then examined for binding to [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD)-receptor complexes by the streptomycin filter assay. Prior to the binding assay, [3H]TCDD-receptor complexes were partially purified by step elution from DEAE-cellulose columns. CP fractions 2, 5, and 7 were found to bind to the Ah receptor with high affinity. Scatchard analysis yielded Kd values in the nanomolar range. Competition with 2-fold excess unlabeled TCDD-receptor complexes was demonstrated, and binding was reduced markedly when the receptor was prepared in the presence of 10 mM molybdate. Such chromatin receptor binding factors (RBFs) may participate in the interaction of receptor with specific DNA sequences resulting in modulation of specific gene expression.

Effects of antiestrogen versus antiprogestin on transformed and nontransformed steroid receptors.

In order to determine if different physicochemical properties exist among antihormone-receptor complexes, we have compared the interaction of the antiprogestin RU486 with progesterone receptor (PR) versus the triphenylethylene antiestrogen H1285 (4-(N,N-diethyl-aminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl-alp ha'- ethylstilbene] with estrogen receptor (ER) from rabbit uterine tissue. Contrary to other reports, we observed no difference in the sedimentation properties of transformed PR (4S) when bound by the antagonist RU486 versus the progesterone agonist R5020 in either cytosol or DEAE partially-purified receptor preparations analyzed on sucrose gradients containing 0.3 M KCl. In addition, we found no difference in the sedimentation properties of these receptor preparations in the presence of 10 mM sodium molybdate: the nontransformed RU486-PR and nontransformed R5020-PR both sedimented as a 6S species. These same results were obtained when the receptor preparation and gradient analysis were performed in the absence of monothioglycerol. Likewise, there was no change in the sedimentation properties of the transformed PR when the receptor, partially purified in the absence of molybdate, was analyzed on sucrose gradients containing 10 mM sodium molybdate to prevent receptor alteration during centrifugation. From DNA-cellulose assays performed with partially purified PR in the absence of molybdate we determined that the 4S form of R5020-PR and RU486-PR is transformed receptor; whereas in the presence of molybdate, the 6S species is nontransformed. In contrast, we found a different pattern of sedimentation when comparing transformed antiestrogen-receptor complexes with transformed estrogen-receptor complexes. In this case, transformed H1285-ER sedimented as 6S and estradiol-ER sedimented as 4S. We conclude from these experiments that these two antihormones, RU486 and H1285, may have different mechanisms of action in their antagonism of steroid hormone action. Antiestrogen stabilizes the salt-transformed ER as a dimer while antiprogestin appears to permit dissociation of the oligomeric form of the receptor to the monomeric form.

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.

Binding of glucocorticoid receptors to mammary chromatin acceptor sites.

We have recently characterized the interaction of mouse mammary estrogen receptors (ER) with mammary chromatin acceptor sites and demonstrated that ER from estrogen resistant lactating mammary glands do not bind to chromatin. In this study we have characterized the chromatin binding of the glucocorticoid receptor from mouse mammary glands isolated from nulliparous and lactating mice in order to better understand the relationship between receptor binding to chromatin and steroidogenic sensitivity of the tissue. Mammary chromatin was linked covalently to cellulose and deproteinized sequentially by 0-8 M Gdn-HCl. Binding to intact chromatin as well as to chromatin deproteinized by Gdn-HCl was determined using partially purified [3H]dexamethasone labelled glucocorticoid-receptor complexes (GR) obtained by fractionation on DEAE-cellulose columns. The binding of [3H]GR from mammary glands of nulliparous mice to chromatin fractions from the same tissue revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 5-6 M Gdn-HCl. Binding of [3H]GR was of high affinity (Kd = 0.2 nM) and saturable. A simultaneous comparison of the chromatin binding patterns for [3H]ER and [3H]GR isolated from mammary glands of nulliparous mice revealed that the chromatin subfractions obtained with 4-6 M Gdn-HCl extraction contained acceptor sites for both [3H]ER and [3H]GR; however, while the [3H]ER bound to a 4.5 M and a 5.5 M site, the [3]GR bound a 5 M and a 6 M site. Competition experiments supported the steroid receptor specificity of the chromatin acceptor sites. Thus, the 4-6 M chromatin fractions contain distinct acceptor sites for the glucocorticoid receptor and for the estrogen receptor. In addition our studies reveal that the binding patterns of [3H]GR isolated from mammary glands of nulliparous and lactating mice to their homologous chromatin is essentially similar. Thus, in contrast to estrogen receptors, glucocorticoid receptors from lactating mammary glands are able to effectively bind to chromatin acceptor sites which supports our previous suggestion that the estrogenic insensitivity of lactating mouse mammary glands may at least be in part due to the impeded interaction of ER with chromatin acceptor sites.

