Figure 6

Inducible oncogenic RAS signaling sensitizes cells to topoisomerase inhibition. (A) In contrast to the comparison between HCT-116 and HKE-3 cells, 4-hydroxy-tamoxifen-inducible oncogenic RAS in an HKE-3 cell background does not confer preferential induction of apoptosis in response to bortezomib treatment (left panel). Conversely, topotecan treatment does still elicit a clear differential apoptosis response when RAS-induced cells are compared to uninduced cells (right panel). Data are relative to control vehicle-treated cells and are represented as mean ± SD. (B) Ratio of apoptosis induced in mutant versus wild-type or ER-RAS-induced versus uninduced cells treated with bortezomib or topotecan (left panel). HKE-3 cells carrying a 4-hydroxy-tamoxifen-inducible oncogenic RAS construct fail to show a preferential induction of apoptosis following treatment with two additional proteasome inhibitors (MG-132 and PI-I) when comparing RAS-induced with uninduced cells. Conversely, treatment with two alternative topoisomerase inhibitors (doxorubicin and camptothecin) still results in a strong preferential induction of apoptosis when comparing RAS-induced with uninduced cells (right panel). (C) Modulation of inducible ER-RAS fusion protein activity, by titration of 4-hydroxy-tamoxifen, shows a gradual decline in apoptosis induction upon treatment with a wide range of camptothecin concentrations to baseline uninduced levels. Differential apoptosis induction is represented by plotting the induced/uninduced ratio. Data are represented as mean ± SD. (D) 24 h induction of oncogenic RAS signaling produces an increase in ROS levels that is elevated further in response to topotecan treatment.