MXPA01006150A - Bioassay for identifying estrogen receptor-&bgr;/&agr;selective modulators - Google Patents

Abstract

The present invention provides novel assay methods for identifying compounds that selectively activate estrogen receptors (ER) of the alpha or beta subtype. In particular, the results from two assays, one measuring ER-&bgr;activity and the other measuring ER-&agr;activity are interpreted. The assay measuring ER-&bgr;activity uses cells comprising endogenous metallothionein-II as well as a DNA plasmid comprising a polynucleotide encoding human ER-&bgr;. The assay monitors expression of metallothionein-II-mRNA in said cells, wherein the level of metallothionein-II expression is regulated when a potential ligand binds to ER-&bgr;. The assay measuring ER-&agr;activity uses cells comprising ER-&agr;as well as DNA plasmid comprising a reporter gene linked to an estrogen response element. The assay monitors expression of the reporter gene, wherein the level of reporter gene is regulated when a potential ligand binds to ER-&agr;. Compounds which modulate activity in one assay but have little or no activity in the other assay are defined as estrogen receptor subtype selective.

Description

BIOASSAY TO IDENTIFY SECRETIVE MODULATORS OF THE RECEIVER-BETA / ALPHA ESTROGENO Field of the Invention. The present invention relates to hormone receptors and more particularly, to methods for the identification of compounds that selectively activate estrogen (ER) receptors of the alpha or beta subtype, as well as a test kit for use in the methods . Background of the Invention The proteins that regulate the expression of genes are essential for the function of cells. A well-studied family of gene regulatory proteins is the superfamily of steroid hormone receptors. These receptor proteins allow cells to respond to various hormones by activating or suppressing specific genes. A member of this family is the ER. Estrogens are classically known as hormones important in sexual development and reproductive function. It is well known that estrogens affect cell proliferation and differentiation in target tissues by binding to target cells in ERs. Estrogen replacement therapy is a well-ref: 129851 treatment established for the prevention and / or reduction of osteoporosis in post-menopausal women (Sagraves, 1995, Lobo, 1995) since these compounds have been shown to prevent the loss of bone and fractures in women. Additionally, estrogen replacement therapy has been associated with a decreased mortality of cardiovascular diseases. Finally, follow-up studies suggest that estrogens may benefit the central nervous system with respect to cognitive improvement and a decrease in Alzheimer's disease. The classic ERs, now designated ER-a, show a modular structure with different domains involved in a ligand binding, dimerization of receptors, DNA binding, and transcriptional activation (Green et al., 1986). The nucleotide sequence of the DNA binding domain is conserved among the steroid hormone receptors. Kuiper et al (1996), exploited this similarity in an attempt to identify additional members of this family. The new member they discovered was identified as an ER because the amino acids in the DNA binding domain were almost identical with those of ER-a, the translated protein in vi t ro, which specifically binds 3H-estradiol with nanomolar affinity , and is able to regulate transcription from a simple element of estrogen response. This protein has been designated ER-ß to distinguish it from the previously known form (now called ER-a). Human and mouse ER-ß have also been cloned (Bhat et al., 1998, Mosselman, 1996, Petersson et al., 1997, Tremblay et al., 1997). After the discovery of the ER-ß's, most of the work has focused on mapping the distribution of their mRNA in normal and neoplastic tissues, characterizing their binding affinity for a wide variety of ligands, and evaluating their interaction with ER -to. The ER-ß mRNA is detectable by RT-PCR and by in situ hybridization in a wide variety of tissues. Since its distribution overlaps with that of ER-a, some tissues express only one type of receptor. For example, both receptors are found in the uterus, ovary and pituitary, whereas ER-β appears more prominent in the prostate, lung and bladder (Kuiper et al., 1996). When examined by in situ hybridization, additional differences can be made between the distribution of the receptor. The ER-β mRNA is expressed in the epithelial cells of the rat prostate; ER-a is found in the stromal compartment (Kuiper et al., 1996). In the ovary of the rat, the ER-β appears in the granulosa cells; ER-a in the stroma (Shughrue et al, 1996, personal communication), in the hypothalamus of the rat, the message of ER-ß but not that of ER-a are expressed in the paraventricular region, while both messages are observed in the preoptic area (Shughrue et al., 1996). Interestingly, important species differences may exist in the relative levels of ER-ß and ER-a of mRNA in certain organs. For example, the mRNA of ER-β is highly expressed in the prostate of the rat, so that more modest levels are detected in the human prostate. ER-β predominates over ER-a in the rat prostate (Kuiper et al., 1996; Lau et al., 1998; Enmark et al., 1997), but the levels are more equivalent in the mouse prostate (Couse et al., 1997). Several groups have characterized the expression of ER in tumor samples and cancer cell lines. Most of the work has been done in the chest where one study reports ER-ß mRNA using RT-PCR in 70% of 40 breast biopsy samples (Dotzlaw et al., 1996). No correlation is observed between the expression of ER-a and ER-β. Another study also reports the heterogeneity of ER-a / β mRNA expression in breast tumor samples, with some tumors having only the ER-a mRNA and others expressing mRNA for both receptor subtypes (Enmark et al. , 1997). Among the breast cancer cell lines, MDA MB 231 has only the ER-β mRNA, and there are conflicting reports about the ER status of MCF-7 cells (Enmark, 1997; Kuiper et al., 1997; Vladusic et al., 1998, R. Henderson, unpublished observations). Although the ligand binding domain of ER-a and human ER-β is 59% identical at the amino acid level (Enmark et al., 1997), the binding affinity of 17β-est radiol is quite similar. Some compounds show a marked selectivity either by ER-a or ER-β. Genistein is a phytoestrogen that binds approximately 10-25 times more affinity in ER-ß (Kuiper 1996, H. Harris unpublished observations). On the other hand, raloxifene binds about 20 times better to ER-a than to ER-β (H. Harris, as unpublished observations). When ER-a and ER-β are coexpressed, the interaction can be measured by various methods including a two-hybrid mammalian system, descent of glutathione S transferase and gel spin / supergiro tests (Cowley et al., 1997; Pettersson et al., 1997; Ogawa et al., 1998). Since these receptors can be heterodimerized, the effect of estrogen in the tissues containing both receptors can be mediated by a complex interaction between ER-a and ER-β. A valuable tool in the determination of the function of ER-β, is the estrogen receptor of the non-transgenic mouse (ERKO) (Lubahn et al., 1993). Because these mice lack functional ER-a, they can help define the physiological roles of ER-a and ER-ß. One study compared the efficiency of treatment with 17β-est radiol to reduce the consequences of artificially induced vascular damage in the carotid arteries in wild-type and ERKO-type mice (Lafrati et al., 1997). In both types of mice, the pharmacological doses of 17β-est radiol suppressed the increase in the medial area and the uniform proliferation of muscle cells observed in the animals treated with the vehicle. It is thought that these responses to endothelial denudation can narrow the passage of the vessel, thus restricting blood flow. Since 17β-estradiol was equally effective in ERKO mice as with wild-type mice, one interpretation is that ER-a is not necessary for these responses. Since the ER-ß mRNA is also expressed in these vessels, it may mediate the action of 17β-est radiol. However, there is a lack of direct evidence to support this hypothesis. Another example of ERKO mice responding to the 17β-radiol substitution was described in a recent poster and summary (Pan et al., 1997, and personal communication). This study reports a loss of femoral bone mineral density and trabecular bone volume in ERKO mice after an ovariotomy and an increase in these parameters with the treatment of 17β-estradiol (but not with dihydrotes terona). Again, based on the indirect evidence, the suggestion is made that the ER-β has a physiological role. Thus, since certain aspects of ER-ß have been characterized, the art fails to provide methods for the identification of ligands, which are functionally selective for ER-a or ER-β. Said assay would be of great advantage for the pharmacological industry to discover the possible therapeutic applications of the ER subtypes. Brief description of the invention. The present invention provides a method for screening a test compound that binds to an ER in a receptor binding assay, wherein the method detects mediated transcription by ER-β polypeptides, the method comprising the steps of: a) supplying a cell comprising at least one estrogen-induced DNA sequence encoding the metallothionein (T-II), and at least one DNA sequence encoding the ER-β polypeptide, wherein the receptor is transcriptionally active; (b) contacting the cell with the test compound that binds to the ER or with a control; (c) detecting the expression of MT-II, wherein the increased expression of MT-II relative to the control indicates that the test compound has estrogen agonist activity. This invention further provides a method for screening a test compound that binds to the ER in a receptor binding assay, wherein the method detects the inhibition of transcription mediated by the ER-β polypeptide, and the method comprises steps of: (a) Providing a cell comprising at least one estrogen-induced DNA frequency encoding the metallothionein (MT-II), and at least one DNA frequency encoding the ER-β polypeptide wherein the receptor is active transcriptionally; (b) Contacting the cell with one or more estrogens in the presence of the test compound known to bind the ER; and (c) and detecting the expression of MT-II, wherein the decreased expression of MT-II in relation to the vision of one or more estrogens only, indicates that the test compound has estrogen antagonistic activity.

