JP2002533098A - Bioassay for identifying estrogen receptor-β / α selective modulators - Google Patents

Abstract

  (57) [Summary] The present invention provides a novel assay method for identifying compounds that selectively activate the estrogen receptor (ER) of the α or β subtype. In particular, it interprets the results from two assays, one measuring the activity of ER-β and the other measuring the activity of ER-α. Assays for measuring ER-β activity use cells comprising a DNA plasmid comprising endogenous metallothionein-II as well as a polynucleotide encoding human ER-β. The assay monitors the expression of metallothionein-II mRNA in the cells, wherein the level of metallothionein-II expression is modulated when the potential ligand binds to ER-β. Assays for measuring ER-α activity use cells comprising a DNA plasmid comprising ER-α and a reporter gene linked to an estrogen response element. The assay monitors reporter gene expression, where the level of the reporter gene is modulated when the potential ligand binds to ER-α. Compounds that modulate activity in one assay but have little or no activity in the other assay are defined as estrogen receptor subtype selective.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD [0001] The present invention relates to methods for identifying compounds that selectively activate hormone receptors, and more specifically, estrogen receptors (ERs) of the α or β subtype, and Test kit for use in the method.

BACKGROUND OF THE INVENTION [0002] Proteins that regulate gene expression are essential for cell function. One well-studied family of gene regulatory proteins is the steroid hormone receptor superfamily. These receptor proteins enable cells to respond to various hormones by activating or repressing specific genes. One member of this family is the ER. Estrogen is classically known as an important hormone in sexual development and reproductive function. It is known that estrogen affects cell proliferation and differentiation in target tissues by binding to ER in target cells. Estrogen replacement therapy is a well-established treatment for prevention and / or amelioration of osteoporosis in postmenopausal women (Sagraves, 1995; Lobo, 1995). This is because these compounds have been proven to prevent bone loss and fracture in women. In addition, estrogen replacement therapy is associated with reduced mortality from cardiovascular disease. Finally, ongoing studies suggest that estrogens may provide benefits to the central nervous system for improving cognition and reducing Alzheimer's disease.

The classical ER, now called ER-α, is known as ligand binding, receptor dimerization,
Represents a modular structure with distinct domains involved in 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 (K
(Uiper) et al. (1996) exploited this similarity in an attempt to identify additional members of this family. The amino acids of the DNA binding domain are almost identical to those of ER-α, and the in vitro translated protein specifically binds [ ³ H] -estradiol with nanomolar affinities and is derived from a simple estrogen response element. Because they were able to regulate transcription, the new member they discovered was identified as ER. This protein has a known form (
(Now called ER-α) and ER-β to identify it. Human and mouse ER-β have also been cloned (Bhat et al. 1998; Mosselman, 1996; Peterson).
Ttersson et al., 1997; Tremblay et al., 19
97). Most studies following the discovery of ER-β have focused on mapping its mRNA distribution in normal and neoplastic tissues, characterizing its binding affinity to a wide range of ligands, And its interaction with ER-α is evaluated.

[0004] ER-β mRNA is detectable by RT-PCR and in situ hybridization in a wide range of tissues. While its distribution overlaps that of ER-α, some tissues express only one receptor type. For example, while both receptors are found in the uterus, ovary and pituitary, ER-β seems more prominent in the prostate, lung and bladder (Kuiper et al., 1996). When tested by in situ hybridization, a further distinction between receptor distributions can be made. ER-β mRNA is expressed in rat prostate epithelial cells; ER-α is found in the stromal compartment (Kuiper (Ku
iper) et al., 1996). In rat ovaries, ER-β is found in granulosa cells;
-Α appears in the interstitium (Shghrue et al., 1996; personal communication).
. In the rat hypothalamus, ER-β (but not ER-α) messages are expressed in the paraventricular region, while both messages are found in the preoptic region (Shughrue et al., 1996). ). Interestingly, there can be considerable species differences in the relative levels of ER-β and ER-α mRNA in certain organs. For example, ER-β mRNA is highly expressed in rat prostate, while modest levels are detected in human prostate. ER
-Β is superior to ER-α in rat prostate (Kuiper et al., 1).
996; Lau et al., 1998; Enmark et al., 199.
7), but in the mouse prostate the levels are more equal (Couse
) Et al., 1997).

[0005] Several groups have characterized the expression of ER in tumor samples and cancer cell lines. Most studies were performed on the breast, and one study included 7 of the 40 breast biopsy samples.
Report ER-β mRNA using RT-PCR at 0% (Dotlow (
Dotzlaw) et al., 1996). No correlation was shown between the expression of ER-α and ER-β. Another study also found that ER-α / β mRNA in breast tumor samples
Heterogeneity of expression was reported, with some tumors having only ER-α mRNA and others expressing mRNAs for both receptor subtypes (Enmark (Enmark
k) et al., 1997). Among the breast cancer cell lines, MDA MB 231 has only ER-β mRNA and there are conflicting reports on the ER status of MCF-7 cells (Enmark, 1997; Kuiper).
r) et al., 1997; Vladusic et al., 1998); R. Henderson, unpublished observations).

[0006] The ligand binding domain of human ER-α and ER-β is 59 amino acids.
% Identical (Enmark et al., 1997) but with 17β-
The binding affinity of estradiol is very similar. Some compounds show significant selectivity for either ER-α or ER-β. Genistein is ER-β
Is a phytoestrogen that binds with approximately 10 to 25-fold greater affinity for (Kuiper 1996, H. Harris unpublished observations). On the other hand, raloxifene binds approximately 20-fold better to ER-α than ER-β (H. Harris unpublished observation).

[0007] When ER-α and ER-β are co-expressed, a mammalian two-hybrid system,
Interactions can be measured by several methods, including glutathione S-transferase pulldown and gel shift / supershift assays (Cowley et al., 1997; Peterson).
Ogawa et al., 1998). Because these receptors can heterodimerize, the effects of estrogen on tissues containing both receptors may be mediated by a complex interaction between ER-α and ER-β. it can.

[0008] One valuable tool in determining the function of ER-β is the estrogen receptor-
α knockout (ERKO) mouse (Lubahn et al., 19
93). Because these mice lack functional ER-α, they may help define the physiological role of both ER-α and ER-β. One study compared the efficacy of 17-β estradiol treatment in improving the consequences of artificially induced vascular injury to the carotid artery of wild-type and ERKO mice (Iafrati et al., 1997). A pharmacological dose of 17-β estradiol is shown in both types of mice in the median area (m
edial area) and increased proliferation of smooth muscle cells. It is believed that these responses to endothelial exposure may narrow the lumen of blood vessels and thus limit blood flow. One interpretation is that ER-α is not required for this response because 17-β estradiol was as effective in wild-type mice as ERKO. ER
Since -β mRNA is also expressed in these vessels, it is likely that 17-β
It may mediate the action of estradiol. However, there is lack of direct evidence to support this hypothesis. ERK responsive to 17-β estradiol substitution
Another example of an O mouse was described in a recent poster and abstract (Pan et al., 1997 and personal communication). This study demonstrates the loss of femoral mineral density and spongy volume in ERKO mice following ovariectomy, as well as 17-β estradiol (
However, these parameters have been increased upon treatment with (but not dihydrotestosterone). Again, based on indirect evidence, suggestions are made that ER-β has a physiological role.

Thus, while certain aspects of ER-β have been characterized, the art
It has failed to provide a method for identifying ligands that are functionally selective for α or ER-β. Such an assay would be of great advantage to the pharmacology industry to discover possible therapeutic applications of ER subtypes.

SUMMARY OF THE INVENTION The present invention provides a method of screening for a test compound that binds to the ER in a receptor binding assay, wherein the method detects ER-β polypeptide mediated transcription, The methods include: (a) at least one estrogen-derived DNA sequence encoding metallothionein (MT-II) and at least one DN encoding ER-β polypeptide.
Providing a cell comprising the A sequence, wherein the receptor is transcriptionally active; (b) contacting the cell with either the test compound or a control that binds ER; And (c) detecting the expression of the MT-II (wherein the MT-II relative to a control is detected).
Elevated expression of II indicates that the test compound has estrogen agonist activity).

The invention further provides a method of screening for a test compound that binds to the ER in a receptor binding assay, wherein the method detects inhibition of ER-β polypeptide-mediated transcription, comprising: a) At least one estrogen-induced DNA sequence encoding metallothionein (MT-II) and at least one DN encoding ER-β polypeptide
Providing a cell comprising the A sequence, wherein said receptor is transcriptionally active; (b) providing said cell in the presence of a test compound known to bind ER Or contacting with one or more estrogen (s); and (c) and detecting expression of the MT-II (where one or more estrogen (s) alone) MT-I for the addition of
Reduced expression of I indicates that the test compound has estrogen antagonist activity).

There is further provided a method of screening test compounds to identify drug candidates that mimic the effects of estrogen on ER-β or ER-α mediated transcription, wherein the method comprises: (I) a first cell comprising at least one DNA sequence encoding a metallothionein (MT-II) and at least one DNA sequence encoding an ER-β polypeptide, wherein said receptor comprises Is transcriptionally active), and (ii) contacting the test compound with a second cell comprising an ERE reporter gene construct, wherein the cell expresses an ER-α polypeptide; (B) increasing the expression of MT-II in the first cell with respect to the control, but having a minimal effect on the expression of the reporter gene in the second cell. To identify compounds having an effect of limit (here, the compound is considered to ER-beta-selective);
Or (c) identifying a compound that increases the expression of the reporter gene in said second cell relative to the control but has minimal effect on the expression of MT-II in said first cell, wherein Said compound is considered to be ER-α selective).

