Figure 8

BimEL expression levels vary between ER⁺ breast cancer cell models and correlate to apoptotic outcome in response to hormonal treatments and MEK1 blockade. (a, b) Cell-number determinations showed that IGF-1 stimulated T-47D cell growth via a MEK1-dependent proliferation pathway. T-47D cells were treated with the indicated hormones in the presence or absence of IGF-1 (20 ng/ml) plus and minus PD 98059 for 216 hours (a) or U0126 for 144 hours (b). (c) Western blot showed BimEL levels relative to the levels of cleaved PARP in T-47D cells treated with hormones in the absence or presence of U0126 for 72 hours. The levels of ER and PR are provided for validation of the ER and PR status of T-47D and MCF-7 cells used in this study. (d) Western blot compared the levels of BimEL in MCF-7 versus T-47D cells treated with hormones plus or minus U0126 for 48 h. (e) ROS levels were determined for T-47D and MCF-7 cells treated with the indicated hormones in the presence or absence of IGF-1 plus or minus MEK1 blockade with U0126. (f) Western blot showed that treatment with MG132 caused an accumulation of phosphorylated Bim EL in MCF-7 cells, but not in T-47D cells. (a through f) As described in Materials and Methods, at the indicated times, cells were harvested and analyzed either for cell counts (a, b), protein expression by SDS/PAGE and immunoblotting for BimEL, pBimEL, pro-caspase-3, pMAPK, and total MAPK or β-actin, which were used as loading controls (c, d), or for ROS determination (e).