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B S Katzenellenbogen

Publications and source records attributed to B S Katzenellenbogen.

At least 73 records · Page 4Linked to original sources
PubMed1996

Identification of amino acids in the hormone binding domain of the human estrogen receptor important in estrogen binding.

The initial step in the regulation of the transcriptional activity of the estrogen receptor (ER) is the binding of hormone. Previous studies have suggested that the region between amino acids 515 and 535 near the C terminus of the human ER is likely to be important in ligand binding. In order to explicitly define which amino acids in this region are critical for ligand recognition and binding, we have utilized alanine-scanning mutagenesis over the complete 515-535 region. The ability of these 21 mutants to activate transcription in response to the natural estrogen, 17beta-estradiol (E2), was evaluated in cell co-transfection assays with estrogen-responsive reporter genes. In addition, their ability to bind E2 was also tested. Mutations at four sites in the 521-528 region had the greatest effects on E2-induced transcription, with L525A reducing responsiveness 250-fold, G521A and H524A 35-fold, and M528A 11-fold. Mutations at other sites had either no effect or a 4-fold or lesser reduction in sensitivity to E2 (M517A, Y526A, N532A, and P535A). Three of the mutants most affected in their transcriptional response, G521A, H524A, and M528A, showed a coordinate reduction in E2 binding affinity. E2 binding by the most affected mutant, L525A, could not be detected. Thus, the altered transcriptional response of these ER mutants appears to derive solely from an alteration in their affinity for the ligand E2. The four sites most affected by alanine substitution, 521, 524, 525, and 528, follow an alpha-helical periodicity, such that they would be positioned on one face of an alpha-helix. Furthermore, they correspond precisely to residues in an alpha-helix shown to be in contact with ligand in the recently described x-ray crystal structures of two other members of the nuclear hormone receptor superfamily, namely the retinoic acid receptor- and thyroid hormone receptor-ligand complexes. Our findings, which broaden observations to the steroid receptor family within the superfamily of nuclear receptors, suggest that this region of the estrogen receptor is in contact with its cognate ligand in a similar fashion.

Amino Acid Sequence↗
PubMed1996

Response-specific antiestrogen resistance in a newly characterized MCF-7 human breast cancer cell line resulting from long-term exposure to trans-hydroxytamoxifen.

To understand better the antiestrogen-resistant phenotype that frequently develops in breast cancer patients receiving tamoxifen, we cultured MCF-7 breast cancer cells long-term (>1 yr) in the presence of the antiestrogen trans-hydroxytamoxifen (TOT) to generate a subline refractory to the growth-suppressive effects of TOT. This subline (designated MCF/TOT) showed growth stimulation, rather than inhibition, with TOT and diminished growth stimulation with estradiol (E2), yet remained as sensitive as the parental cells to growth suppression by another antiestrogen, ICI 164,384. Estrogen receptor (ER) levels were maintained at 40% of that in parent MCF-7 cells, but MCF/TOT cells failed to show an increase in progesterone receptor content in response to E2 or TOT treatment. In contrast, the MCF/TOT subline behaved like parental cells in terms of E2 and TOT regulation of ER and pS2 expression and transactivation of a transiently transfected estrogen-responsive gene construct. DNA sequencing of the hormone binding domain of the ER from both MCF-7 and MCF/TOT cells confirmed the presence of wild-type ER and exon 5 and exon 7 deletion splice variants, but showed no point mutations. Compared to the parental cells, the MCF/TOT subline showed reduced sensitivity to the growth-suppressive effects of retinoic acid and complete resistance to exogenous TGF-beta1. The altered growth responsiveness of MCF/TOT cells to TOT and TGF-beta1 was partly to fully reversible following TOT withdrawal for 16 weeks. Our findings underscore the fact that antiestrogen resistance is response-specific; that loss of growth suppression by TOT appears to be due to the acquisition of weak growth stimulation; and that resistance to TOT does not mean global resistance to other more pure antiestrogens such as ICI 164,384, implying that these antiestrogens must act by somewhat different mechanisms. The association of reduced retinoic acid responsiveness and insensitivity to exogenous TGF-beta with antiestrogen growth resistance in these cells supports the increasing evidence for interrelationships among cell regulatory pathways utilized by these three growth-suppressive agents in breast cancer cells. In addition, our findings indicate that one mechanism of antiestrogen resistance, as seen in MCF/TOT cells, may involve alterations in growth factor and other hormonal pathways that affect the ER response pathway.