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-α mediated transcription, comprising: a) a plurality of said test compounds in the presence and absence of one or more estrogen (s): (i) at least one endogenous D encoding the metallothionein (MT-II) gene
A first cell comprising DNA encoding NA and ER-β polypeptides (
Wherein said receptor is transcriptionally active; and (ii) contacting with a second cell comprising an ERE reporter gene construct, wherein said cell expresses an ER-α polypeptide. And (b) reducing expression of MT-II in said first cell with respect to treatment with one or more estrogen (s) alone, but said reporter gene in said second cell. (C) identifying one or more estrogen (s) alone, wherein said compounds are considered to be ER-β selective; Reduces the expression of the reporter gene in the second cell with respect to treatment with but has a minimal effect on the expression of MT-II in the first cell To identify compounds (wherein said compound is considered ER-alpha selective) comprising the steps of.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an efficient method of screening for a large number of test compounds that selectively activate the ER of the α or β subtype. These compounds include estrogen, including but not limited to cancer (eg, breast, ovary, endometrium, prostate), endometriosis, osteoporosis, and diseases of the cardiovascular and central nervous systems. It can have desirable properties for either treating or preventing a variety of diseases mediated.

Definitions In this description and in the claims, the following terms are defined as set forth below.

“HERβL” is defined as ER-β (cDNA or its translated protein product) in human form as described in Example 1.

“Estrogen” is defined as any ligand that can function like an estrogen agonist.

“Estrogen agonists” substantially mimic 17-β estradiol, as measured in a standard assay for estrogenic activity (eg, a cellular assay as described in Webb et al. (1992)). Defined as a compound.

An “estrogen antagonist” is a substance that substantially inhibits the effects of an estrogen agonist, as measured in a standard assay of estrogen activity (eg, of cells as described in Webb et al. (1992)). Defined as a compound.

“Functional ER” is defined as a receptor capable of transcriptionally activating an endogenous or transfected gene as measured by changes in RNA, protein and / or downstream biological events. .

“Non-functional ER” refers to a receptor that is incapable of transactivating the endogenous or transfected gene as measured by changes in RNA, protein and / or downstream biological events. Define. A “test compound” can include, but is not limited to, any small molecule compound, peptide, polypeptide, natural product, toxin having potential biological activity.

“Ligand” is intended to include any substance that interacts with the receptor.

“Transfection” is defined as any method by which a foreign gene is inserted into cultured cells.

“Reporter” is defined as any substance that can be easily measured and easily distinguished from other cellular components. The reporter can be transfected receptor DNA, transcribed receptor mRNA, enzyme, binding protein or antigen.

A “cell” useful for this purpose is one that has the ability to respond to signal transduction by a given receptor.

A “receptor binding assay” is an assay that measures the amount of a ligand that specifically interacts with a receptor. The ligand can be a radioactive ligand (eg, bound to ³ H or ¹²⁵ I), a fluorescinated ligand (either bound to a fluorochrome or possessing intrinsic fluorescence). Can be, or can be labeled in another way so as to be detectable. Description of the Assay The present invention relies on the discovery that MT-II expression is selectively regulated by ligand interaction with ER-β.

In the methods of the present invention, a screen for selectively detecting the functional activity of both estrogen agonists or antagonists of a ligand following interaction of the ligand with ER-β using modulation of the activity of MT-II. A system can be provided.

The method typically comprises cultured cells that express functional human ER-β and do not express ER-α or express reduced amounts of ER-α. Such cells include, but are not limited to, Saos-2 (ATCC HTB-85).
And LNCaPLN3. Preferred cells for this purpose are
Such as the cells described below, which are recombinantly engineered to overexpress R-β,
Includes cells that overexpress ER-β. The ER-β receptor may be modified in any way, such as in length; these modifications may result in increased ER-β selectivity and increased sensitivity of the assay.

Those skilled in the art will recognize that a variety of recombinant constructs comprising ER-β can be used in conjunction with any cell or line that lacks functional ER-α.

To screen a large number of compounds for estrogen agonist activity, cells expressing ER-β are exposed to a test compound or control solution (used to dissolve the test compound). Cells can be exposed during growth in either separate wells of a multi-well culture dish or in a semi-solid nutrient matrix. MT-II mRNA is measured after treatment for a suitable period of time. The estrogen agonist has an MT when compared to treatment with the control solution alone.
-II mRNA can be increased.

To screen a large number of 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 during growth in either separate wells of a multi-well culture dish or in a semi-solid nutrient matrix. MRN of MT-II after treatment for a suitable period of time
Measure A. Estrogen antagonists can reduce MT-II mRNA when compared to treatment with an estrogen solution alone.

The estrogen or antiestrogen compound identified in the assays of the present invention may be in a standard pharmaceutical composition for the treatment of cancer, either as a component of an oral contraceptive or for which modulation of estrogenic activity is desirable. Can be used in other applications. Pharmaceutical compositions can be manufactured and administered using methods known in the art. The pharmaceutical compositions are generally intended for parenteral, topical, oral or local administration for prophylactic and / or therapeutic treatments. The pharmaceutical compositions can be administered in various unit dosage forms depending on the mode of administration. For example, unit dosage forms suitable for oral administration include powders, tablets, pills, and capsules. Pharmaceutical formulations suitable for use in the present invention include Rem
inton's Pharmaceutical Sciences, Mac Publishing Company
ny), Philadelphia, Philadelphia, 17th Edition (1985). A variety of pharmaceutical compositions can be prepared which comprise a compound of the present invention and a pharmaceutically effective carrier. Applications The methods and compositions of the present invention can be used to identify compounds that either interact with ER-β and either stimulate or inhibit transcriptional activity. Such compounds include, without limitation, co-activator proteins and estrogens and other steroids, steroid-like molecules, or non-steroid-like molecules that act as agonists or antagonists. Screening methods to identify tissue-specific estrogens can also be used.

Identification of compounds that interact with ER-β can be achieved by cell-free or cell-based assays. In one set of embodiments, the purified ER-
β is contacted with a labeled ligand such as, for example, 17-β estradiol in the presence of a test compound to form a test reaction and in the absence of the test compound to form a control reaction. The labeled moiety may include a radiolabel (eg, ³ H or ¹²⁵ I) or a fluorescent molecule. Incubation is allowed to proceed for a period of time sufficient to achieve specific binding and under appropriate conditions, followed by binding of the labeled ligand to ER-β (eg, radioactivity, fluorescence or fluorescence polarization). (By monitoring). In one embodiment, E. coli
The ligand-binding domain of ER-β produced in is adsorbed to the wells of a microtiter dish and incubated with [ ³ H] -17β estradiol in the absence or presence of the test compound. Alternatively, the soluble receptor is incubated with the labeled ligand in the absence or presence of the test compound, and the bound ligand is bound, either by filtration on glass fiber filters or by using dextran-coated charcoal. From the free ligand.

A whole cell binding assay, in which bound ligand is separated from free ligand by rinsing, may also be used. The cells used in these assays may contain endogenous receptors or may overexpress the receptors after stable or transient transfection or infection of ER-β cDNA. Either you can. Non-limiting examples of suitable cells include COS cells, HeL
a cells, CHO cells, human umbilical vein endothelial cells and yeast. Once a compound is identified by its binding activity as a compound that interacts with ER-β, further in vivo and in vitro tests may be performed to determine the nature and extent of the activity, ie, as an agonist or antagonist (see below). Please see).

Compounds that interact with ER-β have also been identified using cell-based assays that measure the activation or repression of transcription of endogenous or transfected estrogen responsive genes. Good. For example, agonists (such as, for example, 17β-estradiol) block interleukin-1β induction of endogenous E-selectin in primary human umbilical vein endothelial cells expressing ER-β. Antagonists (such as, for example, ICI-182780) block the agonist activity of 17β-estradiol. Non-limiting examples of other suitable endogenous estrogen responsive promoter elements include endothelin-1 (ET-1);
Receptors (scavenger receptor type II); and those that regulate enzymes involved in coagulation and fibrinolysis (such as, for example, plasminogen activator inhibitor-1 and complement C3). Any promoter element responsive to estrogen, including, for example, the NFκB binding site or the apolipoprotein A1 gene enhancer sequence, may be used as a suitable target.

In one set of embodiments, a suitable host cell is transfected with an expression vector encoding ER-β and the transfectants are transformed.
ant) is incubated with or without one or more estrogen I in the presence or absence of the test compound. ER-β activity is assessed by measuring transcriptional activation. This may be achieved by detecting mRNA (eg, using Northern blot analysis, reverse transcriptase polymerase chain reaction, RNase protection assay) and / or protein (eg, using immunoassays or functional assays). If activation of the target sequence initiates a biochemical cascade, downstream biological events may also be measured to quantify ER-β activity. Compounds that interact with ER-β are identified as those that positively or negatively affect activation of the target sequence.

In another set of embodiments, a suitable host cell, preferably a mammal, is ER-
Co-transfected with a reporter plasmid containing a reporter gene downstream of an expression vector encoding β and one or more EREs.
The transfected cells are incubated with or without estrogen in the presence or absence of the test compound, and then the activity of ER-β is measured by measuring the expression of the reporter gene. In a preferred embodiment, the activity of ER-β is monitored visually. Non-limiting examples of suitable reporter genes include luciferase, chloramphenicol acetyltransferase (CAT) and green fluorescent protein.

Preferably, the method of the invention is adapted for high-throughput screening, allowing multiple compounds to be tested in a single assay. Candidate estrogens and estrogenic compounds include, without limitation, diethylstilbestrol,
Includes genistein and estrone. Other compounds that interact with ER-β can be found, for example, in natural product libraries, fermentation libraries (including plants and microorganisms), combinatorial libraries, compound files, and synthetic compound libraries. . For example, a synthetic compound library is available from Maybridge Chemical Co.
. ) (Trevillette, Cornwall, UK), Comgenex (Comgen)
ex) (Princeton, NJ), Brandon Associates (Br
and commercially available from Anderson Associates (Merrimack, NH) and Microsource (New Milford, CT). A rare chemical library is available from Aldrich Chemical Company, Inc.
mpany, Inc. ) (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available, for example, from Pan Laboratories.
) (Botel, Washington) or MycoSearch (
North Carolina) or is easily produced. In addition, natural and synthetically produced libraries and compounds are readily modified by conventional chemical, physical and biochemical means (Blond
elle) et al., 1996). The ER-β binding assay of the present invention is advantageous in adapting many different types of solvents and thus allowing for the testing of compounds from many sources.

