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• Wilson VS, Bobseine K, Gray EL. Development and Characterization of a Cell Line That Stably Expresses an Estrogen-Responsive Luciferase Reporter for the Detection of Estrogen Receptor Agonist and Antagonists. Toxicol Sci. 2004 September;81(1):69-77. Available from: http://dx.doi.org/10.1093/toxsci/kfh180.

Used this plasmid

MATERIALS AND METHODS

• Chemicals. (Sigma Chemical Company)

1. 17b-Estradiol (E2, 99%),
2. 17a-ethynylestradiol (EE, >98%),
3. diethylstibestrol (DES, min >99%),
4. 5a-dihydrotestosterone (DHT, min >99%),
5. dexamethasone (DEX, 100%),
6. genestein (Gen, >98%),
7. tamoxifen (Tam, >99%) and
8. methoxychlor (Meth, >98%)

1. 4-Nonylphenol (4-NP, a mixture of branched side chains containing 85% p-isomers) was purchased from Fluka Chemical Corp.(Ronkonkoma, NY).

1. The synthesis of 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane(HPTE), a methoxychlor (Sigma, purity >99%) metabolite, was previously published (Waller et al., 1996).

1. The antiestrogen, ICI 182,780, was supplied by ICI Pharmaceuticals (Macclesfield, England, Lot #C42710).

1. Cadmium chloride (CdCl, purity 99.5%) was purchased from Fisher Chemical

Co. (Fig. 1)

Construction of reporter plasmid pGL2.TATA.Inr.luc.neo.

• Remove Neomycin Gene from puc9neo

1. Neomycin gene was removed from puc9.neo (provided by Phillip Hartig, U.S. EPA, Research Triangle Park, NC) using a BamHI digest.

• Incorporate Neomycin gene into pGL2.TATA.Inr.Luc.ERE(3) plasmid

1. The 1.8 kb fragment was ligated to pGL2.TATA.Inr.luc containing three estrogen-responsive elements(ERE) (provided by Donald McDonnell, Duke University, Durham,NC) that had been linearized with SmaI.

• The resulting plasmid was pGL2.TATA.Inr.luc.neo

Preparing Mammalian cells: T47D Breast Adenocarcinoma

• T47D cells:

1. The T-47 line was isolated by I. Keydar from a pleural effusion obtained from a 54 year old female patient with an infiltrating ductal carcinoma of the breast.
2. This differentiated epithelial substrain (T-47D) was found to contain cytoplasmic junctions and receptors to 17 beta estradiol, other steroids and calcitonin.
3. It will form colonies in soft agar.
4. Purified DNA from this line is available as ATCC 45528 (25 micrograms) and ATCC 45529 (100 micrograms).

• The human breast cancer cell line T-47D (ATCC No. HTB 133), ERa/b 1 /GR 1, was used for transfection.
• Cells were screened for sensitivity to the selection antibiotic, geneticin (Gibco/BRL).
• Concentrations of the antibiotic were selected to produce 100% lethality over a two week culture period (data not shown).
• Growth media was RPMI (Gibco) supplemented with 2.5 g/l glucose, 10 mM HEPES, 1 mM sodium pyruvate, 1.5 g/l NaHCO2, 0.2 U/ml insulin, 10% FBS (fetal bovine serum, Hyclone, characterized), 100 mg/ml penicillin, 100 U/ml streptomycin, and 0.25 mg/ml amphotericin B (Gibco BRL, purchased as a 1003 mixture of penicillin, streptomycin, and amphotericin B).

Transfection procedure.

• T47D Cells were seeded 23105 cells per 60mmculture dish.
• T47D Cells were transfected using Fugene 6 (Roche) per manufacturer protocol with 5 mg pGL2.TATA.Inr.luc.neo per dish.
• T47D cells were placed in selection media (growth media plus 500 mg/ml gentamycin) 24 h after transfection.
• Cells were grown in selection media until colony formation was observed.
• Colonies were transferred by trypsinization to 24-well plates and then to T-25 cm2 flasks for continued culture.

Initial screening of clones.

• For initial screening of colonies, cells were plated at 104 per well in 96-well luminometer plates and allowed to attach 5–6 h.
• After attachment, growth media were replaced with fresh media, except 5% dextran-charcoal treatedFBSwas substituted for 10%regular FBS.
• After 40 to 48h cells were dosed with 100 ml dosing media/well (5% dextran-charcoal treated

FBS media plus test chemical) and incubated for 24 h.

