BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak

Abstract

The BH3-only proteins Bim and Bad bind to the antiapoptotic Bcl-2 proteins and induce apoptosis in wild-type cells and cells from either bax(-/-) or bak(-/-) animals. In contrast, constitutively active forms of Bim and Bad failed to induce apoptosis in bax(-/-)bak(-/-) cells. Expression of Bax restored susceptibility of the cells to Bim and Bad. In addition, Bax but not Bim or Bad sensitized the bax(-/-)bak(-/-) cells to a wide variety of cell death stimuli including UV irradiation, chemotherapeutic agents, and ER stress. These results suggest that neither activation of BH3-only proteins nor suppression of pro-survival Bcl-2 proteins is sufficient to kill cells in the absence of both Bax and Bak. Furthermore, whereas mouse embryo fibroblasts (MEF) expressing only Bax or Bak displayed resistance to transformation, bax(-/-)bak(-/-) MEF were nearly as prone to oncogenic transformation as p53(-/-) MEF. Thus, the function of either Bax or Bak appears required to initiate most forms of apoptosis and to suppress oncogenic transformation.

Figure 1

BimS does not induce apoptosis in bax^−/−bak^−/− MEF. (A) MEF of different genotypes were infected with pBabe–IRES–GFP (vector) or pBabe–BimS–IRES–GFP (BimS). Twenty-four hours after infection, cells were stained with DAPI, and photographed under phase contrast or DAPI filter. (B) Cells from A were collected and subjected to flow cytometric analysis (FACS). The percentage of dead cells was determined by the ratio of DAPI-positive cells to GFP-positive cells. Data shown is the average of three independent experiments. (C) Western blot with anti-Bim of lysates obtained from MEF with different genotypes infected with BimS. Ectopically expressed BimS and endogenous BimEL and BimL are indicated. (D) BimS interacts with Bcl-2 in bax^−/−bak^−/− cells. bax^−/−bak^−/− MEF infected with control vector or BimS were lysed. Immunoprecipitation and immunoblotting as described in Materials and Methods were performed with an anti-Bcl-2 antibody. A cell lysate from wild-type MEF was loaded as a control for Bcl-2 migration. The migration of immunoglobulin light chain (Ig) is also indicated. (E) BimS interacts with Bcl-x_L in bax^−/−bak^−/− cells. MEF infected with BimS or control vector were lysed. Immunoprecipitation and immunoblotting were performed as described in D by use of an anti-Bcl-x_L antibody. A cell lysate from wild-type MEF was loaded as a control for Bcl-x_L migration. The migration of immunoglobulin light chain (Ig) is also indicated. (F) Levels of Bcl-2 and Bcl-x_L are not altered in bax^−/−bak^−/− MEF. A Western blot of MEF lysates from different genotypes was probed with antibodies against Bcl-2, Bcl-x_L, and actin.

Figure 2

BAD(3A) does not induce apoptosis in bax^−/−bak^−/− MEF. (A) MEF of different genotypes were infected with GFP (vector) or BAD(3A)–IRES–GFP [Bad(3A)]. Twenty-four hours after infection, cells were stained with DAPI, and photographed by use of a FITC or DAPI filter. (B) Cells from A were collected and subjected to FACS analysis. The percentage of dead cells was determined by the ratio of DAPI-positive cells to GFP-positive cells. Data shown is the average of three independent experiments.

Figure 3

Bax restores the susceptibility of bax^−/−bak^−/− MEF to undergo apoptosis. (A) Bax restores apoptosis sensitivity to BimS and Bad(3A). bax^−/−bak^−/− MEF were infected with Bax–IRES–GFP together with control (GFP), Bad(3A)–IRES–GFP, or BimS–IRES–GFP. Twenty-four hours after infection, cells were stained with DAPI and photographed by use of a FITC or DAPI filter. (B) bax^−/−bak^−/− MEF were infected with BimS or Bad(3A) together with control or Bax vectors. The percentage of dead cells was determined 24 h later by the number of DAPI-positive cells over that of the GFP-positive cells. (C) Bax, but not Bim or Bad, sensitizes bax^−/−bak^−/− cells to different death-inducing agents. bax^−/−bak^−/− MEF were infected with control (GFP), BimS–IRES–GFP, Bad(3A)–IRES–GFP, or Bax–IRES–GFP. Twenty-four hours later, cells were treated with etoposide (Eto, 100 μM), staurosporine (STS, 4 μM), brefeldin A (BFA, 10 μg/mL), or UV-irradiation (200 J/m²). Cell death was determined 24 h later by the number of DAPI-positive cells over that of GFP-positive cells.

Figure 4

Loss of both Bax and Bak facilitates oncogenic transformation. (A) bax^−/−bak^−/− MEF are resistant to death induced by loss of attachment and serum deprivation. MEF of different genotypes were cultured in uncoated bacterial culture dishes or in the absence of FBS for 48 h. The percentage of cell death was determined by PI-exclusion. (B–D) MEF of different genotypes were infected with viral constructs expressing adenovirus-5 E1A and an activated ras oncogene, and cultured in soft agar. Two weeks after infection, plates were photographed and the number of foci scored. (B) Photographs of representative plates. (C) Photographs of foci. (D) Graph quantifying the number of foci from three experiments.

Figure 5

Model for regulation of apoptosis by Bcl-2 family members. Upon receiving cell death cues, the BH3-only proteins are activated. They activate proapoptotic Bax-like proteins Bax/Bak through neutralization of the anti-apoptotic Bcl-2 proteins.