EP1141405A1 - Bioassay for identifying estrogen receptor-beta/alpha selective modulators - Google Patents
Bioassay for identifying estrogen receptor-beta/alpha selective modulatorsInfo
- Publication number
- EP1141405A1 EP1141405A1 EP99966310A EP99966310A EP1141405A1 EP 1141405 A1 EP1141405 A1 EP 1141405A1 EP 99966310 A EP99966310 A EP 99966310A EP 99966310 A EP99966310 A EP 99966310A EP 1141405 A1 EP1141405 A1 EP 1141405A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- expression
- estrogen
- polypeptide
- compounds
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
- G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the present invention relates to hormone receptors, and, more particularly to methods for identifying compounds that selectively activate estrogen receptors (ER) of the alpha or beta subtype, as well as a test kit for use in the methods.
- ER estrogen receptors
- Estrogens are classically known as important hormones in sexual development and reproductive function. It is well known that estrogens affect cell proliferation and differentiation in target tissues by binding to ERs 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) because these compounds have been demonstrated to prevent bone loss and fractures in women. Additionally, estrogen replacement therapy has been associated with a decreased mortality from cardiovascular disease. Finally, ongoing studies suggest that estrogens may provide benefit to the central nervous system with regard to cognitive improvement and a decrease in Alzheimer's disease.
- the classic ER now designated ER- ⁇ , exhibits a modular structure
- the in vitro translated protein specifically bound [ 3 H]-estradiol with nanomolar affinity and was able to regulate transcription from a simple estrogen response element.
- This protein has been designated ER- ⁇ to
- ER- ⁇ mRNA is detectable by RT-PCR and in situ hybridization in a
- ER- ⁇ mRNA is expressed in the epithelial cells of the rat prostate; ER- ⁇ is found in the stromal compartment (Kuiper et al, 1996). In the rat ovary, ER- ⁇ appears in the granulosa cells; ER- ⁇ in the stroma (Shughrue et al, 1996; personal communication).
- ER- ⁇ In the rat hypothalamus, ER- ⁇ but not ER- ⁇ , message is expressed in the paraventricular region, whereas both messages are seen in the preoptic area (Shughrue et al, 1996). Interestingly, significant species differences may exist in the relative levels of ER- ⁇ and ER- ⁇ mRNA in certain organs. For example, ER- ⁇ mRNA is highly expressed in the rat prostate, whereas more modest levels are detected in the human prostate. ER- ⁇ predominates over ER- ⁇ in the rat prostate (Kuiper et al, 1 996; Lau et al, 1998; Enmark et al, 1997), but the levels are more equal in the mouse prostate (Couse et al, 1997).
- MCF-7 cells (Enmark, 1 997; Kuiper et al, 1 997; Sicilusic et al, 1 998; R. Henderson, unpublished observations).
- Genistein is a phytoestrogen which
- estrogen receptor- ⁇ knockout (ERKO) mouse (Lubahn et al, 1 993).
- mice lack functional ER- ⁇ , they can help define the
- the present invention provides method for screening a test compound that binds to an ER in a receptor binding assay, wherein said method detects ER- ⁇ polypeptide-mediated transcription, said method
- This invention further provides a method for screening a test compound that binds to the ER in a receptor binding assay, wherein said method detects inhibition of ER- ⁇ )- polypeptide-mediated transcription,
- said method comprising the steps of:
- said method comprises the steps of:
- receptor is transcriptionally active and (ii) second cells comprising an ERE reporter gene construct, wherein said cells express ER- ⁇ polypeptide;
- the present invention provides a method of screening test compounds to identify drug candidates which inhibit estrogen's effect on ER- ⁇ - or ER- ⁇ -mediated transcription, said method comprising the steps
- first cells comprising at least one endogenous DNA encoding a metallothionein (MT-II) gene and DNA encoding a ER- ⁇ polypeptide, wherein said receptor is transcriptionally active and
- Figure 1 RT-PCR amplification of ER- ⁇ and ER- ⁇ ⁇ from Saos -2 and
- Figure 2 Sequencing gel separating amplified cDNAs and 1 7- ⁇ estradiol
- Figure 3 (A) Nucleotide sequence of the regulated fragment and its alignment with human MT-II . (B) Translation of fragment sequence from first methionine to stop codon ( *)
- Figure 4 Regulation of MT-II in two cell lines. To determine fold change in mRNA, MT-II signal was normalized to that of GAPDH, and compared to the control cells.