Compounds identified as agonists or antagonists of ER-β using the methods of the present invention have potency, efficacy, uptake, stability, and suitability for use in therapeutic applications and the like. It can be modified to increase. These modifications are achieved and tested using methods known in the art. Examples The present invention is further described by the following examples. The examples are provided merely to illustrate the invention by reference to particular embodiments. These examples illustrate certain aspects of the present invention, but do not delineate or limit the scope of the invention. Example 1 Construction of ER-β Recombinant Adenovirus The coding sequence of human ER-β is described in “Novel Human Estrogen Receptor β (Novel Human Estrogen Re) filed on Aug. 5, 1997.
commonly owned and commonly assigned pending US Ser. No. 08 / 906,365, and Bhat et al. (1998). Essentially, human testis poly A + RNA (1 μg, Clontech, Palo Alto, CA) was combined with 0.5 μg of oligo dT primer (GIBCO) -BRL in a total volume of 10 μl.
, Gaithersburg, MD). After heating the mixture at 70 ° C. for 10 minutes and cooling on ice, 500 μM of each deoxynucleoside triphosphate, 1 × cDNA synthesis buffer and 10 mM DT to a final reaction volume of 20 μl.
T was replenished. The mixture is incubated at 42 ° C. for 2.5 minutes and then
２2 units of reverse transcriptase (GIBCO-BRL, Gittersburg, MD) was supplemented before it was incubated at 45 ° C. for 30 minutes and 50 ° C. for 5 minutes. Then, one-tenth of this mixture containing the cDNA template
(Approximately 2 μl) was added to E using forward and reverse primers as described below.
Used for PCR amplification of R-β.

Using the PCR primers specified in 08 / 906,365 (above), the following components were used: 2 μl of the above-described cDNA template; 1 × PCR buffer; 200 μM of each deoxynucleoside triad. Phosphoric acid, 2 units hot tub (Hot
Tub) The ER-β sequence was amplified in a reaction containing DNA polymerase (Amersham, Arlington Heights, Ill.) And 1 μg of each of the forward and reverse primers. The reaction mixture was heated to 95 ° C. for 2 minutes, annealed at 52 ° C. for 1 minute, and amplified using 36 cycles, then
Incubated at 2 ° C for 1.5 minutes.

One fragment approximately 1500 bp in length was produced. The fragment is
digested with indIII and XbaI (cut at sites present in the forward and reverse primer sequences, respectively, but not in the amplified cDNA sequence) and digested with the pcDNA3 expression vector (Invitrogen (Invitrogen)).
ogen), Carlsbad, CA). This asymmetric cloning strategy involves the addition of the 5 'end of the ER-β cDNA to pcDNA.
3 under the control of the CMV promoter of the virus. This clone is called "short
ERβ ”or hERβT.

To confirm the amino terminal and upstream sequences of human ERβ, two independent approaches were taken as described below.

(1) 10 μl of human ovary 5 ′ stretch (Stretch) cDNA library (Clontech, Palo Alto, Calif.) Was combined with 50 μl of 1 × K buffer (1 × PCR buffer (GIBCO)- BRL
Mix, 2.5 mM MgCl ₂ , 0.5% Tween- ₂₀ , 100 μg / ml proteinase K) and the reaction mixture at 56 ° C. for 2 hours, then at 99 ° C. Incubated for 10 minutes. Thereafter, 5 μL of this reaction mixture was used as a template in a nested PCR reaction using the PCR primers specified in 08 / 906,365 (above). The reaction was performed with 1 × Klentaq PCR reaction buffer (40 mM Tricine-KOH, 15 mM KOAc, 3.5 mM Mg).
(OAc) ₂ , 75 μg / ml bovine serum albumin); 0.2 μM of each dNTP
0.2 μM of each of the above primers and 1 unit of Klentac (Kle
ntq) polymerase mixture (Clontech, Palo Alto, CA). Touchdown PCR
The conditions were as follows: 5 cycles of 94 ° C. for 2 seconds and 72 ° C. for 4 minutes, followed by 30 cycles of 94 ° C. for 2 seconds and 67 ° C. for 3 minutes.

Excess nucleotides and primers were removed from the first PCR reaction by purification using a Wizard PCR column (Promega, Madison, WI). Then, on 08/906, 36
A second PCR reaction was performed using 2 μl of the purified first reaction mixture, using the PCR primers specified in No. 5 (above). The PCR reaction and circulation conditions were identical to those used in the first round. The product was purified with pCR2.1 (Invitrogen, Carlsbad, CA).
And the three resulting clones were sequenced. All three clones (designated L1, L2 and L3) contained ERβ inserts of different lengths, all of which were ERβ and mutually homologous.

(2) Marathon Ready (M) for 5 ′ Rapid Amplification of cDNA End (RACE)
arathon Ready thymus cDNA kit (Clontech
ech), Palo Alto, CA) was also used to isolate the 5 'clone of ERβ. In the first nested PCR reaction, 5 μl of human thymic marathon-
Ready-made (Marathon-Ready) cDNA (Clontech
ech), Palo Alto, CA) as a template and
The primers specified in 906, 365 (above) were used. The PCR reaction and cycling conditions were identical to those described in (1) above.

Excess nucleotides and primers were removed from the first PCR reaction by purification on a Wizard PCR column (Promega, Palo Alto, CA). Using the primers specified in 08 / 906,365 (above), using 2 μl of the purified first reaction, 2
A second PCR reaction was performed. The conditions of the second round PCR reaction and circulation were the same as those used in the first round. The product was cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA) and two clones were sequenced. The two clones contain ERβ, mutual, and variable length inserts that are homologous to sequences isolated from a human ovarian cDNA library as described above.

The entire ERβ sequence isolated by the above methods (1) and (2)
It contained 110 nucleotides corresponding to the T sequence, as well as 228 additional nucleotides at the 5 'end.

The hERβT and the 5 ′ end sequence are ligated and the resulting cDNA is ligated with pCDNA3 (in vitrogen) under the control of the cytomegalovirus IE promoter.
Invitrogen), Carlsbad, CA);
This expression vector was designated as “long hER-β” or hERβL. The full length hERβL cDNA sequence encodes a polypeptide having 530 amino acid residues as found in SEQ ID NO: 1.

The hERβL sequence contained the optimal translation initiation sequence CCACC immediately upstream of the start codon, and the sequence was under the control of the cytomegalovirus IE promoter. The Ad5ΔE1a vector plasmid containing the adenovirus sequence from map unit 0-17 with a deletion of the E1a region between map units 1.4-9.1 (Davis AR et al., 1985, Glusman).
zman, Y .; ) Et al., 1982) introduced the coding sequence for hERβL.
HERβL transcription units in Ad5ΔE1a plasmid contained the cytomegalovirus of 1E promoter, tripartite leader Ad5, the coding sequence and the SV40 late polyadenylation signal sequence hERβ _L. Then, Ad
HERβL in the 5ΔE1a plasmid was linearized with the BstEII enzyme and 293 cells (transformed primary human embryonic kidney, ATCC CRL 1573). Virus plaques generated by homologous recombination were isolated, amplified, and analyzed by restriction DNA analysis and A549.
Characterized by a cell lysis assay in cells (human lung carcinoma, ATCC CCL 185). Confirming tests showed that the recombinant Ad5 hERβL virus contained the expected DNA fragment and was replication defective. The virus was further purified by re-plaquing. The isolated plaque was amplified, tested, and used as a seed stock to generate large amounts of virus in 293 cells. Virus was titrated in 293 cells by plaque assay and the stock was 1.28 × 10 ⁹ PFU
/ Ml. Example 2: Evaluation of the level of endogenous ER mRNA in Saos-2 and LNCaPLN3 cells Unless otherwise indicated, cell culture reagents were obtained from Gibco BRL (Gaithersburg, MD). LNCaPLN3 cells are 10% F
RPMI supplemented with BS, 2 mM GlutaMAX-1, 100 U / ml penicillin g and 100 μg / ml streptomycin sulfate
Grow in 1640 medium. Saos-2 cells (ATCC, Manassas, VA) contain 10% fetal bovine serum (FBS), 2 mM glutamax (Glut).
aMAX) -1, 100 U / ml penicillin g, 100 μg / ml streptomycin sulfate supplemented with McCoy 5A medium and maintained in monolayer culture. Total RNA was isolated from LNCaPLN3 and Saos-2 cells using RT-PCR TRIzol (Gibco BRL, Gatorsburg, MD) according to the manufacturer's instructions. Thereafter, the sample was collected at 1 unit / μ.
g of DNase I without RNase (Gibco BRL, Gatorsburg, MD) for 30 minutes at 37 ° C. RN Easy (RN
RNA was purified from the reaction using an easy column (Qiagen, Hilden, Germany) and the amount recovered was estimated by UV spectrophotometry.

The reverse transcription reaction was performed with 0.5 μg of RNA in a 20 μl reaction. For ER-α, the reaction was performed with 1 × PCR buffer (Gibco BRL, Gaithersburg, MD), 5 mM MgCl ₂ , 1.25 μM ERα-specific reverse primer (5′-CCAGCAGCCATGTCGAAGATCC-3 ′). ), 0.
5 μM GAPDH-specific reverse primer (5′-CACCCTGTTGCT
GTAGCCAAATTC-3 '), 0.5 mM dNTPs, 20 units of RNasin (Promega, Madison, WI) and 200 units of Superscript II.
Reverse transcriptase (Gibco BRL, Gatorsburg, MD)
Was contained. The ER-β reaction was performed with the following exceptions: 2.5 mM MgCl ₂ and 1.25 μM ERβ-specific reverse primer (5′-GCAGAGTGTG
AGCATCCCTCTTTG-3 ′) and contained the same components as ER-α. Duplicate reactions, which were identical for all reagents except that they did not contain Superscript II reverse transcriptase, to ensure that RNA samples were not contaminated by DNA Was performed on each sample as a negative control. The reaction was incubated at 42 ° C. for 15 minutes, then at 99 ° C. for 5 minutes and on ice for 5 minutes prior to amplification.