A method for screening test compounds to identify drug candidates that mimic the effect of estrogens on ER-β or ER-a mediated transcription is also provided, wherein the method comprises the steps of: a) contacting the test compound with a plurality of: (i) first cells comprising at least one DNA sequence encoding methotothione (MT-II) and at least one DNA sequence encoding an ER polypeptide -β, wherein the receptor is transcriptionally active and (ii) second cells comprising a construct of the ERE reporter gene, wherein the cells express the ER-a polypeptide; (b) identify compounds that increase the expression of MT-II in the first cells in relation to the control, but that have a minimal effect on the expression of the reporter gene in the second cells, where the compounds are considered selective to the ER- H.H; or (c) identify compounds that increase the expression of the reporter gene in the second cells relative to the control but that have a minimal effect on the expression of MT-II in the first cells, where the compounds are considered selective to the ER-a . Finally, the present invention provides a method for screening test compounds to identify drug candidates that inhibit the effect of estrogen on ER-β or ER-a mediated transcription, the method comprising the steps of: (a) contacting the test compound in the presence and absence of one or more estrogens with a plurality of: (i) first cells comprising at least one Endogenous DNA that encodes a gene - metallothionein (MT-II) and a DNA encoding an ER-β polypeptide, where the receptor is transcriptionally active (ii) second cells comprising a construct of the ERE reporter gene, wherein the cells express the ER-a polypeptide; and (b) identifying compounds that decrease the expression of MT-II in the first cells in relation to treatment with one or more estrogens alone, but that have a minimal effect on the expression of the reporter gene in the second cells, where compounds are considered selective for ER-β; or (c) identifying compounds that decrease the expression of the reporter gene in the second cells in relation to treatment with one or more estrogens alone but that have a minimal effect on the expression of MT-II in the first cells, where the compounds they are considered selective for ER-a.

Brief Description of the Figures Figure 1: It is the RT-PCR amplification of the ER-ß and ER-a of the Saos-2 and LNCaPLN3 cells. Figure 2: Amplified cDNAs separate the sequence-forming gel and the upregulation of the 17-β estradiol from fragment 6a. Figure 3: (A) nucleotide sequence (SEQ ID Num.12) of the regulated fragment and its alignment with human MT-II, SEQ ID Num.13. (B) translation of the fragment sequence of the first methionine to stop the stop codon (*) SEQ ID Num.14 Figure 4: Regulation of MT-II in two cell lines. To determine the incremental change in the mRNA, the MT-II signal was normalized with that of the GAPDH, and compared with the control cells. Figure 5: Dose response of 17-ß-estradiol regulation of T-II in Saos-2 cells. The results are shown for four different experiments and the EC50 shown for each. Figure 6: Time course of the regulation of methotreotomy-I I in Saos-2 cells. The data is normalized with the GAPDH and the incremental change is calculated from the control of the vehicle in time equal to 0 hours. Figure 7: Specificity of the receptor for the regulation of MT-II in Saos-2 cells. Figure 8: Regulation of MT-II in Saos-2 cells by various components. Figure 9: Regulation of metallothionein-I I in Saos-2 cells treated with cycloheximide.

(A) Measurement of protein synthesis during treatment with cycloheximide. (B) assay of ER binding in whole cells after 8 hours of treatment with cycloheximide. (C, D) regulation of the ionein I-metalotine after 8 and 24 hours of treatment respectively. Figure 10: MT is regulated by estrogen in the rat prostate. Figure 11: The induction of MT-II in the rat prostate requires at least two days of dosing. Figure 12: The screening strategy for ligands that selectively activate ER-ß and / or ERa. Detailed Description of the Invention The present invention provides an efficient way to screen large numbers of test compounds that selectively activate ERs of subtype a or β. These compounds may have desirable properties either for the treatment or prevention of various estrogen-mediated diseases, including but not limited to cancers (e.g., breast, ovaries, endometrium, prostate), endometriosis, osteoporosis, and nervous system diseases. central and cardiovascular. Definitions In the description and current claims, the following terms will be defined as indicated below. "hERßL" is defined as the human form of the ER-β described in Example 1 (the cDNA or its translated protein product). "Estrogen" is defined as any ligand that can function as an estrogen agonist. "Estrogen agonist" is defined as a compound that substantially mimics 17-ß-estradiol as measured in a standard assay for estrogenic activity, for example, cell assays as described in Webb et al. (1992).

"Estrogen antagonist" is defined as a compound that substantially inhibits the effect of estrogen agonists as measured in a standard assay for estrogenic activity, eg, cellular as described in ebb et al. (1992). "A functional ER" is defined as a receptor capable of transcriptional activation of endogenous or transfected genes as measured by changes in RNA, proteins, and / or biological events in the 3 'direction. "A non-functional ER" is defined as a receptor incapable of transcriptional activation of endogenous or transfected genes as measured by changes in RNA, proteins and / or biological events in the 3 'direction. A "test compound" includes but is not limited to any small molecule compound, peptide, polypeptide, natural product, toxin with potential biological activity. A "ligand" is intended to include any substance that interacts with a receptor. "Transfection" is defined as any method by which a foreign gene is inserted into a cultured cell.

An "informant" is defined as any substance that can be easily measured and distinguished from other cellular components. The informant can be the DNA of the transfected receptor, the mRNA of the transcribed receptor, an enzyme, a binding protein or an antigen. A "cell" useful for the current purpose, is one that has the ability to respond to signal transduction through a given receiver. A "receptor binding assay" is a test that measures the amount of ligand that specifically interacts with a receptor. The ligand can be a radio ligand (for example conjugated to 3 H or 125 I), a fluoresced ligand (either conjugated or with a fluorochrome or having an inherent fluorescence) or otherwise labeled to be detectable. Description of the test The present invention is supported by the discovery that the expression of MT-II is selectively regulated by the interaction of a ligand with ER-β. In the methods of the invention, the regulation of MT-II activity can be used to provide a screening system that selectively detects the estrogen agonist or antagonist functional activity of a ligand that follows its interaction with ER-β. The methods typically comprise cultured cells expressing functional human ER-β and none, or a decreased amount of ER-a. Such cells include but are not limited to Saos-2 (ATCC HTB-85) and LNaCPLN3. Preferred cells for this purpose include cells that over-express ER-β, such as the cells described below that are manipulated recombinantly to overexpress ER-β. The ER-β receptor can be modified in any way, such as in length; these modifications may result in an increased selectivity of the ER-β and an increased sensitivity of the assay. Someone with skill will recognize that various recombinant constructs comprising ER-β can be used in combination with any cell or line lacking functional ER-a. To screen various compounds for estrogen agonist activity, cells expressing ER-β are exposed to a test compound or to a control solution (which is used to dissolve the test compound). The cells can be exposed while growing in separate wells of a multi-well culture dish or in a semi-solid nutrient matrix. After treatment for an appropriate period of time, the mRNA of MT-II is measured. An estrogen agonist will increase the mRNA of MT-1I when compared to treatment with the control solution alone. To screen various compounds for estrogen antagonist activity, cells expressing ER-β are exposed to one or more estrogens in the presence or absence of a test compound. Cells can be exposed while growing in separate wells of a culture dish or in a matrix of semi-solid nutrients. After treatment for an appropriate period of time, the mRNA of MT-II is measured. An estrogen antagonist will decrease the mRNA of MT-II when compared to treatment with the estrogen solution alone. The estrogenic or antigenic compounds identified in the assays of the invention can be used in standard pharmaceutical compositions for the treatment of cancer, as components of oral contraceptives, or in any other application in which modulation of activity is desired. of estrogen. The pharmaceutical compositions can be prepared and administered using methods well known in the art. The pharmaceutical compositions are generally intended for parenteral, topical, oral or local administration, for prophylactic and / or therapeutic treatment. The pharmaceutical compositions can be administered in a variety of dosage unit forms depending on the method of administration. For example, the unit dosage forms suitable for oral administration include powders, tablets, pills and capsules. Pharmaceutical formulations suitable for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17a. Ed. (1985). A variety of pharmaceutical compositions comprising the compounds of the present invention and pharmaceutically effective carriers can be prepared.