- Figure 7 Receptor specificity of MT-II regulation in Saos-2 cells.
- Figure 8 Regulation of MT-II in Saos-2 cells by various compounds.
- Figure 9 Metallothionein-ll regulation in cycloheximide-treated Saos-2 cells.
- A Measurement of protein synthesis during treatment with cycloheximide.
- B Whole cell ER binding assay after 8 hours of cycloheximide treatment.
- C,D Metallothionein-ll regulation after 8 and 24 hours of treatment respectively.
- Figure 10 MT is regulated by estrogens in the rat prostate.
- Figure 1 1 Induction of MT-II in the rat prostate requires at least two days of dosing.
- Figure 1 2 Screening strategy for ligands which selectively activate ER- ⁇ and/or ER- ⁇ .
- the present invention provides an efficient way to screen large numbers of test compounds which selectively activate ERs of the ⁇ or ⁇
- estrogens may have desirable properties for either the treatment or the prevention of various diseases mediated by estrogens, including but not limited to cancers (e.g. breast, ovarian, endometrial, prostate), endometriosis, osteoporosis and cardiovascular and central nervous system diseases.
- cancers e.g. breast, ovarian, endometrial, prostate
- endometriosis e.g., endometrial, prostate
- osteoporosis e.g., osteoporosis
- cardiovascular and central nervous system diseases e.g., central nervous system diseases.
- hER ⁇ L is defined as the human form of ER- ⁇ 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- ⁇ estradiol as measured in a standard assay for estrogenic
- Estrogen antagonist is defined as a compound that substantially inhibits the effect of estrogen agonists as measured in a standard assay for estrogenic activity, for example, cellular as described in Webb et al. ( 1 992)
- a functional ER is defined as a receptor capable of transcriptional activation of endogenous or transfected genes as measured by changes in RNA, protein and/or downstream biological events.
- a nonfunctional ER is defined as a receptor incapable of transcriptional activation of endogenous or transfected genes as measured by changes in RNA, protein and/or downstream biological events.
- 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.
- reporter is defined as any substance that can be readily measured and distinguished from other cellular components.
- the reporter may be the transfected receptor DNA, the transcribed receptor mRNA, an enzyme, a binding protein or an antigen.
- a “cell” useful for the present purpose is one which has the ability to respond to signal transduction through a given receptor.
- a “receptor binding assay” is an assay measuring the amount of ligand specifically interacting with a receptor.
- the ligand can be a radioligand (e.g. conjugated to 3 H or 125 l), a fluorescinated ligand (either conjugated to a fluorochrome or possessing inherent fluorescence) or otherwise labeled so as to be detectable. Description of the Assay
- the present invention relies on the discovery that the expression of MT-II is selectively regulated by the interaction of a ligand with ER- ⁇ . .
- the regulation of MT-II activity may be used to provide a screening system that selectively detects both estrogen agonist or antagonist functional activity of a ligand following its interaction with ER- ⁇ .
- the methods typically comprise cultured cells that express functional human ER- ⁇ and no, or a diminished amount of ER- ⁇ .
- Such cells include but are not limited to Saos-2 (ATCC HTB-85) and LNCaPLN3.
- Preferred cells for this purpose include cells which over- express ER- ⁇ , such as the cells described below which are recombinantly
- the ER- ⁇ receptor may be modified in
- MT-II mRNA is measured.
- An estrogen agonist will increase MT-II mRNA when compared to treatment with the control solution alone.
- cells expressing ER- ⁇ are exposed to one or more estrogens in the
- the cells can be exposed while either growing in separate wells of a multi-well culture dish or in a semi- solid nutrient matrix. After treatment for a suitable period of time, MT-II mRNA is measured. An estrogen antagonist will decrease MT-II mRNA when compared to treatment with the estrogen solution alone.
- Estrogenic or antiestrogenic 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 any other application in which the modulation of estrogen activity is desired.
- 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 unit dosage forms depending upon the method of administration.
- unit dosage forms suitable for oral administration include powder, tablets, pills, and capsules.
- Suitable pharmaceutical formulations for use in the present invention arefound in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 1 7th ed. ( 1 985).
- a variety of pharmaceutical compositions comprising compounds of the present invention and pharmaceutically effective carriers can be prepared.