ER-α specific forward primer (5′-GGAGACATGAGAGCTG
CCAAC-3 ′) or ER-β specific forward primer (5′-CAGC
ATTCCCAGCAATGTCAC-3 ') and a GAPDH-specific forward primer (5'-GACATCAAGAAGGTGGTGAAGCAG-3')
PCR was started by directly adding 80 μl of the master mixture containing to the 20 μl reverse transcriptase reaction. The final concentration of the reagents in the 100 μl PCR reaction of ER-α was as follows: 0.25 μM of each ER specific primer,
0.1 μM of each GAPDH primer, 1 × PCR buffer (Gibco
) BRL, Gatorsburg, MD), 0.2 mM dNTPs, 2 mM MgCl ₂ and 0.5 units of Taq DNA polymerase (Gibco
co) BRL, Gatorsburg, MD). ER-β reaction is 1
containing reagents same amount except mM MgCl _2. The two-step PCR was performed as follows: 95 ° C. for 30 seconds, 64 ° C. for 1.5 minutes, PE 9600 for 25 cycles.
It was carried out in. After amplification, the samples were incubated at 64 ° C. for 10 minutes.

Separate 20 microliters of each sample using a 1.5% agarose gel and Hybond-N + (Amersham Pharmacia) by capillary alkaline southern blotting in 0.4N NaOH, 0.6M NaCl. (Amersham Pharmacia), Piscataway, NJ). Rapid-Hyb buffer (Amersham Pharmacia)
The blots were prehybridized at 42 ° C. for 30 minutes in Piscataway, NJ. Polynucleotide kinase (Gibco BRL)
ER-α (5'-TGAAC) using Gaitersberg, MD.
CAGCCTCCCTGTCTGCCCAGGTTGGT-3 '), ER-β (5'
-CCGCATACAGATGTGTATAACTGGCGATGGA-3 ') and GAPDH (5'-GCTGTTGAAGTCACAGGAGACAACC
TGGT-3 ') oligonucleotide probe specific for the fragment ³² P-
End-labeled with γATP. Probes were added to the blot at 3.0 × 10 ⁶ CPM / ml and incubated at 42 ° C. for 1 hour. Hybridization of ER and GAPDH was performed independently. Blots were run in 2 × SSC, 0.1% SD
S once at room temperature in S for 15 minutes, then 1x at 42 ° C. in 0.2 × SSC, 0.1% SDS
Washed twice for 5 minutes. Thereafter, the blot was exposed to film.

Saos-2 cells expressed endogenous ER-β (but not ER-α) mRNA as assessed by PCR (FIG. 1). GAP as positive control
DH mRNA was co-amplified in these reactions. Both cell lines have the GAPDH mR
Only Saos-2 cells contained ER-β, while containing NA. Similar results were obtained for LNCaPLN3 cells (data not shown). Example 3: ER-β is MT-II in Saos-2 and LNCaPLN3 cells
Cell culture and infection Due to low levels of ER-β messages, Saos-2 and LNCa
PLN3 cells were engineered to overexpress hERβL by transient infection with a recombinant adenovirus (see Example 1) prior to differential display.

LNCaPLN3 and Saos-2 cells were cultured as described above. FBS (HyClone, Logan, Utah) treated with 10% charcoal / dextran 16 hours prior to infection, 2 mM GlutaMAX-1, 100 U / ml penicillin g, 100
Phenol red-free R supplemented with μg / ml streptomycin sulfate
Cells were plated in PMI 1640 medium. This medium was used for the rest of the experiment.

The cells were transformed with 2% containing antibiotics and GlutaMAX-1
Using a phenol red-free medium of the stripped FBS of
20-fold dilution of Ad5 hERβL virus (see Example 1) at 37 ° C.
For 2 hours. The medium containing the virus was aspirated and the cells were washed with medium. Fresh medium was added and cells were allowed to recover at 37 ° C. overnight.

The next day, cells were treated with 10 nM 17β-estradiol or vehicle for 24 hours, and total RNA was used for differential display using TRIzol reagent (Gibco BRL, Gaithersburg, MD). Prepared. Remove one sample to remove residual DNA
Unit / μg RNase-free DNase I (Gibco BRL
(Gatorsburg, Md.) At 37 ° C. for 30 minutes. RNA was purified from the reaction using an RNeasy column (Qiagen, Hilden, Germany) and the amount recovered was estimated by UV spectrophotometry. Rapid Analysis of Differential Expression (RADE) Reverse Transcription (RT) After DNase I treatment, 6 micrograms of total RNA was added to a final volume of 600 μL in 1 × RT buffer (25 mM Tris-Cl, pH 8.3). , 37.6 mM KCl, 3 mM MgCl ₂ and 5 mM DTT, Gen Hunter (Gen
hunter, from Nashville, TN), 20 μM dNTPs (dA,
C, G and TTP 2'-deoxynucleoside 5 'triphosphate, Gibco (Gi
bco) from BRL (Gatorsburg, MD) with 0.2 μM HT ₁₁ C (oligonucleotide AAGCTTTTTTTTTTTTC). The reaction mixture was incubated at 65 ° C for 5 minutes to denature the secondary structure, then incubated at 37 ° C for 10 minutes. At this point, 30 μl of Superscript II reverse transcriptase (200 U / μl,
Gibco BRL (Geatersburg, MD) was added to the reaction and the incubation was allowed to proceed at 37 ° C. for 1 hour. The enzyme was inactivated by heating at 75 ° C. for 5 minutes. An aliquot of this reaction is then
Was used for second-strand synthesis. Polymerase chain reaction (PCR) For 2 μl of RT reaction, 1 × PCR buffer (10 μl for a total reaction volume of 20 μl)
mM Tris-Cl, pH 8.4, 100 mM KCl, 1.5 mM MgCl ₂
And 0.001% gelatin), 2 μM dNTP, 15 nM ³³ P dATP
(NEN, Boston, MA) 1 unit of AmpliTac (Ampl
iTaq) DNA polymerase (Perkin-Elme
r), Norwalk, Conn.) as well as 1 μM of primer 5 ′ AAGCTTGCCATGG-3 ′. The reaction mixture was then heat cycled using the following parameters: 92 ° C for 2 minutes, 1 cycle 92 ° C for 15 seconds, 40 ° C for 2 minutes, 72 ° C for 30 seconds, 40 cycles 72 ° C for 5 minutes. Gel electrophoresis Duplicate samples of the PCR products were run in 1 × TBE buffer (0.1 M Tris, 0.0
6% denaturing polyacrylamide gel (5.7% acrylamide, 0.3% bisacrylamide, 42% at 2000 volts for 3 hours in 9M boric acid, 1 mM EDTA)
% Urea and 51% H ₂ O). The gel was then filtered through filter paper (Schleicher & Schu).
ell), Keene, NH), dried under vacuum at 80 ° C. for 1 hour, and exposed to X-ray film for 24 hours.

One band, designated 6a, was produced by Saos- 17-β estradiol.
2 and LNCaPLN3 cells were clearly upregulated (FIG. 2). Thereafter, the developed film is overlaid on the dried gel and
The corners of the band were marked using a 2 gauge syringe needle. Gel slices within these boundaries were cut with a razor blade and immersed in 100 μl H ₂ O. The sample was boiled in a water bath for 15 minutes, centrifuged for 2 minutes, and the supernatant solution was transferred to a new tube. 5 μl of 10 mg / ml glycogen, 1
0 μl of 3M sodium acetate and 450 μl of 100% ethanol were added. The samples were mixed, precipitated overnight at -20 ° C, and centrifuged at 10,000 g for 10 minutes. The supernatant solution was removed, the pellet was washed with 200 μl of 85% ethanol, dried and resuspended in 10 μl of H ₂ O. Aliquot 3 μl aliquots into 1 × PCR buffer, 20 μl using the same cycling parameters as the PCR reaction above
MdNTP was used in a reamplification PCR reaction in the presence of 0.2 μM discretionary primer and 0.2 μM oligonucleotide HT ₁₁ C, and 2 units of AmpliTaq polymerase. 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. Fragment Cloning Band 6a was cloned using a TA cloning kit (Invitrogen, Carlsbad, CA). After lysis, colonies were screened by PCR for the correct insert size. Colonies were selected from 20 mM Tris-HCl, pH 8, 50 mM KCl, 2.5 mM
mM MgCl ₂ , 0.5% Tween 20 and 100 μg /
Lysis was performed by incubating for 30 minutes at 56 ° C. in ml proteinase K for 30 minutes, then at 99 ° C. for 10 minutes to inactivate proteinase K. 2 [mu] l of this reaction was mixed with 20 mM Tris-HCl, pH 8, 50 mM KCl, 2.
5 mM MgCl ₂ , 75 μM dNTPs, 375 nM M-13 forward and reverse primers, and 2.5 U Taq polymerase (Gibco)
(BRL, Gatorsburg, MD). The reaction is as follows: 95 ° C. for 30 seconds; 64 ° C. for 30 seconds;
Circulated for 0 cycles. 6a. One clone, designated 2 was selected for sequencing. Fragment sequencing Applied Biosystems (
Using the recommended protocol from Foster City, California)
ABI Prism [PRISM] ™ Dye Terminator Circulation Sequencing Immediate Reaction Kit (AmpliTaq) DNA Polymerase (D
ye Terminator Cycle Sequencing Ready
Reaction Kit), clone 6a. 2 was sequenced. After circular sequencing, unincorporated dye-labeled nucleotides were removed using a spin column (AGTC). Using an ABI 373 DNA sequencer, an automated DNA sequencing grade 4.75% polyacrylamide gel was loaded with all D
Runs on NA sequencing samples. Sequencing data was edited using Sequence Navigator,
And assembled using DNAStar (DNAStar, Madison, WI). Trim the sequence from the RADE primer (trimm
ed of) and Millennium Software
are) (BLAST search using Boston, Mass.). Clone 6a. A search for nucleotide homology between the two sequences showed 98% identity with human MT-II. However, no amino acid mismatch was present across the coding sequence (FIG. 3). Northern blot and quantitative reverse transcriptase polymerase chain reaction (qRT-PC
Confirmation of regulation using R): To confirm the results obtained by PCR amplification of cellular mRNA, the effect of 17-β estradiol or other compounds on message levels was determined using Northern blot and qRT-PCR. evaluated. Unless otherwise indicated,
All experiments used cells that transiently overexpress hERβL in response to adenovirus infection as described above, and were treated with compounds for 24 hours. In some cases, cells that transiently overexpress ER-α were used for comparison. E
The method of adenovirus-mediated overexpression of R-α is identical to that described for ER-β. Northern Blot Poly A + RNA was isolated from total RNA using the Oligotex mRNA isolation kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Six micrograms of mRNA or 10 μg of total RNA was added to 1.5% agarose, 0.22 M formaldehyde, 10 mM H
Separated on EPES, 1 mM EDTA gel. 20 × SSC (Gibco
RNA by Hybond-N (Amersham Pharmacia (co) BRL, Gatorsburg, MD) by capillary action.
(Amersham Pharmacia), Piscataway, NJ) overnight. After transfer, the membrane was crosslinked with UV and dried at 80 ° C. for 10 minutes. Northern blots were run in a Rapid Hyb solution (
Prehybridized in Amersham Pharmacia (Piscataway, NJ) for 30 minutes.