• Stock chemicals were prepared in 95% ethanol.
• Dosing solutions were prepared by diluting the chemical stock with fresh dosing media to the desired concentration. In no case did the ethanol concentration exceed 0.2%.
• Negative control wells were dosed with media plus 0.1% ethanol.
• Positive control wells were dosed with 0.1 nM or 1.0nM 17b-estradiol (E2).
• Both controls (vehicle and E2) were also competed with 1mM ICI, an ERantagonist, to assess ER-specific responsiveness and background.
• Cells were washed with phosphate buffered saline at room temperature and then 25 ml lysis buffer (Ligand Pharmaceuticals) was added per well and incubated until cells were lysed (15–30 min).
• Relative light units per well were determined using a 96-wellMLXLuminometer (Dynex, Chantilly, VA).
• The final clone was chosen based on

1. appropriate ligand responsiveness and
2. genetic stability over time and
3. renamed T47D-KBluc.

Chemical screening.

• Stock cells from the chosen clone, T47D-KBluc, were maintained in standard growth media as detailed above.
• Cells were placed in growth media modified by replacement of 10% FBS with 10% dextran-charcoal

treated FBS (Hyclone) without antibiotic supplement one week prior to assay.

• Dosing media was further modified by reduction of dextran-charcoal treated FBS to 5%.
• Cells were seeded at 104 cells per well in 96-well luminometer plates and allowed to attach overnight.
• Media was then replaced with 100 ml/well of dosing media and the test chemical and incubated 24 h. Ethanol vehicle did not exceed 0.2%.
• Cells were washed with phosphate buffered saline at room temperature, then harvested in 25ml lysis buffer (Ligand Pharmaceuticals) per well.
• Luciferase activity was determined using an MLX microtiter plate luminometer (Dynex, Chantilly, VA) and quantified as relative light units (RLU).
• Each well received 25ml reactionbuffer (25mMglycylglycine, 15mMMgCl2, 5mMATP, 0.1 mg/ml

BSA, pH 7.8), followed by 25 ml 1 mMD-luciferin 5 s later.

• Each chemical was assayed independently at least three times (three replicate assay plates) with a

minimum of four wells per each replicate assay unless otherwise noted in the text.

• Cells were screened with a battery of chemicals using agonist positive (E2), negative (vehicle only), antagonist (E2 plus ICI), and background (vehicle plus ICI) controls on every plate.
• Each chemical was tested both alone and in the presence of an appropriate competitor such as 0.1 nM E2 (suspected antagonist) or ICI (suspected agonists).
• E2 positive controls were monitored over time as an assessment of the stability of the cells line. * In instances where cytotoxicity of a chemical was suspected, duplicate plates were dosed in parallel.
• Luciferase activity was assayed in one plate as described above and the second plate was

tested for cell toxicity.

• Cytotoxicity was evaluated by determining the mitochondrial function of the cells using the tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) following treatment with the compound.
• MTT is a yellow vital dye that is actively converted by mitochondrial oxidation-reduction reactions into blue formazan crystals. The formation of the blue MTTcrystals within the cell decreases in direct proportion to the viability of cells (Li et al., 1999).

Statistical analysis.

• These data were collected from several independent experiments, with three or more replicates (plates) per experiment.
• A replicate was a 96-well plate which included 4–8 independent observations (wells) each of

the vehicle control, positive control (either 0.1 or 1.0 nM E2), antiestrogen control (E2 plus ICI), background control (vehicle plus ICI), and all other treatment groups.

• Data were analyzed by two-way ANOVA (main effects being replicate [a nuisance or blocking factor] and treatment) using Proc GLM available from SAS version 6.09 (SAS Institute, Cary, NC.) on the U.S. EPA IBM mainframe computer.
• Relative light units were converted to fold induction above the vehicle control value or as percent of E2 positive control response for each replicate for statistical analysis.
• Data were analyzed in a GLM model that included the concentrations and replicates.
• Statistically significant effects ( p < 0.05) were examined using the least squares means (LSMEANS) procedure available on SAS.
• Means and standard errors were calculated using PROC means.
• For agonists, which stimulate luciferase expression, treatments were compared to the vehicle (media plus ETOH) control group or to the relative response of their respective E2 control (either 0.1 or 1.0 nM E2).
• Estrogen antagonist, ICI, which blocks E2-induced luciferase expression, was compared to the E2 positive control group.
• Graphs were prepared using Origin scientific graphing software (OriginLab, Northampton, MA). Best fit curves were generated using logistic fit of the data.