U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, MD-72, Research Triangle Park, North Carolina 27711
Received August 17, 2001; accepted November 19, 2001
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ABSTRACT |
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Key Words: stable cell line; androgen receptor; glucocorticoid receptor; endocrine screening; antiandrogen; vinclozolin; p`,p-DDE; linuron, procymidone; HPTE.
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INTRODUCTION |
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A stable cell line could potentially be used to identify compounds that bind AR and, unlike standard receptor binding assays, have utility in discrimination of androgen agonists from antagonists, thus aiding in defining mechanisms of action. Transient transfection assays, in which both the AR and an androgen-responsive reporter are cotransformed into naive cells (Kemppainen, et al., 1999; Lambright et al., 2000
; Lobaccaro, et al., 1999
), can supply similar information. However, transient transfection assays may not reflect endogenous levels of receptor, as quantities of expressed receptor might vary greatly from assay to assay, and give responsiveness limited in time, since the transgenes are usually lost within 72 h. Stably transformed cells would eliminate the need for repetitious transient transfections, reduce the variability associated with this technique, and be attractive for high throughput or semi-high throughput screening. However, until now, such an androgen-responsive transcriptional activation assay has not been widely available.
The goal of this study was to develop a cell line stably expressing an androgen-responsive reporter gene and to evaluate its utility for screening compounds for androgenic or antiandrogenic activity, using chemicals known to act as AR agonists, GR agonists, or AR antagonists. The parent cell line, MDA-MB-453 human breast cancer cells, was chosen for transformation because this cell line has been shown to express high levels of functional, endogenous androgen receptor (AR). Using binding assays, the level of AR in MDA-MB-453 cells was determined to be about 240 fmol of AR per mg of protein (Hall et al., 1992). While in contrast, estrogen receptor alpha (ER
) and progesterone receptor (PR) are not detectable at the mRNA level (Hall et al., 1992
), and ERß is expressed only at very low levels in these cells (Vladusic et al., 2000
). This cell line does, however, contain glucocorticoid receptor (GR). (Sutherland, 1999
). Therefore, since the suite of endogenous steroid receptors is limited to AR and GR, potentially confounding responses mediated through steroid receptors other than the androgen or glucocorticoid receptors would be eliminated. Cells were transformed with an androgen-responsive luciferase reporter plasmid driven by the mouse mammary tumor virus promoter (MMTV). The MMTV promoter was chosen for transformation because it is a robust viral promoter and is well characterized as being androgen responsive. In addition to AR, both PR and GR have a high affinity for, and can activate the MMTV promoter. As mentioned above, MDA-MB-453 cells do not contain PR but both AR and GR are present in the cells. Although both AR and GR agonists drive the MMTV luciferase reporter, they can be distinguished by co-administration of the potent AR antagonist hydroxyflutamide (OHF).
The stable cell line derived from MDA-MD-453 and designated MDA-kb2 is a useful tool for studying the activation of AR or GR by hormone agonists and antagonists. Since cells were derived from a single stable clone, inter- and intra-assay variability would be reduced, making the assay easier to standardize and validate. Model compounds with well defined mechanisms of action were used to evaluate the utility of this assay system (Table 1). In addition, a novel chemical with unknown activity, neburon, was also tested, and with the use of selective inhibitors, this cell line is also useful for detecting activation of the MMTV luciferase reporter by glucocorticoid agonists.
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MATERIALS AND METHODS |
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Cells were grown in selection medium, which was changed twice weekly until colony formation was observed. Using cloning rings, individual colonies were then transferred by trypsinization to 24-well plates, still in selection medium. When cell numbers were sufficient, clones from individual wells were transferred to T-25 cm2 flasks. Cells remained in selection medium until screened, and one or more vials of cells were frozen. In order to screen clones for responsiveness to an androgen agonist, cells grown from individual colonies were plated at 1 x 104 cells per well in 96-well luminometer plates and allowed to attach. When cells were attached, the medium was replaced with freshly prepared dosing medium containing ethanol only (negative control), an agonist (dihydrotestosterone, DHT; 1 nM), or the agonist plus a known competitor (hydroxyflutamide, OHF; 1 M). Chemical stocks were prepared in 100% ethanol and diluted into fresh medium to give the desired final concentration in the dosing medium. Final ethanol concentration in each medium preparation did not exceed 0.1%. Cells were dosed with 100 µl of medium/well and incubated for 2024 h, at which time cells were harvested with 25 µl/well of lysis buffer (Ligand Pharmaceuticals) and assayed for luciferase reporter activity as described below. Relative light units per well were determined using a 96-well MLX Luminometer (Dynex, Chantilly, VA). Clones with a minimum 2-fold response to 1 nM DHT were retained in culture. The final clone (MDA-kb2) was chosen based on appropriate ligand responsiveness and genetic stability over time.