Applications The methods and compositions of the present invention can be used to identify compounds that interact with ER-β, either to stimulate or to inhibit transcriptional activity. Such compounds include, without limitation, coactivating proteins, as well as oestrogens and other spheroids, and spheroidal-like molecules, or non-spheroidal-like molecules that act as agonists or antagonists. Sifting methods can also be used to identify tissue-specific estrogens. The identification of interactive ER-β compounds can be achieved by cell-based or cell-free assays. In a set of embodiments, the purified ER-ß is contacted with a labeled ligand, such as for example 17-β estradiol, in the presence of test compounds to form the test reactions, and in the absence of the compounds of test to form control reactions. The labeled portion may comprise a labeled radio (such as for example 3H or 125I) or a fluorescent molecule. Incubation is allowed to proceed for a sufficient time under appropriate conditions to reach the specific binding, after which, the labeled ligand bond is measured to the ER-β (by monitoring, eg, radioactivity, fluorescence or fluorescence polarization). In one embodiment, the ligand binding domain of ER-β produced in E. coli is absorbed into the wells of the microtiter plate and incubated with [3 H] -17β estradiol in the absence or presence of compounds. the soluble receptor is incubated with the tagged ligand in the absence or presence of test compounds, and the bound ligand is separated from the free ligand either by filtration in glass fiber filters or by using carbon coated with dextran. whole can also be used where the bound ligand is separated from the free ligand by rinsing.The cells used in these assays can contain the endogenous receptor or can over-express the receptor subsequent to transfection or stable or transient infection of an ER-β cDNA Non-limiting examples of appropriate cells include COS cells, HeLa cells, CHO cells, yeast from the endothelial cell of the human umbilical vein. has identified a compound as an interactive compound ER-β for its binding activity, additional tests can be carried out in vivo and in vitro to determine the nature and degree of activity, that is, as an agonist or antagonist (see below). ). Interactive ER-β compounds can also be identified using cell-based assays that measure transcriptional activation or suppression of transgenered or endogenous estrogen response genes. For example, agonists (such as for example 17-β-estradiol) block the induction of interleukin-lβ from endogenous E-selectin in endothelial cells of the human umbilical vein that express ER-β. Antagonists (such as for example ICI-182780) block the agonist activity of 17β-est radiol. Non-limiting examples of other elements that promote the response to appropriate endogenous estrogens include those that regulate endothelin-1 (ET-1); the HDL receptor (type 2 sequestering receptor); and the enzymes involved in coagulation and fibrinolysis (such as, for example, plasminogen activator inhibitor-1 and C3 complements). Any promoter element responsive to estrogen can be used as an appropriate target, including for example, the NFkB binding site or the apolipoprotein Al gene enrichment sequence. In a set of embodiments, the appropriate host cells are transfected with a vector of expression encoding the ER-β and the transfectants are incubated with or without one or more estrogens I in the presence or absence of test compounds. The ER-β activity is evaluated when measuring transcriptional activation. This can be achieved by the detection of the mRNA (using for example, Northern blot analysis, reverse transcriptase polymerase chain reaction, RNase protection assays) and / or detection of the protein (using for example immunoassays or functional assays). If activation of the target sequence initiates a biochemical cascade, biological events can also be measured in the 3 'direction to quantify ER-β activity. Interactive ER-β compounds are identified as those that have a positive or negative influence on the activation of the target sequence. In another set of embodiments, the appropriate host cells (preferably from mammals) are co-transfected with an expression vector encoding ER-β and a reporter plasmid containing a reporter gene in the 3 'direction of one or more EREs . The transfected cells are incubated with or without an estrogen in the presence or absence of the test compounds, after which the ER-β activity is determined by measuring the expression of the reporter gene. In a preferred embodiment, the ER-β activity is monitored visually. Very limiting examples of appropriate reporter genes include luciferase, acetyl chloramphenicol transferase (CAT), and green fluorescence protein. Preferably, the methods of the present invention are adapted to a high production screening, allowing a multiplicity of compounds to be tested in a simple assay. Candidate estrogens and estrogen-like compounds include, without limitation, diethylsterolysterol, genistein, and estrone. Other interactive ER-β compounds can be found in eg natural product collections, fermentation collections (encompassing plants and microorganisms), combinatorial collections, compound files and collections of synthetic compounds. For example, collections of synthetic compounds are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical collection is available from Aldrich Chemical Company, Inc. (Milwaukee, Wl) Alternatively, collections of natural compounds in the form of animal and plant extracts, fungi and bacteria are available from for example the Pan Laboratories (Bothell, WA) or MycoSearch (NC) or are easily produced. Additionally, the collections produced naturally and synthetically and the compounds, are easily modified through conventional chemical, physical and biochemical means (Blondelle et al., 1996). The ER-β binding assays according to the present invention are advantageous in accommodating different types of solvents and thus allow the testing of compounds from many sources. The compounds - identified as ER-β agonists or antagonists, which use the methods of the present invention, can be modified to increase potency, efficacy, ingestion, stability and suitability for use in therapeutic applications. These modifications are achieved and tested using methods well known in the art. ' EXAMPLES The present invention is further described by the following examples. The examples are provided solely to illustrate the invention with reference to the specific embodiments. These examples, while illustrating certain specific aspects of the invention, do not show the limitations or circumscribe the scope of the invention. Example 1 Construction of recombinant adenovirus ER-β. The human ER-β coding sequence was obtained as described in the pending co-proprietary and co-assigned US Serial Application 08 / 906,365 entitled "Novel Human ß-estrogen Receptor" filed on August 5, 1997, and in Bhat et al., (1998). Essentially, human poly A + RNA tests (1 μg, Clontech, Palo Alto CA) were mixed with 0.5 μg of an oligo dT primer (GIBCO-BRL, Gaithersburg MD) in a total volume of 10 μl. The mixture was heated at 70 ° C for 10 minutes and after cooling on ice was supplemented with 500 μM of deoxynucleoside triphosphate., synthesis buffer of cDNA IX, and 10 mM DTT up to a final reaction volume of 20 μl. The mixture was incubated at 42 ° C for 2.5 minutes and then supplemented with 1-2 units of reverse transcriptase (GIBCO-BRL, Gaithersburg MD), after which it was incubated at 45 ° C for 30 minutes and 50 ° C for 5 minutes. One-tenth of this mixture (approximately 2 μl) containing the cDNA template was then used in the PCR amplification of the ER-β using reverse and forward primers as described below. PCR primers designated in the No. 08/906, 365 series (supra) were used to amplify the ER-β sequence in a reaction containing the following components: 2 μl of the cDNA template described above; PCR buffer solution IX; 200 μm of each of deoxynucleoside triphosphate, 2 units of hot-run DNA polymerase (Amersham, Arlington Heights IL), and one μg of each of the reverse and forward primers. The reaction mixture was heated at 95 ° C for 2 minutes, strengthened at 52 ° C for 1 minute and amplified using 36 cycles followed by incubation at 72 ° C for 1.5 minutes. A fragment of approximately 1500 bp in length was produced. The fragment digested with HindIII and Xbal (which is split at the sites present in the reverse and forward primer sequences respectively, but not in the main body of the amplified cDNA sequence) and cloned into the corresponding sites of the pcDNA3 expression vector ( Invitrogen, Carlsbad CA). This asymmetric cloning strategy places the 5 'end of cDNA ER-β under the control of the viral CMV promoter in pcDNA3. This clone was designated as "truncated hERβ" or hERβT. To verify the amino terminal and the sequence in the 5 'direction of human ERβ, two independent approaches were taken as described below. (1) 10 μl of an extended 5 'cDNA collection from the human ovary (Clontech, Palo Alto CA) was mixed with 50 μl of a K IX solution (PCR buffer IX (GIBCO-BRL, Gaithersburg MD), 2.5 mM of MgCl2, and 0.5% of Tween-20, 100 μg / ml of proteinase K), and the reaction mixture was incubated at 56 ° C for 2 hours, then at 99 ° C for 10 minutes. Five μL of this reaction mixture were then used as a template in an incubated PCR reaction using the PCR primers designated in serial No. 08 / 906,365 (supra). The reaction contained a slow PCR reaction buffer IX (40 mM Tricine-KOH, 15 mM KAOc, 3.5 mM Mg (Oac) 2, 75 μg / ml bovine serum albumin, 0.2 μM each dNTP, 0.2 μM each of the above primers and a Klentaq polymerase mixing unit (Clontech, Palo Alto CA) The PCR break conditions were as follows: 5 cycles at 94 ° C for 2 seconds and 72 ° C for 4 minutes, followed for 30 cycles of 94 ° C for 2 seconds and 67 ° C for 3 minutes.