Relationships between mammary estrogen receptor and estrogenic sensitivity. II. Binding of cytoplasmic receptor to chromatin.

Mammary glands from nulliparous mice are responsive to estradiol, whereas mammary glands from lactating mice are unresponsive, despite the presence of high concentrations of estrogen receptors. This study examined the relation between mammary estrogenic sensitivity and ability of mammary estrogen receptors to bind to intact chromatin as well as to partially deproteinized chromatin. Mammary chromatin was prepared from nulliparous and lactating mice, linked covalently to cellulose and deproteinized sequentially by 0-8 M guanidine chloride (Gdn X HCl). The binding of receptors to these various chromatin preparations was determined using partially purified [3H]estradiol-receptor complexes ([3H]ER) obtained by fractionation on diethylaminoethyl cellulose columns. The binding pattern of [3H]ER from nulliparous mice to chromatin fractions from either nulliparous or lactating mice revealed maximal binding activity with chromatin previously extracted with 4-6 M Gdn X HCl. Binding was of high affinity [dissociation constant (Kd) 3.6 X 10(-10) M], saturable and steroid receptor and species specific. However, mammary [3H]ER preparations from lactating mice bound poorly to intact chromatin as well as to the Gdn X HCl extracted chromatin fractions isolated from either mammary gland of nulliparous or lactating mice. In mixing experiments the estrogen receptor preparation from lactating mice decreased substantially the binding activity of [3H]ER from nulliparous mice to chromatin suggesting the presence of an inhibiting factor. Thus, these studies reveal that the unresponsiveness of lactating mammary glands to estradiol coexists with the inability of estrogen receptors from lactating mice to interact with specific high affinity sites on mammary chromatin and also that this impeded interaction of estrogen receptors with chromatin may be due to some inhibitor(s) present in the cytosol of lactating mammary glands.

Acceptor sites on chromatin for receptor bound by estrogen versus antiestrogen in antiestrogen-sensitive and -resistant MCF-7 cells.

We examined the chromatin binding characteristics of estrogen receptor from MCF-7 cells when bound by [3H] estradiol vs. the high affinity antiestrogen [3H]H1285 [4-(N,N-diethylaminoethoxy) 4'-methoxy-alpha-(p-hydroxyphenyl)alpha-ethylstilbene]. Two sublines of MCF-7 cells were used: E-3, which is sensitive to antiestrogens, and RR, which is antiestrogen resistant and was selected for its ability to grow in the presence of tamoxifen. Chromatin was prepared from both E-3 and RR cells, linked covalently to cellulose and deproteinized sequentially by 0-8 M guanidine hydrochloride (Gdn X HCl). The chromatin acceptor activity unmasked by Gdn X HCl was determined using partially purified (30-fold) activated [3H]estradiol- or [3H] H1285-receptor complexes obtained by KCl step elution from DEAE-cellulose columns. With chromatin from E-3 cells, maximal binding (acceptor activity) for [3H]estradiol-receptor complexes prepared from either type of MCF-7 cells (E-3 or RR) was unmasked by 1 and 6 M Gdn X HCl, whereas [3H]H1285-receptor complexes exhibited maximal binding to 1 and 4 M Gdn X HCl-extracted chromatin subfractions. Chromatin prepared from RR cells was similar to that from E-3 cells in its binding activity for [3H]estradiol-receptor complexes. It differed, however, in that [3H]H1285-receptor complexes showed less chromatin acceptor site binding in general to 1-8 M Gdn X HCl-deproteinized RR chromatin, and the binding peak unmasked by 4 M Gdn X HCl was absent in chromatin from these cells. Receptor binding to chromatin was stable and was competitively inhibited by radioinert estradiol- or H1285-receptor complexes (but not by denatured receptors), demonstrating the saturability and specificity of these acceptor sites. Thus, estrogen receptor binds differently to chromatin depending on whether estradiol or an antiestrogen is bound to it. In addition, the acquisition of antiestrogen resistance by the RR subline of MCF-7 cells appears to result from alterations in the state of its chromatin rather than changes in the receptor itself. Finally, the observation that the chromatin from the resistant cells differs from that of the sensitive cells suggests that antiestrogens may be able to inhibit the growth of MCF-7 and other antiestrogen-sensitive cells not only by antagonizing the stimulatory effect of estrogens, but also by exerting some separate effect of their own.