Maintenance of cultures and transcriptional activation assays.
MDA-kb2 cells stably transformed with the pMMTV.neo.luc reporter gene construct were maintained in L-15 media (Gibco BRL) supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C, without CO2. For experiments, cells were plated at 1 x 104 cells per well in 100 µl of medium in 96-well luminometer plates (Costar). When cells were attached (46 h), medium was removed and replaced with dosing medium. Stock solutions for each chemical were prepared at 1000x in ethanol. Dosing medium was prepared at the time of treatment by aliquoting 1 µl of stock solution into 1 ml of maintenance medium. Ethanol concentrations in media never exceeded 0.1%. Vehicle control wells contained 100 µl medium/ well with1 µl of ethanol/ml of medium. Each plate also contained either 0.1 or 1.0 nM DHT, or DHT/plus 1 µM OHF as agonist and antagonist controls respectively. Cells were incubated overnight at 37°C without CO2. Prior to lysis, the cells were visually examined under a phase-contrast microscope for signs of cytotoxity such as detachment, vacuolization, membrane degradation, or lack of phase brightness. In some cases where cell toxicity was suspected, duplicate plates were dosed in parallel. Luciferase activity was assayed in one plate as described below, and the second plate was tested for cell toxicity by trypan blue exclusion. Dosing media were then removed from each plate by inversion and gentle shaking. Cells were washed once with phosphate-buffered saline at room temperature and then 25µl lysis buffer (Ligand Pharmaceuticals) was added per well and incubated for 15 min or until cells were lysed. Plates were either stored at 80°C or assayed immediately. 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 of reaction buffer (25 mM glycylglycine, 15 mM MgCl2, 5 mM ATP, 0.1 mg/ml BSA, pH 7.8), followed by 25 µl of 1 mM D-luciferin 5 s later. Each chemical was assayed independently at least 3 times (3 replicate assays) with a minimum of 4 wells per each replicate assay.
Statistical analysis.
These data were collected from several independent experiments, with 3 or more replicates (plates) per experiment. A replicate was a 96-well plate, which included 48 independent observations (wells) each of vehicle control, positive control (either 0.1 or 1.0 nM DHT), antiandrogen control (DHT plus OHF), and all other treatment groups. Data were analyzed by two-way ANOVA (main effects being replicate and treatment) using Proc GLM available from the SAS version 6.09 (SAS Institute, Cary, NC) on U.S. EPA's IBM mainframe computer. Relative light units were converted to fold induction above the vehicle control value for each replicate for statistical analysis. Fold data was 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. In this regard, the standard errors (SE) in the figures are not corrected for replicate variation. For agonists, which stimulate luciferase expression, treatments were compared to the vehicle (media plus ETOH) control group. Androgen antagonists, which block DHT-induced luciferase expression, were compared to the DHT positive control group.
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RESULTS |
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Response to Other Steroid Agonists
Dexamethasone, corticosterone, and progesterone were also assayed for their ability to activate the luciferase reporter gene (Fig. 4). The MDA-MB-452 parent cell line contains both endogenous AR and GR, and both of these receptors can bind to and activate the MMTV.neo.luc response element. Dexamethasone (DEX) was a potent activator of luciferase activity, producing from 1.319.5-fold induction at concentrations from 1 to 1000 nM. Corticosterone (CORT) and progesterone (PROG), although less potent agonists than DEX, induced luciferase activity in a dose-dependent manner. To distinguish between AR and GR agonist activity, compounds were measured against the known antiandrogen, hydroxyflutamide (OHF). OHF specifically competed with AR- but not with GR-mediated responses (Fig. 5
). Cotreatment of DHT with 1 µM OHF significantly reduced DHT luciferase activity. OHF treatment also reduced MPA-induced luciferase activity although to a lesser extent. The activity of DEX and PROG were not affected by cotreatment with OHF.
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Several compounds were tested for antiandrogenic activity (Table 1) and results are presented in Figures 7 and 8
. Vinclozolin and its metabolites, M1 and M2, were measured against 1 nM DHT (Fig. 7
). Vinclozolin competed poorly with DHT, as significant decreases in luciferase activity were detectable only at the highest concentration of 10 µM. As expected, M1 and M2 were more potent competitors than vinclozolin (Kelce et al., 1994
). Bis-OH-DDE, a compound previously reported to be antiandrogenic in vitro (Gaido et al., 2000
) significantly reduced 1 nM DHT-induced luciferase activity at concentrations of 0.2 µM and greater (Fig. 7
). Antiandrogens such as p,p`-DDE and linuron, which have been confirmed to be antiandrogenic in vivo (Gray, 1999; Kelce et al., 1995
) were coadministered with 1.0 nM DHT. Since significant decreases were detectable only at high concentration (20 µM, Fig. 7
), these antiandrogens were compared with 0.1 nM DHT and their antiandrogenic activity became much more apparent. In this case, significant dose-dependent decreases in luciferase activity compared to DHT were detectable at concentrations of 5 µM and greater for both p`,p-DDE and linuron.