Nucleotides and primers were removed in excess of the first round of PCR reactions by purification using Wizard columns for PCR (Promega, Madison Wl). A second round PCR reaction was then carried out using 2 μl of the purified first round reaction mixture using the PCR primers designated in serial No. 08 / 906,365 (supra). The PRC reaction and the cyclization conditions were identical to those used in the first round. The products were cloned into pCR2.1 (Invitrogen, Carlsbad CA) and three resulting clones were sequenced. All three clones (designated Ll, L2 and L3) contained the ER-β inserts of different lengths, all of which were homologous to the ER-β and one with the other. (2) A Marathon Ready cDNA thymus kit (Clontech, Palo Alto CA) for fast 5 'amplification of the cDNA terminals (RECE) was also used to isolate the 5' clones from the ER-β. In the first round of an incubated PCR reaction, 5 μl of the Marathon-ready human thymus cDNA (Clontech, Palo Alto CA) was used as a template and the primers designated in serial No. 08 / 906,365 (supra) were used. . The PCR reaction and the cyclization conditions were identical to those described in (1) above. Excessive primers and nucleotides were separated from the first round of PCR reactions by purification on Wizard PCR columns (Promega, Palo Alto CA). A second round of PCR reaction was carried out using 2 μl of the purified first round reaction using the primers designated in serial No. 08 / 906,365 (supra). The PCR reaction of the second round and the cyclization conditions were identical to those used in the first round. The products were cloned into the vector pCR2.1 (Invitrogen, Carlsbad CA) and two clones were sequenced. The two clones contain sequences of inserts of different lengths that are homologous to the ER-β, one with the other and the sequences isolated from the cDNA collection of the human ovary as described above. All the ER-β sequences isolated by methods 1 and 2 above contained 110 nucleotides corresponding to the hERβT sequences, as well as 228 additional nucleotides at the 5 'terminal. The hERβT and 5 'end sequences were joined and the resulting cDNA was cloned into pCADN3 (Invitrogen, Carlsbad CA) under the control of the cytomegalovirus IE promoter; this expression vector was designated as "long hER-β" or hERβL. The full-length sequence of the hERβL cDNA encodes a polypeptide having 530 amino acid residues. The hERßL sequence contained an optimal CCACC translation initiation sequence immediately in the 5 'direction to the initiation codon and the sequence was under the control of the cytomegalovirus IE promoter. The coding sequence of hERßL was then transferred to a plasmid of an Ad5? Vector containing the adenovirus sequences of mapping unit 0-17 with a deletion of the Ela region between mapping units 1.4-9.1 (Davis AR et al., 1985, Gluzman, Y. et al., 1982). The hERßL transcription unit in the Ad5? Plasmid contained the cytomegalovirus 1E promoter, the tripartite leader Ad5, the hERβL coding sequence and the SV40 late polyadenylation signal sequences. The hERßL in plasmid Ad5? El was then linearized with the BstEII enzyme and transfected with the ClaI A fragment of the Ad5 virus with the removal of the E3 region (80-88 mapping units) in 293 cells (primary human embryonic kidney) transformed, ATCC CRL 1573). Viral plaques generated by homologous recombination were isolated, amplified and characterized by DNA restriction analysis and cell lysis assay in A549 cells (human lung carcinoma, ATCC CCL 185). Confirmatory tests indicated that the recombinant virus of Ad5 hERßL contained the expected fragments of DNA and were defective in replication. The virus was further purified by a new plaque formation. The isolated plates were amplified, tested and used as a stock inventory to generate large amounts of the virus in 293 cells. The virus was titrated in 293 cells per plaque assay and the inventory contained 1.28xl09 PFU / ml. Example 2: Evaluation of the endogenous levels of ER mRNA in Saos-2 and LNCaPLN3 cells. Unless indicated otherwise, the reagents for the Gibco BRL cell culture (Gaithersburg MD) were obtained. The LNCaPLN3 cells were grown in RPMI 1640 medium supplemented with 10% FBS, 2 mM GlutaMAX-1, 100 U / ml penicillin g, and 100 μg / ml sulfate is reptil omocin. Saos-2 cells (ATCC, Manassas VA) were maintained in a monolayer culture using a McCoy 5A medium supplemented with 10% fetal bovine serum (FBS), 2 mM GlutaMAX.l, 100 U / ml g penicillin, and 100 μg / ml streptomycin sulfate. RT-PCR. Total RNA was isolated from LNCaPLN3 and Saos-2 cells using TRIzol (Gibco BRL, Gaithersburg MD) according to the manufacturers' instructions. The samples were then treated with RNase-free DNase I (Gibco BRL, Gaithersburg MD) at 1 unit / μg for 30 minutes at 37 ° C. RNA was purified from the reaction using RNeasy columns (Qiagen, Hilden Germany) and the amount recovered was estimated by UV spectrophotometry. The reverse transcription reactions were carried out in 0.5 μg of RNA in a 20 μl reaction. For the ER-a reaction containing the IxPCR buffer (Gibco BRL, Gaithersburg MD), 5 mM MgC12, ERa specific reverse primer 1.25 μM (5'-CCAGCAGCATGTCGAAGATC-3 ', SEQ ID NO: 1), specific reverse primer of GAPDH 0.5 μM (5'-CACCCTGTTGCTGTAGCCAAATTC-3 ', SEQ ID NO: 2), 0.5 mM dNTPs, 20 units of RNasin (Promega, Madison Wl) and 200 units of Superscript II reverse transcriptase (Gibco BRL, Gaithersburg MD) . The ER-β reaction contained the same components as ER-a with the following exceptions: 2.5 mM MgS12 and ERβ specific reverse primer (5 '-GCAGAAGTGAGCATCCCTTTG-3', SEQ ID NO: 3). A duplicated reaction that was identical in all the reagents except that it did not contain the Superscript II reverse transcriptase was carried out for each sample as a negative control to ensure that the RNA samples were not contaminated by DNA. The reactions were incubated at 42 ° C for 15 minutes, followed by 5 minutes at 99 ° C and 5 minutes on ice before amplification. PCR was initiated by adding 80 μl of original mixture containing the specific forward primer ER-a (5 '-GGAGACATGAGAGCTGCCAAC-3', SEQ ID NO: 4) or the specific forward primer ER-β (5 '-CAGCATTCCCAGCAATGTCAC-3 ', SEQ ID NO: 5) and the specific forward primer GAPDH (5'-GACATCAAGAAGGTGGTGAAGCAG-3', SEQ ID NO: 6) directly to the 20 μl reverse transcriptase reaction. The final concentration of the reagents in the PCR reaction of 100 μl of ER-a was as follows: 0.25 μM each for the specific ER primer, 0.1 μM for each primer GAPDH, lx PCR buffer (Gibco BRL, Gaithersburg MD), 0.2 mM of dNTPs, 2 mM of MgC12 and 0.5 units of Taq DNA polymerase (Gibco BRL, Gaithersburg MD). The ER-β reaction contained the same amount of reagents except for 1 mM MgCl 2. A two-step PCR was carried out in a PE 9600 for 25 cycles as follows: 95 ° C for 30 seconds, 64 ° C for 1.5 minutes. The samples were incubated at 64 ° C for 10 minutes after amplification. 20 microliters were separated from each sample using a 1.5% agarose gel and transferred to Hybond-N + (Amersham Pharmacia, Piscataway NJ) by an alkaline capillary Southern stain in NaOH, 0.4 N, 0.6M NaCl. The stains were prehybridized at 42 ° C for 30 minutes in a Rapid-Hyb buffer solution (Amersham Pharmacia, Piscataway NJ). Oligonucleotide probes specific for ER-a fragments (5 '-TGAACCAGCTCCCTGTCTGCCAGGTTGGT-3', SEQ ID NO: 7), ER-β (5 '-CCGCATACAGATGTGATAACTGGCGATGGA-3', SEQ ID NO: 8) and GAPDH (5 ' -GCTGTTGAAGTCACAGGAGACAACCTGGT-3 ', SEQ ID NO: 9) were labeled in the terminal with 32P-? ATP using the polynucleotide synthase (Gibco BRL, Gaithersburg MD). The probes were added to the spot at 3.0 x 106 CPM / ml and incubated at 42 ° C for 1 hour. Hybridizations of ER and GAPDH were done independently. The smears were washed once in 2x SSC, 0.1% SDS at room temperature for 15 minutes then twice in 0.2x SSC, 0.1% SDS at 42 ° C for 15 min. The stains were then exposed to the film. Saos-2 cells expressed endogenous ER-β but not ER-a mRNA when evaluated by PCR (Figure 1) . As a positive control, the GAPDH mRNA was co-amplified in these reactions.

Although both cell lines contained the GAPDH mRNA, only the Saos-2 cells contained the ER-β. Similar results were obtained for LNCaPLN3 cells (data not shown).

Example 3: Discovery of ER-ß upregulates MT-II- in Saos-2 and in LNCaPLN3 cells Cultivation of cells and infection Because the message levels for ER-β were low, engineering preparations were prepared Saos-2 and LNCaPLN3 cells to over express hERßL by transient infection with a recombinant adenovirus (see example 1) prior to differential deployment. The cells of LNCaPLN3 and Saos-2 were cultured as described above. 16 hours before infection, the cells were plated in a medium free of phenol red RPMI 1640 supplemented with 10% carbon / treated with dextran ("depleted") FBS (HyClone, Logan UT), 2 mM GlutaMAX-1 , 100 U / ml of penicillin g, and 100 μg / ml of streptomycin sulfate. This medium was used for the rest of the experiment. The cells were infected with a 1/20 dilution of an Ad5 hERßL virus (see Example 1) using a phenol red free medium FBS depleted with antibiotics and GlutaMAX-1 for 2 hours at 37 ° C. The virus containing the medium was aspirated and the cells were washed with the medium. Fresh medium was added and the cells were allowed to recover overnight at 37 ° C. The next day, the cells were treated with 10 nM 17β-radiol or vehicle for 24 hours and the total RNA was prepared for differential deployment -using the TRIzol reagent (Gibco BRL, Gaithersburg MD). To remove the residual DNA, the samples were treated with Rnasa-free DNase I (Gibco BRL, Gaithersburg MD) at one unit / μg for 30 minutes at 37 ° C. RNA was purified from the reaction using RNeasy columns (Qiagen, Hilden Germany) and the amount recovered was estimated by UV spectrophotometry. Rapid differential expression analysis (RADE) Reverse transcription (RT) After treatment with DNase I, 6 micrograms of total RNA were incubated with a buffer solution lx RT (25 mM Tris-Cl, pH 8.3, 37.6 mM KCl, 3 mM MgCl2 and 5 mM DTT, from Genhunter, Nashville TN), 20 μm dNTP's (dA, C, G and 2 'TTP triphosphates-5' deoxynucleosides from Gibco BRL (Gaithersburg MD), 0.2 μM HT11C (oligonucleotide AAGCTTTTTTTTTTTC, SEQ ID NO: 15) in a final volume of 600 μL This reaction mixture was incubated at 65 ° C for five minutes to denature the secondary structures, followed by a 10 minute incubation at 37 ° C.

At this time, 30 μl of Superscript II reverse transciptase (200U / μl, Gibco BRL, Gaitersburg MD) to the reaction and the incubation proceeded for 1 hour at 37 ° C. The enzyme is inactivated when heating at 75 ° C for five minutes.

An aliquot of this reaction was then used for the second strand synthesis by PCR. Polymerase Chain Reaction (PCR) To 2 μl of the RT reaction, buffer solution lx PCR (10 mM Tris-Cl, pH 8. 4, 100 mM KCl, 1.5 mM MgCl2 and 0.001% gelatin), 2 μM dNTO's, 15nM 33P dATP (NEN, Boston MA), 1 unit of AmpliTag DNA polymerase (Perkin-Elmer, Norwalk CT) and arbitrary primer 1 μM 5 'AAGCTTGCCATGG-3' for a total reaction volume of 20 μl. This reaction mixture was then put on a thermal cycle using the following parameters: 92 ° C for 2 min, 1 cycle. 92 ° C for 15 sec, 40 ° C for 2 minutes, 72 ° C for 30 sec, 40 cycles. 72 ° C for 5 minutes. Gel electrophoresis Duplicate mixtures of PCR products were separated by gel electrophoresis on a 6% denatured polyacrylamide gel (5.7% acrylamide, 0.3% bisacrylamide, 42% urea and 51% H20) in a buffer solution lx TBE (tris) 0.1 M 0.09 M boric acid, 1 mM EDTA) for 3 hours at 2000 volts. The gel was then transferred to a filter paper (Schleicher &Schuell, Keene NH), dried under vacuum at 80 ° C for 1 hour and exposed to an X-ray film for 24 hours.

