Life or Death in Cells

The Bcl-2 protein family members Bax and Bak play an important role in regulating apoptosis.1,2 But following their discovery in the 1990s, they did not take center stage because researchers didn't anticipate their role as "critical effectors of programmed cell death," says Craig Thompson, scientific director of the Abramson Family Cancer Research Institute at the University of Pennsylvania. But the limelight is now upon them as Thompson and his colleagues at the Abramson Institute, in collaboration with researchers at the Dana-Farber Cancer Institute in Boston and Emory University School of Medicine in Atlanta, have published a series of recent papers3-5 showing how Bax and Bak are "central and key in the pathway," and how "other Bcl-2 family members don't have an effect unless Bak and Bax are present," says Thompson. "It is a terribly important step in the process," comments Douglas Green, an expert in the field of apoptosis and head of the cellular immunology division at the La Jolla Institute for Allergy and Immunology in Ontario, Calif. Of the newly discovered function of Bak and Bax, Green explains, "It will be one of the keys to the control of life and death in cells." Many Bcl-2 protein family members have been implicated as major components of the mammalian apoptotic pathway. The anti-apoptotic members block apoptosis, whereas others, called the pro-apoptotic members, accelerate the process. Anti-apoptotic Bcl-2 (B-cell lymphocyte/leukemia-2 gene) was the first Bcl-2 family member identified.6 A whole suite of members have been identified since, including pro-apoptotic Bax (Bcl-2 associated protein X)1 and Bak (Bcl-2 homologous antagonist/killer).2 But virtually nothing was known about their underlying mechanisms. Many scientists study the function of individual Bcl-2 proteins, including Bax and Bak, by deleting these genes from mice. Scientists have found that even though Bax knockout mice show some developmental abnormalities, like male sterility, the mice are still viable,7 as are Bak knockout mice.5 "Neither have much wrong with them when we eliminate only one or the other gene," says Thompson, suggesting that either that Bak and Bax aren't essential to apoptosis or that they somehow compensate for each other when just one is eliminated. Redundant Proteins? Courtesy of Abramson Family Research Inst.Craig Thompson Thompson and his collaborators decided to see what would happen if both proteins were deleted. If the proteins were redundant (both serving the same regulatory function), then the mice would show evidence of a disrupted apoptotic pathway. And indeed, "when we bred the Bak-/- and Bax-/- mice together," Thompson says, "there were problems." Fewer than 10 percent of the double knockout mice reached adulthood, and those that did displayed severe phenotypic abnormalities, the most striking of which was inter-digital webbing on their paws.3 Inter-digital webbing is a tell-tale sign of the absence of apoptosis, notes Thompson. The double knockout mice also exhibited several other anatomic and neurological abnormalities associated with dysfunctional apoptotic pathways.3 Furthermore, cells that were isolated from the double knockout mice were resistant to apoptosis in response to various external cell death stimuli, including radiation, one of the classic assays for apoptosis.3 The fact that the double knockout mice and the cells derived from them showed signs of disrupted apoptosis, whereas single knockout mice and cells derived from them seemed normal, suggests that Bax and Bak are indeed redundant.3 Because they can compensate for each other when just one is eliminated could partially account for why these proteins' central role in the apoptotic pathway is just now being uncovered. While recognizing the importance of the work, Bruno Antonsson, a senior scientist in protein biochemistry at Serono Pharmaceutical Research Institute in Geneva, raises a question about the redundancy factor. "I am not convinced that the multi-domain proteins, like Bax and Bak, are simply redundant forms," he says. "I believe they have specific functions, although, at the moment, these functions are largely unknown." In a second study, Thompson's group again used murine cells depleted of Bax and Bak to test cellular resistance to additional forms of apoptotic stimuli, including ultraviolet radiation and growth factor deprivation.4 They found that whereas cells deficient in one or the other protein were susceptible to the apoptotic stimuli, the cells from the double knockout mice were resistant, again pointing to the central role of Bax and Bak in regulating the cell death pathway. Without one or the other, apoptosis simply does not happen. This is true even of apoptotic pathways known as "death receptor pathways" previously believed to