- compositions of the present invention can be used to identify compounds that interact with ER- ⁇ , either to stimulate or to
- Such compounds include, without limitation, co-activator proteins, as well as estrogens and other steroids, steroid-like molecules, or non-steroid-like molecules that act as agonists or antagonists. Screening methods can also be used to identify tissue-specific estrogens. Identification of ER- ⁇ -interactive compounds can be achieved by
- purified ER- ⁇ is
- a labeled ligand such as, e.g., 1 7- ⁇ estradiol
- the labeled moiety may comprise a radiolabel (such as, e.g., 3 H or 125 l) or a fluorescent molecule. Incubation is allowed to proceed for a sufficient time and under appropriate conditions to achieve specific binding, after which binding of labeled ligand to ER- ⁇ is measured (by monitoring, e.g., radioactivity, fluorescence, or
- the ligand binding domain of ER- ⁇ produced in E. coli is adsorbed to the wells of a microtiter dish and
- soluble receptor is incubated with the labeled ligand in the absence or presence of test compounds, and bound ligand is separated from free ligand, either by filtration on glass fiber filters or by using dextran-coated charcoal
- Whole cell binding assays may also be used in which bound ligand is separated from free ligand by rinsing. Cells used in these assays may either contain endogenous receptor, or may overexpress the receptor subsequent to stable or transient transfection or infection of an ER- ⁇ cDNA.
- suitable cells include COS cells, HeLa cells, CHO cells, human umbilical vein endothelial cellsand yeast.
- ER- ⁇ -interactive compounds may also be identified using cell-based
- agonists such as, e.g., 17 ⁇ -estradiol
- Antagonists (such as, e.g., ICI-1 82780) block the agonist
- endogenous estrogen-responsive promoter elements include those that regulate endothelin-1 (ET-1 ); HDL receptor (scavenger receptor type II); and enzymes involved in coagulation and fibrinolysis (such as, e.g., plasminogen activator inhibitor- 1 and complement C3).
- Any promoter element that responds to estrogen may be used as an appropriate target, including, e.g., the NFkB binding site or the apolipoprotein A1 gene enhancer sequence.
- appropriate host cells are transfected with an expression vector encoding ER- ⁇ and the transfectants are incubated with or without one or more estrogens I in the presence or absence of test compounds.
- ER- ⁇ activity is assessed by measuring transcriptional activation. This may be achieved by detection of mRNA (using, e.g., Northern blot analysis, reverse transcriptase polymerase chain reaction, RNase protection assays) and/or by detection of the protein
- ER- ⁇ -interactive compounds are identified as those that positively or negatively influence target sequence activation.
- appropriate host cells are co-transfected with an expression vector encoding ER- ⁇ and a reporter plasmid containing a reporter gene downstream of one or more ERE s.
- Transfected cells are incubated with or without an estrogen in the presence or absence of test compounds, after which ER- ⁇ activity is determined by measuring expression of the reporter gene.
- ER- ⁇ activity is monitored visually.
- suitable reporter genes include luciferase, chloramphenicol acetyl transferase (CAT), and green fluorescence protein.
- the methods of the present invention are adapted to a high-throughput screen, allowing a multiplicity of compounds to be tested in a single assay.
- Candidate estrogens and estrogen-like compounds include without limitation diethylstilbesterol, genistein, and estrone.
- interactive compounds may be found in, for example, natural product libraries, fermentation libraries (encompassing plants and microorganisms), combinatorial libraries, compound files, and synthetic compound libraries.
- synthetic compound libraries 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 library is available from Aldrich Chemical Company, Inc. (Milwaukee, Wl).
- libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from, for example, Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily produced .
- natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., 1 996).
- methods of the present invention may be modified to enhance potency, efficacy, uptake, stability, and suitability for use in therapeutic applications, etc. These modifications are achieved and tested using methods well-known in the art.
- human testis Poly A + RNA (1 ⁇ g, Ciontech, Palo Alto CA) was mixed with 0.5 ⁇ g oligo dT primer (GIBCO-BRL, Gaithersburg MD) in a total volume of 10 ⁇ l.
- the mixture was heated at 70°C for 10 minutes, and, after cooling on ice, was supplemented with 500 ⁇ M of each deoxynucleoside triphosphate, 1 X cDNA synthesis buffer, and 10 mM DTT to a final reaction volume of 20 ⁇ l.
