Presence Of 4-hydroxytamoxifen Research Articles

Regulated control of virus replication by 4-hydroxytamoxifen-induced splicing.

Designing a modified virus that can be controlled to replicate will facilitate the study of pathogenic mechanisms of virus and virus-host interactions. Here, we report a universal switch element that enables precise control of virus replication after exposure to a small molecule. Inteins mediate a traceless protein splicing-ligation process, and we generate a series of modified vesicular stomatitis virus (VSV) with intein insertion into the nucleocapsid, phosphoprotein, or large RNA-dependent RNA polymerase of VSV. Two recombinant VSV, LC599 and LY1744, were screened for intein insertion in the large RNA-dependent RNA polymerase of VSV, and their replication was regulated in a dose-dependent manner with the small molecule 4-hydroxytamoxifen, which induces intein splicing to restore the VSV replication. Furthermore, in the presence of 4-hydroxytamoxifen, the intein-modified VSV LC599 replicated efficiently in an animal model like a prototype of VSV. Thus, we present a simple and highly adaptable tool for regulating virus replication.

Autonomous BCR Signaling and Genetic Aberrations in CLL-Phenotype Monoclonal B Lymphocytosis in Siblings of CLL Patients

Clonal expansion of CD5-expressing B cells, termed monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL) with an increased incidence in CLL siblings. In app. 1% of MBL carriers, MBLprogresses eventually to clinically overt CLL. The mechanisms driving benign MBL expansion and subsequent malignant progression are unknown. Autonomous signaling of the clonotypic BCR of CLL cells, i.e. signaling without stimulation by cognate antigen, is an indispensable oncogenic signal in CLL. In order to investigate the early stages in CLL ontogeny, we sought to clarify whether MBL cells express BCR with autonomous signaling capacity and to compare the prevalence of inherited risk loci and acquired genetic aberrations in MBL-CLL sibling pairs. Peripheral blood of 191 siblings of CLL patients was screened with informed consent by six-color flow cytometry. A discrete CD19+CD5+CD20lowCD79low CLL-phenotype population of 0.2 to 1863 cells / µL blood was detected in 34 siblings (17.8%) of 26 CLL patients; 32 siblings (94%) had <500 clonal CLL-phenotype cells / µL. Clonal BCR were identified from highly purified MBL cells of 17 CLL siblings by ARTISAN PCR. Five MBL clones could be assigned to CLL stereotyped subsets, including two MBL BCR belonging to CLL subset #2 with expression of the IGLV3-21 gene with a G110R mutation and one IgG-expressing MBL belonging to CLL subset #4. Eleven MBL BCR, including all 5 assigned to a CLL stereotyped subset, were transduced into murine TKO cells arrested at the pro-B-cell stage by genetic deficiency of rag2, lambda5, and slp65. Slp65 function is reconstituted by a slp65-ERT2 fusion gene that enables BCR signaling in the presence of 4-hydroxytamoxifen. Calcium mobilization was measured in Indo-1 AM-loaded, live-gated TKO cells by flow cytometry as the ratio of signal intensities (SIR) at 485 and 405 nm prior and after tamoxifen supplementation and after BCR crosslinking with isotype-specific antibody. Robust calcium mobilization was observed in all MBL BCR-transduced TKO cells in the absence of external antigen and without experimental cross-linking, regardless of assignment to a CLL subtype. Calibrated quantification of this autonomous BCR signal, however, revealed lesser intensity in MBL BCR compared to the BCR of their CLL sibling (p=0.015, Wilcoxon's matched-pair signed rank test). BCR signaling strength was correlated to the degree of MBL clonal expansion (p=0.034). According to genotyping by single nucleotide polymorphism array, whole exome sequencing (WES), and targeted panel sequencing, a polygenic CLL risk score (PRS) of 24 risk loci was higher in both CLL and MBL siblings than in reference population data, but we found no evidence for a higher PRS in CLL cases in a matched-pair comparison to their MBL siblings. With respect to acquired genetic changes, quantitative analysis of SNP array data revealed recurrent CLL-associated copy number variations (CNV) at clonal levels in all of 11 informative CLL cases. In contrast, individual CNV were present in only a minor fraction of the highly purified MBL cells in six of ten samples (p=0.003; Fisher's exact test). Likewise, variant allele frequencies (VAF) of non-shared variants as detected by WES was significantly lower in MBL samples than in CLL cases (p<0.0001). In particular, subclonal variants defined by a VAF of 0.1-0.33 were more prevalent in MBL compared to CLL siblings (p=0.014), whereas clonal variants (VAF 0.43-0.55) were equally abundant (p=0.65) in both conditions. In this comprehensive comparison of MBL and CLL in siblings, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, suggesting that CLL risk loci predispose to clonal expansion of CLL phenotype cells in both low-count MBL and CLL. Vice versa, we did not find evidence for a particular association of CLL risk alleles with malignant progression from MBL to clinical CLL. Autonomous BCR signaling operates in both MBL and CLL as predicted by BCR stereotype, albeit at lesser strength in MBL. Acquired CLL-genetic changes, both copy number and sequence variants, are frequently subclonal in MBL but not in CLL. These findings support a stepwise CLL pathogenetic model wherein autonomous BCR signaling causes a non-malignant expansion of clonal CD5+ B cells in genetically predisposed individuals, followed by malignant progression to CLL after gradual acquisition of pathogenic genetic variants.

