RESUMO
The crystallization of the ligand-binding domain (LBD) of the estrogen receptor (ER) with 17beta-estradiol and raloxifene [A. M. Brzozowski et al., Nature (Lond.), 389: 753-758, 1997] now provides a molecular basis for the biological activity of complexes as either agonists or antagonists. It is well established that the critical structural feature of antiestrogens is a correctly positioned alkylaminoethoxy side chain. The X-ray crystallography clearly shows that the alkylaminoethoxy side chain of raloxifene causes a specific and inappropriate molecular perturbation of the LBD and that the nitrogen in the side chain must hydrogen bond with aspartate 351 in the LBD of ER. We previously identified and characterized a naturally occurring mutation in the ER from a tamoxifen-stimulated transplantable human breast tumor line. The mutation is at AA351 of LBD, where the aspartate is changed to tyrosine (Asp351Tyr). In this report, we compared and contrasted the pharmacology of raloxifene to block or induce E2-stimulated increase in TGF-alpha mRNA in stable transfectants of ER-negative human breast cancer cells with the cDNAs from wild-type, mutant-amino acid (AA) 400 ER and mutant-AA 351 ER. Our results show that the mutation at AA 351 that replaces aspartate by tyrosine specifically changes the pharmacology of raloxifene from an antiestrogen to an estrogen. By contrast, a mutation at AA 400 does not, and the antiestrogenic properties of raloxifene are retained. These data and the fact that the nitrogen in the side chain must specifically interact with aspartate 351 makes this the key to the antiestrogenic activity of raloxifene.
Assuntos
Ácido Aspártico , Antagonistas de Estrogênios/farmacologia , Piperidinas/farmacologia , Receptores de Estrogênio/metabolismo , Northern Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Estradiol/agonistas , Estradiol/farmacologia , Antagonistas de Estrogênios/química , Feminino , Humanos , Mutagênese Sítio-Dirigida , Piperidinas/química , Mutação Puntual , RNA Mensageiro/metabolismo , Cloridrato de Raloxifeno , Receptores de Estrogênio/genética , Transfecção , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Estrogens are involved in a multiplicity of programmed events in target tissues e.g.: uterus, breast, and pituitary gland, and hormone-responsive tumors occur at these target sites. We have addressed the possibility that all of the estrogens do not produce the same conformation of estrogen receptor alpha (ER). A novel assay in vitro was used to activate the transforming growth factor alpha (TGF-alpha) gene in situ in MDA-MB-231 cells stably transfected with cDNA for D351 ER or D351G ER. Three estrogen types were used: estradiol, diethylstilbestrol, and a triphenylethylene (TPE) derivative of tamoxifen without the antiestrogenic side chain. Computer molecular modeling was used to interpret data. A flat estrogen such as estradiol or diethylstilbestrol can induce TGF-alpha through a correctly positioned activating function 2 (AF2) and bind SRC-1. The TPE did not activate AF2 but activated the TGF-alpha gene through AF2b. This was demonstrated because D351 but not D351G ER activated the TGF-alpha gene with the TPE. We propose two classes of estrogens with different ER complexes that may incorporate different coactivators to function. Phytoestrogens and environmental xenoestrogens will fall into different classes based on structure and may exhibit selective actions and carcinogenic potential based on different ER conformations.
Assuntos
Estrogênios/classificação , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Congêneres do Estradiol/química , Congêneres do Estradiol/classificação , Congêneres do Estradiol/farmacologia , Estrogênios/química , Estrogênios/fisiologia , Humanos , Modelos Moleculares , Conformação Proteica , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Transfecção , Fator de Crescimento Transformador alfa/genética , Células Tumorais CultivadasRESUMO
Knowledge of the mechanism of action and pharmacology of tamoxifen and raloxifene, for the prevention of breast cancer and osteoporosis respectively, has opened the door for the discovery of multifunctional medicines. There is now the potential to prevent osteoporosis, coronary heart disease, breast and endometrial cancer in postmenopausal women with elevated risk factors.
Assuntos
Neoplasias da Mama/prevenção & controle , Cloridrato de Raloxifeno/uso terapêutico , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Tamoxifeno/uso terapêutico , Feminino , HumanosRESUMO
Knowledge of the mechanism of action and pharmacology of tamoxifen and raloxifene, for the prevention of breast cancer and osteoporosis respectively, has opened the door for the discovery of multifunctional medicines. There is now the potential to prevent osteoporosis, coronary heart disease, breast and endometrial cancer in postmenopausal women with elevated risk factors.