- the mixture was incubated at 42°C for
- PCR primers designated in Serial No. 08/906,365 were used to amplify ER- ⁇ sequences in a reaction containing the following
- reaction mixture was heated to 95 °C for 2 minutes, annealed at 52 °C for 1 minute, and amplified using 36 cycles, followed by an incubation at 72°C for 1 .5 minutes.
- a fragment of approximately 1 500 bp in length was produced.
- the fragment digested with Hindlll and Xbal (which cleave at sites present in the forward and reverse 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).
- Hindlll and Xbal which cleave at sites present in the forward and reverse primer sequences, respectively, but not in the main body of the amplified cDNA sequence
- This asymmetric cloning strategy places the 5' end of ER- ⁇ cDNA under the
- the reaction contained 1 X Klentaq PCR reaction buffer (40 mM Tricine-KOH, 1 5 mM KOAc, 3.5 mM Mg(OAc)2, 75 ⁇ g/ml bovine serum albumin); 0.2 ⁇ M of each dNTP; 0.2 ⁇ M
- Touchdown PCR 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.
- the hER ⁇ L sequence contained an optimal translation initiation sequence
- CCACC immediately upstream to the initiation codon and the sequence was under the control of the cytomegalovirus IE promoter.
- the coding sequence of hER ⁇ L was then transferred into an Ad5 ⁇ E1 a vector plasmid
- plasmid contained cytomegalovirus 1 E promoter, Ad5 tripartite leader, coding sequence of hER ⁇ L and SV40 late polyadenylation signal
- Ad5 virus with E3 region deletion 80-88 map units
- 293 cells transformed primary human embryonic kidney, ATCC CRL 1 573.
- Viral plaques generated by homologous recombination were isolated, amplified and characterized by restriction DNA analysis and cell lysis assay in A549 cells (human lung carcinoma, ATCC CCL 185). Confirmatory tests indicated that the recombinant Ad5 hER ⁇ L virus contained the expected DNA fragments and
- cell culture reagents were obtained from Gibco BRL (Gaithersburg MD).
- LNCaPLN3 cells were grown in RPMI 1 640 medium supplemented with 10% FBS, 2 mM GlutaMAX-1 , 100 U/ml penicillin g, and 100 //g/ml streptomycin sulfate.
- Saos-2 cells ATCC, Manassas VA
- McCoy's 5A medium supplemented with 1 0% fetal bovine serum (FBS), 2 mM GlutaMAX-1 , 100 U/ml penicillin g, 100 g/ml streptomycin sulfate.
- FBS fetal bovine serum
- Reverse transcription reactions were performed on 0.5 ⁇ g of RNA in a 20 ⁇ reaction.
- the reaction contained 1 x PCR Buffer (Gibco).
- ER- ⁇ reaction contained the same components as ER- ⁇ with the following
- PCR was initiated by adding 80/71 of master mix containing ER- ⁇ -specific forward primer (5'-GGAGACATGAGAGCTGCCAAC-3') or ER- ⁇ - specific forward primer (5'-CAGCATTCCCAGCAATGTCAC-3') and GAPDH-specific forward primer (5'-GACATCAAGAAGGTGGTGAAGCAG- 3') directly to the 20 /I reverse transcriptase reaction.
- ER- ⁇ -specific forward primer 5'-GGAGACATGAGAGCTGCCAAC-3'
- ER- ⁇ -specific forward primer 5'-CAGCATTCCCAGCAATGTCAC-3'
- GAPDH-specific forward primer 5'-GACATCAAGAAGGTGGTGAAGCAG- 3'
- each ER-specific primer 0.25 ⁇ M each ER-specific primer, 0.1 /M each GAPDH primer, 1 x PCR Buffer (Gibco BRL, Gaithersburg MD), 0.2 mM dNTPs, 2 mM MgCl2 and
- PCR PCR PCR was carried out in a PE 9600 for 25 cycles as follows: 95°C for 30 sec, 64°C for 1 .5 min. Samples were incubated at 64°C for 10 min after amplification.
- Probes were added to the blot at 3.0 x 10 6 CPM/ml and incubated at 42°C for 1 hour. ER and GAPDH hybridizations were
- Saos-2 cells expressed endogenous ER- ⁇ but not ER- ⁇ mRNA when
- LNCaPLN3 and Saos-2 cells were cultured as described above. Sixteen hours prior to infection, the cells were plated in phenol red-free RPMI 1 640 medium supplemented with 10% charcoal/dextran-treated ("stripped") FBS (HyClone, Logan UT), 2 mM GlutaMAX-1 , 100 U/ml penicillin g, 100 ⁇ g/ml streptomycin sulfate. This medium was used for the remainder of the experiment.