Generation of c-MycERTAM-transduced human late-adherent olfactory mucosa cells for potential regenerative applications

Human olfactory mucosa cells (hOMCs) have been transplanted to the damaged spinal cord both pre-clinically and clinically. To date mainly autologous cells have been tested. However, inter-patient variability in cell recovery and quality, and the fact that the neuroprotective olfactory ensheathing cell (OEC) subset is difficult to isolate, means an allogeneic hOMC therapy would be an attractive “off-the-shelf” alternative. The aim of this study was to generate a candidate cell line from late-adherent hOMCs, thought to contain the OEC subset. Primary late-adherent hOMCs were transduced with a c-MycERTAM gene that enables cell proliferation in the presence of 4-hydroxytamoxifen (4-OHT). Two c-MycERTAM-derived polyclonal populations, PA5 and PA7, were generated and expanded. PA5 cells had a normal human karyotype (46, XY) and exhibited faster growth kinetics than PA7, and were therefore selected for further characterisation. PA5 hOMCs express glial markers (p75NTR, S100ß, GFAP and oligodendrocyte marker O4), neuronal markers (nestin and ß-III-tubulin) and fibroblast-associated markers (CD90/Thy1 and fibronectin). Co-culture of PA5 cells with a neuronal cell line (NG108-15) and with primary dorsal root ganglion (DRG) neurons resulted in significant neurite outgrowth after 5 days. Therefore, c-MycERTAM-derived PA5 hOMCs have potential as a regenerative therapy for neural cells.

Loss of function of NF1 is a mechanism of acquired resistance to endocrine therapy in lobular breast cancer.

BackgroundInvasive lobular carcinoma (ILC) as a disease entity distinct from invasive ductal carcinoma (IDC) has merited focused studies of the genomic landscape, but those to date are largely limited to the assessment of early-stage cancers. Given that genomic alterations develop as acquired resistance to endocrine therapy, studies on refractory ILC are needed.Patients and methodsTissue from 336 primary-enriched, breast-biopsied ILC and 485 estrogen receptor (ER)-positive IDC and metastatic biopsy specimens from 180 ILC and 191 ER-positive IDC patients was assayed with hybrid-capture-based comprehensive genomic profiling for short variant, indel, copy number variants, and rearrangements in up to 395 cancer-related genes.ResultsWhereas ESR1 alterations are enriched in the metastases of both ILC and IDC compared with breast specimens, NF1 alterations are enriched only in ILC metastases (mILC). NF1 alterations are predominantly under loss of heterozygosity (11/14, 79%), are mutually exclusive with ESR1 mutations [odds ratio = 0.24, P < 0.027] and are frequently polyclonal in ctDNA assays. Assessment of paired specimens shows that NF1 alterations arise in the setting of acquired resistance. An in vitro model of CDH1 mutated ER-positive breast cancer demonstrates that NF1 knockdown confers a growth advantage in the presence of 4-hydroxy tamoxifen. Our study further identified a significant increase in tumor mutational burden (TMB) in mILCs relative to breast ILCs or metastatic IDCs (8.9% >20 mutations/mb; P < 0.001). Most TMB-high mILCs harbor an APOBEC trinucleotide signature (14/16; 88%).ConclusionsThis study identifies alteration of NF1 as enriched specifically in mILC. Mutual exclusivity with ESR1 alterations, polyclonality in relapsed ctDNA, and de novo acquisition suggest a role for NF1 loss in endocrine therapy resistance. Since NF1 loss leads to RAS/RAF kinase activation, patients may benefit from a matched inhibitor. Moreover, for an independent subset of mILC, TMB was elevated relative to breast ILC, suggesting possible benefit from immune checkpoint inhibitors.

Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.

Signaling transmitted by the Ras family of small GTPases (H-, N-, and K-Ras) is essential for proliferation of mouse embryonic fibroblasts (MEFs). However, constitutive activation of the downstream Raf/Mek/Erk pathway can bypass the requirement for Ras proteins and allow cells to proliferate in the absence of the three Ras isoforms. Here we describe a protocol for a colony formation assay that permits evaluating the role of candidate proteins that are positive or negative regulators of cell proliferation mediated via Ras-independent Raf/Mek/Erk pathway activation. K-Raslox (H-Ras -/-, N-Ras -/-, K-Ras lox/lox, RERTert/ert) MEFs are infected with retro- or lentiviral vectors expressing wild-type or constitutively activated candidate cDNAs, shRNAs, or sgRNAs in combination with Cas9 to ascertain the possibility of candidate proteins to function either as an activator or inhibitor of Ras-independent Raf/Mek/Erk activation. These cells are then seeded in the absence or presence of 4-Hydroxytamoxifen (4-OHT), which activates the resident CreERT2 alleles resulting in elimination of the conditional K-Ras alleles and ultimately generating Rasless cells. Colony formation in the presence of 4-OHT indicates cell proliferation via Ras-independent Raf/Mek/Erk activation.

Dynamics of Protein Expression Reveals Primary Targets and Secondary Messengers of Estrogen Receptor Alpha Signaling in MCF-7 Breast Cancer Cells

Estrogen receptor alpha (ERα)-mediated proliferation of breast cancer cells is facilitated through expression of multiple primary target genes, products of which induce a secondary response to stimulation. To differentiate between the primary and secondary target proteins of ERα signaling, we measured dynamics of protein expression induced by 17β-estradiol in MCF-7 breast cancer cells. Measurement of the global proteomic effects of estradiol by stable isotope labeling by amino acids in cell culture (SILAC) resulted in identification of 103 estrogen-regulated proteins, with only 40 of the corresponding genes having estrogen response elements. Selected reaction monitoring (SRM) assays were used to validate the differential expression of 19 proteins and measure the dynamics of their expression within 72 h after estradiol stimulation, and in the absence or presence of 4-hydroxytamoxifen, to confirm ERα-mediated signaling. Dynamics of protein expression unambiguously revealed early and delayed response proteins and well correlated with presence or absence of estrogen response elements in the corresponding genes. Finally, we quantified dynamics of protein expression in a rarely studied network of transcription factors with a negative feedback loop (ERα-EGR3-NAB2). Because NAB2 protein is a repressor of EGR3-induced transcription, siRNA-mediated silencing of NAB2 resulted in the enhanced expression of the EGR3-induced protein ITGA2. To conclude, we provided a high-quality proteomic resource to supplement genomic and transcriptomic studies of ERα signaling.