Antineoplastic Agents↗
PubMed1996

Nuclear hormone receptors: ligand-activated regulators of transcription and diverse cell responses.

Signal transduction via nuclear hormone receptors is unusual in that the hormone ligand forms an integral part of the protein complex involved in DNA binding and transcriptional activation. New structural and biochemical results have begun to unravel how these receptors produce different effects in different cells, and the structural changes involved in transcriptional activation.

Animals↗
PubMed1996

Estrogen receptors: bioactivities and interactions with cell signaling pathways.

Estrogens regulate the growth, differentiation, and functioning of diverse target tissues, both within and outside of the reproductive system. Most of the actions of estrogens appear to be exerted via the estrogen receptor (ER) of target cells, an intracellular receptor that is a member of a large superfamily of proteins that function as ligand-activated transcription factors, regulating the synthesis of specific RNAs and proteins. To understand how the ER discriminates between estrogen ligands, which activate the ER, and antiestrogen ligands, which fail to effectively activate the ER, we have generated and analyzed human estrogen receptors with mutations in the ER hormone binding domain. These studies provide evidence for the promoter-specific and cell-specific actions of the estrogen-occupied and antiestrogen-occupied and antiestrogen-occupied ER, highlight a regional dissociation of the hormone binding and transcription activation functions in domain E of the receptor, and indicate that some of the contact sites of estrogens and antiestrogens in the ER are likely different. In addition, multiple interactions among different cellular signaling pathways are involved in the regulation of gene expression and cell proliferation by the ER. In several cell types, protein kinase activators and some growth factors enhance the transcriptional activity of the ER. Cyclic AMP also alters the agonist/antagonist balance of some antiestrogens. Estrogens and, to a lesser extent, antiestrogens, as well as protein kinase activators and growth factors increase phosphorylation of the ER and possibly other proteins involved in the ER-specific response pathway, suggesting that changes in cellular phosphorylation state will be important in determining the biological activity of the ER and the effectiveness of antiestrogens as estrogen antagonists. The ER also has important interrelationships with the progesterone receptor (PR) system in modulation of biological responses. Liganded PR-A and PR-B can each suppress estradiol-stimulated ER activity, with the magnitude of repression dependent on the PR isoform, progestin ligand, promoter, and cell type. These findings underscore the mounting evidence for the importance of interactions between members of the steroid hormone receptor family.

Animals↗
PubMed1996

Constitutively active human estrogen receptors containing amino acid substitutions for tyrosine 537 in the receptor protein.

To better understand structure-activity relationships in the human estrogen receptor (ER), we examined the role of tyrosine 537 in the transcriptional response of the receptor, since this residue is close to a region of the hormone-binding domain shown previously to be important in hormone-dependent transcriptional activity and because this amino acid has been proposed to be a tyrosine kinase phosphorylation site important in the activity of the ER. We substituted five amino acids at this position (alanine, phenylalanine, glutamic acid, lysine, or serine) and screened these mutants for their biological activities in the presence and absence of estradiol. Two of the ER mutants, Y537A and Y537S, displayed estrogen-independent constitutive activity that was approximately 20% or 100%, respectively, of the activity of the wild type receptor with estradiol, when assessed in two different cell backgrounds using three different estrogen-responsive promoters. In some circumstances, the Y537E and Y537K proteins also exhibited some low level of constitutive activity. The constitutive activity of the mutants, as well as their activity in the presence of E2, was fully suppressed by antiestrogen. The extent of interaction of the constitutively active ERs with the steroid receptor coactivator-1 (SRC-1) closely parallel the magnitude of transcriptional activity of the receptor. Whereas wild type ER showed interaction with SRC-1 only in the presence of estrogen, Y537A and Y537S ER showed moderate or full interaction in the absence of ligand, an interaction that was blocked by antiestrogen, and the magnitude of interaction was increased to or remained at 100% upon estradiol treatment, implying that the ability of an ER to associate with SRC-1 is a good indicator of a transcriptionally active conformational state of the receptor. Our findings indicate that tyrosine 537 is in a region important in the ligand regulation of ER transcriptional activity and that the presence of certain amino acids at this position can shift ER into a conformation that is active even without ligand. However, tyrosine is not required at this site for estrogen binding or transcriptional response to estrogen in the systems investigated. Our findings, interpreted in light of the recently published x-ray crystal structure of the ligand-binding domains of three related receptors of the nuclear receptor superfamily, suggest that some of the amino acid substitutions introduced at position 537 may facilitate the shift of helix 12 of the ER into an active conformation and/or allow for differential stabilization of the receptor in its active form.