Using PCR as described above for screening colonies, clone 6a. The insert from 2 was isolated from the plasmid. PCR products were purified from agarose gels using Wizard Prep (Promega, Madison, WI). According to the manufacturer's instructions, a Redi-prime kit (Amersham)
Using Amersham Pharmacia, Piscataway, NJ, reamplified RADE fragment of band 6a or clone 6a. Fragments from 2 were random primer labeled.
Nap-5 column (Amersham Pharma)
macia), Piscataway, NJ) to remove unincorporated nucleotides and incorporation of [ ³² P] -dCTP was determined by liquid scintillation counting.

The probe was denatured at 100 ° C. for 10 minutes and 1.5 × 10 ⁶ CPM / ml
Rapid-Hyb hybridization solution was added to the membrane. The blot was hybridized at 65 ° C. for 5 hours, and once as follows in 2 × SSC, 0.1% SDS at 65 ° C. for 15 minutes; 0
. Washed twice in 2 × SSC, 0.1% SDS at 65 ° C. for 15-30 minutes. Blots are screened with a film and a PhosphorImager screen (Molecular Dynamics).
cs), Sunnyvale, CA). 6a RADE fragment or 6a. After probing with two cloned fragments, the blot was probed with cDNA homologous to GAPDH as above. 6a
. Hybridization signals from the two fragments were analyzed by PhosphorImager (Molecular Dynamics (Mo
G. (Regular Dynamics), Sunnyvale, CA)
The fold induction was determined by normalizing to the signal of APDH. qRT-PCR clone 6a. except that it contained a 63 bp deletion. MT same as 2
-II fragment was replaced with pcDNA. 3 (Invitrogen
en), Carlsbad, CA). Large Scale Transcription Kit for T7 Promoter (T7 Promoter Large Sca)
le Transcription Kit) (Novagen
), Madison, Wis.). Phenol
RNA synthesized after chloroform extraction and ethanol precipitation was quantified and analyzed using UV spectrophotometry and gel electrophoresis.

Reverse transcription reactions were performed with 200 ng and 300 ng of DNase-treated Saos-2 total RNA containing known amounts of MT-II standard RNA in a 20 μl reaction. Reactions were performed in 1 × PCR buffer (Gibco BRL, Gatorsburg, MD), 3.75 mM MgCl ₂ , 1.25 μM MT-II.
Specific reverse primer (5'-GGAATATAGCAAACGGTCAGG
GTC-3 '), 0.5 mM dNTPs, 1 mM DTT, 20 units of RNasin (Promega) and 200 units of Superscript II reverse transcriptase (Gibco
) BRL, Gatorsburg, MD). The reaction was incubated at 42 ° C. for 15 minutes prior to amplification, then at 99 ° C. for 5 minutes and on ice for 5 minutes.

An MT-II-specific forward primer (5′-GGCTCCTGGCAATG
The PCR was started by adding 80 μl of the master mixture containing CAAAGAG-3 ′) directly to 20 μl of the reverse transcriptase reaction. The final concentrations of the reagents in the 100 μl PCR reaction were as follows: 0.25 μM of each MT-II
Specific primers, 1 × PCR buffer (Gibco BRL, Gatorsburg, MD), 0.1 mM dNTPs, 1.5 mM MgCl ₂ and 0.5 units of Taq DNA polymerase (Gibco BRL),
Gatorsburg, MD). The two-step PCR was performed in PE 9600 (Perkin-Elmer, Norwalk, Conn.) For the following: 40 cycles, such as 95 ° C. for 30 seconds and 64 ° C. for 1.5 minutes. The samples were incubated at 64 ° C. for 10 minutes after amplification.

Reverse phase ion pair high performance liquid chromatography DNA Sep column (Sarasep (
The PCR products were separated and analyzed on a Sarasep (San Jose, CA). The elution system was an acetonitrile gradient in 0.1 M triethylammonium acetate (Fluka, Ronconcoma, NY) at a flow rate of 0.7 ml / min. The acetonitrile gradient was increased from 14.6% to 16.6% over 5 minutes. The amount of product from standard and native RNA was determined by UV absorbance detection at 254 nm, and the signal was analyzed by an on-line integrator. From the chromatogram, the ratio of Saos-2 R
Input MT-II standard RN for amount of natural MT-II message in NA
The ratio of the amounts of A was determined.

The magnitude of MT-II induction by 17-β estradiol in LNCaPLN3 cells is approximately 6-fold (FIG. 4). In Saos-2 cells, 10 nM of 17
β-estradiol up-regulates MT-II mRNA approximately 14-fold (FIG. 4). This up-regulation in Saos-2 cells has been repeated in a minimum of 20 experiments and the range of induction is 3.5-14 fold. The EC _{50 for} this regulation is 4.6 ± 2.7 nM as assessed by qRT-PCR (FIG. 5). When Saos-2 cells are treated with 10 nM 17β-estradiol and RNA is prepared at various times after treatment, the first discernable increase in MT-II message occurs after 8 hours, and a response occurs by 24 hours. The highest point is reached (FIG. 6).

Since the samples prepared for differential display overexpressed hERβL, the regulation of MT-II was evaluated in native Saos-2 cells and those overexpressing ERa. As illustrated in FIG. 7, endogenous levels of ER-β or overexpressed levels of ER-α were ineffective at mediating the regulation of 17β-estradiol MT-II.

Other compounds were tested for their ability to up-regulate MT-II in Saos-2 cells. 10 nanomolar concentration of diethylstilbestrol (
Both Sigma (St. Louis, Mo.) and 0.1 μM genistein (RBI, Natick, Mass.) Increased MT-II mRNA. One micromolar antiestrogen ICI-182780 (Zeneca, Wilmington, Del.) Completely blocked the induction by 17β-estradiol and genistein, but had no effect when given alone ( (FIG. 8). 1 μM antiprogesterone / antiglucocorticoid, RU486 (ligand Pharmaceuticals (Ligand)
Pharmaceuticals, La Jolla, Calif.)
Did not block regulation by 17β-estradiol (data not shown)
. Example 4: Treatment of Saos-2 cells with cycloheximide To determine whether increasing MT-II mRNA requires new protein synthesis, after overexpression of hERβL and before treatment with 17β-estradiol. Translation was stopped using cycloheximide. Confirmation of the Effect of Cycloheximide on Protein Synthesis Cells were plated and infected with the hERβL virus as described above, followed by pretreatment with 10 μg / mL cycloheximide or vehicle for 1 hour at 37 ° C. After this pre-incubation, 10
μg / mL cycloheximide, 10 nM 17-β estradiol and 5
0 μCi / ml ³⁵ S-methionine (NEN, Boston, Mass.) Was added and incubation was continued at 37 ° C. for 8 or 24 hours.
Cells were washed three times in cold PBS and then scraped from the plate in 500 μl of PBS. The cells are pelleted and 200 μl of RIPA buffer (1
Resuspended in 50 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS and 50 mM Tris, pH 8.0). Methionine incorporation was measured by TCA precipitation. 5 and 10 microliters of each sample were spotted on separate Whatman filters. The filters were boiled in 10% trichloroacetic acid for 10 minutes, then washed 3 times for 10 minutes in deionized water, 3 times for 10 minutes in 95% ethanol, and once for 10 minutes in acetone. The dried filters were placed in scintillation fluid and counted for 1 minute. Treatment of cells with 17-β estradiol Cells were plated and infected with the hERβL virus as described above. Cells were pretreated with 10 μg / mL cycloheximide or vehicle at 37 ° C. for 1 hour. After this preincubation, 10 nM 17-β estradiol or vehicle was added and the incubation continued at 37 ° C for 8 or 24 hours. RNA was isolated from cells as described above and gene expression was assessed by Northern blot analysis or qRT-PCR as described in Example 3. Confirmation of functional ER-β protein after cycloheximide treatment Duplicate cultures were prepared and processed as described in the previous section.
After an 8-hour incubation, the cells were washed four times with DMEM to remove 17-β estradiol. Cycloheximide (10 μg / mL) was added to all samples, as was 1 nM [ ³ H] -estradiol. Some cells were co-treated with 0.3 μM diethylstilbestrol to estimate non-specific binding. After a 2.5 hour incubation at 37 ° C., the cells were washed with DMEM and lysed with 0.1% sodium dodecyl sulfate. DPM was measured by liquid scintillation counting.