A band designated as 6a was apparently upregulated in Saos-2 and LNCaPLN3 cells by 17-ß-estradiol (Figure 2). The developed film was superimposed on the dry gel and the corners of the band were marked using a 22 gauge syringe needle. The gel portion within these borders was cut with a razor and immersed in 100 μl H20. This sample was boiled in a water bath for 15 minutes, centrifuged for 2 minutes and the supernatant solution transferred to a new tube. Five μl of 10 mg / ml glycogen, 10 μl of 3 M sodium acetate and 450 μl of 100% ethanol were added to this sample. The sample was mixed, allowed to precipitate overnight at -20 ° C and 10 000 g was centrifuged for ten minutes. The solution was separated from the supernatant, the pellet was washed with ethanol with 200 μl of 85% ethanol, dried and resuspended in 10 μl of H20. A 3 μl aliquot was used in a PCR re-amplification reaction in the presence of a lx PCR buffer, 20 μM dNTPrs, an arbitrary 0.2 μm primer and the 0.2 μm HTnC oligonucleotide and 2 units of AmpliTaq polymerase, using the same parameters of cycle as in the previous PCR reaction. The resulting product was then used as a probe in a Northern hybridization assay to confirm regulation and cloned into a bacterial plasmid for sequence analysis. Cloning of the fragment The 6a band was cloned using the TA cloning kit (Invitrogen, Carlsbad CA). After lysis, the colonies were screened by PCR for the correct size of the insert. Colonies were used in 20 mM Tris-HCl, pH 8.50 mM KCl, 2.5 mM MgCl2, 0.5% Tween-20 and 100 μg / ml proteinase K, by incubation for 30 minutes at 56 ° C, then minutes at 99 ° C to inactivate proteinase K. From this reaction, 2 μl was used in a PCR reaction of 20 mM Tris-HCl, pH 8, 50 mM KCl, 2.5 mM MgCl2, 75 μM dNTPs, 375 nM M-13 primers posterior and anterior and 2.5 U Taq polymerase (Gibco BRL, Gaithersburg MD). The reactions were cycled as follows: 95 ° C for 30 seconds; 64 ° C for 30 seconds; 72 ° C for 45 seconds for 30 cycles. A clone designated as 6a.2 was chosen to form a sequence. Formation of the fragment sequence The sequence of clone 6a.2 was formed according to the reaction kit for easy formation of ABI PRISM colored terminator cycle sequences, with AmpliTaq DNA polymerase using the recommended protocol of Applied Biosystems (Foster City CA). Rotation columns (AGTC) were used to remove nucleotides labeled with unincorporated dye after cycle sequence formation. Polyacrylamide gels 4.75% grade were circulated for the formation of automatic DNA sequences for all DNA sequence formation samples using ABI 373 DNA sequence formers. The sequence formation data were edited using the Sequence Navigator and assembled using the DNAStar (DNAStar, Madison Wl). The RADE primers were adjusted to the sequence and used in a BLAST search using Millenium software (Boston MA). A search for nucleotide homology of the clone 6a2 sequence revealed 98% identity with the human MT-II. On the coding sequence, however, there were no anti-mismatches of ammonia (Figure 3). Confirmation of regulation using Northern spotting and quantitative polymerase chain reaction and reverse transcriptase (qRT-PCR): To confirm the results obtained with the PCR amplification of the cellular mRNA, Northern blot and qRT-PCR were used to evaluate the effect of the 17-ß estradiol or other compounds at message levels. Unless indicated otherwise, all experiments utilize cells that transiently overexpress hERßL in response to adenovirus infection as described above and were treated with compound for 24 hours. In some cases, cells transiently overexpressing the ER-a were used for comparison. Methods for overexpression mediated by the ER-a adenovirus, are identical to those described for ER-ß. Northern blotting Poly A + RNA was isolated from the total RNA using the Oligotex mRNA isolation kit (Qiagen, Hilden Germany) according to the manufacturer's instructions. 6 micrograms of mRNA or 10 μg of total RNA were separated in 1.5% agarose, 0.22 M formaldehyde, 10 mM HEPES, 1 mM EDTA gel. The RNA was transferred to a Hybond-N (Amersham Pharmacia, Piscataway NJ) by capillary action in 20X SSC (Gibco BRL, Gaithersburg MD) overnight. After the transfer, the membrane was UV-crosslinked and dried at 80 ° C for 10 minutes. The Northern blots were prehybridized in a Rapid Hyb solution (copied for 30 minutes) The clone 6a.2 insert was isolated from the plasmid using PCR as described above for colony screening The PRC product was purified from a agarose gel using Wizard Preps (Promega, Madison Wl). RADE reamplified fragments for band 6a or clone fragment 6a .2 were randomly labeled using a Redi-prime kit (Amersham Pharmacia, Piscataway NJ) according to the manufacturers' instructions. The unincorporated nucleotides were separated using a Nap-5 column (Amersham Pharmacia, Piscataway NJ) and the incorporation of [32 P] -dCTP as measured by a liquid scintillation count. The probes were denatured at 100 ° C for 10 minutes and a Rapid-Hyb hybridization solution of 1.5 x 10 6 CPM / ml was added to the membrane. The smears were hybridized at 65 ° C for 5 hours and washed as follows: Once in 2X SSC, 0.1% SDS at 65 ° C for 15 minutes; twice in 0.2X SSC, 0.1% SDS at 65 ° C for 15-30 minutes. The stains were exposed to the film and to a Phosphorlmager sieve (Molecular Dynamics, Sunnyvale CA). After probing with a 6a RADE fragment or a cloned 6a2 fragment, those stained with a cDNA homologous to GAPDH as above were probed. The hybridization signal of fragment 6a2 was normalized with that of GAPDH in a Phosphorlmager (Molecular Dynamics, Sunnyvale CA) to determine the induction of the fold. QRT-PCR.

An MT-II fragment identical to clone 6a2 was subcloned except that it contained a 63 bp deletion, within pcDAN3 (Invitrogen, Carlsbad CA). The RNA was transcribed using the T7 large-scale promoter transcription kit (Novagen, Madison Wl). After extraction with phenol-chloroform and precipitation with ethanol, the synthesized RNA was quantified and analyzed using UV spectrophotometry and gel electrophoresis. The reverse transcription reactions were carried out at 200 ng and 300 ng of Saos-2 total DNase with RNA plus a known amount of standard RNA with MT-II in a 20 μl reaction. The reaction contained PCR Ix buffer (Gibco BRL, Gaithersburg MD), 3.75 mM MgCl2, reverse primer specific for MT-II 1.25 μM (5 '-GGAATATAGCAAACGGTCAGGGTC-3', SEQ ID NO.10), 0.5 mM dNTPs, 1 mM DTT, 20 units of Rnasina (Promega) and 200 units of Superscript II reverse transcriptase (Gibco BRL, Gaithersburg MD). The reactions were incubated at 42 ° C for 15 minutes followed by 5 minutes at 99 ° C and 5 minutes on ice before amplification. PCR was initiated by adding 80 μl of the original mixture containing the MT-II specific forward primer (5 '-GGCTCCTGCAAATGCAAAGAG-3', SEQ ID NO: 11) directly to the 20 μl reverse transcriptase reaction. The final concentration of the reagents in the 100 μl PCR reaction was as follows: 0.25 μM of each MT-II specific primer, lx PCR buffer (Gibco BRL, Gaithersburg MD), 0.1 mM dNTPs, 1.5 mM MgCl2 and 0.5 Taq DNA polymerase units (Gibco BRL, Gaithersburg MD). PCR was carried out in two steps in a PE 9600 (Perkin-Elmer, Norwalk CT) for 40 cycles as follows: 95 ° C for 30 seconds' 64 ° C for 1.5 minutes. The samples were incubated at 64 ° C for 10 minutes after amplification. The PCR products were separated and analyzed on a DNAsep column of high performance liquid chromatography of ion-pair and reverse phase (Sarasep, San José CA). The elution system was a gradient of acetonitrile in 0.1M triethylammonium acetate (Fluka, Ronkonkoma NY) at a flow rate of 0.7 ml / min. The acetonitrile gradient was increased from 14.6% to 16.6% for 5 minutes. The amount of standard and natural RNA product was determined by the detection of UV absorbance at 254 nm and the signal was analyzed by an in-line integrator. From the chromatograms, the ratio of the area under each peak was used to determine the ratio of the amount of standard input RNA MT-II to the amount of the natural MT-II message in the Saos-2 RNA. In LNCaPLN3 cells, the magnitude of MT-II induction by 17-ß-estradiol is approximately 6-fold (Figure 4). In Saos-2 cells, 10 nM 17-β estradiol upregulates MT-II mRNA as much as 14 times (Figure 4). This overregulation in cells Saos-2 has been repeated in at least 20 experiments in the induction range is 3.5-14 times. The EC50 of this regulation is 4.6 + 2.7 as assessed by qRT-PCR (Figure 5). If the cells Saos-2 are treated with 10 nM 17-β estradiol and the RNA is prepared at different times after treatment, the first increment that is distinguished in the MT-II message happens after 8 hours and the peaks of response in 24 hours ( Figure 6). Since the samples prepared for differential deployment overexpressed the hERßL, the regulation of MT-II in natural cells of Saos-2 and in those that overexpress the ERa was evaluated. As illustrated in Figure 7, the endogenous levels of ER-β or the overexpressed levels of ER-a were not effective in mediating the regulation of 17-β-estradiol from MT-II. Other compounds were tested for their ability to upregulate MT-II in Saos-2 cells. The ten-nanomolar ilestilbesterol diet (Sigma, St. Louis MO) and the 0.1μm genistein (RBI, Natick MA) both increased the MT-II mRNA. A micromolar of the anti-estrogen ICI-182780 (Zeneca, Wilmington DE), completely blocked induction by 17β estradiol and genistein but had no effect when given separately (Figure 8). The co-treatment with 1 μm of antiprogesterone / antiglucocorticoid, RU486 (Ligand Pharmaceuticals, La Jolla CA), did not block the regulation by 17β estradiol (data not shown). Example 4: Treatment of Saos-2 cells with cycloheximide. To determine if the increase in mRNA of MT-I1 requires a new protein synthesis, cycloheximide was used after overexpression of hERßL and before treatment with 17β estradiol to stop translation.