Alanine↗
PubMed1996

A transcriptionally active estrogen receptor mutant is a novel type of dominant negative inhibitor of estrogen action.

We have characterized a human estrogen receptor (ER) mutant, V364E, which has a single amino acid substitution in its hormone-binding domain. This ER mutant is fully active or even superactive at saturating levels of estradiol (10(-8) M E2) yet has the capacity to act as a strong dominant negative inhibitor of the wild type ER. In transient transfection assays using ER-negative Chinese hamster ovary (CHO) cells and two different estrogen response element (ERE)-containing promoter reporter genes, V364E treated with 10(-8) M E2 exhibited approximately 250% and 100% of the activity of the wild type ER with these two promoter contexts, respectively. Despite the high activity of V364E when present alone in cells, coexpression of both V364E and wild type ER causes a significant decrease in overall ER-mediated transcriptional activity. On the TATA promoter, where V364E was more inhibitory, estrogen-stimulated activity was reduced by approximately 50% at a 1:1 ratio of mutant to wild type ER expression vector, and at a 10:1 ratio, 75% of ER activity was inhibited. V364E was expressed at lower levels than wild type ER and has a approximately 40-fold lower affinity for E2 compared with wild type ER. In promoter interference assays, V364E exhibited a strict dependence upon E2 for binding to an ERE. Surprisingly, even when V364E was unable to bind to ERE DNA (i.e. either at low E2 concentration or by mutation of its DNA-binding domain), this mutant retained full dominant negative activity. This highly active ER mutant is, thus, able to repress ER-mediated transcription when the mutant and wild type ER are present together in cells, even without DNA binding. Since competition for ERE binding and the formation of inactive heterodimers cannot fully account for the dominant negative activity of V364E, it is probable that altered interactions with proteins important in ER-mediated transcription play a key role in the repression of transcription by V364E. The properties and probable mechanism of action of V364E distinguish it from other previously described dominant negative inhibitors, in which competition for cis-acting DNA elements by transcriptionally inactive receptors played a large role in the resultant dominant negative phenotype.

Animals↗
PubMed1996

Human estrogen receptor ligand activity inversion mutants: receptors that interpret antiestrogens as estrogens and estrogens as antiestrogens and discriminate among different antiestrogens.

The estrogen receptor (ER) is a transcription factor whose activity is normally activated by the hormone estradiol and inhibited by antiestrogen. It has been found that certain mutational changes in the activation function-2 region in the hormone-binding domain of the human ER result in ligand activity inversion mutants, i.e. receptors that are now activated by antiestrogen and inhibited by estrogen. The ER point mutant L540Q is activated by several antiestrogens (the more pure antiestrogens ICI 164,384 and RU 54,876 or the partial antiestrogen trans-hydroxytamoxifen) but not by estradiol. The presence of the F domain and an intact activation function-i in the A/B domain are required for this activity, as is the DNA-binding ability of the receptor. This inverted ligand activity is observed with several estrogen-responsive promoters, both simple and complex; however, the activating ability of antiestrogens is observed only in some cells, highlighting the important role of cell-specific factors in ligand interpretation. The introduction of two additional amino acid changes close to 540 results in receptors that are still not activated by estradiol but are now able to distinguish between partial antiestrogens (which remain agonistic) and pure antiestrogens (which show a greatly reduced stimulatory activity). These ligand activity inversion mutants remain stable in cells in the presence of the antiestrogen ICI 164,384, as does a related ER mutant receptor that shows the normal, wild type ER ligand activity profile in which ICI 164,384 is transcriptionally inactive. Thus, the presence of adequate levels of mutant ER may be necessary but not sufficient for ICI 164,384 to elicit transcriptional activity. These findings highlight the means by which the carboxyl-terminal region in domain E functions to interpret the activity of a ligand, and they demonstrate that rather minimal changes in the ER can result in receptors with inverted response to antiestrogen and estrogen. Such point mutations, if present in estrogen target cells, would result in antiestrogens being seen as growth stimulators, rather than suppressors, with potentially detrimental consequences in terms of breast cancer treatment with antiestrogens.