Estrogen and antiestrogen binding to rat uterine and pituitary estrogen receptor: evidence for at least two physicochemical forms of the estrogen receptor.

Our laboratory has previously reported that calf uterine cytosol prepared in buffer containing 10 mM molybdate and chromatographed on DEAE-Sephadex contains two forms of the unactivated estrogen receptor, Peak I and Peak II; however, cytosol receptor bound to the high-affinity antiestrogen, H1285 (4-(N,N-diethylaminoethoxy)-4'-methoxy-alpha- (p-hydroxyphenyl)-alpha'-ethylstilbene), eluted only as Peak I. We have extended these studies to the rat uterus and pituitary in order to determine the organ and species specificity of this phenomenon. Cytosol prepared in Tris-molybdate buffer from immature and adult rat uteri or pituitaries was labelled with 10 nM [3H]estradiol or [3H]H1285 and chromatographed on QAE-Sephadex. Uterine estrogen receptors bound to either [3H]estradiol or [3H]H1285 eluted from QAE-Sephadex as a large Peak I (approximately 0.21 M KCl) and a smaller Peak II (approximately 0.25 M KCl). Analyses of these partially purified estrogen receptor fractions using high-salt sucrose density gradients showed that Peak I [3H]estradiol-receptor complexes sedimented predominantly as a lighter form (4.0S). In contrast, Peak I [3H]H1285-receptor complexes sedimented primarily as a heavier form (5.5S) often accompanied by a smaller lighter form (4.0S). Peak II [3H]estradiol- and [3H]H1285-receptor complexes sedimented as the heavier form (5.3-5.5S). These data suggest a monomer-dimer relationship between estrogen receptor forms with antiestrogen binding favoring the formation of the dimeric form. Further analysis of these Peak I receptor complexes by gel filtration chromatography yielded molecular forms of approx 70 KDaltons for [3H]estradiol-receptor complexes and 73 KDaltons and 165 KDaltons for [3H]H1285-receptor complexes, supporting the monomer-dimer concept. Data from experiments with the pituitary also suggest that H1285 causes the formation of the dimeric receptor form whereas estradiol interaction with the receptor results only in the monomeric form. These differences in estrogen receptor forms when bound by estrogen versus antiestrogen may be related to the different biological responses induced by these ligands.

Antiestrogenic potency and binding characteristics of the triphenylethylene H1285 in MCF-7 human breast cancer cells.

The antiestrogenic character and potency of 4-(N,N-diethylaminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl)-alpha' -ethylstilbene (H1285) and its binding to estrogen receptor and to estrogen-noncompetible antiestrogen binding sites have been studied in MCF-7 human breast cancer cells. H1285 has an affinity for the estrogen receptor (Kd 0.23 nM) which is comparable to that of estradiol (Kd 0.25 nM), and the binding of these two compounds to estrogen receptor is mutually competitive. On high salt sucrose gradients, the sedimentation profiles of nuclear receptor complexes with H1285 and estradiol are different. While the sedimentation profile of the complex with estradiol varies with the buffer composition, being 4.1S in phosphate:thioglycerol: glycerol and predominantly 5.5S in Tris:EDTA buffered gradients, the H1285 receptor complex shows the same sedimentation (5.5S) regardless of the buffer composition. H1285 also binds to estrogen-noncompetable antiestrogen binding sites that are distinct from the estrogen receptor with a low affinity, only 15% that of the antiestrogen tamoxifen. The biological character and potency of H1285 were examined by determining its effects on cell proliferation, cellular progesterone receptor levels, and plasminogen activator activity. In MCF-7 cells, H1285 was a 30- to 100-fold more potent inhibitor of cell proliferation than was the antiestrogen tamoxifen, and it was approximately equipotent with the higher affinity antiestrogen trans-hydroxytamoxifen. H1285 evoked very minimal increases in cellular progesterone receptor levels, and no increase in plasminogen activator activity over a broad range of concentrations (10(-10)-10(-6)M), and it suppressed plasminogen activator activity stimulated by estradiol. Therefore, by the criteria we have used, we conclude that H1285 is a potent and very effective antiestrogen in MCF-7 cells. The ability of estradiol to reverse the suppression of cell proliferation by H1285, and the high affinity of H1285 for estrogen receptor and its low affinity for estrogen-noncompetible antiestrogen binding sites suggest that H1285 exerts its antiestrogenic effects via interaction with the estrogen receptor of these breast cancer cells.

