Cell death in the absence of Bax and Bak

When the Korsmeyer laboratory reported that a B-cell-specific bcl-2 transgene led to an expansion in murine B-cells by conferring a survival advantage to the cells, it ushered in a new era in cancer research.¹ This paper complemented a finding published previously by the Adams' laboratory showing that overexpression of bcl-2 in growth factor-dependent cells could keep these cells alive following growth factor withdrawal in vitro.² These two observations initiated the study of the role of cell death regulation in the pathogenesis and treatment of cancer. The early years of apoptosis research saw the cloning of a number of bcl-2-related genes that appear to play key roles in the regulation of programmed cell death (apoptosis).³ Surprisingly, this bcl-2-related family of genes contained both proapoptotic and antiapoptotic members. The roles of these genes in development and tissue homeostasis in the mouse have been delineated in large part owing to the techniques of targeted gene deletion and tissue-specific transgenesis. Collectively, these studies suggest that the Bcl-2 family regulates the ability of damaged and/or excess cells to be deleted from the host by apoptosis. Overexpression of antiapoptotic family members can act as oncogenes by promoting the survival of partially transformed cells. In contrast, the proapoptotic family members appear to have evolved to suppress cell autonomous survival in adult animals. The proapoptotic family members consist of Bax and Bak, which show ubiquitous tissue expression, Bok, which is mainly expressed in reproductive organs.⁴ Two other proapoptotic family members, Bid⁵ and BCL2L13,⁶ which share overall homology to other Bcl-2 family members appear to function only as initiators of Bax- and /or Bak-dependent killing. The targeted deletions of the two major proapoptotic genes bak and bax produced mice with no overt phenotype⁷ or with a relatively mild phenotype, respectively.⁸ However, when both bax and bak were deleted, the resulting mouse was found to have a dizzying array of defects.⁷ This commentary describes the major phenotypes of the bax^−/−bak^−/− mouse and considers cell survival and cell death in the absence of Bax and Bak.

Although Bax and Bak share an overall structural homology with the antiapoptotic Bcl-2-related proteins, there exists a second group of proapoptotic proteins, the so-called BH3-only proteins, which contain only a single domain of homology with Bcl-2. These proteins/genes are inactive in viable cells as a result of post-translational modifications or transcriptional repression. One early insight gained from the study of cell lines from the bax^−/−bak^−/− mice came from experiments aimed at defining the proapoptotic functions of BH3-only proteins. When the proapoptotic form of the BH3-only protein Bim (BimS) was introduced into bax^−/−bak^−/− mouse embryo fibroblasts (MEF), the cells remained viable whereas single bax^−/−, bak^−/−, or wild-type cells died rapidly.¹³ The introduced BimS was functional as it was able to co-precipitate both endogenous Bcl-2 and Bcl-x_L. Similar results were obtained following transfection with Bad and Noxa.^{13, 14} These data demonstrated that inhibitors of antiapoptotic Bcl-2 proteins do not promote cell death in the absence of Bax and Bak. Similarly, a second group of BH3-only proteins such as tBid, defined by the ability to bind Bax and Bak, is also incapable of initiating apoptosis in bax^−/−bak^−/− cells.¹⁵ Furthermore, bax^−/−bak^−/− MEF became as susceptible as wild-type cells when Bax and BH3-only proteins were introduced at the same time. bax^−/−bak^−/− MEF also became sensitive to other death-inducing agents such as etoposide, staurosporine, brefeldinA, and UV treatment, when Bax was re-introduced. These experiments demonstrated that Bax and Bak are required for the initiation of apoptotic cell death. In the absence of Bax and Bak, BH3-only proteins are not able to induce cell death. Thus, in a healthy, viable cell, the proapoptotic proteins Bax and Bak are kept in check by the antiapoptotic molecules Bcl-2 and Bcl-x_L and the BH3-only proteins are inactive. When the cell is subjected to death-inducing stimuli such as DNA damage, growth factor deprivation, or ER stress, the BH3-only proteins are induced. Depending on the individual BH3-only protein, it can either bind to an antiapoptotic Bcl-2 protein and inhibit their activity or bind to Bax and/or Bak to induce their effector function. Either action results ultimately in the loss of the integrity of the outer mitochondrial membrane and apoptotic cell death.

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