The MT promoter contains glucocorticoid and metal response elements, but the ERE has not been described. The effect of 17-β estradiol on MT-II mRNA expression may be indirect. After overexpression of ER-β
Aos-2 cells were treated with cycloheximide to severely limit new protein synthesis (FIG. 9A). Comparable amounts of receptor protein were expressed with and without cycloheximide treatment as measured by whole cell binding assay (FIG. 9B), but no induction of MT-II occurred (FIG. 9C, D). Example 5: Treatment of rats with 17-β estradiol 17-β estradiol is MT-I in the prostate cancer cell line LNCaPLN3 cells
To upregulate I, a similar response in the rat prostate was sought.

All animals were processed according to institutional guidelines using approved protocols. Adult (15-19 weeks, about 375 g) castrated Sprague-Dawley
ue-Dawley rats are from Taconic Fas.
rms) (Germantown, NY). Ten days after castration,
Rats were given vehicle, 16 μg 17-β estradiol, 16 μg diethylstilbestrol (Sigma, St. Louis, Mo.) or 16 μg 17-β estradiol and 1.6 mg raloxifene (synthesized in-house) for 3 days. One subcutaneous injection per day. Approximately 24 hours after the last dose, the rats were euthanized by CO ₂ asphyxiation and the prostate was removed. Total RNA was prepared and analyzed for MT-II mRNA as described above. In another experiment, rats were administered 16 μg of 17-β estradiol for 1, 2, or 3 days before collecting prostate tissue.

The metallothionein-II mRNA increased 5-fold after 17-β estradiol treatment. A similar response occurred when rats were administered diethylstilbestrol, but this up-regulation was blocked by co-administration of the estrogen antagonist, raloxifene hydrochloride, with a 100-fold excess (FIG. 10).
. While the first experiment used tissue from rats administered for 3 days, up-regulation of MT-II in rat prostate was first seen 2 days after administration of 17-β estradiol (FIG. 11). Example 6: ERE-luciferase reporter assay using MCF-7 cells. Cell-based assays because the regulation of MT-II in Saos-2 cells is mediated by ER-β (and not ER-α). An additional assay was needed to compare the selectivity of the compounds for these two receptors. MCF-7 is an estrogen-responsive breast cancer cell line, and in our hands, RT-
Expresses only ER-α as measured by PCR (data not shown)
. Reporter gene activity can be measured when MCF-7 cells are transiently infected with an ERE-reporter gene construct and treated with 17-β estradiol.

MCF-7 (HTB 22, ATCC, Manassas, VA) cells were grown in growth medium [10% (v / v) heat-inactivated fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin sulfate, 2 mM Glutamax (Glu
D-MEM / F-12 medium containing (taMAX) -1] twice a week. Cells are maintained inside vented flasks at 37 ° C. inside a 5% CO ₂ /95% humidified air incubator. One day prior to treatment, cells are plated in growth wells at 25,000 cells per well in a 96-well plate and incubated at 37 ° C overnight.

Cells were treated with experimental medium [fetal bovine serum stripped with 10% (v / v) heat-inactivated charcoal, 100 U / ml penicillin, 100 μg / ml streptomycin sulfate, 2 mM glutamax (GlutaMAX). -1 in phenol red-free DMEM / F-12 medium containing 1 mM sodium pyruvate], 50 μl per well, 10-fold diluted adenovirus 5-ERE-TK.
-Infect luciferase (Bodyne et al., 1997) at 37 ° C for 2 hours. The wells are then washed once with 150 μl of experimental medium. Finally, 150 μl vehicle (≦ 0.1 v / v) with 8 well replicates per treatment
v% DMSO) or compounds diluted ≧ 1000-fold in experimental medium at 37 ° C. for 24 hours.

After treatment, 25 μl per well of 1 × cell culture lysis reagent (Promega (Pr
omega), Madison, WI) for 15 minutes on a shaker.
Lyse cells. Cell lysate (20 μl) was applied to a 96-well luminometer (luminometer)
) Transfer to plates and use 100 μl per well of luciferase substrate (Promega, Madison, WI) with a MicroLumat LB 96P luminometer (EG & G Berthold, Bad Wild, Germany) The luciferase activity is measured in birds). A 1 second background measurement is taken for each well before substrate injection. After injection of the substrate, luciferase activity is measured for 10 seconds after a 1 second delay. The average and standard deviation are calculated after subtracting the background subtraction.

Compounds that induce MT-II in Saos-2 cells and do not stimulate reporter gene activity in MCF-7 cells are therefore selective for ER-β. In addition, the results from these two assays can also be used to select compounds that are selective for ER-α, as shown in FIG. Discussion MT-I in two cell lines in which ER-β was treated with 17-β estradiol
Increasing I mRNA was unexpectedly discovered using differential display. To our knowledge, this is the first gene found to be regulated by this new form of ER. This response has been extensively characterized in the human osteosarcoma cell line Saos-2. Various estrogens are MT-
II may be up-regulated and this response is blocked by co-treatment with the estrogen antagonist ICI-182780. The EC ₅₀ of 17-β estradiol is approximately 5 nM, and this response is mediated by ER-β, which is acted on by a previously unknown protein.

The effect of ER-β on MT-II is not a general phenomenon. Investigation of more than a dozen cell lines failed to show this induction in samples other than Saos-2 and LNCaPLN3 cells. However, MT-II
The fact that is regulated by 17-β estradiol in the rat prostate raises the possibility that this is a physiologically relevant response and not an artifact of receptor overexpression in cancer cell lines. We believe this prostate response is mediated by the ER for two reasons. That is, the non-steroidal estrogen (diethylstilbestrol) up-regulates MT-II and the estrogen agonist / antagonist, raloxifene,
-Block the action of β-estradiol. However, at this point it is not clear whether this induction reflects the activity of ER-β and / or ER-α. While ER-β is the predominant form of ER in rat prostate, ER-α is also present. Current research is ongoing to define the type of receptor responsible for this in vivo response.

Metallothionein is a low molecular weight cysteine-rich protein that binds metals such as cadmium, copper and zinc. The first metallothioneins were discovered more than 40 years ago (Vallee, 1957), but controversy continues regarding their function. Several proposals have been made and include protection from metal toxicity, regulation of zinc and copper homeostasis, and protection against oxidative stress. Regulation of energy balance is also relevant. Because MT
(-I and -II) knockout mice become obese after reaching sexual maturity (Beattie et al., 1998).

Recently, studies have detailed how MT can act to regulate zinc homeostasis in cells. Two recent publications using purified zinc-dependent enzymes, such as E. coli alkaline phosphatase, bovine carboxypeptidase A and ovine sorbitol dehydrogenase, disclose that when the agent is MT Shows how it may facilitate exchange between zinc complexed with apoenzymes (metal depleted) (Jacob (Jaco)
b) et al., 1998; Jiang et al., 1998). Citrate and glutathione can influence the direction of zinc movement and thus regulate enzyme activity depending on the redox state of the cell. Although not an enzyme, ER also requires zinc for activity, and ER from MCF-7 cells is capable of reversibly exchanging zinc in vitro with purified MT (Cano-Gauci).
) Et al., 1996).

A myriad of agents, including metals such as glucocorticoids and cadmium, can modulate MT levels (for review, see Hamer).
, 1986). Estrogen is not a classic modulator of MT,
However, two intriguing papers appear in the literature. First, one of the female rats
Treatment with 7-β estradiol for 2 weeks upregulated intestinal mucosal copper binding protein, which reduced the amount of copper absorbed into plasma. Since the molecular weight of this protein was about 10K, the authors suggest it is MT (Cohen et al., 1979). Recently, MT was identified in a subtractive hybridization screen of regulated uterine mRNA following a single injection of 17α ethinylestradiol (Rivera-G.
onzalez et al., 1998). Although no isoform has been identified, 17
MT transcripts increased 3-fold between 4 and 8 hours after -α ethinyl estradiol stimulation. In addition, it is not clear which receptor type achieves this regulation. Because ER-α (but not ER-β) is the most abundant ER in the uterus (Cuiper (K
uiper) et al., 1996; Couse et al., 1997).

Since the function of MT has only begun to be elucidated, and the complete unknown of ER-β, it is not possible at this point to understand the significance of their association. However, even when the relationship between these two proteins is uncertain, E
It is still possible to explore regulatory observations to help design R-β or ER-α specific ligands. The fact that genistein up-regulates MT-II in Saos-2 cells similarly or better than 17β-estradiol indicates that ER-β selective compounds may effectively direct transcription. Show. In addition, several structurally different compounds that bind to ER-β may also up-regulate this message in Saos-2 cells. When data on the ability of a compound to modulate MT-II in Saos-2 cells is combined with information on how it modulates reporter gene activity in MCF-7 cells, ER-β and ER-α Can be evaluated. Thus, these two assays are tools that, when used together, help to design selective compounds of either ER type. Finally, if it is possible to show that regulation in the prostate is also mediated by ER-β, the in vivo activity of the compounds can be assessed, providing another valuable tool for drug discovery. provide.

[0078]

[Table 1]

[0079]

[Table 2]

[0080]

[Table 3]

[0081]

[Table 4]

[0082]

[Table 5]

[0083]

[Table 6]

[0084]

[Table 7]

[Brief description of the drawings]

FIG. 1. RT-PCR amplification of ER-β and ER-α from Saos-2 and LNCaPLN3 cells according to the invention.