Binding of [3H]triamcinolone acetonide-receptor complexes to chromatin from the B-cell leukemia line, BCL1.

The binding characteristics of partially purified glucocorticoid receptor complexes from hormone sensitive, non-differentiating BCL1 cells to sequentially deproteinized BCL1 chromatin-cellulose was investigated. [3H]Triamcinolone acetonide (TA)-receptor complexes were purified (approx. 30-fold) from DEAE-cellulose columns by salt elution which allowed receptor activation only in the absence of molybdate. Addition of 10 mM molybdate completely blocked salt activation. The binding pattern of the activated [3H]TA-receptor complexes to chromatin-cellulose extracted with 0-8 M guanidine hydrochloride revealed three regions of increased binding activity (acceptor sites), at 2, 5 and 7 M guanidine hydrochloride. Acceptor site binding was markedly reduced for chromatin extracted with 3, 6 and 8 M guanidine hydrochloride. Non-activated receptor complexes demonstrated very low binding to deproteinized chromatin. It was also shown that chromatin binding required glucocorticoid receptors and that free ligand or ligand bound to other proteins did not bind significantly to chromatin. In addition, binding of [3H]TA-receptor complexes to partially deproteinized chromatin was competable by unlabeled TA-receptor complexes. Scatchard analysis demonstrated that chromatin from non-differentiating BCL1 cells possesses multiple, high-affinity binding sites which differ in their affinity for the glucocorticoid receptor. Partially deproteinized chromatin from lipopolysaccharide-stimulated BCL1 cells demonstrated a different pattern of receptor binding, i.e., receptor binding was significantly greater to chromatin previously extracted with 6-8 M guanidine hydrochloride. These results suggest that differentiation alters the state of chromatin and the interaction of non-histone protein/DNA acceptor sites with glucocorticoid receptors. These alterations may play a role in the acquisition of hormone resistance.

Interaction of the antiestrogen [3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor.

The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.

Analysis of two forms of molybdate-stabilized estrogen receptor.

We recently reported that the calf uterine estrogen receptor prepared in 10-mM molybdate elutes as two components (peaks I and II) from DEAE-Sephadex columns with a linear KCl gradient. We have extended these studies and report that unoccupied cytosol receptors also elute as two peaks (0.21 and 0.25 m KCl), as determined by postlabeling experiments with [3H]estradiol. Since molybdate-stabilized cytosol estrogen receptors incubated in the presence of 0.3 M KCl as well as estrogen receptors in peaks I and II did not demonstrate receptor binding to DNA-cellulose, we conclude that both receptor forms are nonactivated. Also, [3H]estradiol-receptor complexes from cytosol prepared in 10 mM molybdate sediment as 6.7S in 5-20% sucrose density gradients containing 0.3 M KCl, whereas receptor complexes from cytosol prepared without molybdate sediment as 4.5S. Both peaks I and II were eluted from DEAE-Sephadex columns prepared with either 10 mM molybdate or 10 mM tungstate, with both phosphatase inhibitors having effectively blocked salt activation of the receptor. However, receptor preparations in the presence of 10 mM arsenate were not eluted from DEAE-Sephadex, and arsenate was unable to inhibit receptor activation by KCl. Sucrose density gradient analysis of peaks I and II indicates that peak I sediments at approximately 4.8S, whereas peak II sediments at approximately 6.3S. If pooled fractions of the leading portion of peak I are rechromatographed on another DEAE-Sephadex column, both peaks I and II are recovered. Likewise, if pooled fractions of the descending portion of peak II are rechromatographed, both peaks I and II were measured. Therefore, we conclude that there may be an equilibrium between two forms of the molybdate-stabilized calf uterine estrogen receptor.

Activation of [3H]estradiol--and [3H]H1285-receptor complexes: effect of salt versus ATP on molybdate-stabilized estrogen receptors.