3T3 Cells↗
PubMed1995

Analysis of mechanisms that determine dominant negative estrogen receptor effectiveness.

To analyze the mechanisms by which estrogen receptor (ER) activity is suppressed by dominant negative mutants, we examined the role of specific ER functions and domains in transcriptional repression. We previously described three transcriptionally inactive human ER mutants (the frameshift mutant S554fs, the point mutant L540Q, and the truncated receptor ER1-530), which act as effective dominant negative mutants, inhibiting the activity of wild type ER when they are coexpressed in mammalian cells. After additional mutational modifications, the ability of the ER mutants to suppress the activity of wild type ER was analyzed in cotransfection assays of the dominant negative mutants and wild type ER and an estrogen-responsive reporter gene (2ERE-TATA-CAT or 2ERE-pS2-CAT). Eliminating the ability of the three dominant negative mutants to bind to estrogen response element (ERE) DNA (by introducing three point mutations in their DNA binding domains) dramatically reduced, but did not completely abolish, the dominant negative activity of the ER mutants. The mutation G521R, which rendered the three mutants incapable of binding estradiol, also reduced, but did not abolish, their dominant negative activity. Immunoprecipitation with monoclonal or flag antibodies followed by Western blotting demonstrated that each of the original dominant negative ER mutants formed heterodimers with wild type ER. Rendering the dominant negative mutants dimerization deficient by the mutation L507R strongly reduced, but did not eliminate, their dominant negative activity. Deletion of the N-terminal A/B domain resulted in the nearly complete loss of inhibitory activity of the three dominant negative mutants. However, these double mutants retained their ability to heterodimerize with wild type ER, suggesting that dominant negative interference also occurs at an additional step beyond dimerization. Our data indicate that competition for ERE binding, formation of inactive heterodimers, and specific transcriptional silencing can all contribute to the dominant negative phenotype and that these receptors suppress the activity of wild type ER by acting at multiple steps in the ER-response pathway.

Animals↗
PubMed1995

Ligand-dependent, transcriptionally productive association of the amino- and carboxyl-terminal regions of a steroid hormone nuclear receptor.

The estrogen receptor (ER), a 66-kDa protein that mediates the actions of estrogens in estrogen-responsive tissues, is a member of a large superfamily of nuclear hormone receptors that function as ligand-activated transcription factors. ER shares a conserved structural and functional organization with other members of this superfamily, including two transcriptional activation functions (AFs), one located in its amino-terminal region (AF-1) and the second located in its carboxyl-terminal, ligand-binding region (AF-2). In most promoter contexts, synergism between AF-1 and AF-2 is required for full ER activity. In these studies, we demonstrate a functional interaction of the two AF-containing regions of ER, when expressed as separate polypeptides in mammalian cells, in response to 17 beta-estradiol (E2) and antiestrogen binding. The interaction was transcriptionally productive only in response to E2, and was eliminated by point or deletion mutations that destroy AF-1 or AF-2 activity or E2 binding. Our results suggest a definitive mechanistic role for E2 in the activity of ER--namely, to alter receptor conformation to promote an association of the amino- and carboxyl-terminal regions, leading to transcriptional synergism between AF-1 and AF-2. The productive re assembly of two portions of ER expressed in cells as separate polypeptides demonstrates the evolutionarily conserved modular structural and functional organization of the nuclear hormone receptors. The ligand-dependent interaction of the two AF-containing regions of ER allows for the assembly of a complete activation function from two distinct regions within the same protein, providing a mechanism for hormonally regulated transcription.

3T3 Cells↗
PubMed1995

Antiestrogens: mechanisms and actions in target cells.