FIG. 2. Sequencing gel separating the amplified cDNA and up-regulation of 17-β estradiol of fragment 6a according to the invention.

FIG. 3 (A) Nucleotide sequence of a regulated fragment and human MT-I according to the invention
Its alignment with I.

FIG. 3 (B) Translation of the fragment sequence from the first methionine to the stop codon (*) according to the invention

FIG. 4. Modulation of MT-II in two cell lines according to the invention. To determine the fold change in mRNA, the MT-II signal was normalized to GAPDH signal and compared to control cells.

FIG. 5. Dose response of 17-β estradiol modulation of MT-II in Saos-2 cells according to the invention. The results shown for four different experiments, also shows an EC ₅₀ for each.

FIG. 6: Time course of the regulation of metallothionein-II in Saos-2 cells according to the invention. Data are normalized with GAPDH and fold change is calculated from vehicle control at time = 0 hours.

FIG. 7. Receptor specificity of MT-II regulation in Saos-2 cells according to the invention.

FIG. 8: Modulation of MT-II in Saos-2 cells by various compounds according to the invention.

FIG. 9. Modulation of metallothionein-II in Saos-2 cells treated with cycloheximide according to the invention. (A) Measurement of protein synthesis during treatment with cycloheximide. (B) Whole cell ER binding assay 8 hours after cycloheximide treatment.
(C, D) Modulation of metallothionein-II 8 and 24 hours after treatment, respectively.

FIG. 10. According to the invention, MT is regulated by estrogen in rat prostate.

FIG. 11. Induction of MT-II in rat prostate according to the invention requires a minimum of 2 days of administration.

FIG. 12. Screening strategy for ligands that selectively activate ER-β and / or ER-α according to the invention.

[Procedural Amendment] Submission of translation of Article 34 Amendment

[Submission date] August 26, 2000 (2000.8.26)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0050

[Correction method] Change

[Contents of correction]

The reverse transcription reaction was performed with 0.5 μg of RNA in a 20 μl reaction. For ER-α, the reaction was performed with 1 × PCR buffer (Gibco BRL, Gaithersburg, MD), 5 mM MgCl ₂ , 1.25 μM ERα-specific reverse primer (5′-CCAGCAGCCATGTCGAAGATCC-3 ′). , SEQ ID NO 1), GAPDH-specific reverse primer 0.5μM (5'-CACCCT
GTTGCTGTAGCCAAATTC-3 ′ , SEQ ID NO: 2 ), 0.5 mM d
NTP, 20 units RNasin (RNasin) (Promega)
, Madison, WI) and 200 units of Superscript (Su
perscript II reverse transcriptase (Gibco BRL, Gatorsburg, MD). The ER-β reaction was performed with the following exceptions: 2.5 mM MgCl ₂ and 1.25 μM ERβ-specific reverse primer (
5'-GCAGAGAGTGAGCATCCCTCTTTG-3 ' , SEQ ID NO: 3 )
And contained the same components as ER-α. It is a super script (Sup
erscript) Duplicate reactions that were identical for all reagents except that they did not contain reverse transcriptase were used as negative controls to ensure that RNA samples were not contaminated with DNA. Performed on samples. The reaction was incubated at 42 ° C. for 15 minutes, then at 99 ° C. for 5 minutes and on ice for 5 minutes prior to amplification.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0051

[Correction method] Change

[Contents of correction]

ER-α specific forward primer (5′-GGAGACATGAGAGCTG
CCAAC-3 ′ , SEQ ID NO: 4 ) or an ER-β specific forward primer (5
'-CAGCATCCCAGCAATGTCAC-3' , SEQ ID NO: 5 ) and a GAPDH-specific forward primer (5'-GACATCAAGAAGGTGG
80 μl of the master mixture containing TGAAGCAG-3 ′ (SEQ ID NO: 6 )
PCR was started by adding 0 μl directly to the reverse transcriptase reaction. ER-
The final concentration of the reagents in a 100 μl PCR reaction of α was as follows:
. 25 μM of each ER specific primer, 0.1 μM of each GAPDH primer,
1 × PCR buffer (Gibco BRL, Gatorsburg, MD), 0.2 mM dNTPs, 2 mM MgCl ₂ and 0.5 units of Taq DNA polymerase (Gibco BRL, Gittersburg, MD) )Met. The ER-β reaction contained the same amount of reagent except for 1 mM MgCl ₂ . The two-stage PCR was performed as follows: 95 ° C. for 30 seconds, 64 ° C. 1
. Performed on PE 9600 for 5 minutes, 25 cycles. 64 ° C after amplification
For 10 minutes.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0052

[Correction method] Change

[Contents of correction]

Separate 20 microliters of each sample using a 1.5% agarose gel and Hybond-N + (Amersham Pharmacia) by capillary alkaline southern blotting in 0.4N NaOH, 0.6M NaCl. (Amersham Pharmacia), Piscataway, NJ). Rapid-Hyb buffer (Amersham Pharmacia)
The blots were prehybridized at 42 ° C. for 30 minutes in Piscataway, NJ. Polynucleotide kinase (Gibco BRL)
ER-α (5'-TGAAC) using Gaitersberg, MD.
CAGCTCCCTGTCTGGCAGGTTGGT-3 ′ , SEQ ID NO: 7 ), E
R-β (5′-CCGCATACAGATGTGTATAACTGGCGATGG
A-3 ′ , SEQ ID NO: 8 ) and GAPDH (5′-GCTGTTGAAGTCA)
CAGGAGACAACCTGGT-3 ' , SEQ ID NO: 9 ) An oligonucleotide probe specific to the fragment was end-labeled with ³² P-γATP. 3 probes
. It was added to the blot at 0 × 10 ⁶ CPM / ml and incubated at 42 ° C. for 1 hour. Hybridization of ER and GAPDH was performed independently. The blot was washed once in 2 × SSC, 0.1% SDS at room temperature for 15 minutes, then 0.2 times
Washed twice in × SSC, 0.1% SDS at 42 ° C. for 15 minutes. Thereafter, the blot was exposed to film.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0056

[Correction method] Change

[Contents of correction]

The next day, cells were treated with 10 nM 17β-estradiol or vehicle for 24 hours.
Time treated and total RNA was prepared using TRIzol reagent (Gibco BRL, Gatorsburg, MD) for differential display. To remove residual DNA, the sample was treated with 1 unit / μg RNase-free DNase I (Gibco BR
L, Gatorsburg, MD) at 37 ° C for 30 minutes. RNeasy columns (Qiagen, Hilden, Germany)
RNA was purified from the reaction using and the amount recovered was estimated by UV spectrophotometry. Rapid Analysis of Differential Expression (RADE) Reverse Transcription (RT) After DNase I treatment, 6 micrograms of total RNA was added to a final volume of 600 μL in 1 × RT buffer (25 mM Tris-Cl, pH 8.3). , 37.6 mM KCl, 3 mM MgCl ₂ and 5 mM DTT, Gen Hunter (Gen
hunter, from Nashville, TN), 20 μM dNTPs (dA,
C, G and TTP 2'-deoxynucleoside 5 'triphosphate, Gibco (Gi
bco) from BRL (Gatorsburg, MD) with 0.2 μM HT ₁₁ C (oligonucleotide AAGCTTTTTTTTTTCTC , SEQ ID NO: 15 ). The reaction mixture was incubated at 65 ° C for 5 minutes to denature the secondary structure, then incubated at 37 ° C for 10 minutes. At this point, 30 μl of Superscript II reverse transcriptase (2
00U / μl, Gibco BRL, Gatorsburg, MD) was added to the reaction and the incubation proceeded at 37 ° C for 1 hour. 7
The enzyme was inactivated by heating at 5 ° C. for 5 minutes. An aliquot of this reaction was then used for second strand synthesis by PCR. Polymerase chain reaction (PCR) For 2 μl of RT reaction, 1 × PCR buffer (10 μl for a total reaction volume of 20 μl)
mM Tris-Cl, pH 8.4, 100 mM KCl, 1.5 mM MgCl ₂
And 0.001% gelatin), 2 μM dNTP, 15 nM ³³ P dATP
(NEN, Boston, MA) 1 unit of AmpliTac (Ampl
iTaq) DNA polymerase (Perkin-Elme
r), Norwalk, Conn.) as well as 1 μM of primer 5 ′ AAGCTTGCCATGG-3 ′. The reaction mixture was then heat cycled using the following parameters: 92 ° C for 2 minutes, 1 cycle 92 ° C for 15 seconds, 40 ° C for 2 minutes, 72 ° C for 30 seconds, 40 cycles 72 ° C for 5 minutes. Gel electrophoresis Duplicate samples of the PCR products were run in 1 × TBE buffer (0.1 M Tris, 0.0
6% denaturing polyacrylamide gel (5.7% acrylamide, 0.3% bisacrylamide, 42% at 2000 volts for 3 hours in 9M boric acid, 1 mM EDTA)
% Urea and 51% H ₂ O). The gel was then filtered through filter paper (Schleicher & Schu).
ell), Keene, NH), dried under vacuum at 80 ° C. for 1 hour, and exposed to X-ray film for 24 hours.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0060

[Correction method] Change

[Contents of correction]

Reverse transcription reactions were performed with 200 ng and 300 ng of DNase-treated Saos-2 total RNA containing known amounts of MT-II standard RNA in a 20 μl reaction. Reactions were performed in 1 × PCR buffer (Gibco BRL, Gatorsburg, MD), 3.75 mM MgCl ₂ , 1.25 μM MT-II.
Specific reverse primer (5'-GGAATATAGCAAACGGTCAGG
GTC-3 ′ , SEQ ID NO: 10 ), 0.5 mM dNTP, 1 mM DTT, 20
Units of RNasin (Promega) and 20
Contains 0 units of Superscript II reverse transcriptase (Gibco BRL, Gatorsburg, MD). The reaction was incubated at 42 ° C. for 15 minutes prior to amplification, then at 99 ° C. for 5 minutes and on ice for 5 minutes.

