Figure 3

AEE788 in combination with 4-hydroxytamoxifen (4-OH) tamoxifen or letrozole enhances G1 arrest compared with monotherapy. (A) Steroid-depleted MCF-7 A2 and (B) BT474 A3 cells were treated for 72 h with vehicle, androstenedione (10 nM), 10 nM 4-OH tamoxifen or 10 nM letrozole±AEE788 (5 μ M for MCF-7 A2, or 0.5 μ M for BT474 A3 cells). Cell cycle was monitored by fluorescence-activated cell sorting analysis of cells stained with propidium iodide (PI). Data are representative of two individual experiments. Bars represent ±s.e.m, ^*P<0.05, ^**P<0.01 are derived from the comparison of endocrine agent alone vs the combination with AEE788 by Student's unpaired t-test (C). Duplicate plates treated with drug combinations were harvested after 24 h treatment. Whole-cell extracts were probed for total and phosphorylated p27^Kip1 and cyclin D1. (D) Steroid-depleted MCF-7 A2 and BT474 A3 cells were treated for 24 h with vehicle, androstenedione (10 nM), 10 nM 4-OH tamoxifen or 10 nM letrozole±AEE788 (5 μ M for MCF-7 A2, or 0.5 μ M for BT474 A3 cells). Apoptosis was measured using a Cell Death Detection ELISA PLUS kit (Roche). Data are expressed as fold increase in apoptosis compared with that in the steroid-treated control. Bars represent ±s.e.m of triplicate wells ^*P<0.05, ^**P<0.01 are derived from the comparison of endocrine agent alone vs the combination with AEE788 by Student's unpaired t-test. Data are representative of two individual experiments.