Antiestrogens, acting via the estrogen receptor (ER) evoke conformational changes in the ER and inhibit the effects of estrogens as well as exerting anti-growth factor activities. Although the binding of estrogens and antiestrogens is mutually competitive, studies with ER mutants indicate that some of the contact sites of estrogens and antiestrogens are likely different. Some mutations in the hormone-binding domain of the ER and deletions of C-terminal regions result in ligand discrimination mutants, i.e. receptors that are differentially altered in their ability to bind and/or mediate the actions of estrogens vs antiestrogens. Studies in a variety of cell lines and with different promoters indicate marked cell context- and promoter-dependence in the actions of antiestrogens and variant ERs. In several cell systems, estrogens and protein kinase activators such as cAMP synergize to enhance the transcriptional activity of the ER in a promoter-specific manner. In addition, cAMP changes the agonist/antagonist balance of tamoxifen-like antiestrogens, increasing their agonistic activity and reducing their efficacy in reversing estrogen actions. Estrogens, and antiestrogens to a lesser extent, as well as protein kinase activators and growth factors increase phosphorylation of the ER and/or proteins involved in the ER-specific response pathway. These changes in phosphorylation alter the biological effectiveness of the ER. Multiple interactions among different cellular signal transduction systems are involved in the regulation of cell proliferation and gene expression by estrogens and antiestrogens.

Cyclic AMP↗
PubMed1995

Inhibitory cross-talk between steroid hormone receptors: differential targeting of estrogen receptor in the repression of its transcriptional activity by agonist- and antagonist-occupied progestin receptors.

Although estrogen receptor (ER) and progestin receptor (PR) are members of different steroid hormone receptor subfamilies, there is considerable biological evidence for cross-talk between the estrogen and progestin hormone-receptor signaling pathways. We have developed a model system to analyze the mechanisms underlying this cross-talk, specifically the repression of ER-mediated transcriptional activity by PR complexed with agonistic or antagonistic ligands. Estrogen- and progestin-responsive reporter vectors containing a variety of promoters were transfected into primary cultures of rat uterine cells and 3T3 mouse fibroblasts with expression vectors for PR (the A and/or B isoforms) as well as ER. Our results demonstrate that both PR isoforms can act as potent ligand-dependent repressors of ER activity. The magnitude of the repression was dependent on the PR isoform (i.e., PR A or PR B), ligand type (i.e., agonist or antagonist), PR levels, and ligand concentration but was unaffected by the ER levels. The promoter context was important in determining both the magnitude and PR isoform specificity of the repression for agonist-occupied PR but not for antagonist-occupied PR. Ligand-occupied PR A was a stronger repressor of ER-mediated transcriptional activity than was ligand-occupied PR B, and antagonist-occupied PR was a more effective repressor than agonist-occupied PR. Mechanistic studies suggest that liganded PR represses ER activity by interfering with its ability to interact productively with the transcriptional machinery, a process known as quenching. The data do not support competitive repression, direct repression, or squelching as the mechanism of PR's inhibitory effect. Experiments with ER mutants demonstrated that the N-terminal portion of ER was required for repression by agonist-occupied PR but not by antagonist-occupied PR. These results, as well as other differences between the two PR-ligand complexes, suggest that they differentially target ER when repressing ER transcriptional activity. These findings underscore the mounting evidence for the importance of interactions between members of the steroid hormone receptor family.

3T3 Cells↗
PubMed1995

Repression of endogenous estrogen receptor activity in MCF-7 human breast cancer cells by dominant negative estrogen receptors.

