MDA-kb2 Cell Line

The MDA-kb2 cell line was used for the identification of GR agonists and antagonists. This cell line was developed by Wilson et al. (2002), and it was derived from a breast cancer MDA-MB-453 cell line that constitutively expressed high levels of functional GR and AR. The MDA-kb2 cell line was prepared by stable transfection of the MDA-MB-453 cell line with a murine mammalian tumor virus luciferase neo reporter gene construct, which expresses firefly luciferase on exposure to GR and AR agonists. To discriminate against AR-mediated increases in the luciferase production, these cells were concomitantly treated with an AR antagonist FLU in the GR agonist assays as described below. The MDA-kb2 cell line was purchased from American Type Culture Collection (ATCC CRL-2,713) and maintained in Leibovitz’s L-15 medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum (Gibco), 100U/mL penicillin and 100μg/mL streptomycin (both from Sigma-Aldrich). The test medium was prepared with the Leibovitz’s L-15 medium supplemented with 10% dextran-charcoal-stripped fetal bovine serum (Gibco), 100U/mL penicillin and 100μg/mL streptomycin (both from Sigma-Aldrich). The assays were carried out according to Wilson et al. (2002). Briefly, 1×104 cells in 100μL/well were seeded in 96-well plates in test medium and preincubated for 24 h before the treatments. The control and preservative stock solutions were serially diluted in 1mL test medium. The medium from the wells was then removed. For the glucocorticoid agonist assays, the AR was blocked with 10μM androgen antagonist FLU with an incubation for 30 min; then 50μL was added to each well, as triplicates. Similarly for the glucocorticoid antagonist assay (but without FLU), 50μL was added to each well, as triplicates, and incubated for 30 min, and then 50μL 1μM HC in medium was added. The cells were incubated for 24 h, followed by cell lysis with 20μL Luciferase Cell Culture Lysis Reagent (Promega). Then, 35μL firefly luciferase reagent ONE-Glo (Promega) was added, and luciferase luminescence was recorded (2-s medium shaking step followed by luminescence end point measurement; no light source or emission filters) using a microplate reader (Synergy 4 Hybrid Multi-Mode; BioTek). Cell viability assays were run in parallel, as described above for the AR-EcoScreen cell line.