Verification of the effect of cycloheximide on protein synthesis. Cells were plated and infected with the hERßL virus as described above, then pretreated with 10 μg / mL cycloheximide or vehicle for 1 hour at 37 ° C. After this pre-incubation, 10 μg / mL of cycloheximide, 10 nM 17-β estradiol and 50 μCi / mL of 35 S-met ionin (NEN, Boston MA) were added in methionine-deficient medium, and incubation was continued 37 ° C for 8 or 24 hours. The 3x cells were washed in cold PBS and then scraped from the plates in 500 μl of PBS. The cells were pelleted and resuspended in 200 μl of RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, and 50 mM Tris, pH 8.0). The methionine incorporation was measured by precipitation with TCA. Five to ten microliters of each sample were labeled on individual Whatman filters. The filters were boiled in 10% trichloroacetic acid for 10 minutes, then washed 3 times for 10 minutes in deionized water, three times for 10 minutes in 95% ethanol and once for 10 minutes in acetone. The dry filters were placed in scintillation fluid and counted for 1 minute. Treatment of cells with 17-ß-estradiol Cells were plated and infected with the hERßL virus as described above. The cells were pretreated with 10 μg / mL of cycloheximide or vehicle for 1 hour at 37 ° C. After this pre-incubation, 17-ß-estradiol or 10 nM vehicle was added and incubation was continued at 37 ° C for 8 or 24 hours. RNA was isolated from the cells as described above and gene expression was assessed by a Northern blotting analyzer qRT-PCR as described in Example 3. Verification of the functional protein ER-β after treatment with cycloheximide. Duplicate cultures were prepared and treated as described in the previous section. After the 8 hour incubation, the cells were washed 4 times with DMEM to remove 17-β estradiol. Cycloheximide (10 μg / mL) was added to all samples, such as 1 nM [3 H] -estradiol. Some cells were co-treated with 0.3 μM t i lbesterol diets to estimate the non-specific binding. After incubation for 2.5 hours at 37 ° C, the cells were washed with DMEM and used with 0.1% sodium dodecyl sulfate. The DPM was measured by liquid scintillation counting. The MT promoter contains glucocorticoid and metal response elements, but EREs have not been described. It is possible that the effect of 17-ß-estradiol on mRNA expression of MT-II is indirect. After over expression of ER-β, Saos-2 cells were treated with cycloheximide to severely limit the new protein synthesis (Figure 9A). Although comparable amounts of the receptor protein were expressed with and without the cycloheximide treatment as measured by a whole cell binding assay (Figure 9B), the induction of MT-II did not occur (Figure 9C, D). Example 5: Treatment of rats with 17-ß-estradiol. Because 17-β estradiol upregulates MT-II in the LNCaPLN3 cell, a prostate cancer cell line, a similar response was sought in the rat prostate.

All animals were treated according to institutional guidelines using the approved protocols. Adult castrated Sprague-Dawley rats (15-19 weeks, 375g) were purchased from Taconic Farms (Germantown NY). Ten days after castration, the rats were injected with the vehicle, 16 μg of 17-ß-estradiol, 16 μg of diethylstilbesterol (Sigma, St. Louis MO) or 16 μg of 17-ß. estradiol plus 1.6 mg raloxifene (synthesized in laboratory) subcutaneously once a day for three days. Approximately 24 hours after the last dose, euthanasia was applied to the rats by asphyxia with C02 and the prostate was removed. The total RNA was prepared and analyzed by mRNA MT-II as described above. In another experiment, the rats were dosed with 1, 2 or 3 days with 16 μg of 17-ß-estradiol before the tissue of the prostate was harvested. The mRNA in the metallotein-I I was increased five-fold after treatment with 17-ß-estradiol. A similar response occurred when the rats were dosed with diethylstilbesterol, but this overregulation was blocked by the coadministration of a 100-fold excess of raloxifene hydrochloride, an estrogen antagonist (Figure 10). Although the initial experiments used tissues from rats dosed for three days, upregulation of MT-II in the rat prostate was first observed after two days of dosing with 17-ß-estradiol (Figure 11). Example 6: ERE-luciferase reporter assay using MCF-7 cells. Since the regulation of MT-II in Saos-2 cells is mediated by ER-β and not by ER-a, another assay was needed to compare the selectivity of the compounds of these two receptors in cell-based assays . MCF-7 is a breast cancer cell line responsible for estrogen that expresses only ER-a as measured by RT-PCR (data not shown). When the MCF-7 cells are transiently infected with an ERE reporter gene construct, and treated with 17-β estradiol, the activity of the reporter gene can be measured. The MCF-7 HTB 22 cells, ATCC, Manasas VA) are passed twice weekly with a growth medium [D-MEM / F-12 medium containing 10% (v / v) of heat inactivated fetal bovine serum, 100 U / ml penicillin g, 100 μg / ml streptomycin sulfate, 2 mM glutaMAX-1]. The cells are kept in flasks vented at 37 ° C in an air incubator and humidified at 95% / 5% C02. One day before treatment, the cells are plated with a growth medium at 25,000 / well into 96 well plates and incubated at 37 ° C overnight. The cells are infected for 2 hours at 37 ° C with 50 μl / well of a 1:10 dilution of the adenovirus of 5-ERE-TK-luci ferase (Bodine et al., 1997) in an experimental medium [medium phenol red free DMEM / F-12 containing 10% (v / v) of heat-inactivated fetal bovine serum depleted with heat, 100 U / ml penicillin g, 100 μg / ml streptomycin sulfate, GlutaMAX-1 2 mM, 1 mM sodium pyruvate]. The wells are then washed once with 150 μl of experimental medium. Finally, the cells are treated for 24 hours at 37 ° C in 8-well repeats / treatment with 150 μl / well vehicle (<0.1% v / v DMSO) or the compound that is diluted > _ 1000 times within the experimental medium.

After treatment, the cells are lysed on a shaker for 15 minutes with 25 μl / well of a cell culture lysis reagent IX (Promega, Madison Wl). The cell lysates (20 μl) are transferred to a 96-well luminometric plate and measure the activity of the luciferase in a MicroLumat LB 96 P luminometer (EG &G Berthold, Bad Wildbad Germany) using 100 μl / well of luciferase substrate (Promega, Madison Wl). Prior to the injection of the substrate, a second background measurement is made for each well. After injection of the substrate, the luciferase activity is measured for 10 seconds after a delay of 1 second. After subtracting the background measurements, the mean and the standard deviation are calculated. Compounds that induce MT-II in Saos-2 cells, and that do not stimulate the activity of the reporter gene in MCF-7 cells, are thus selective for ER-β. In addition, the results of these two assays can also be used to select a compound that is selective for ER-a as shown in Figure 12.