Gfi1 Is Repressed by p53 and Inhibits DNA Damage-Induced Apoptosis

Abstract 1228Gfi1 is a transcriptional repressor that plays a critical role in hematopoiesis. Gfi1 has also been implicated in lymphomagenesis when aberrantly activated as a result of deregulated expression. It is still poorly understood how Gfi1 expression is regulated and how it acts in the hematopoietic system. We show here that Gfi1 transcription was repressed by the tumor suppressor p53 in hematopoietic cells. Treatment of cells with doxorubicin (Doxo), which induces topoisomerase II-mediated DNA double strand breaks (DSB), led to a steady increase in p53 protein level, which was accompanied by a gradual decline in Gfi1 expression at both protein and mRNA levels. Knockdown of p53 resulted in increased Gfi1 expression and also abolished Doxo-induced Gfi1 downregulation. In contrast, Gfi1 expression was reduced and its downregulation in response to DNA damage was restored in p53-deficient cells transfected with the p53/estrogen receptor ligand binding domain fusion protein (p53ERTAM) in the presence of 4-hydroxytamoxifen (4-OHT). In luciferase reporter assays, Doxo treatment inhibited the activity of a ∼2.4-kb Gfi1 promoter fragment in p53+/+ cells, but not in p53−/− cells. Consistent with this, the wild type p53, but not a DNA binding-defective p53 mutant, repressed the Gfi1 promoter fragment. Chromatin immunoprecipitation (ChIP) assays demonstrated that p53 bound to the proximal region of the Gfi1 promoter. Detailed mapping of the Gfi1 promoter indicated that the core promoter region of Gfi1 spanning −33 to +6 bp is sufficient for p53-mediated repression. This core promoter region contains a putative p53 repressive response element (RRE) and, when the RRE was mutated, p53 failed to bind to and repress the Gfi1 promoter. Significantly, apoptosis induction by Doxo treatment was inhibited upon Gfi1 overexpression, but augmented following Gfi1 knockdown. Together, these data establish for the first time that Gfi1 is repressed by p53 and indicate that Gfi1 acts to protect hematopoietic cells from DNA damage-induced apoptosis. Our findings have important implications for understanding the roles of Gfi-1 in normal hematopoiesis and lymphomagenesis. Disclosures:No relevant conflicts of interest to declare.

Abstract 1582: Identification of BCAR genes relevant for breast cancer progression and endocrine therapy resistance

Abstract Endocrine therapy of breast cancer is effective and widely applied. In advanced disease, about half of the patients will experience a clinical benefit from tamoxifen treatment, but ultimately the disease will progress into a resistant phenotype. The molecular basis of this general failure of endocrine therapy is not understood yet. Although gene expression profiling experiments provide gene lists to identify patients who may benefit from specific treatments, these genes mostly do not define the underlying molecular mechanisms. We have executed a functional screen for the identification of BCAR genes causing anti-estrogen resistance in a human breast cancer model and evaluated their role in a large breast cancer cohort. Estrogen-dependent ZR-75-1 human breast cancer cells were subjected to insertion mutagenesis with replication-defective retroviruses and selected for proliferation in the presence of 4-hydroxy tamoxifen. In our panel of 79 tamoxifen-resistant cell lines, the target genes of the retroviruses have been identified and were functionally tested following transfection into ZR-75-1 cells. Seven BCAR genes (AKT1, AKT2, BCAR1, BCAR3, EGFR, GRB7 and TRERF1) causing the tamoxifen-resistant phenotype were identified. Furthermore, additional putative BCAR genes and loci have been pinpointed. To determine the relevance of the identified genes in clinical breast cancer, we have studied 561 estrogen receptor-positive primary breast cancers for tumor aggressiveness and tamoxifen therapy resistance. Quantitative RT-PCR of ten genes was performed to measure the tumor mRNA levels. The endpoint for tumor aggressiveness was metastasis-free survival (MFS) of a cohort of lymph node-negative patients. Therapy resistance was evaluated in a group of patients treated with tamoxifen as first-line therapy for recurrent disease and the endpoint of this analysis was progression-free survival (PFS). Statistically significant associations with MFS were found for AKT2, EGFR, TLE3 and TRERF1 mRNA levels. In multivariate analyses including the traditional prognostic factors, AKT2, EGFR and TRERF1 were independent prognostic factors for MFS and thus are associated with tumor aggressiveness. In the analyses of PFS, mRNA levels of BCAR3, ERBB2, GRB7, TLE3 and TRERF1 were associated with the duration of clinical benefit of tamoxifen therapy, independently of the classical predictive factors. Our functional screen identified a set of clinically relevant BCAR genes offering insight in the molecular and cellular pathways of breast tumor aggressiveness and tamoxifen resistance, and thereby provides novel targets for personalized treatment and prevention strategies. Supported by the Dutch Cancer Society, the Susan G Komen Breast Cancer Foundation and the Association for International Cancer Research.