Assuntos
Neoplasias da Mama/química , Receptores de Estrogênio/fisiologia , Neoplasias da Mama/etiologia , Neoplasias da Mama/prevenção & controle , Feminino , Humanos , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/químicaRESUMO
We have shown that 4-hydroxytamoxifen (4-OHT) has estrogen-like effects on induction of TGFalpha mRNA in estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells, transfected with either wildtype (S30 cells) or a codon 351asp-->tyr mutant ER (BC-2 cells). The mutant receptor used to produce the stable transfectants was identified in a tamoxifen-stimulated human breast tumor. We have also demonstrated that raloxifene exhibits a gene-specific estrogen-like effect with mutant ER (BC-2 cells) but not with wildtype ER (S30 cells) (Levenson, A.S., Catherino, W.H. and Jordan, V.C. (1997) Estrogenic activity is increased for an antiestrogen by a natural mutation of the estrogen receptor. J. Steroid Biochem. Mol. Biol., 60, 261-268). We now describe the regulation of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) endogenous gene expression by estradiol (E2) and different antiestrogens in BC-2 cells. Northern blot analyses revealed that 4-OHT and raloxifene have concentration-dependent agonistic (E2-like) effects on the regulation of these genes. In contrast, the pure antiestrogen ICI 182780 alone had no effect but could block the action of E2, 4-OHT and raloxifene. The E2-like effects of non-steroidal antiestrogens in this model system cannot be explained by the mutation in the ER alone because 4-OHT acts as an agonist with wildtype receptor as well. We propose that the clear cut biological expression of estrogen-like qualities with different antiestrogens will in the future serve as an important model to dissect the signal transduction pathway.
Assuntos
Neoplasias da Mama/metabolismo , Antagonistas de Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica , Inibidor 1 de Ativador de Plasminogênio/genética , Receptores de Estrogênio/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Feminino , Humanos , Piperidinas/farmacologia , RNA Mensageiro/análise , Cloridrato de Raloxifeno , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Células Tumorais CultivadasRESUMO
Estrogen responsiveness of breast tumors can be correlated with the presence or absence of the estrogen receptor (ER). Breast cancer cells that contain ER are, in general, responsive to stimulation by estrogen both in vivo and in vitro; therefore hormonal control is possible. Breast tumors that lose the ER, and become hormone-independent are refractory to the direct effect of estrogens and antiestrogens. It is therefore of interest to determine whether the re-expression of the ER will be sufficient to make ER-negative cells sensitive to the growth effect of estrogen. Transfection experiments with wild type and mutant ER cDNAs into different mammalian cell lines have been performed to re-establish hormonal control over hormone-independent cells. Paradoxically, introduction of exogenous ER into ER-negative cells and treatment with estrogen leads to growth inhibition rather than growth promotion. The activation of a number of estrogen-regulated genes has been examined in ER-transfectants but gene regulation is often variable. It is clear that the transfection of the ER gene into cells lacking this protein does not simply re-create the native ER-positive phenotype. Studies need to be extended to identify either the transcription factors that interact with ER to cause the negative effects of estrogen indirectly ("squelching") or the precise target genes that cause growth inhibition directly.
Assuntos
Divisão Celular/fisiologia , Estrogênios/fisiologia , Receptores de Estrogênio/genética , Transfecção , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Complementar , Regulação da Expressão Gênica/fisiologia , Humanos , Dados de Sequência Molecular , Mutação , Receptores de Estrogênio/fisiologiaRESUMO
The estrogen receptor (ER) functions as a ligand-activated transcription factor which mediates the actions of estrogens and antiestrogens in target tissues. Other investigators have shown that artificial point mutations in the transcriptional activation domain AF-2 of the ligand binding domain (LBD) of the ER can increase the estrogenic properties of antiestrogens, determined by transcriptional activation of estrogen-responsive reporter constructs cotransfected into cells. Although these data provide valuable information about ER function there is no evidence that these mutations occur naturally. We have taken a different approach and examined the naturally occurring codon 351 asp --> tyr mutation in the LBD of ER to stimulate the expression of an endogenous target gene. This approach avoids dependence on artificial reporter constructs and their idealized estrogen response elements (EREs). In this report we describe the regulation of transforming growth factor alpha (TGF alpha) mRNA by estradiol and the antiestrogens keoxifene and ICI 182,780 in our stable transfectants of ER-negative MDA-MB-231 breast cancer cells, which express either the wild-type (S30 cells) or codon 351 asp --> tyr mutant ER (BC-2 cells). The mutant receptor was identified in a tamoxifen-stimulated human breast tumor. Our results demonstrate, for the first time, that a naturally occurring mutation in the ER changes the pharmacology of the antiestrogen keoxifene by increasing estrogenic activity, and that keoxifene exhibits a gene-specific estrogen-like effect with mutant ER but not with wild-type ER. The pure antiestrogen ICI 182,780 maintained complete antagonistic activities in both ER transfectants, demonstrating that its action is unaffected by the mutation.