- Example 1 using 2% stripped FBS phenol red-free medium with antibiotics and GlutaMAX-1 for 2 hours at 37° C. Medium containing virus was aspirated and the cells were washed with medium. Fresh medium was added and the cells allowed to recover overnight at 37°C.
- RNA was purified from the reaction using RNeasy columns (Qiagen, Hilden Germany) and amount recovered estimated by UV spectrophotometry. Rapid Analysis of Differential Expression (RAPE) Reverse transcription (RT)
- transcriptase (200U/ /I, Gibco BRL, Gaithersburg MD) was added to the reaction and incubation proceeded for 1 hr at 37°C.
- the enzyme was
- reaction 2 ⁇ l was used in a PCR reaction of 20 mM Tris-HCI, pH 8, 50 mM KCI, 2.5 mM MgCI 2 , 75 ⁇ M dNTPs, 375 nM M-1 3 forward and reverse primers and 2.5 U of Taq polymerase (Gibco BRL, Gaithersburg MD). The reactions were cycled as follows: 95°C for 30 sec; 64°C for 30 sec;
- Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase using the recommended protocol from Applied Biosystems (Foster City CA). Spin columns (AGTC) were employed to remove unincorporated dye-labeled nucleotides after cycle sequencing. Automated DNA sequencing grade 4.75 % polyacrylamide gels were run for all the DNA sequencing samples using ABI 373 DNA sequencers. Sequencing data was edited using Sequence Navigator and assembled using DNAStar (DNAStar, Madison Wl). The sequence was trimmed of RADE primers and used in a BLAST search using Millennium Software (Boston MA). A nucleotide homology search of clone 6a.2 sequence revealed a 98% identity with human MT-II-. Over the coding sequence, however, there were no amino acid mismatches (Figure 3) Confirmation of regulation using Northern blots and Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR):
- mediated overexpression of ER- ⁇ are identical to those described for ER- ⁇ .
- Hyb solution (Amersham Pharmacia, Piscataway NJ) for 30 minutes.
- the insert from clone 6a.2 was isolated from the plasmid using PCR as described above for colony screen.
- the PCR product was purified from an agarose gel using Wizard Preps (Promega, Madison Wl).
- Re- amplified RADE fragments for band 6a or fragment from clone 6a.2 were random primer labeled using Redi-prime kit (Amersham Pharmacia, Piscataway NJ) according to manufacturers instructions. Unincorporated nucleotides were removed using a Nap-5 column (Amersham Pharmacia, Piscataway NJ) and incorporation of [ 32 P]-dCTP measured by liquid scintillation counting.
- the probes were denatured at 100°C for 10 min and 1 .5 x 10 6
- Reverse transcription reactions were performed on 200 ng and 300 ng of DNased Saos-2 total RNA plus a known amount of MT-II standard RNA in a 20 ⁇ l reaction.
- the reaction contained 1 x PCR Buffer (Gibco BRL, Gaithersburg MD), 3.75 mM MgCl2, 1 .25 ⁇ M MT-ll-specific reverse
- primer (5'-GGAATATAGCAAACGGTCAGGGTC-3'), 0.5 mM dNTPs, 1 mM DTT, 20 units of RNasin (Promega) and 200 units of Superscript II Reverse Transcriptase (Gibco BRL, Gaithersburg MD). Reactions were incubated at 42°C for 1 5 minutes followed by 5 minutes at 99°C and 5 minutes on ice prior to amplification PCR was initiated by adding 80 ⁇ l of master mix containing MT-II specific forward primer (5'-GGCTCCTGCAAATGCAAAGAG-3') directly to the 20 ⁇ l reverse transcriptase reaction.
- MT-II specific forward primer 5'-GGCTCCTGCAAATGCAAAGAG-3'
- PCR products were separated and analyzed on a reverse-phase ion- pair high performance liquid chromatography DNASep column (Sarasep, San Jose CA).
- the elution system was a gradient of acetonitrile in 0.1 M triethylammonium acetate (Fluka, Ronkonkoma NY) at a flow rate of 0.7 ml/min. The acetonitrile gradient increased from 14.6% to 1 6.6% over 5 minutes.
