U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle Park, North Carolina 27711
Received March 17, 2004; accepted May 25, 2004
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ABSTRACT |
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Key Words: estrogenicity; in vitro screening assay; estrogen-responsive luciferase reporter.
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INTRODUCTION |
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In comparison to other existing in vitro assays, reporter gene assays based on stably transfected cell lines provide the most specific, responsive, and relatively quick means to screen substances for potential estrogenic and antiestrogenic activity (Legler et al., 1999). Recently, a few such cell lines have been developed (Legler et al., 1999
; Pons et al., 1990
). These cell lines are sensitive and responsive to estrogens but are not freely available. Consequently, the goal of this project was to develop an estrogen-dependent stable cell line by stably transfecting an estrogen-responsive luciferase reporter gene construct into T47D human breast adencarcinoma cells expressing endogenous estrogen receptors alpha and beta and to make the cell line easily available to other laboratories. T47D cells express a relatively high number of endogenous ER, reportedly 67.6 ± 6.2 fmol/mg cytosolic protein (Watanabe et al., 1990
). Western blot analysis indicated that these cells contain both the alpha and beta isoforms of the ER protein with very slightly higher levels of beta than alpha (Power and Thompson, 2003
). The reporter gene construct consists of three estrogen response elements (ERE) upstream from a TATA box that regulates the expression of a luciferase reporter gene. Stable transfection of this promoter-reporter construct into T47D cells resulted in a sensitive, responsive clone. In very simple terms, compounds enter the cell; estrogen receptor ligands bind to the ER; two ligand-bound receptors dimerize and bind coactivators; then the dimer binds to the ERE on the reporter gene construct and activates the luciferase reporter gene. The presence of the luciferase enzyme can then be assayed by measuring the light produced when the enzyme substrate, luciferin, and appropriate cofactors are added. The amount of light produced is relative to the degree of estrogenic activity of the test chemical. Model compounds, with well-defined mechanisms of action, were used to evaluate the utility of this cell line, T47D-KBluc, and those results are presented here. When testing chemicals using the T47D-KBluc cells, an estrogen was defined as a chemical that induced dose-dependent luciferase activity which could be specifically inhibited by the antiestrogen ICI. Plans include making these cells widely available by depositing the cell line with ATCC for maintenance and distribution.
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MATERIALS AND METHODS |
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Mammalian cells. The human breast cancer cell line T-47D (ATCC No. HTB 133), ER/ß + /GR +, 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 µg/ml penicillin, 100 U/ml streptomycin, and 0.25 µg/ml amphotericin B (Gibco BRL, purchased as a 100x mixture of penicillin, streptomycin, and amphotericin B).
Transfection procedure. Cells were seeded 2 x 105 cells per 60 mm culture dish. Cells were transfected using Fugene 6 (Roche) per manufacturer protocol with 5 µg pGL2.TATA.Inr.luc.neo per dish. The cells were placed in selection media (growth media plus 500 µg/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 56 h. After attachment, growth media were replaced with fresh media, except 5% dextran-charcoal treated FBS was substituted for 10% regular FBS. After 40 to 48 h cells were dosed with 100 µl 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 1.0 nM 17ß-estradiol (E2). Both controls (vehicle and E2) were also competed with 1 µM ICI, an ER antagonist, to assess ER-specific responsiveness and background. Cells were washed with phosphate buffered saline at room temperature and then 25 µl lysis buffer (Ligand Pharmaceuticals) was added per well and incubated until cells were lysed (1530 min). Relative light units per well were determined using a 96-well MLX Luminometer (Dynex, Chantilly, VA). The final clone was chosen based on appropriate ligand responsiveness and genetic stability over time and 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 µl/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 25 µl 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 25 µl reaction buffer (25 mM glycylglycine, 15 mM MgCl2, 5 mM ATP, 0.1 mg/ml BSA, pH 7.8), followed by 25 µl 1 mM D-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 MTT crystals 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 48 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.
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RESULTS |
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DISCUSSION |
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The performance of this assay was evaluated using known ER agonists and antagonists. We have shown, herein, that the responsiveness of this stable cell line to potent estrogens, including E2, EE, and DES, is in the pM to nM range, which would imply sufficient sensitivity for most applications. At E2 concentrations between 0.1 and 1 pM luciferase activity rapidly increased, illustrating the ability of the assay to detect very low E2 concentrations. The EC50 of E2 following a 24-h exposure was approximately 3 pM, with maximum induction occurring near 100 pM (0.1 nM) and above. These results are similar to those obtained in the ER-CALUX assay for E2, which also used T47D cells as the parent cell line, where an EC50 of 6 pM was reported (Legler et al., 1999). They reported maximum induction at 30 pM, while we report maximum induction near 100 pm. We chose to test concentrations at log intervals; however, it is possible that if intermediate doses were tested that number could be refined. Fold induction due to estrogens in our stable cell line is less than that obtained with the ER-CALUX assay, but the sensitivity and predictability of the assays is very similar when testing both potent estrogens and weaker environmental estrogenic compounds.
Similar results were obtained for both EE and DES, as would be expected for potent estrogenic compounds. EE-induced luciferase activity dramatically increased at concentrations greater than 1 pM, with an EC50 of approximately 8.5 pM. The EC50 value for DES was estimated at 14 pM. Maximal induction was achieved for all three compounds, E2, EE, and DES, at 0.1 nM. Together these results indicate their similar potency in this assay. To test the specificity of the assay, both DEX and DHT were tested. DEX did not activate gene transcription at any of the concentrations tested (1 pM to 10 nM). DHT did activate gene transcription to about half the maximal E2 response, but only at very high concentrations (statistically significant at 200 nM and above). The response to DHT was reduced by the addition of ICI, indicating it was likely ER mediated. T47D breast cancer cells have been shown to express mRNA for aromatase (Sadekova et al., 1994); however, since DHT cannot be aromatized to estrogen, this is an unlikely explanation for the induction of luciferase activity by DHT at high concentrations.