Development of a human neural stem cell line for use in recovery from disability after stroke

A clonal human neural stem cell line (ReN001) has been developed for clinical use in the treatment of stable disability after stroke. This cell line has been conditionally immortalized using the fusion transgene c-mycERTAM to allow controlled expansion when cultured in the presence of 4-hydroxytamoxifen. The cell line has been banked and fully characterized to assure there is genetic stability and no phenotypic drift with extended passages. In vivo studies determined the ability of the cell line to survive after implantation into damaged brain and its efficacy in the reduction of chronic behavioural dysfunction after implantation into a rodent model of stroke damage. A further study was conducted in this model and a dose-dependent effect was observed on behavioural recovery. No safety or toxicology issues were identified in in vivo studies with this cell line, which made REN001 a strong candidate for one of the first cell-based IND applications to be submitted to the Food and Drug Administration in the United States for consideration for the treatment of stroke in humans.

Reversible Heart Failure in Gαq Transgenic Mice

For many patients with cardiac insufficiency, the disease progresses inexorably to organ dilatation, pump failure, and death. Although there are examples of reversible heart failure in man, our understanding of how the myocardium repairs itself is limited. A well defined animal model of reversible heart failure would allow us to better investigate these restorative processes. Receptors that activate Galpha(q), a signal transduction molecule in the heterotrimeric G protein superfamily, are thought to play a key role in the development of heart failure. We demonstrated previously that mice expressing a recombinant Galpha(q) protein, the activity of which can be turned on or off at will in cardiac myocytes, develop a dilated cardiomyopathy with generalized edema and heart failure following activation of the protein (Fan, G., Jiang, Y.-P., Lu, Z., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (2005) J. Biol. Chem. 280, 40337-40346). Here we report that the contractile dysfunction and pathological structural changes in the myocardium improved significantly after termination of the Galpha(q) signal, even in animals with overt heart failure. Abnormalities in two proteins that regulate Ca(2+) handling in myocytes, phospholamban and the voltage-dependent L-type Ca(2+) channel, were also reversed, as was the increased expression of genes that are associated with heart failure. These results indicate that the heart has a substantial reparative capacity if the molecular signals responsible for the myocardial dysfunction can be identified and blocked.

Delineation of a unique protein-protein interaction site on the surface of the estrogen receptor.

Recent studies have identified a series of estrogen receptor (ER)-interacting peptides that recognize sites that are distinct from the classic coregulator recruitment (AF2) region. Here, we report the structural and functional characterization of an ERalpha-specific peptide that binds to the liganded receptor in an AF2-independent manner. The 2-A crystal structure of the ER/peptide complex reveals a binding site that is centered on a shallow depression on the beta-hairpin face of the ligand-binding domain. The peptide binds in an unusual extended conformation and makes multiple contacts with the ligand-binding domain. The location and architecture of the binding site provides an insight into the peptide's ER subtype specificity and ligand interaction preferences. In vivo, an engineered coactivator containing the peptide motif is able to strongly enhance the transcriptional activity of liganded ERalpha, particularly in the presence of 4-hydroxytamoxifen. Furthermore, disruption of this binding surface alters ER's response to the coregulator TIF2. Together, these results indicate that this previously unknown interaction site represents a bona fide control surface involved in regulating receptor activity.

Pharmacologic activation of p53 elicits Bax-dependent apoptosis in the absence of transcription

Recent efforts to develop pharmacologic agents that restore function to mutant forms of p53 hold significant promise in cancer therapy. Here, we examine the effects of such pharmacologic activation of p53 function using a small molecule, PRIMA-1, and a model system employing a p53 protein fused to a mutant steroid binding domain of the murine estrogen receptor (p53ER tam) that renders it responsive only in the presence of 4-hydroxytamoxifen. In either case, p53 activation triggered apoptosis that was not inhibited by the presence of macromolecular synthesis inhibitors. This p53-induced, transcription-independent apoptosis is Bax dependent, proceeds in the absence of a nucleus, and involves Bax translocation and cytochrome c release. Hence, pharmacologic p53 modulators can activate a transcription-independent apoptotic program.