The activation by salt or ATP of [3H]estradiol- and [3H]H1285-receptor complexes from rabbit uterus and their binding capacity to DNA-cellulose, phosphocellulose and ATP-Sepharose has been studied. The estrogen-receptor was prepared in 1 mM molybdate which stabilized the receptor; but both salt- and ATP-transformation of estrogen receptors occurred. The binding of molybdate-stabilized cytosol [3H]estradiol-receptor complexes to the various resins revealed that salt-activation by 0.3 M KCl caused the greatest binding (5-6-fold) to DNA-cellulose as compared to other resins. However, 5 mM ATP-dependent activation of receptor-complexes resulted in preferential binding to ATP-Sepharose. Activated cytosol [3H]H1285-receptor complexes bound all the resins to a lesser degree when compared to [3H]estradiol-receptor complexes. Partially purified receptor complexes also showed different resin-binding patterns for salt- and ATP-mediated activation. These findings suggest that salt-activation is different than ATP-activation. Further, the differential magnitude of [3H]estradiol- and [3H]H1285-receptor activation suggests that estrogen-receptor complexes are "fully" activated as compared to "partially" activated antiestrogen-receptor complexes.

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin.

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [3H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [3H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physiochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.

Binding of the estradiol-receptor complex to reconstituted nucleoacidic protein from calf uterus.

Non-histone protein-DNA complexes with acceptor activity for estradiol-receptor complexes were reconstituted from fractionated calf uterine chromatin. Acceptor activity had tissue specificity with target tissue binding exceeding non-target tissue binding. The binding of estradiol-receptor complexes to acceptor sites was dependent on intact non-histone protein-DNA complexes, reconstituted select non-histone proteins, and protein equivalent: DNA reconstitution ratios. [3H]Estradiol-receptor complexes were bound to reconstituted non-histone protein-DNA complexes (i.e., nucleoacidic protein) with a high affinity and with a limited number of binding sites. Fractionation of uterine chromatin non-histone proteins identified two major sets of non-histone proteins which had acceptor activity when reconstituted with DNA. Thus, it seems possible to reconstitute nucleoacidic protein fractions with specific acceptor activity for the calf uterine estrogen receptor.

Interaction of the radiolabelled high-affinity anti-oestrogen [3H]H1285 with the cytoplasmic oestrogen receptor.

The high-affinity triarylethylene anti-oestrogen H1285 [4-(NN-diethylaminoethoxy)-beta-ethyl-alpha-(p-hydroxyphenyl) -4'-methoxystilbene] was tritiated to high specific radioactivity (35 Ci/mmol). Competition experiments between [3H]H1285 and H1285 or oestradiol demonstrated that both compounds would compete with [3H]H1285 for oestrogen-specific binding sites in rat uterine cytosol. [3H]H1285 had at least 10 times the affinity for the receptor compared with oestradiol at the 50% competition level. [3H]H1285 appeared to have at least twice the association rate for the oestrogen receptor compared with [3H]oestradiol. In addition, the dissociation half-life (t1/2) of specific binding of [3H]H1285 to oestrogen receptors at 0 degrees C was about 220 h compared with a value of 60 h for [3H]oestradiol. Because of the extremely slow dissociation of [3H]H1285 from the oestrogen receptor, we were able to compare the sedimentation profiles of [3H]H1285-receptor complexes with those of [3H]oestradiol-receptor complexes in the presence of 0.4 M-KCl on 5-20% sucrose density gradients. [3H]Oestradiol-receptor complexes had a major peak at 4.4 S with a smaller peak at 5.6 S, whereas with [3H]H1285-receptor complexes the 5.6 S peak was always higher than the 4.4 S peak. There was significant variation between the dissociation behaviour at 20 degrees C of [3H]H1285-receptor complexes and [3H]oestradiol-receptor complexes pre-activated at 25 degrees C for 30 min in the presence and in the absence of 10 mM-sodium molybdate. The dissociation t1/2 of [3H]oestradiol-receptor complexes at 20 degrees C decreased from 1.5 h to 0.5 h when molybdate was present during heat treatment whereas the dissociation t1/2 for [3H]H1285-receptor complexes was 5 h for both conditions. These observations indicate that there are fundamental differences in the initial interaction of H1285 and oestradiol with the oestrogen receptor.