AD 198 suppressed c-Myc expression in TRAF3
tumor B cells. (A) Dose-dependent effects of AD 198 on c-Myc protein levels. Mouse or human tumor B cells were cultured in the absence or presence of various concentrations of AD 198 for 6 h. Cytosolic and nuclear extracts were prepared as described in the Methods. (B) Time-dependent effects of AD 198 on c-Myc protein levels. Mouse or human tumor B cells were cultured in the absence or presence of AD 198 (1 μM for 105–8 cells and 4 μM for 8226 cells). Total cellular lysates were prepared at indicated time points. Proteins were immunoblotted for c-Myc, followed by actin. Immunoblots of actin were used as loading control. Results of A and B are representative of three independent experiments. Similar results were also obtained with other TRAF3-/- cell lines. (C) c-Myc transcript levels analyzed by real time PCR. Cells were treated with AD 198 for indicated time periods. Drug concentrations used: 1 μM AD 198 for 105–8 cells; 2 μM AD 198 for 27–9 cells; 4 μM AD 198 for 8226 and KMS11 cells. Total cellular RNA was extracted and reverse transcribed. Quantitative real-time PCR was performed using TaqMan primers and probe (FAM-labeled) specific for c-Myc. Each PCR reaction also included primers and the probe (VIC-labeled) specific for β-actin mRNA, which served as endogenous control. Relative mRNA expression levels of c-Myc were analyzed using the Sequence Detection Software and the comparative Ct (ΔΔCt) method. Graphs depict the results of three independent experiments with duplicate samples in each experiment (mean ± SEM).