Estrogen receptor beta activates the human retinoic acid receptor alpha-1 promoter in response to tamoxifen and other estrogen receptor antagonists, but not in response to estrogen.

Human estrogen receptor-alpha (hERalpha) or -beta (hERbeta) transfected into Hep G2 or COS1 cells each responded to estrogen to increase transcription from an estrogen-responsive element (ERE)-driven reporter vector with similar fold induction through a classical mechanism involving direct receptor binding to DNA. ER antagonists inhibited this estrogen induction through both hERalpha and hERbeta, although raloxifene was more potent through ERalpha than ERbeta, and tamoxifen was more potent via ERbeta than ERalpha. We have shown previously that estrogen stimulated the human retinoic acid receptor-alpha-1 (hRARalpha-1) promoter through nonclassical EREs by a mechanism that was ERalpha dependent, but that did not involve direct receptor binding to DNA. We show here that in contrast to hERalpha, hERbeta did not induce reporter activity driven by the hRARalpha-1 promoter in the presence of estrogen. While hERbeta did not confer estrogen responsiveness on this promoter, it did elicit transcriptional activation in the presence of 4-hydroxytamoxifen (4-OH-Tam). Additionally, this 4-OH-Tam agonist activity via ERbeta was completely blocked by estrogen. Like ERalpha, transcriptional activation of this promoter by ERbeta was not mediated by direct receptor binding to DNA. While hERalpha was shown to act through two estrogen-responsive sequences within the promoter, hERbeta acted only at the 3'-region, through two Sp1 sites, in response to 4-OH-Tam. Other ER antagonists including raloxifene, ICI-164,384 and ICI-182,780 also acted as agonists through ERbeta via the hRARalpha-1 promoter. Through the use of mutant and chimeric receptors, it was shown that the 4-OH-Tam activity via ERbeta from the hRARalpha-1 promoter in Hep G2 cells required the amino-terminal region of ERbeta, a region that was not necessary for estrogen-induced ERbeta activity from an ERE in Hep G2 cells. Additionally, the progesterone receptor (PR) antagonist RU486 acted as a weak (IC50 >1 microM) antagonist via hERalpha and as a fairly potent (IC50 approximately 200 nM) antagonist via hERbeta from an ERE-driven reporter in cells that do not express PR. Although RU486 bound only weakly to ERalpha or ERbeta in vitro, it did bind to ERbeta in whole-cell binding assays, and therefore, it is likely metabolized to an ERbeta-interacting compound in the cell. Interestingly, RU486 acted as an agonist through ERbeta to stimulate the hRARalpha-1 promoter in Hep G2 cells. These findings may have ramifications in breast cancer treatment regimens utilizing tamoxifen or other ER antagonists and may explain some of the known estrogenic or antiestrogenic biological actions of RU486.

Ligand-Dependent Repression of the Erythroid Transcription Factor GATA-1 by the Estrogen Receptor

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

Identification of residues in the estrogen receptor that confer differential sensitivity to estrogen and hydroxytamoxifen.

We have generated mutant mouse estrogen receptors which differ in their sensitivity to estrogen and the antiestrogen 4-hydroxytamoxifen. Mutation of the glycine at position 525 and the methionine and/or serine at positions 521/522 virtually abolishes the ability of the receptor to bind estradiol and stimulate transcription. In contrast, the mutant receptors retain the partial agonist activity exhibited by the wild-type receptor in the presence of 4-hydroxytamoxifen. The mutations do not affect the expression and DNA-binding activity of the receptor, but do abolish the estrogen-induced increase in the mobility of the receptor-DNA complex observed with the wild-type receptor. Other mutant receptors that were able to bind and stimulate transcription in the presence of estradiol also failed to show the agonist-induced increase in the mobility of the receptor-DNA complex, suggesting that it is unlikely to reflect the formation of a hormone-dependent transcriptional activation function.