- the amount of product from the standard and the native RNA was determined by UV absorbance detection at 254 nm and signal was analyzed by an on-line integrator. From the chromatograms, the ratio of the area under each peak was used to determine the ratio of the amount of input MT-II standard RNA to the amount of native MT-II message in the Saos-2 RNA.
- estradiol is approximately 6 fold (Figure 4). In Saos-2 cells, 10 nM 17 ⁇ -
- methionine-deficient media was added and incubation continued at 37°C
- RIPA buffer 150 mM NaCI, 1 % NP-40, 0.5% DOC, 0.1 % SDS, and
- the MT promoter contains glucocorticoid and metal response elements, but EREs have not been described. It is possible the effect of 17- ⁇ estradiol on MT-II mRNA expression is indirect.
- prostate cancer cell line we looked for a similar response in the rat prostate.
- estradiol 16 ⁇ g diethylstilbesterol (Sigma, St. Louis MO), or 16 ⁇ g 17- ⁇ estradiol plus 1.6 mg raloxifene (synthesized in-house) subcutaneously once per day for three days.
- rats were euthanized by C0 2 asphyxiation and the prostate excised.
- Total RNA was prepared and analyzed for MT-II mRNA as described above.
- rats were dosed for 1 , 2 or 3 days with 16 ⁇ g 17- ⁇ estradiol before prostate tissue was collected.
- Metallothionein-ll mRNA increased five-fold after 17- ⁇ estradiol
- MCF-7 is an estrogen- responsive breast cancer cell line and, in our hands, expresses only ER- ⁇
- MCF-7 HTB 22, ATCC , Manassas VA cells are passaged twice a week with growth medium [D-MEM/F-1 2 medium containing 10% (v/v) heat-inactivated fetal bovine serum 1 00 U/ml penicillin g, 1 00 ⁇ g/ml
- the cells are plated with growth medium at
- the cells are infected for 2 hr at 37°C with 50 ⁇ l/well of a 1 : 10
- lysates (20 ⁇ l) are transferred to a 96 well luminometer plate, and luciferase activity is measured in a MicroLumat LB 96 P luminometer (EG & G Berthold, Bad Wildbad Germany) using 100 ⁇ l/well of luciferase substrate (Promega, Madison Wl). Prior to the injection of substrate, a 1 second background measurement is made for each well. Following the injection of substrate, luciferase activity is measured for 10 seconds after a 1 second delay. After subtracting background subtracts the mean and standard deviation are calculated.
- ER- ⁇ increases MT-II mRNA in two cell lines treated with 17- ⁇ estradiol. To our knowledge, this is the first gene discovered to be regulated by this new form of the 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 cotreatment with the estrogen antagonist ICI-182780.
- the EC 50 for 17- ⁇ estradiol is approximately 5 nM and this response is mediated by ER- ⁇ acting through as yet unknown proteins.
- the action of ER- ⁇ on MT-II is not a general phenomenon as a
- a nonsteroidal estrogen (diethylstilbesterol) upregulates MT-II and raloxifene, an estrogen agonist/antagonist, blocks 1 7- ⁇ estradiol's action.
- ER- ⁇ is the predominant form of the ER in the rat prostate, ER- ⁇
- Metallothioneins are low molecular weight, cysteine rich proteins that bind metals such as cadmium, copper and zinc. Although the first metallothionein was discovered over forty years ago (Vallee, 1 957), debate continues as to their function. Several proposals have been made and these include protection form metal toxicity, regulation of zinc and copper homeostasis and defense against oxidative stress. Regulation of energy balance has also been implicated because, after reaching sexual maturity, MT (-I and -II) knockout mice become obese (Beattie et al, 1 998). Recently, studies have detailed how MT may act to regulate zinc homeostasis in cells. Using purified zinc-dependent enzymes such as E.
- estradiol A myriad of agents can regulate MT levels, including glucocorticoids and metals such as cadmium (for review see Hamer, 1986). Estrogens are not a classical regulators of MT, but two interesting papers appear in the literature. First, a two week treatment of female rats with 1 7- ⁇ estradiol upregulated a copper binding protein in intestinal
- Blondelle SE Houghten RA. 1 996. 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, Ltd B. 1997. Functional properties of a conditionally phenotypic, estrogen- responsive, human osteoblast cell line. J Cell Biochem 65:368-387.
- Estrogen inhibits the vascular injury response in estrogen receptor- ⁇ -deficient mice. Nature
- 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 ⁇ . Mol Endocrinol 1 1 :353-365.
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