Assuntos
Neoplasias da Mama/genética , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptores de Estrogênio/genética , Fator de Crescimento Transformador alfa/biossíntese , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estradiol/análogos & derivados , Feminino , Fulvestranto , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Piperidinas/farmacologia , RNA Mensageiro/análise , RNA Neoplásico/análise , Cloridrato de Raloxifeno , Receptores de Estrogênio/agonistas , Transfecção , Células Tumorais CultivadasRESUMO
Tamoxifen is a valuable therapeutic agent with applications in the treatment and prevention of breast cancer. However, the development of drug resistance limits the usefulness of tamoxifen therapy. One form of drug resistance in breast cancer is tamoxifen-stimulated growth. We have addressed a mechanism how the tamoxifen-estrogen receptor (ER) complex can convert from being a blocking to stimulatory signal in breast cancer. We have described an effective assay system to study the action of antiestrogen-ER complex through the activation of transforming growth factor alpha gene in situ. The MDA-MB-231 breast cancer cells were stably transfected with cDNAs for wtER (D351), mutant Asp351Tyr ER (D351Y) and mutant Asp351Gly ER (D351G). The D351Y ER can enhance the estrogenic properties of 4OHT and change the pharmacology of raloxifene by converting it from antiestrogen to estrogen. We hypothesized that alterations in the charge of amino acid (aa) 351, and changes in the interaction with the side chain of an antiestrogen, are critical for the subsequent estrogenicity of the complex. Our goal was (1) to modulate the estrogenicity of the antiestrogen-ER complex by different aa substitutions at position 351 and (2) to examine the role of alterations in the side chain of antiestrogens on the estrogenicity of the complex. Substitution of tyrosine for aspartate at aa351 results in increased estrogenicity for a series of tamoxifen derivatives-ER complexes and the conversion of EM 652-ER and GW 7604-ER complexes from antiestrogenic to estrogen-like. Substitution of glycine for aspartate at aa 351 results in the conversion of 4OHT-ER complex from estrogen-like to antiestrogenic. We propose that the side chain of antiestrogens either neutralizes or displaces the charge at aspartate 351 thereby removing a charged site for the opportunistic binding of a novel coactivator. If no charge is present (D351G) then no coactivator can bind and the complex with any antiestrogen is not estrogen-like. However, if the charge is extended beyond the reach of an antiestrogen side chain (D351Y), then the coactivators bind and compounds are estrogen-like. The establishment of a relationship between the structure of the antiestrogen-ER complex and its function will enhance the development of novel compounds with unique biological activities and potentially avoid premature drug resistance.
Assuntos
Aminoácidos/metabolismo , Antineoplásicos Hormonais/metabolismo , Estrogênios/fisiologia , Receptores de Estrogênio/metabolismo , Tamoxifeno/metabolismo , Aminoácidos/química , Northern Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Humanos , RNA Mensageiro/genética , Receptores de Estrogênio/química , Fator de Crescimento Transformador alfa/genética , Células Tumorais CultivadasRESUMO
The selective estrogen receptor modulator, 4-hydroxytamoxifen (4-OHT) is a full agonist at the transforming growth factor (TGF) alpha gene in ER negative breast cancer cells stably transfected with ER alpha cDNA (Levenson et al., Br. J. Cancer 77 (1998) 1812-1819). E(2) and 4-OHT increase TGF alpha mRNA and protein in a concentration dependent manner. The responses to E(2) and 4-OHT are blocked by the pure antiestrogen ICI 182,780, which does not induce TGF alpha. Transfected MDA-MB-231 cells contain functional ER alpha but no ER beta function was detected. Neo transfected cells that did not express ER alpha or cells stably transfected with the DNA binding domain mutant C202R/E203V which prevents gene activation did not induce TGF alpha mRNA after either E(2) or 4-OHT treatment. An examination of the time course for either 10 nM E(2) or 1 microM 4-OHT for MDA-MB-231 cells stably transfected with cDNA for ER alpha showed increases in TGF alpha mRNA within 2 or 3 h respectively. Cells pretreated with cycloheximide (1 microg/ml) showed induced TGF alpha mRNA in response to E(2) or 4-OHT but TGF alpha mRNA induction was blocked by actinomycin D (1 microg/ml). We conclude that both E(2) and 4-OHT induce TGF alpha by direct interaction of ER alpha with DNA and that ER beta is not involved in the estrogen-like response to 4-OHT in the MDA-MB-231 cells.