Discussion Using a differential display, it was unexpectedly discovered that ER-β increases the mRNA of the MT-II in two cell lines treated with 17-ß-estradiol. To our knowledge, this is the first discovered gene that is regulated by this new form of ER. This response has been extensively characterized in the human osteosarcoma cell line Saos-2. A variety of estrogens can upregulate MT-II, and this response is blocked by co-treatment with the estrogen antagonist ICI-182780. The EC50 for 17-ß-estradiol is approximately 5 nM and this response is mediated by the action of ER-β through still unknown proteins. The action of ER-ß in MT-II is not a general phenomenon, when an investigation of more than a dozen cell lines failed to reveal this induction in samples other than Saos-2 and LNCaPLN3 cells. However, the fact that MT-II is regulated by 17-ß estradiol in the rat prostate strengthens the possibility that it is a physiologically relevant response and not an artifact of receptor overexpression in cell lines with cancer. There is confidence that this prostate response is mediated by the ER for two reasons. A non-spheroidal estrogen (diethylstilbesterol) upregulates MT-II and raloxifene, an estrogen agonist / antagonist, blocks the action of 17-ß-estradiol. However, at this time it is not clear whether this induction reflects the activity of ER-ß and / or ER-a. Although ER-ß is the predominant form of ER in the prostate of the rat, ER-a is also present. Current studies are on track to define the type of receptor responsible for this in vivo response. Metallic salts are proteins rich in low molecular weight cysteine that bind to metals such as cadmium, copper and zinc. Although the first metalline ionein was discovered more than forty years ago (Valle, 1957) the debate continues as to its function. Various proposals have been made and these include the toxicity of the metal in the form of protection, the regulation of homeostasis by zinc and copper and the defense against oxidative attention. Regulation of the energy balance has also been implicated because after reaching sexual maturity, non-transgenic mice with MT (-1 and -II) became obese (Beattie et al., 1998). Recently, studies have detailed how MT can act to regulate zinc homeostasis in cells. Using purified zinc-dependent enzymes such as alkaline phosphatase from E. coli, carboxypept idase A from bovine and sorbitol dehydrogenase from sheep, two recent publications show how agents at the cellular site can facilitate the exchange between complex-forming apoenzymes. of zinc with MT and those without metal (Jacob et al, 1998; Jiang et al., 1998). Citrate and glutathione can influence the direction of zinc transfer and thus regulate the activity of the enzyme depending on the oxidation state of the cell. Although not an enzyme, ER also requires zinc for activity, and the ER of MCF-7 cells can reversibly exchange zinc with purified MT in vitro (Cano-Gauci et al., 1996). A multitude of agents can regulate MT levels, including glucocorticoids and metals such as cadmium (for review see Hamer, 1986). Estrogens are not a classic regulator of MT, but two interesting documents appear in the literature. First, a two-week treatment of female rats with 17-ß-estradiol, over-regulated a copper-binding protein in the intestinal mucosa which reduced the amount of copper absorbed into the plasma. Since the molecular weight of this protein was around 10K, the authors suggest that it is MT (Cohen et al., 1979). Recently, the MT was identified in a subtractive hybridization screen of the regulated uterine mRNA after a simple injection of 17-ethinyl estradiol (Rivera-González et al., 1998). Although the isoform is not identified, an MT transcript increased three-fold between four and eight hours after stimulation with 17-a-ethinyl estradiol. In addition, it is not clear which type of receptor this regulation makes since the ER-a, not the ER-ß, is the most abundant ER in the uterus (Kuiper et al., 1996; Couse et al., 1997). Since the function of MT is just beginning to be clear and is completely unknown to the ER-ß, understanding the importance of its association is impossible at this time. However, even if the connection between these two proteins is unclear, observation of regulation can still be exploited to help design a specific ligand for ER-β or ER-α. The fact that genistein upregulates MT-II in Saos-2 cells as well as better than 17-β estradiol, indicates an ER-β selective compound that can effectively direct transcription. In addition, various structurally varied compounds that bind to ER-β can also upregulate this message in Saos-2 cells. When data of the ability of a compound to regulate MT-II in Saos-2 cells is coupled with information about how the activity of the reporter gene is regulated in a MCF-7 cell, the selectivity of the ER can be evaluated. -ß and ER-a. Thus, when used together, these two assays are tools to help design selective compounds for any type of ER. Finally, regulation in the prostate can be shown to be mediated by ER-ß, so in vivo activity of compounds can be assessed, providing another valuable tool for drug discovery. References Beattie JH, Wood AM, Newman AM, Bremner I, Choo KHA, Michalska AE, Duncan JS, Trayhurn P. 1998. Obesity and hyperlept inemia in metallothionein (-1 and -II) nuil mice. Proc Nati Acad Sci (USA) 95: 358-363. Bhat RA, Harnish DC, Stevis PE, Lyttle CR, Komm BS. 1998. A novel human estrogen receptor: identification and functional analysis of additional N-terminal amino acids. J Steroid Molec Biol, in press. Blondelle SE, Houghten RA. 1996. Novel antimicrobial compounds identified using synthetic combinatorial library technology. Trends Biotechnol 14 (2): 60-65. Bodine PVN, Green J, Harris HA, Bhat R, Stein GS, Lian JB, Komm B. 1997. Functional properties of a condi tional and phenotypic, estrogen-responsive, human osteoblast cell line. J Cell Biochem 65: 368-387. Cano-Gauci DF, Sarkar B. 1996. Reversible exchange between metallothionein and the estrogen receptor zinc finger. FEBS Letters 386: 1-14.

Cohen Di, Illowsky B, Linder MC. 1979. Altered copper absorption in tumor-bearing and estrogen-treated rats. Am J Physiol 236 (3): E309-15. Couse JF, Lindzey J, Grandien K, Gustafsson J, Korach, KS. Tissue distribution and quantitative analysis of estrogen receptor-a (ERa) and estrogen receptor-ß (ERß) messenger ribonucleic acid in the wild type and ERa-knockout mouse. 1997. Endocrinology 138 (11): 4613-4621. Cowley SM, Hoare S, Mosselman S, Parker MG. 1997. Estrogen receptors a and ß form heterodimers on DNA. J Biol Chem 272 (32): 19858-19862. Davis AR, Kostek B, Mason B, Hsiao CL, Morin J, Dheer SK, Hung P. 1985. Expression of hepatitis B surface antigen with a recombinant adenovirus. Proc Nati Acad Sci (USA) 82: 7560-7564. Dotzlaw H, Leygue E, Watson PH, Murphy LC. 1996. Expression of estrogen receptor-beta in human breast tumors. J Clin Endocrinol Metab 82 (7): 2371-2374. Enmark E, Pelto-Huikko M, Grandien K, Lagercrantz S, Lagercrantz J, Fried G, Nordenskjold M, Gustafsson J. 1997. Human estrogen receptor-ß gene structure, chromosomal localization, and expression pattern. J Clin Endocrinol Metab 82 (12): 4258-4265. Green S, Kumar V, Walter KP, Chambon P. 1986. Structural and functional domains of the estrogen receptor. Cold Spring Harbor Symposia on Quantitative Biology L1.751-758. Gluzman Y, Reichl H, Solnick D. 1982. Helper-free adenovirus type-5 vectors. In Eukaryotic Viral Vectors, and Gluzman ed., (Cold Spring Harbor Laboratory), pp. 187, 192. Hamer DH. 1986. Metallothionein. Ann Rev Biochem 55: 913-951. Iafrati MD, Karas RH, Aronovitz M, Kim S, Sullivan TR, Lubahn DB, O'Donnell Jr. TF, Korach KS, Mendelsohn ME. 1997. Estrogen inhibits the vascular injury response in estrogen receptor-a-deficient mice. Nature Med 3 (5): 545-548. Jacob C, Maret W, Vallee BL. 1998. Control of zinc transfer between thionein, metallothionein and zinc proteins. Proc Nati Acad Sci (USA) 95: 3489-3494. Jiang L, Maret W, Valee BL. 1998. The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase. Proc Nati Acad Sci (USA) 95: 3483-3488. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. 1996. Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Nati Acad Sci (USA) 93: 5925-5930. Kuiper GGJM, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson, JA. 1997. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors a and ß. Endocrinology 138 (3): 863-870. Lau K, Leav I, Ho S. 1998. Rat estrogen receptor-a and -β, and progesterone receptor mRNA expression in various prostatic lobes and microdissected normal and dysplastic epithelial tissues of the Noble rats. Endocrinology 139 (1): 424-427. Lobo R. 1995. Benefits and risks of estrogen replacement therapy. Am J Obstet Gynecol 173 (3): 982-989. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach, KS. 1993. Alteration of reproductive function but not sexual prenatal development after insertional disruption of mouse estrogen receptor gene. Proc Nati Acad Sci (USA) 90: 11162-11166. Mosselman S, Polman J, Dijkema R. 1996. ERß: identification and characterization of a novel human estrogen receptor. FEBS Letters 392: 49-53. Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, Ouchi Y, Muramatsu M. 1998. The complete primary structure of human estrogen receptor ß (hERß) and its heterodimerizat ion with ER a in vivo and in vitro . Biochem Biophys Res Comm 243: 122-126. Pan LC, Ke HZ, Simmons HA, Crawford DT, Chidsey-Fink KL, McCurdy SP, Schafer JR, Kimbro KS, Taki M, Korach KS, Thompson DD. Estrogen receptor-alpha knockout (ERKO) mice lose trabecular and cortical bone following ovariectomy. 1997. J Bone Mineral Res 12 (Supplement 1): S134. Pettersson K, Grandien K, Kuiper GGJM, Gustafsson J. 1997. Mouse estrogen receptor ß forms estrogen response element-binding heterodimers with estrogen receptor-a. Mol Endocrinol 11: 1486-1496. Rivera-Gonzalez R, Peterson DN, Tkalcevic G, Thompson DD, Brown TA. 1998. It is a gene-induced genes in the uterus of ovariectomized rats and their regulation by droloxifene and tamoxifen. J Steroid Biochem Molec Biol 64 (1/2): 13-24. Sagraves R. 1995. Estrogen therapy for postmenopausal symptoms and prevention of osteoporosis. J Clin Pharmacol 35: 2s-10s. Shughrue PJ, Komm B, Merchenthaler I. 1996. The distribution of estrogen receptor-ß mRNA in the rat hypothalamus. Steroids 61: 678-681. Trembley GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V. 1997. Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor ß. Mol Endocrinol 11: 353-365. Vallee BL. 1957. A cadmium protein from equine kidney cortex. J Am Chem Soc 79: 4813. Vladusic EA, Hornby AE, Guerra-Vladusic FK, Lupu R. 1998. Expression of estrogen receptor ß messenger RNA variant in breast cancer. Cancer Res 58: 210-214. Webb P, López GN, Greene GL,. Baxter JD, Kushner PJ. 1992. The limits of the cellular capacity to mediate an estrogen response. Mol Endocrinology. 6 (2): 157-67.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