operate independently of the mitochondria, a surprising finding given that Bak and Bax operate through these organelles. Again using cells from the double knockout mice, researchers tested the cells' resistance to apoptosis that was induced by the Fas death receptor pathway component tBID. (TBID is the caspase-activated form of the Bcl-2 family member Bid). They found that cells from the double knockout mice are completely resistant to tBID-induced apoptosis,4 which, says Thompson, suggests that "under most physiological conditions, it [the Fas death receptor pathway] doesn't work without going through the Bak/Bax pathway." "But that's only half the story," Thompson says of the first two studies. The question then becomes, 'Which are the real effectors?' Bcl-2, for example, is an anti-apoptotic family member that works in opposition to Bak and Bax. "Does Bcl-2 keep you alive," Thompson asks, "or does it just keep Bax and Bak from killing you by binding up with them?" In the third published work,5 Thompson's group evaluated the ability of two other pro-apoptotic Bcl-2 family members, Bid and Bad, to induce apoptosis in cells from Bax-and-Bak deleted mice. In theory, accelerated cell death should occur due to Bid and Bad binding to anti-apoptotic Bcl-2 and Bcl-XL. But, the researchers found that binding does not occur unless either Bak or Bax are present, suggesting that Bcl-2 and Bcl-XL inhibit Bak and Bax, not the other way around. If the latter were true, the mice would have shown signs of apoptosis, since there was nothing stopping them-the anti-apoptotic members were bound up. So Bcl-2 keeps Bax and Bak from initiating death. Bax and Bak are the dominant players, not Bcl-2, Bcl-XL or any of the other pro-apoptotic Bcl-2 family members. This means that "Bax and Bak aren't just thermostats that set the sensitivity of the mitochondrial switch in apoptosis," says Green, whose team studies mitochondria involvement in cellular stress-induced apoptosis. "They're core components of the apoptotic machinery. The striking information contained in this current work is that the other pro-apoptotic Bcl-2 family members, such as Bid, Bad, Bim, and others, must work via Bax and Bak." "This brings us to really making some strong and definitive statements about the apoptotic pathway," says Thompson. "It doesn't work when Bak and Bax are gone." The next stop for Thompson and collaborators is to "figure out what Bax and Bak do biochemically to the mitochondria to cause cells to die." It is known that Bax and Bak promote the release of cytochrome c from the mitochondria8,9 but it is not known how. Green suggests that there are more steps along the apoptotic pathway yet to be discovered. "Some are sure there will be, and most of us hope there will be, because that keeps it fun. The answer is almost certainly yes. What we can't predict right now is whether any of these will turn out to be fundamentally important." When scientists do "fully understand exactly what Bax and Bak do to the mitochondria and how Bcl-2 and Bcl-XL can interfere with this, we will have an extremely important target for drug development," says Green. "This step doesn't only control whether or not caspases will be activated, in many settings it controls whether or not a cell dies, regardless of whether caspases are involved. If we can truly control this, there are amazing implications for medical advance." Leslie Pray can be contacted at lpray@nasw.org. References 1. Z.N. Oltvai et al., "Bcl-2 heterodimers in vivo with a conserved homolog, Bax, that accelerates programmed cell death," Cell, 74:609-19, 1993. 2. T. Chittenden et al., "Induction of apoptosis by the Bcl-2 homoloue Bak," Nature, 374:733-6, 1995. 3. T. Lindsten et al., "The combined functions of proapoptotic Bcl-2 family members Bak and Bax are essential for normal development of multiple tissues," Molecular Cell, 6[6]:1389-99, December 2000. 4. M.C. Wei et al., "Proapoptotic Bax and Bak: a requisite gateway to mitochondria dysfunction and death," Science, 292:727-30, April 27, 2001. 5. W.-X. Zong et al., "BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak," Genes and Development, June 15, 2001. [in press] 6. Y. Tsujimoto et al., "Involvement of the bcl-2 gene in human follicular lymphoma," Science, 228:1440-3, 1985. 7. C.M. Knudson et al., "Bax-deficient mice with lymphoid hyperplasia and male germ death," Science, 270:96-99, 1995. 8. R. Eskes et al., "Bax-induced cytochrome c release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions," Journal of Cellular Biology, 143:217-24, 1998. 9. J.M. Jurgensmeier et al., "Bax directly induces release of cytochrome c from isolated mitochondria," Proceedings of the National Academy of Science, 95:4997-5002, 1998.

Life or Death in Cells

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