Assuntos
Estradiol/farmacologia , Receptores de Estrogênio/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Fator de Crescimento Transformador alfa/metabolismo , Northern Blotting , Western Blotting , Cicloeximida/farmacologia , DNA Complementar/metabolismo , Dactinomicina/análogos & derivados , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Antagonistas de Estrogênios , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Humanos , Luciferases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Receptores de Estrogênio/biossíntese , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
In a breast tumor xenograft model, the MCT-1 oncogene increases the in vivo tumorgenicity of MCF7 cells by promoting angiogenesis and inhibiting apoptosis. Increases in the tumor microvascular density are accompanied by a strong reduction in the levels of the angiogenesis inhibitor thrombospondin-1 (TSP1), but the mechanisms underlying this process are unknown. We show that TSP1 expression is controlled, at least in part, by post-transcriptional events. Using RNA interference to knock down the expression of the RNA-binding protein HuR in MCF7 cells as well as HuR overexpression, we demonstrate that HuR plays an important role in translation of the TSP1 mRNA. Furthermore, employing the RIP-Chip assay yielded 595 transcripts with significantly altered binding to HuR in the more tumorigenic breast cancer clones compared with the weakly tumorigenic clones. These mRNAs clustered in several pathways implicated in the transformed phenotype, such as the RAS pathway (involved in mitogenesis), the PI3K pathway (evasion of apoptosis) and pathways mediating angiogenesis and the cellular response to hypoxia. These findings demonstrate for the first time that global changes in HuR-bound mRNAs are implicated in the evolution to a more tumorigenic phenotype in an in vivo tumor model and underscore the role of global mRNA-protein interactions toward tumor progression.
Assuntos
Antígenos de Superfície/fisiologia , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a RNA/fisiologia , Trombospondina 1/genética , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Feminino , Humanos , Proteínas Oncogênicas/fisiologia , Fenótipo , Fosfatidilinositol 3-Quinases/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Transdução de Sinais , Transcrição GênicaRESUMO
Oestrogens and antioestrogens modulate the synthesis of transforming growth factor alpha (TGF-alpha) in breast cancer cells. The purpose of the present report was to examine regulation of TGF-alpha gene expression by oestradiol (E2) and antioestrogens in MDA-MB-231 breast cancer cells transfected with either the wild-type or mutant oestrogen receptor (ER). We recently reported the concentration-dependent E2 stimulation of TGF-alpha mRNA in MDA-MB-231 ER transfectants (Levenson et al, 1997). We now report that 4-hydroxytamoxifen (4-OHT) shows oestrogen-like effects on the induction of TGF-alpha gene expression in our transfectants. Accumulation of TGF-alpha mRNA in response to both E2 and 4-OHT but not in response to the pure antioestrogen ICI 182,780 suggests that E2-ER and 4-OHT-ER complexes can bind to an oestrogen response element (ERE), located in the promoter region of the TGF-alpha gene and can activate transcription of the gene. Surprisingly, no activation of luciferase expression was observed after transient transfection of the TGF-alpha ERE/luciferase reporter constructs. Possible activation of an alternative ER-mediated pathway responsible for the regulation of TGF-alpha gene expression in the ER transfectants is discussed.
Assuntos
Neoplasias da Mama/metabolismo , Antagonistas de Estrogênios/farmacologia , Estrogênios/farmacologia , RNA Mensageiro/análise , Receptores de Estrogênio/fisiologia , Tamoxifeno/análogos & derivados , Fator de Crescimento Transformador alfa/genética , Neoplasias da Mama/patologia , Relação Dose-Resposta a Droga , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Fulvestranto , Humanos , Tamoxifeno/farmacologiaRESUMO
The characterization of growth arrest-associated genes has revealed that cells actively suppress mitotic growth in response to extracellular signals. Mouse 3T3-L1 cells growth arrest at multiple distinct points during terminal differentiation to adipocytes. We examined the expression of growth arrest-specific (gas) and growth arrest- and DNA damage-inducible (gadd) genes as a function of 3T3-L1 growth arrest and adipocyte development. These growth arrest-associated genes are differentially expressed throughout adipocyte development. Some of the gas/gadd genes are preferentially expressed in a subset of growth arrest states. In contrast, gas1 and gas3 are expressed in serum-starved adipoblasts, contact-inhibited adipoblasts, and post-mitotic adipocytes. However, in post-mitotic adipocytes, gas1 and gas3 are induced in response to nutrient deprivation, not altered growth status. gas6 is an exception to the general concordance of mitotic growth arrest and gas/gadd expression in that gas6 is preferentially expressed during the clonal expansion of postconfluent adipoblasts. Combined, the expression patterns indicate that growth arrest-associated genes are regulated by numerous signal transduction pathways throughout a discrete developmental transition.
