Cells were then washed twice with serum and phenol red free RPMI before 5 μM 7-ethoxyresorufin and 20 μM dicumarol were SP600125 added to the wells. Subsequent fluorescence readouts were recorded continuously at 37 °C
in a Synergy HT microplate reader (λex = 530 nm, λem = 585 nm) and the respetive activity of CYP1 enyzmes was calculated based on a calibration curve with resorufin. Protein concentrations were measured using a BCA protein kit (Thermo Scientific, Waltham, MA, USA). All measurements were performed as 6-fold replicates. Protein stability of firefly luciferase (Promega, Madison, WI, USA) was assayed by fluorescent thermal shift (Niesen et al., 2007 and Vedadi et al., 2006). Assays were performed in 96-well PCR plates using 3.6 μM of protein, 2 mM ATP and SYPRO orange in 50 mM tris–acetate, pH 7.6. Ligands, such as TCC, were added as indicated. Subsequent thermal shifts ranged from 25 °C to 99 °C at an increment of 1 °C/min and were recorded using the ROX filter set of a HT7500 PCR cycler (Applied Biosystems, Foster City, CA, USA). Data were analysed and the temperature of half-maximal denaturation (Tm) was calculated using the MS Excel spreadsheets as provided by (available at ftp:// ftp.sgc.ox.ac.uk /pub /biophysics) ( Niesen www.selleckchem.com/products/3-methyladenine.html et al., 2007 and Vedadi et al., 2006. All experiments were done in triplicate at least. Plotted error bars refer to
the standard error of the mean (SEM) and a two-tailed Student’s t-test was used to assess significance. Respective p-values of mafosfamide p < 0.05 are indicated by an asterisk as appropriate. Concerns about an androgenic potential of TCC are mainly
fuelled by results obtained from luciferase-based reporter screens (Ahn et al., 2008; Christen et al., 2010). However, the suitability of such systems as sole indicators for potential endocrine activity is disputed (Diel et al., 1999, Baker, 2001 and Thorne et al., 2010). To investigate the androgenic potential of TCC this study therefore supplemented a commonly used AR-sensitive cellular luciferase assay with quantitative RT-PCR. The concentration of TCC used in the assays was 1 μM as this corresponds to the maximal levels realistically expected in human blood (SCCP, 2005; Schebb et al., 2012). Initially the reported androgen mediated amplification of luciferase-activity by TCC was reproduced using a MDA-kb2 reporter cell line (Fig. 2). In contrast to the T47D-ARE cell line used previously (Ahn et al., 2008) this cell line originates from human MDA-MB-453 breast cancer cells which were transfected with a MMTV.luciferase.neo reporter construct. The respective reporter is negative for ER but maintains endogenous expression of the AR (Christen et al., 2010). The molecular background thus allows the monitoring of AR-responsive genes, while the luciferase reporter will be responsive to stimulation of the AR as well as the GR.