Several environmental estrogens were also tested in the T47D-KBluc cells, and results were generally as expected. The lowest observed effect concentrations (LOEC) for genistein and 4-NP were 10 nM and 0.5 nM, respectively. This is somewhat lower that the LOEC (100 nM for both compounds) reported for the ER-CALUX assay. This is an interesting observation, given that the two assays are similar and were developed from the same parent cell line, T47D cells. Interestingly, some compounds such as genistein and 4-NP elicited luciferase induction higher than the maximal response attained by E2. Similar responses have been seen by others when these compounds are tested at high concentrations (Legler et al., 1999; Routledge and Sumpter, 1996
). Even though these are "simplified" systems compared to the whole animal, they still contain complex biological processes. The mechanism behind this action is not clear, but some have hypothesized that it may be due to stimulated receptor and/or coactivation factor renewal or to effects on luciferase stability (Legler et al., 1999
). Most importantly, the goal was to correctly identify estrogenic compounds, and as an initial first-pass screen, both compounds would have been correctly identified for their estrogenic activity and their relative potency based upon their EC50. Before one could conclude that a compound truly had some sort of supramaximal activity, estrogenic activity would need to be confirmed in vivo.
Recently, studies have shown that the heavy metal cadmium mimics the effects of E2 both in vitro and in vivo (Johnson et al., 2003; Stoica et al., 2000
), and our results confirm the in vitro estrogenic activity of cadmium chloride (CdCl). The metal appears to bind to the ER with high affinity with estrogenic activity detected at very low concentrations of CdCl (10 fM). Activity continued to increase up to the highest concentration tested (10 µM) and reached near 100% of the 0.1 nM E2 response at 1 µM CdCl. The fact that this response can be blocked by ICI reinforces the indication that the effect is ER mediated. Work done by Stoica et al. (2000)
indicated that the effect is mediated through ER
, and the interaction of cadmium with the receptor appears to involve several specific amino acids in the ligand binding domain of the receptor.
A comprehensive summary of the published data on in vitro assays used to test substances for their ability to initiate ER-dependent gene transcription was recently compiled by ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) at the request of the U.S. EPA as part of the methods validation process for the Endocrine Disruptor Screening Program (EDSP). These background review documents along with the Expert Panel review are available on the ICCVAM web site (http://iccvam.niehs.nih.gov/docs/docs.htm). The ICCVAM document for estrogen-dependent transcriptional activation assays and the expert panel review recommended testing a range of doses for test chemicals at log intervals over at least seven orders of magnitude when using transcriptional activation assays for screening purposes. As they recommended, we conducted most of these assays using doses at log intervals. This is a good approach when using transcriptional activation assays for screening of chemicals, especially when little or no information on the relative potency of the test chemical is available, as the probability of detecting a positive is increased when a broad range of concentrations is tested. Admittedly, the determination of EC50 values could be refined by using several intermediate concentrations within the linear section of the dose-response curve, but further refinement is not always necessary when used only for screening purposes.
We found that the use of appropriate controls on every assay plate was essential for monitoring the function of the cells and for defining which data sets are appropriate to include in a response analysis. Always of concern when utilizing estrogen-dependent gene transcriptional activation assays is the issue of high background activation levels. We found that increased background could be variable and influenced by many factors. Because of this, we recommend always testing levels of background activity on every assay plate by adding a vehicle plus ICI control in addition to the standard positive and negative controls. Several factors can influence background activation, such as serum that is improperly stripped of steroid hormones. Each new batch of dextran-coated charcoal stripped fetal bovine serum needs to be tested for any residual steroid activity. We also found that some antibiotic mixtures, added to help avoid contamination, can contribute to high background levels of estrogenicity. Figure 7 illustrates the difference in background levels of luciferase activity with and without antibiotic/antifungals in the medium. High background levels of estrogenicity blunt the responsiveness of the cells and may lead to false conclusions about a compound. During any assay, if background was unusually high on a particular plate, those assay results were discarded and the assay repeated. The portion of the graph shown in Figure 7 without antibiotics illustrates what we considered to be acceptable levels of background luciferase activity. In addition, for the purpose of screening, runs/plates were discarded when the stimulation induced by the 0.1 nM E2 control was less than 23 fold when compared to the vehicle control. Once high background problems were reduced by weaning cells from the antibiotic/antimycotic mixture for 1 week prior to conducting an assay, lack of responsiveness to E2 was only a minor concern (less than 1% of plates were discarded).
In summary, in vitro assays are becoming increasingly attractive as screening tools because they are rapidly done and fairly easy to perform, some are amenable to high-throughput systems, and they have the potential to reduce the number of animals needed for chemical testing. Stably transfected cell lines offer several advantages in comparison to other in vitro systems such as binding assays, repeated transient transfections, yeast based assays, and cell proliferation assays. They are also an excellent aid in defining mechanism of action. Assays utilizing this ER stable cell line are sensitive and generate reproducible data. Our plans include making the T47D-KBluc cell line widely available by depositing it with ATCC as we have done with the MDA-kb2 cells (an AR dependent stable cell line) (Wilson et al., 2002)
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NOTES |
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1 To whom correspondence should be addressed at U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, MD-72, Research Triangle Park, NC 27711. Fax: (919) 541-4017. E-mail: wilson.vickie{at}epa.gov.
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