Assuntos
Adipócitos/citologia , Ciclo Celular , Regulação da Expressão Gênica , Glicoproteínas de Membrana/biossíntese , Proteínas da Mielina/biossíntese , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae , Células 3T3 , Adipócitos/fisiologia , Animais , Sequência de Bases , Northern Blotting , Proteínas de Ciclo Celular , Meios de Cultura Livres de Soro , Dano ao DNA , Primers do DNA , Sondas de DNA , Fase G1 , Proteínas Ligadas por GPI , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/genética , Proteínas de Membrana , Camundongos , Mitose , Dados de Sequência Molecular , Proteínas da Mielina/genética , Proteínas/genética , Fatores de Tempo , Transcrição Gênica , Proteínas GADD45RESUMO
To understand the hormonal regulation of the components of the plasminogen-plasmin system in human breast cancer, we examined the oestradiol (E2) regulation of plasminogen activators (PAs), namely urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1) and uPA receptor (uPAR), in our model system. We used stable transfectants of the MDA-MB-231 human breast cancer cells that express either the wild-type (S30 cells) or the mutant 351asp-->tyr oestrogen receptor (ER) (BC-2 cells). Northern blot analysis showed that there was a concentration-dependent down-regulation of uPA, tPA and PAI-1 mRNAs by E2. In contrast, uPAR mRNA was not modulated by E2. The pure anti-oestrogen ICI 182,780 was able to block E2 action, indicating that the regulation of these genes is ER mediated. The E2 also inhibited the expression and secretion of uPA, tPA and PAI-1 proteins as determined by enzyme-linked immunosorbent assay (ELISA) in cell extracts (CEs) and conditioned media (CM). Zymography of the CM confirmed the inhibitory effect of E2 on uPA activity. Thus, we now report the regulation of uPA, PAI-1 and tPA by E2 in both mRNA and protein levels in ER transfectants. The association between down-regulation of the uPA by E2 and known E2-mediated growth inhibition of these cells was also explored. Our findings indicate that down-regulation of uPA by E2 is an upstream event of inhibitory effects of E2 on growth of these cells as the addition of exogenous uPA did not block the growth inhibition by E2.
Assuntos
Estradiol/farmacologia , Proteínas de Neoplasias/metabolismo , Ativadores de Plasminogênio/metabolismo , Receptores de Estrogênio/fisiologia , Neoplasias da Mama/metabolismo , Divisão Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Proteínas de Neoplasias/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativadores de Plasminogênio/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tecidual/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
The purpose of this study was to classify selective oestrogen receptor modulators based on gene expression profiles produced in breast cancer cells expressing either wtERalpha or mutant(351)ERalpha. In total, 54 microarray experiments were carried out by using a commercially available Atlas cDNA Expression Arrays (Clontech), containing 588 cancer-related genes. Nine sets of data were generated for each cell line following 24 h of treatment: expression data were obtained for cells treated with vehicle EtOH (Control); with 10(-9) or 10(-8) M oestradiol; with 10(-6) M 4-hydroxytamoxifen; with 10(-6) M raloxifene; with 10(-6) M idoxifene, with 10(-6) M EM 652, with 10(-6) M GW 7604; with 5 x 10(-5) M resveratrol and with 10(-6) M ICI 182,780. We developed a new algorithm 'Expression Signatures' to classify compounds on the basis of differential gene expression profiles. We created dendrograms for each cell line, in which branches represent relationships between compounds. Additionally, clustering analysis was performed using different subsets of genes to assess the robustness of the analysis. In general, only small differences between gene expression profiles treated with compounds were observed with correlation coefficients ranged from 0.83 to 0.98. This observation may be explained by the use of the same cell context for treatments with compounds that essentially belong to the same class of drugs with oestrogen receptors related mechanisms. The most surprising observation was that ICI 182,780 clustered together with oestrodiol and raloxifene for cells expressing wtERalpha and clustered together with EM 652 for cells expressing mutant(351)ERalpha. These data provide a rationale for a more precise and elaborate study in which custom made oligonucleotide arrays can be used with comprehensive sets of genes known to have consensus and putative oestrogen response elements in their promoter regions.