LIST OF SEQUENCE < 110 > American Home Proaac's Corporation Harris, Heatner Bhat, Ramesh A < 120 > Bioassay for the identification of selective beta / alpha estrogen receptors < 130 > 97400seq < 140 > PCT US 99/29856 < 141 > 2000-12-17 < 150 > 60 / 112,790 < 151 > 1998-12-18 < 160 > 15 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial sequence: Primer / oligonucleotidoa < 400 > 1 ccagcagcat. gtcgaagatc 20 < 210 > 2 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial sequence: Primer / oligonucleotides < 400 > 2 caccctgttg ctgtagccaa attc 24 < 210 > 3 < 223 > Description of the Artificial sequence: Primer / oligonucleotides < 400 > 10 ggaata agc aaacggtcag sgtc < 210 > 11 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer / oligonucleotides < 400 > 11 ggctcctgca aatgcaaaga g < 210 > 12 < 211 > 196 < 212 > DNA < 213 > Homo sapiens < 400 > 12 atcccaactg ctcctgcgcc gccggtgact cctgcacctg cgccggctcc tgcaaatgca 6C aagagtgcaa atgcacctcc tgcaagaaaa gctgctgctc ctgctgccct gtgggctgtg 120 ccaagtgtgc ccagggctgc atctgcaaag gggcgtcgga caagtgcagc tgctgcgcct 180 gatgctggga cagccc 196 < 210 > 13 < 211 > 196 < 212 > DNA < 213 > Homo sapiens < 400 > 13 accccaactg ctcgtgcgcc gccggtgact cctgcacctg cgccggctcc tgcaaatgca 60 aagagtgcaa atgcacctcc tgcaagaaaa gctgctgctc ctgctgccct gtgggctgtg 120 ccaagtgtgc ccagggctgc atctgcaaag gggcgtcgga caagtgcagc tgctgcgcct 180 gatgctggga cagccc 196 < 210 > 14 < 211 > 61 < 212 > PRT < 213 > Hom sapiens gacatcaaga aggtggtgaa gcag 24 < 210 > 7 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial sequence: Primer / ol gonucleot two < 400 > 7 tgaaccact ccctgtctgc caggttggt 29 < 210 > 8 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial sequence: Primer / oligonucleotides < 4C0 > e ccgcatacag atgtgataac tggcgatgga 30 < 210 > 9 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial sequence: Primer / ol gonucleotides < 400 > 9 gctgttgaag tcacaggaga caacctggt 29 c210 > 10 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial sequence: Primer / ol gonucleotides < 400 > 3 gcagaagtga gcatccctct ttg 2 < 210 > 4 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial sequence: Primer / oligonucleotides < 400 > 4 ggagacatga gagctgccaa c < 210 > 5 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Sequence description Artificia: Primer / oligonucleotides < 40C > 5 cagcattcc agcaatgtca c 21 < 210 > 6 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial sequence: Primer / oligonucleotides < 400 > 6 < 400 > 14 Met Asp Pro Asn Cys Ser Cys Ala Ala Gly Asp Ser Cys ~ ye Ala 1 5 10 Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Se: 20 25 30 Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cye 35 40 45 lie Cys Lys Gly Ala Ser Asp Lys Cys Ser Cys Cys Ala 50 55 60 < 210 > 15 < 211 > 16 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial sequence: oligonucleotide < 400 > 15 aagctttttt; ttttc 16

Claims (32)

1. Claims Having described the invention as above, the content of the following claims is claimed as property. 1. A method for screening a test compound that binds to an ER in a receptor binding assay, characterized in that the method detects mediated transcription by ER-β polypeptides, the method comprising the steps of: (a) Providing a cell comprising at least one DNA sequence regulated by estrogens that encode MT-II and at least one DNA sequence encoding the ER-β polypeptide, wherein the receptor is cross-reactively active; (b) Contacting the cell with any of the test compounds that are linked to the ER or to a control; and (c) Detecting the expression of T-II wherein the enriched expression of MT-II relative to a control indicates that the test compound has estrogen agonist activity.
2. 2. The method according to claim 1, characterized in that the DNA sequence encoding the ER-β polypeptide is incorporated into an adenovirus.
3. 3. The method according to claim 2, characterized in that the adenovirus is an Ad5 virus defective in replication.
4. 4. The method according to claim 1, characterized in that the cells endogenously express the mRNA of ER-β at a higher level than the mRNA of ER-a.
5. 5. The method according to claim 4, characterized in that the cells are transformed with a recombinant RNA plasmid comprising a polynucleotide encoding a Human ER-ß which is operatively linked to an appropriate promoter, wherein the transformed cells express human ER-β at higher levels than the cells that have not been trans formed.
6. 6. The method according to claim 1, characterized in that the cells are Saos-2 or LNCaPLN3 cells.
7. 7. The method according to claim 1, characterized in that the cells do not have a functional ER-a.
8. 8. A method for screening a test compound that binds to an ER in a receptor binding assay, characterized in that the method detects the inhibition of transcription mediated by the ER-β polypeptide, the method comprises the steps of : (a) Providing a cell comprising at least one estrogen DNA sequence encoding MT-II and at least one DNA sequence encoding the ER-β polypeptide, wherein the receptor is transcriptionally active; (b) Contacting the cell with one or more estrogens in the presence of the test compound known to bind the ER and (c) Detecting the expression of MT-II where the decreased expression of MT-II relative to the addition of one or more estrogens alone indicates that the test compound has an estrogen antagonist activity.
9. The method according to claim 8, characterized in that the ER-β polypeptide encoding the DNA is incorporated into an adenovirus.
10. 10. The method according to claim 9, characterized in that the adenovirus is an Ad5 virus defective in replication.
11. 11. The method according to claim 8, characterized in that the cells endogenously express the ER-β mRNA at a level, higher than the mRNA ER-a.
12. The method according to claim 11, characterized in that the cells are transformed with a recombinant DNA plasmid comprising a polynucleotide encoding a human ER-β operably linked to an appropriate promoter wherein the transformed cells express the ER-β human to higher levels than cells that have not been transformed.
13. The method according to claim 12, characterized in that the cells are Saos-2 or LNCaPLN3 cells.
14. 14. The method according to claim 13, characterized in that the cells do not have a functional ER-a.
15. 15. A method for screening test compounds to identify candidates for drugs that mimic the effect of estrogen on ER-β or ER-a mediated transcription, the method characterized in that it comprises the steps of (a) contacting the test compound with a plurality of: (i) first cells comprising at least one endogenous DNA sequence encoding the MT-II and at least one sequence of DNA encoding an ER-β polypeptide, wherein the receptor is transcriptionally active and (ii) second cells comprising a reporter gene construct ERE wherein the cells express the ER-a polypeptide; (b) Identify compounds that increase the expression of MT-II in the first cells relative to the control but that have a minimal effect on the expression of the reporter gene or in the second cells, where the compounds are considered selective for ER-β; or (c) Identify compounds that increase the expression of the reporter gene in the second cells relative to the control, but that have a minimal effect on the expression of MT-II in the first cells, where the compounds are considered selective for ER -to.
16. 16. The method according to claim 15, characterized in that the ER-β polypeptide encoding the DNA of the first cells is incorporated into an adenovirus.
17. 17. The method according to claim 16, characterized in that the adenovirus is an Ad5 virus defective in replication.
18. 18. The method according to claim 15, characterized in that the first cells endogenously express the ER-β mRNA at a higher level than the ER-a mRNA.
19. 19. The method according to claim 15, characterized in that the first cells are transformed with a recombinant DNA plasmid comprising a polynucleotide encoding a human ER-ß operably linked to an appropriate promoter, wherein the first transformed cells express the Human ER-ß at higher levels than the second cells.
20. 20. The method according to claim 15, characterized in that the first cells are Saos-2 or LNCaPLN3 cells.
21. 21. The method according to claim 15, characterized in that the first cells do not have a functional ER-a and the second cells do not have a functional ER-β. .
22. 22. The method according to claim 15, characterized in that the second _ cells were transformed with a DNA polynucleotide comprising an ERE operably linked to a reporter gene.
23. 23. The method according to claim 22, characterized in that the reporter gene is a luciferase gene.
24. 24. A method for screening test compounds to identify drug candidates that inhibit the effect of estrogen on transcription mediated by ER-β or ER-a, The method characterized in that it comprises the steps of: (a) Putting contacting the test compound in the presence or absence of one or more estrogens with a plurality of: (i) first cells comprising at least one endogenous DNA sequence encoding a metallotein (T-II) gene and at least one DNA sequence encoding an ER-β polypeptide, wherein the receptor is transcriptionally active and (ii) second cells comprising a reporter gene construct ERE, where the cells express an ER-a polypeptide and (b) identify compounds that increase the expression of T-II in the first cells in relation to the treatment with one or more estrogens but that have a minimal effect on the expression of the reporter gene in the second cells, where the are considered selective for ER-ß or (c) Identify compounds that decrease the expression of the reporter gene in the second cells in relation to treatment with one or more estrogens but that have a minimal effect on the expression of MT-II in the first cells, where the compounds are considered selective to ER-a.
25. 25. The method according to claim 24, characterized in that the ER-β polypeptide encoding the DNA of the first cells is incorporated into an adenovirus.
26. 26. The method according to claim 25, characterized in that the adenovirus is an Ad5 virus defective in replication.
27. 27. The method according to claim 24, characterized in that the first cells endogenously express the ER-β mRNA at a higher level than the ER-a mRNA.
28. The method according to claim 24, characterized in that the first cells are transformed with a recombinant DNA plasmid comprising a polynucleotide encoding human ER-ß linked operatively to an appropriate promoter., wherein the first transformed cells express human ER-β at higher levels than the second cells.
29. 29. The method according to claim 24, characterized in that the first cells are Saos-2 or LNCaPLN3 cells.
30. 30. The method according to claim 24, characterized in that the first cells do not have a functional ER-a and the second cells do not have a functional ER-β.
31. 31. The method according to claim 24, characterized in that the second cells were transformed with a DNA polynucleotide comprising an ERE operably linked to a reporter gene.
32. 32. The method according to claim 31, characterized in that the reporter gene is a luciferase gene.