Figure 4
Figure 4. Loss of Bim increases resistance of Eμ-Myc lymphomas lacking both Puma and Noxa to DNA-damaging drugs both in vitro and in vivo. (A) Eμ-Myc/Noxa−/−Puma−/− cell lines, transduced with either shBim-GFP, GFP, or shCaspase-12-GFP retroviral vectors, were treated with etoposide (0.04, 0.2, or 1 μg/mL). Percent of drug-specific apoptosis in cells transduced with the shBim-GFP vector, compared with control vectors, at 6 (left-hand panel) and 24 hours (right-hand panel). Mean ± SEM from 3 independent experiments; representative of 5 Eμ-Myc/Noxa−/−Puma−/− cell lines analyzed. (B) Percent specific apoptosis for Eμ-myc (shown for 2 independently generated lymphomas), Eμ-myc/Puma−/− (derived from n = 4 independently generated lymphomas) and Eμ-myc/Noxa−/−Puma−/− (derived from n = 5 independently generated lymphomas) cell lines retrovirally transduced with a shBim-GFP vector (black bars) or a control-GFP vector (white bars) and treated with etoposide (0.2 μg/mL) for 6 (left-hand panel) or 24 hours (right-hand panel). Mean ± SEM from 4-5 independent cell lines per genotype (for Eμ-myc/Puma−/−, Eμ-myc/Noxa−/−Puma−/−), with each cell line analyzed in at least 3 independent experiments. (C) Data from (B) represented as the percent reduction in apoptosis after 6 hours (left panel) or 24 hours (right panel) of etoposide treatment of lymphoma cell lines transduced with a shBim-GFP vector, compared with that observed for control-GFP vector. Mean ± SEM from 4-5 independent cell lines per genotype (for Eμ-myc/Puma−/−, Eμ-myc/Noxa−/−Puma−/−), with each cell line analyzed in at least 3 independent experiments. (D-F) Primary Eμ-Myc/Noxa−/−Puma−/− lymphomas transduced with shBim-GFP or GFP retroviral vectors were injected into recipient Rag-1−/− mice (see supplemental Figure 6C). When spleens were palpable, mice were treated with CTX (250 mg/kg), monitored, and culled if unwell. Kaplan-Meier curves for 2 independent lymphomas: (D) shBim-GFP vs. GFP-control vector: median survival = 25 days, 20 days respectively, n = 13 mice; P = .0007; (E) shBim-GFP vs. GFP-control vector: median survival 15 and 24.5 days, respectively, n = 10 mice; P < .0001); and (F) shBim-GFP vs. GFP-control vector: median survival = 18 days and 42.5 days, respectively, n = 10 mice, P = .0042.

Loss of Bim increases resistance of Eμ-Myc lymphomas lacking both Puma and Noxa to DNA-damaging drugs both in vitro and in vivo. (A) Eμ-Myc/Noxa−/−Puma−/− cell lines, transduced with either shBim-GFP, GFP, or shCaspase-12-GFP retroviral vectors, were treated with etoposide (0.04, 0.2, or 1 μg/mL). Percent of drug-specific apoptosis in cells transduced with the shBim-GFP vector, compared with control vectors, at 6 (left-hand panel) and 24 hours (right-hand panel). Mean ± SEM from 3 independent experiments; representative of 5 Eμ-Myc/Noxa−/−Puma−/− cell lines analyzed. (B) Percent specific apoptosis for Eμ-myc (shown for 2 independently generated lymphomas), Eμ-myc/Puma−/− (derived from n = 4 independently generated lymphomas) and Eμ-myc/Noxa−/−Puma−/− (derived from n = 5 independently generated lymphomas) cell lines retrovirally transduced with a shBim-GFP vector (black bars) or a control-GFP vector (white bars) and treated with etoposide (0.2 μg/mL) for 6 (left-hand panel) or 24 hours (right-hand panel). Mean ± SEM from 4-5 independent cell lines per genotype (for Eμ-myc/Puma−/−, Eμ-myc/Noxa−/−Puma−/−), with each cell line analyzed in at least 3 independent experiments. (C) Data from (B) represented as the percent reduction in apoptosis after 6 hours (left panel) or 24 hours (right panel) of etoposide treatment of lymphoma cell lines transduced with a shBim-GFP vector, compared with that observed for control-GFP vector. Mean ± SEM from 4-5 independent cell lines per genotype (for Eμ-myc/Puma−/−, Eμ-myc/Noxa−/−Puma−/−), with each cell line analyzed in at least 3 independent experiments. (D-F) Primary Eμ-Myc/Noxa−/−Puma−/− lymphomas transduced with shBim-GFP or GFP retroviral vectors were injected into recipient Rag-1−/− mice (see supplemental Figure 6C). When spleens were palpable, mice were treated with CTX (250 mg/kg), monitored, and culled if unwell. Kaplan-Meier curves for 2 independent lymphomas: (D) shBim-GFP vs. GFP-control vector: median survival = 25 days, 20 days respectively, n = 13 mice; P = .0007; (E) shBim-GFP vs. GFP-control vector: median survival 15 and 24.5 days, respectively, n = 10 mice; P < .0001); and (F) shBim-GFP vs. GFP-control vector: median survival = 18 days and 42.5 days, respectively, n = 10 mice, P = .0042.

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