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Mixed Agonist/Antagonist Activity
As reported, some compounds displayed mixed agonist and antagonist activity, depending on their concentration and the presence of competitor (Kemppainen and Wilson, 1996; Wong et al., 1995
). At higher concentrations, OHF reportedly acts as an androgen agonist. In this cell line, OHF alone acted as an AR agonist only at 10 µM, but was an effective antagonist in competition with DHT at lower concentrations, especially at 1 µM (Fig. 9A
). OHF decreased 1 nM DHT-induced luciferase activity in a dose-dependent manner at concentrations from 0.05 to1 µM. Ten µM OHF also significantly reduced DHT-mediated transcription compared to 1 nM DHT alone, but less dramatically than at 1 µM. As a result, 1 µM OHF was chosen to run as an antagonist control against either 0.1 or 1.0 nM DHT on each assay plate. The vinclozolin metabolite, M2, also acts as an androgen agonist at 10 µM. In the presence of competitor, M2 inhibits DHT-induced luciferase activity in a dose-dependent manner, with statistically significant decreases at concentrations equal to or greater than 0.2 µM.
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DISCUSSION |
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As shown herein, these cells display appropriate hormonal sensitivity to agonists and antagonists. The potent, classical AR agonist, DHT, significantly induced luciferase activity at concentrations of 0.1 nM and greater with DHT-induced luciferase activity leveling off at concentrations over 10 nM. This response was highly reproducible between replicates over an extended time and number of passages. Because each cell in this stable cell line is derived from a single clone, responses are much less variable than those seen with transient transfection systems. Compared to our experience with transient transfection systems, which often produce highly variable results from one assay to the next, interassay variability was greatly reduced in the MDA-kb2 assay. In transient transfection cell assays conducted in our lab, the interassay CV (8 replicates) for DHT-induced luciferase activity was found to be 145% or about 2.8 fold higher than the CV of 52.7% across 28 replicate plates, as seen in the MDA-kb2 cell line. We believe that one major source of variability in the transient transfection assay arises from differences in transfection efficiencies from replicate to replicate, a source of variation that does not exist in the stably transformed MDA-kb2 cells.
The progestin, MPA, reported to have AR agonist activity both in vitro and in vivo in primates and rats (Barden et al., 1984; Bentel et al., 1999; Mowszowicz et al., 1974
; Poulin et al., 1991
; Raynaud et al., 1980
), was also an AR agonist in the MDA-kb2 assay. The reduction in agonist activity of MPA by 1 µM OHF indicates that it is an AR agonist in these cells. The reduction in MPA-induced luciferase activity, however, was less dramatic than that seen in DHT. In addition, MPA-induced luciferase activity did not level off as seen with DHT, but continued to increase up to 10 µM (the highest concentration tested). Reportedly, at low concentrations in vitro, the action of MPA is primarily mediated through the AR, but at high concentrations, MPA also can activate the GR (Poulin et al., 1991
; Selman et al., 1996
). Since both AR and GR can activate the MMTV-luc reporter, this may explain the additional increase in luciferase activity seen with MPA when compared to DHT, which only activates the AR. Taken together, these data indicate that MPA may be acting through both the AR and the GR in this system, but additional tests with a selective GR antagonist would be needed to confirm that hypothesis.
Since AR, PR, and GR share a common hormone response element (HRE) in the MMTV promoter (Lieberman et al., 1993; Nordeen et al., 1990
; Roche et al., 1992
), and the parent cell line MDA-MB-453 contains both AR and GR but not PR, ER
or significant levels of ERß response to other hormone agonists was tested to access cellular specificity. These included DEX, CORT, PROG, and E2 (Figs. 4 and 6
). The nonsteroidal, monospecific androgen antagonist, OHF, was employed to determine that DEX, CORT, and PROG were acting through the GR and not the AR in this system. Conversely, luciferase activity induced by 17ß-estradiol in this system was likely mediated through AR. This result was not surprising since 17ß-estradiol has been shown to bind to the AR, albeit with lower affinity than either DHT or testosterone (Raynaud et al., 1980
) and with much lower affinity than it has for either ER
or ERß.