We have investigated the ability of several transcriptionally inactive estrogen receptor (ER) mutants to block endogenous ER-mediated transcription in MCF-7 human breast cancer cells. In transient transfections of MCF-7 cells, two of the mutants, a frame-shifted ER (S554fs) and a point-mutated ER (L540Q), strongly inhibit the ability of endogenous wild-type ER to activate transcription of estrogen-regulated reporter plasmids. A third mutant, ER1-530, which is missing 65 residues from its carboxy-terminus, is a weaker repressor of estradiol-stimulated transcription. When an estrogen response element (ERE)-thymidine kinase-chloramphenicol acetyltransferase reporter gene is used, S554fs, L540Q, and ER1-530 suppress the transcriptional activity of endogenous MCF-7 ER by 87%, 97%, and 62%, respectively. The magnitude of dominant negative repression is promoter specific; when an ERE-pS2-chloramphenicol acetyltransferase reporter is employed, inhibition of endogenous ER activity by equivalent amounts of S554fs, L540Q, and ER1-530 ranges from 85-97%. Dose-response studies show the S554fs mutant to be the most potent of the three ER mutants as a repressor of estrogen action in these cells. In addition, elevated levels of intracellular cAMP, achieved by the addition of 3-isobutyl-1-methylxanthine plus cholera toxin to cells, fail to compromise the effectiveness of these mutants as dominant negative ERs despite the cAMP-enhanced transcriptional activity of ER. The mutants are also powerful repressors of the agonist activity of trans-hydroxytamoxifen-stimulated ER transcription. The dominant negative activity of the three mutants is lost when the A/B domain of these receptors is deleted, implying an important role for this N-terminal region of the ER in the ability of these mutants to inhibit endogenous wild-type ER activity. All in all, the data suggest that S554fs in particular is a reasonable candidate for studies designed to use a dominant negative ER to inhibit the estrogen- and tamoxifen-stimulated growth of human breast cancer cells.

Breast Neoplasms↗
PubMed1995

Estrogen receptor mutants which do not bind 17 beta-estradiol dimerize and bind to the estrogen response element in vivo.

To investigate the stage in estrogen receptor (ER) action at which hormone functions, we prepared human ER mutants unable to bind 17 beta-estradiol. In transfected Chinese Hamster Ovary (CHO) cells, two of the ER mutants exhibited less than 5% of the ability to activate transcription shown by wild type ER. Immunoprecipitation followed by Western blotting with monoclonal antibodies was used to examine the ability of the ER mutants to form heterodimers with a truncated form of wild type ER. The non-hormone-binding mutants formed heterodimers with the truncated ER as efficiently as wild type ER. We used a promoter interference assay to measure the interaction of the ER with the estrogen response element (ERE) in vivo. Expression plasmids encoding the ER mutants and wild type ER were transfected into CHO cells across a range of concentrations, resulting in both high and low levels of promoter interference. The ER mutants and wild type ER elicited similar levels of promoter interference, indicating that although they were unable to bind ligand, the ER mutants bound to the ERE in vivo as effectively as wild type ER. Additional evidence that the non-hormone-binding ER mutants are not in a functionally inactive complex comes from their ability to suppress the activity of wild type ER, when they were coexpressed in the same cells. These data support a model for ER action in which the unliganded ER is free to dimerize and bind to the ERE. In this model, the primary role of 17 beta-estradiol in ER action is to induce a conformational change which activates the ligand-dependent transactivation domain.

Amino Acid Sequence↗
PubMed1995

The carboxy-terminal F domain of the human estrogen receptor: role in the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists.

Of the steroid hormone receptor family members, the estrogen receptor (ER) is notable in containing a sizable (42-amino acid) C-terminal region, denoted domain F. This F region differs from its adjacent hormone-binding domain, domain E, in that it is not well conserved among different vertebrate ER species, and its role in the biological activity of the ER is not well defined. We report an important role for the F domain of the ER in modulating the magnitude of gene transcription by estrogen and antiestrogen, and in determining the effectiveness of antiestrogens in suppressing estrogen-stimulated gene transcription. Using transient transfections, we have examined, in several cell types, the transcriptional activity of the full-length wild type human ER and ER lacking the carboxy-terminal F domain (delta F ER, containing amino acids 1-554) or ER altered in the F domain by point mutations. In some cells, namely Chinese hamster ovary (CHO) cells and MDA-MB-231 human breast cancer cells expressing wild type ER or delta F ER, estradiol (E2) stimulates equally transcription of several estrogen-responsive promoter-reporter gene constructs [estrogen ca-18119 element, (ERE)2-TATA-CAT, (ERE)2-pS2-CAT, (ERE)2-progesterone receptor(distal)-CAT]; however, the antiestrogens trans-hydroxytamoxifen and ICI 164,384, which stimulate transcription of some of these reporter constructs with the wild type ER, were unable to stimulate transcription with delta F ER. In addition, these antiestrogens were more effective antagonists of E2-stimulated transcription by delta F ER than by wild type ER. By contrast, in HeLa human cervical cancer cells and 3T3 mouse fibroblast cells, the delta F ER exposed to E2 is much less effective than wild type ER in stimulating transcription, and antiestrogens were less potent in suppressing E2-stimulated transcription by the delta F ER. These differences in response of the delta F and wild type ER to estrogen or antiestrogen do not appear to be due to a change in receptor expression level, binding affinity for ligands, or binding to estrogen response element DNA. Our data support the supposition that the conformation of the receptor-ligand complex is different with estrogen vs. antiestrogen and with wild type vs. delta F ER, such that its potential for interaction with protein cofactors or transcription factors is different and is markedly influenced by cell context. Thus, the F domain of the ER has a specific modulatory function that affects the agonist/antagonist effectiveness of antiestrogens and the transcriptional activity of the liganded ER in cells.

