Genetically defining the mechanism of Puma- and Bim-induced apoptosis

Abstract

Using genetically modified mouse models, we report here that p53 upregulated modulator of apoptosis (Puma) and Bcl-2 interacting mediator of cell death (Bim), two pro-apoptotic members of the B-cell lymphoma protein-2 (Bcl-2) family of proteins, cooperate in causing bone marrow and gastrointestinal tract toxicity in response to chemo and radiation therapy. Deletion of both Puma and Bim provides long-term survival without evidence of increased tumor susceptibility following a lethal challenge of carboplatin and ionizing radiation. Consistent with these in vivo findings, studies of primary mast cells demonstrated that the loss of Puma and Bim confers complete protection from cytokine starvation and DNA damage, similar to that observed for Bax/Bak double knockout cells. Biochemical analyses demonstrated an essential role for either Puma or Bim to activate Bax, thereby leading to mitochondrial outer membrane permeability, cytochrome c release and apoptosis. Treatment of cytokine-deprived cells with ABT-737, a BH3 mimetic, demonstrated that Puma is sufficient to activate Bax even in the absence of all other known direct activators, including Bim, Bid and p53. Collectively, our results identify Puma and Bim as key mediators of DNA damage-induced bone marrow failure and provide mechanistic insight into how BH3-only proteins trigger cell death.

Figure 1

Co-deletion of Bim and Puma protects mice from lethal myelosuppression and is associated with recovery of the hematopoietic system. (a) Survival of wild-type (WT) (squares; n=15), Bim^−/− (triangles; n=14), Puma^−/− (circles; n=15) and Puma^−/−/Bim^−/− (diamonds; n=7) mice treated with a myelosuppressive regimen of 80 mg/kg carboplatin and 7.5 Gy TBI. (b) The effects of 80 mg/kg carboplatin and 7.5 Gy TBI on white blood cell count (WBC; left panel), platelet count (middle panel) and hemoglobin concentration (right panel) in each mouse, from (a), determined at the indicated time points. Each data point in (b) represents the mean±S.E.M.

Figure 2

Puma^−/−/Bim^−/− primary CTMCs are resistant to p53-dependent and p53-independent cell death. (a and b) WT (squares), Bim^−/− (triangles), Puma^−/− (inverted triangles), Puma^−/−/Bim^−/− (diamonds) and conditionally deleted Bax/Bak double knockout (circles) cells were deprived of cytokines for 0–48 h (a) or in combination with 5 Gy IR (b). Apoptosis was assessed by flow cytometry of AnnexinV/7AAD-positive cells. (c) WT and Puma^−/−/Bim^−/− cells were cultured in complete media with and without cytokines (+/− Cyt) for 48 h, treated with 5 Gy ionizing radiation (IR) at time zero, or restimulated with cytokines for 24 h following Cyt deprivation with. DNA synthesis was monitored by BrdU incorporation and FACS analysis, and quantified as the percent of BrdU-positive cells. Data are presented as the mean±S.E.M. of at least three independent experiments, each performed with bone marrow-derived cells isolated from (≥2) mice for each genotype

Figure 3

Cytokine withdrawal modulates the expression of both pro- and anti-apoptotic Bcl-2 family members in myeloid cells. WT, Bim^−/−, Puma^−/− and Puma^−/−/Bim^−/− cells were deprived of cytokines for 0–16 h and Bcl-2-related proteins were evaluated by western blot analysis. Representative blot from at least three independent experiments is presented

Figure 4

Bim and Puma are required for Bax activation in response to p53-dependent and p53-independent cell death. (a) WT, Bim^−/−, Puma^−/− and Puma^−/−/Bim^−/− primary CTMCs were deprived of cytokines for 16 h with or without 5 Gy IR. Bax activation was determined by immunoprecipitation with antibody 6A7, which detects Bax in its active confirmation, and subsequent western blot analysis. Actin levels were assessed in cell lysates before immunoprecipitation (n=2). (b) Western blot analysis of activated Bax in WT and Puma^−/−/Bim^−/− cells treated with cytokine withdrawal and 5 Gy IR for the indicated times (n=2)

Figure 5

Bax activation and apoptosis in response to Bcl-2 and Bcl-X_L inhibition is dependent upon Bim and Puma. (a) WT, Bim^−/−, Puma^−/− or Puma^−/−/Bim^−/− primary CTMCs were treated with 1 μM ABT-737 for the indicated times in fully supplemented media (left panel) and under conditions of cytokine withdrawal (middle panel), or treated with cytokine withdrawal alone for 16 h (right panel). Apoptosis was determined by flow cytometric evaluation of AnnexinV/7AAD-positive population. (b) WT, Bim^−/−/Bid^−/− or p53^−/−/Bim^−/−/Bid^−/− cells were treated with 1 μM ABT-737 for the indicated times in fully supplemented media (left panel) and under conditions of cytokine withdrawal (middle panel) or treated with cytokine withdrawal alone for 16 h (right panel). (c) IP/western blot analysis of Bax activation in cells deprived of cytokines and treated with ABT-737 as described in Materials and Methods (n=2). Data in (a and b) are presented as the mean±S.E.M. of three independent experiments

Figure 6

ABT-737 induced cytochrome c release is Bim and Puma dependent. Primary WT, Puma^−/−/Bim^−/− and Bax^−/−/Bak^−/− CTMCs were cultured in the presence or absence of cytokines for 6 h, permeablized in 0.015% digitonin and treated with C8-Bid (25 nM), Puma or Bim peptide (10 μM) or ABT-737 (0.75 μM) for 75 min. Cytochrome c release was determined by western blot analysis and actin was used as a loading control. Representative blot from three independent experiments is presented