To detect androgen antagonist activity, compounds were evaluated for their ability to decrease DHT-induced luciferase activity. For these experiments, concentrations of DHT within the linear concentration-response range were used. Antagonist activity can usually be detected in competition against 1 nM DHT; however, high concentrations of antagonist were sometimes required to detect antiandrogenic activity. Therefore, for weaker androgen antagonists, 0.1 nM DHT was chosen. Using lower concentrations of DHT resulted in a more sensitive assay. Reductions in DHT-induced activity could still be easily detected and solubility issues associated with the use of higher concentrations of some antagonists were avoided.
The mixed agonist/antagonist activity of some compounds (such as M2 and OHF) was not unexpected. It has been previously reported that some androgen antagonists can act as agonists depending on ligand binding affinity, concentration of ligand, and the presence of competing natural ligands (Wong et al., 1995). The reason for this mixed androgen agonist/antagonist activity remains inconclusive. Wong et al. suggested that, depending on the conformational changes induced in the ligand bound receptor, mixed ligand dimers (i.e., an agonist-bound receptor dimerized with an antagonist bound receptor) may be required for antagonism, whereas same-ligand dimers (which would be found at high concentrations) may promote gene activation. Research conducted with other steroid hormone receptors supports this hypothesis (Allan et al., 1992
; Garcia et al., 1992
; Jiang et al., 1992
).
In vitro assays each have their own utility for defining mechanism of action and screening. Some of the advantages and limitations of in vitro AR assays are listed in Table 2. Binding assays, such as the COS whole-cell binding assay, are useful for assessing the ability of a chemical to compete for binding to the receptor. However, they give no insight into the chemical's ability to initiate or inhibit gene expression. Transient transformation assays, such as the CV-1 assay, address the ability of the test chemical to interfere with transcriptional activation and can also give more control over the specificity of response. For example, androgen receptors or glucocorticoid receptors can be transfected into cells separately and responses assessed independently. Using this method, Parks et al. detected AR-mediated activity in Kraft mill effluent samples from the Fenholloway River in Florida but demonstrated that the water initiated no GR-mediated transcriptional activation (Parks et al., 2001
). Although fold induction attained over media controls can be quite large in transient transformation assays due to the high expression levels of receptor, variable transfection efficiency and other factors cause interassay variability to be relatively high. Other limitations of transient transfection include limited cellular responsiveness and the fact that that these assays can be more labor intensive. Adenoviral transduction assays, which are also being developed in our lab, are similar to transient transfection, in that new cells need to be transduced for each set of assays, and fold induction is considerably higher than in the stable cell line. However, viral infection appears more efficient than transient transfection, as the CVs are similar to that seen in the stable cell line (Hartig et al., 2001).
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As with other in vitro assays, the use of stable cell lines has its limitations. In comparison to traditional receptor binding assays using cytosolic preparations, cell culture equipment, techniques, and training are required. The presence of more than one receptor in the cells that can activate the MMTV-driven luciferase reporter could also be viewed as a limitation, since that requires additional assays using selective competitors to differentiate individual receptor activity. This cell line may be viewed as less sensitive (on a fold-induction basis) than transient transfection, since the cellular responsiveness is limited by the levels of endogenous androgen receptor present in the parent cell line, but this has not been a problem, due to the consistency and low variability of responsiveness. In other words, a 100-fold induction is not better than a 5-fold induction if the standard deviations also are comparably higher and the CVs are equal.
Before this cell line could be used routinely to screen chemicals, especially if a high-throughput mode is desirable, the issue of more reliable and quantitative measures of cell toxicity needs to be addressed. This is an important parameter that has not received much attention in some in vitro systems. The ability to incorporate cell toxicity tests into routine chemical testing, however, is an important extension of the method. We have, in the past, used trypan blue exclusion as a measure of cellular toxicity following some chemical treatments, but the use of this method essentially doubles the workload since duplicate plates need to be run in parallel for each chemical tested. Work is continuing in our lab to incorporate a more quantitative measure of cell toxicity into this test system.
In summary, in vitro assays are becoming increasingly attractive as screening tools because many are rapidly done and easy to perform, and they reduce the number of animals needed for testing. They are also an excellent aid in defining potential mechanisms of action of endocrine-disrupting compounds. Each type of assay has its own particular usefulness when used appropriately. Assays utilizing the MDA-kb2 stable cell line are sensitive and generate highly reproducible data, and the cell line is easy to grow. Our plans include making this stable cell line widely available. It is an efficient and accurate tool for the determination of androgen agonists and antagonists and, as such, fills the need for an effective screening tool.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (919) 541-4017. E-mail: wilson.vickie{at}epa.gov.
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