3T3 Cells↗
PubMed1994

Alterations in transforming growth factor-alpha and -beta production and cell responsiveness during the progression of MCF-7 human breast cancer cells to estrogen-autonomous growth.

Hormonal management of breast cancer is confounded by an almost inevitable progression of cell growth from a steroid-regulated to a steroid-autonomous state. We have experimentally induced this progression in the estrogen growth-responsive MCF-7 human breast cancer cell line by long-term culture in the absence of steroids. After an initial period (10-12 weeks) of slowed growth in response to steroid deprivation, rapid, steroid-independent growth rates were consistently established. In these cells, which contained 3-fold elevated, functional estrogen receptor levels (as determined by induction of PgR and transactivation of a transiently transfected estrogen-responsive gene construct), antiestrogens still effectively suppressed cell proliferation, although estrogens only minimally increased the proliferation rate. Depletion of steroids from the growth media also resulted in a marked (70-80%) transient decrease in transforming growth factor (TGF) alpha mRNA and TGF-alpha protein production at 2 weeks that was followed by a progressive, partial return to the initial parental TGF-alpha mRNA and protein levels. In contrast, the mRNAs for TGF-beta 1, -beta 2, and -beta 3 and bioactive TGF-beta proteins transiently increased (3-10-fold) at 2 to 10 weeks of steroid deprivation and then returned by 24 weeks to the lower levels of the parental MCF-7 cells. These results suggest that the cells acquired steroid-independent means to regulate the production of these peptides. The long-term steroid-deprived sublines showed a loss of regulation of proliferation by TGF-alpha or anti-TGF-alpha antibodies and a 10-fold decrease in sensitivity to the growth-suppressive effects of TGF-beta 1, despite little change in receptor levels for these factors. The diminished contributions of TGF-alpha and TGF-beta s to the regulation of cell proliferation in long-term steroid-deprived MCF-7 breast cancer cells suggest that the TGFs do not act as major growth regulators in these estrogen-autonomous sublines. However, the marked, transient alterations in the levels of these growth factors indicate that they may play a role in the events which accompany the progression from estrogen-responsive to estrogen-autonomous growth. In addition, continued exposure to estrogen may be needed for the long-term maintenance of cell responsiveness to these TGFs.

Breast Neoplasms↗
PubMed1994

Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription.

Estrogenic hormones, believed to exert most of their effects via the direct interaction of their receptors with chromatin, are found to increase cAMP in target breast cancer and uterine cells in culture and in the intact uterus in vivo. Increases in intracellular cAMP are evoked by very low concentrations of estradiol (half maximal at 10 pM) and by other physiologically active estrogens and antiestrogens, but not by an inactive estrogen stereoisomer. These increases in cAMP result from enhanced membrane adenylate cyclase activity by a mechanism that does not involve genomic actions of the hormones (are not blocked by inhibitors of RNA and protein synthesis). The estrogen-stimulated levels of cAMP are sufficient to activate transcription from cAMP response element-containing genes and reporter plasmid constructs. Our findings document a nongenomic action of estrogenic hormones that involves the activation of an important second-messenger signaling system and suggest that estrogen regulation of cAMP may provide an additional mechanism by which this steroid hormone can alter the expression of genes.

Adenylyl Cyclases↗