[Procedure amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0061

[Correction method] Change

[Contents of correction]

An MT-II-specific forward primer (5′-GGCTCCTGGCAATG
The PCR was started by directly adding 80 μl of the master mixture containing CAAAGAG-3 ′ , SEQ ID NO: 11 ) to a 20 μl reverse transcriptase reaction. 100
The final concentration of reagents in the μl PCR reaction was as follows: 0.25 μM
Of each MT-II specific primer, 1 × PCR buffer (Gibco B
RL, Gatorsburg, Md., 0.1 mM dNTPs, 1.5 mM MgCl ₂ and 0.5 units of Taq DNA polymerase (Gibco
co) BRL, Gatorsburg, MD). PE 9600 (
The two-step PCR was performed in Perkin-Elmer (Norwalk, Conn.) For the following 40 cycles: 95 ° C. for 30 seconds, 64 ° C. for 1.5 minutes. The samples were incubated at 64 ° C. for 10 minutes after amplification.

[Procedure amendment 7]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Change

[Contents of correction]

[Brief description of the drawings]

FIG. 1. RT-PCR amplification of ER-β and ER-α from Saos-2 and LNCaPLN3 cells according to the invention.

FIG. 2. Sequencing gel separating the amplified cDNA and up-regulation of 17-β estradiol of fragment 6a according to the invention.

FIG. 3 (A) Nucleotide sequence of a regulated fragment according to the invention (SEQ ID NO: 12) and its alignment with human MT-II (SEQ ID NO: 13) .

FIG. 3 (B): Fragment sequence from the first methionine to the stop codon (*) according to the present invention
(SEQ ID NO: 14)Translation of

FIG. 4. Modulation of MT-II in two cell lines according to the invention. To determine the fold change in mRNA, the MT-II signal was normalized to GAPDH signal and compared to control cells.

FIG. 5. Dose response of 17-β estradiol modulation of MT-II in Saos-2 cells according to the invention. The results shown for four different experiments, also shows an EC ₅₀ for each.

FIG. 6: Time course of the regulation of metallothionein-II in Saos-2 cells according to the invention. Data are normalized with GAPDH and fold change is calculated from vehicle control at time = 0 hours.

FIG. 7. Receptor specificity of MT-II regulation in Saos-2 cells according to the invention.

FIG. 8: Modulation of MT-II in Saos-2 cells by various compounds according to the invention.

FIG. 9. Modulation of metallothionein-II in Saos-2 cells treated with cycloheximide according to the invention. (A) Measurement of protein synthesis during treatment with cycloheximide. (B) Whole cell ER binding assay 8 hours after cycloheximide treatment.
(C, D) Modulation of metallothionein-II 8 and 24 hours after treatment, respectively.

FIG. 10. According to the invention, MT is regulated by estrogen in rat prostate.

FIG. 11. Induction of MT-II in rat prostate according to the invention requires a minimum of 2 days of administration.

FIG. 12. Screening strategy for ligands that selectively activate ER-β and / or ER-α according to the invention.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference) 4B024 AA11 BA63 CA04 DA02 DA03 EA02 FA02 FA10 GA11 HA01 HA11 4B063 QA01 QA18 QQ79 QR32 QR55 QR77 QS34 QX02

Claims (32)

[Claims] 1. A method for screening a test compound that binds to the ER in a receptor binding assay, said method comprising detecting ER-β polypeptide mediated transcription, and Providing a cell comprising one estrogen-regulated DNA sequence and at least one DNA sequence encoding an ER-β polypeptide, wherein the receptor is transcriptionally active; (B) contacting the cells with either the test compound or a control that binds ER; and (c) detecting the expression of the MT-II, wherein the MT-II relative to a control is detected.
Elevated expression of II indicates that the test compound has estrogen agonist activity). 2. The method of claim 1, wherein the DNA sequence encoding the ER-β polypeptide has been incorporated into an adenovirus. 3. The adenovirus is a replication-defective Ad5 virus.
The described method. 4. The method according to claim 1, wherein the cells are ER-β mR faster than ER-α mRNA.
2. The method of claim 1, wherein the NA is expressed endogenously. 5. The cell is transformed with a recombinant DNA plasmid comprising a polynucleotide encoding human ER-β operably linked to a suitable promoter, and the transformed cell is transformed. 5. The method of claim 4, wherein said cell expresses human ER- [beta] at a higher level than non-expressed cells. 6. The method of claim 1, wherein said cells are Saos-2 or LNCaPLN3 cells. 7. The method of claim 1, wherein said cells do not have functional ER-α. 8. A method of screening for a test compound that binds to the ER in a receptor binding assay, wherein the method detects inhibition of ER-β polypeptide-mediated transcription, and (a) encodes MT-II. At least one estrogen DNA sequence and ER-
providing a cell comprising at least one DNA sequence encoding a β polypeptide, wherein the receptor is transcriptionally active; (b) binding the cell to the ER. Contacting with one or more estrogens in the presence of a known test compound; and (c) detecting the expression of said MT-II (wherein with respect to the addition of one or more estrogens alone, Reduced expression of MT-II indicates that the test compound has estrogen antagonist activity). 9. The method of claim 8, wherein the DNA encoding the ER-β polypeptide is integrated into an adenovirus. 10. The method of claim 9, wherein the adenovirus is a replication-defective Ad5 virus. 11. The method according to claim 10, wherein said cells are ER-β mRNA faster than ER-α mRNA.
9. The method of claim 8, wherein the RNA is expressed endogenously. 12. The cell is transformed with a recombinant DNA plasmid comprising a polynucleotide encoding human ER-β operably linked to a suitable promoter, and the transformed cell is transformed. 12. The method of claim 11, wherein the cell expresses human ER- [beta] at a higher level than non-expressed cells. 13. The method of claim 12, wherein said cells are Saos-2 or LNCaPLN3 cells. 14. The method of claim 13, wherein said cells do not have functional ER-α. 15. A method for screening test compounds to identify drug candidates that mimic the effects of estrogen on ER-β or ER-α mediated transcription, comprising: (a) a plurality of: (i) MT A first cell comprising at least one endogenous DNA sequence encoding -II and at least one DNA sequence encoding an ER-β polypeptide, wherein
Contacting said test compound with a second cell comprising said ERE reporter gene construct, wherein said cell expresses an ER-α polypeptide; and said receptor is transcriptionally active. (B) identifying a compound that increases the expression of MT-II in said first cell relative to a control but has minimal effect on expression of said reporter gene in said second cell ( Wherein the compound is considered ER-β selective);
Or (c) identifying a compound that increases the expression of the reporter gene in said second cell relative to the control but has minimal effect on the expression of MT-II in said first cell, wherein Wherein the compound is considered to be ER-α selective. 16. The DN encoding the first cell ER-β polypeptide.
16. The method of claim 15, wherein A is incorporated into an adenovirus. 17. The method of claim 16, wherein the adenovirus is a replication-defective Ad5 virus. 18. The method according to claim 18, wherein the first cell is ER-α at a higher rate than ER-α mRNA.
16. The method of claim 15, wherein the β mRNA is expressed endogenously. 19. The first cell is transformed with a recombinant DNA plasmid comprising a polynucleotide encoding human ER-β operably linked to a suitable promoter. 16. The method of claim 15, wherein said cells express human ER-beta at a higher level than said second cells. 20. The method according to claim 19, wherein the first cell is Saos-2 or LNCaPLN3.
16. The method of claim 15, which is a cell. 21. The method of claim 15, wherein said first cell does not have functional ER-α and said second cell does not have functional ER-β. 22. The method according to claim 15, wherein the second cell has been transformed with a DNA polynucleotide comprising an ERE operably linked to a reporter gene.
The described method. 23. The method according to claim 22, wherein said reporter gene is a luciferase gene. 24. A method for screening a test compound for identifying a drug candidate that inhibits the effect of estrogen on ER-β or ER-α-mediated transcription, comprising: (a) using one type of the test compound; (I) at least one endogenous D encoding the metallothionein (MT-II) gene in the presence and absence of or more estrogen;
A first cell comprising an NA sequence and at least one DNA sequence encoding an ER-β polypeptide, wherein the receptor is transcriptionally active; and (ii) an ERE reporter gene construct. Contacting with a second cell comprising said ER-α polypeptide; and (b) treating said first cell with respect to treatment with one or more estrogens. Identifying a compound that reduces the expression of MT-II but has minimal effect on expression of the reporter gene in the second cell, wherein the compound is considered to be ER-β selective Or (c) reducing the expression of a reporter gene in said second cell with respect to treatment with one or more estrogens but not in said first cell. MT-II
Identifying a compound that has minimal effect on the expression of said compound, wherein said compound is considered to be ER-α selective. 25. A DN encoding the first cell ER-β polypeptide.
25. The method of claim 24, wherein A is incorporated into an adenovirus. 26. The method of claim 25, wherein the adenovirus is a replication-defective Ad5 virus. 27. The method according to claim 27, wherein the first cell is faster than ER-α mRNA.
25. The method of claim 24, wherein the β mRNA is expressed endogenously. 28. The first cell, wherein the first cell is transformed with a recombinant DNA plasmid comprising a polynucleotide encoding human ER-β operably linked to a suitable promoter. 25. The method of claim 24, wherein said cells express human ER- [beta] at higher levels than said second cells. 29. The method according to claim 29, wherein the first cell is Saos-2 or LNCaPLN3.
26. The method of claim 24, wherein the method is a cell. 30. The method of claim 24, wherein said first cell has no functional ER-α and said second cell has no functional ER-β. 31. The second cell has been transformed with a DNA polynucleotide comprising an ERE operably linked to a reporter gene.
The described method. 32. The method according to claim 31, wherein said reporter gene is a luciferase gene.