Death in the Balance

In 1997, to the surprise of many researchers, mitochondria reclaimed the limelight of apoptosis research when several groups observed that cytochrome c released from the mitochondria could induce apoptosis in cell-free systems.1,2 A hot research topic decades ago, mitochondria excitement cooled in the 1970s when researchers agreed on the mechanisms of oxidative phosphorylation (ox-phos). Now, mitochondria-mediated apoptosis is believed to be central to a number of major debilitating diseases, including Parkinson's disease and cancer.3,4 Just why mitochondria assume this seemingly disparate role is the subject of speculation. Neil Blackstone, associate professor of biological sciences, Northern Illinois University, DeKalb, and Douglas Green, head of the division of immunology at the La Jolla Institute for Allergy and Immunology, Ontario, Calif., believe that it is an evolutionary issue, one that goes back to the mitochondria's roots as an endosymbiont.5 They suggest that after ancient eukaryotes took in protomitochondria, mitochondria were forced to interact with their environment through their hosts. The protomitochondria weren't completely at the eukaryotes' mercy, however; they could manipulate the host cell phenotype with by-products of ox-phos, and, in particular, reactive oxygen species (ROSs). ROSs raise the mutation rate and, some think, sexual recombination along with it.6 In good times, that is, times of rapid growth, the mitochondria would crank out adenosine triphosphate (ATP), keeping the redox state of the mitochondria fully oxidized. However, during times of slow growth, the equilibrium would shift away from ox-phos and toward ROS production, which could, because of their penchant to induce mutations, lead to novel and potentially more advantageous phenotypes. Later, the genomes of the protomitochondria and their host became integrated, and Green hypothesizes that at this point, the system was streamlined to involve just cytochrome c. 'A Pore Way to Die' Despite the confusing literature about mitochondria's role in apoptosis, one fact is indisputable: cytochrome c release is key to the current research excitement, says Dave Nicholls, professor of mitochondrial physiology at the Buck Institute for Age Research in Novato, Calif. An area that is still under investigation and has generated some conflicting theories concerns how cytochrome c escapes from the mitochondria. While the outer mitochondrial membrane contains pores, normally only small molecules, 1,000 daltons (Da) molecular weight or less, can pass through, far smaller than the 10,000 Da cytochrome c. As with apoptosis itself, more than one mechanism likely exist. One theory says cytochrome c release is mediated by a permeability transition, brought about by severe oxidative stress. When cells are stressed, either by injury or certain toxins, mitochondrial swelling occurs, which can lead to the outer mitochondrial membrane rupturing and the release of protein from the inter-membrane space. Such a permeability transition also collapses the mitochondrial membrane potential, which is vital to ATP production through the respiratory chain. (Herein lies a cause-and-effect question: whether cytochrome c release occurs before or after mitochondrial dysfunction). Under these conditions, an orderly apoptosis, which requires ATP and hence functional mitochondria, is not possible. Andrew Halestrap, professor of biochemistry at the University of Bristol, compares this to a horrific car accident, in which the car breaks apart and its contents are thrown everywhere. From this, there is no return, and rather than apoptosis, necrosis (an entirely different but related process) ensues. A second, more regulated mechanism for cytochrome c release could involve the formation of specific channels. The Bcl-2 family, a well studied and ubiquitous family of proteins, appears to be in control here. One member keeps apoptosis in check; the other is thought to promote cytochrome c release. But it's not quite that simple; other proteins and factors exist both in the cytoplasm and mitochondria that modulate the apoptotic response. Cytoplasmic anti-apoptotic factors prevent cytochrome c from initiating apoptosis, while the mitochondria has a second line of attack, SMAC (Second Mitochondria-derived Activator of Caspase), that shifts the balance back toward apoptosis. Nicholls likens it to an arms race between the mitochondria and cytoplasm for survival or death. Nicholls says, "These check-and-control mechanisms are very important, because cytochrome c can very easily dribble out of mitochondria--it just needs a slight tear in the membrane--and the last thing you would want to do is kill the cell." In addition, some researchers see mitochondria in a supportive rather than a starring role in apoptosis.7 Andreas Strasser, a research fellow at the Walter and Eliza Hall Institute of Medical Research in Australia, says, "We believe that physiological cell death uses the mitochondria as an amplification mechanism, but not as an initiating event. Certainly, some nonphysiological death stimuli, such as certain chemotherapeutic drugs, can, amongst other things, damage mitochondria and thereby activate this amplification loop directly." Attacking Apoptosis Companies are gearing up to attack apoptosis, because the list of diseases where apoptosis plays a role includes many major health problems, such as cancer and stroke, and neurodegenerative disorders Parkinson's and Huntington's diseases. Until the ambiguities of the pathways' initial stages are cleared up, some companies, like Idun Pharmaceuticals in San Diego are concentrating on down-stream events. Currently, Idun has an apoptosis inhibitor (a broad spectrum caspase inhibitor) in Phase I clinical trials, where the hope is to stem the tide of cell death during acute organ failure. Also, Idun and Maxim Pharmaceuticals, also in San Diego, are trying to unleash apoptosis in cancer by targeting the apoptosis-inhibitor Bcl-2. And Genentech, in South San Francisco, Calif., and Immunotech of Seattle, Wash., two biotech giants, are teaming up to develop a cancer therapy based on TRAIL/Apo2 Ligand, an inducer in the mitochondrial-independent pathway of apoptosis (See "New Hope in Finding the Magic Bullet"). A company well-positioned to capitalize on mitochondria's role in disease is San Diego-based MitoKor. Since 1996, MitoKor scientists have been developing technology to assess the effects of potential drugs on mitochondrial function. Now that they have several cell- and mitochondria-based assays in place, they have entered the drug discovery phase, using their technology platform as the engine, according to Anne Murphy, director of mitochondrial biology. While not revealing exactly what molecules MitoKor is targeting, Murphy points out that mitochondria present all the same opportunities as the cell does in terms of drug design and discovery. Pharmaceutical companies have long targeted signaling pathways, transporters and ion channels for therapeutic products. Now, since the discoveries that they play a role in mitochondria-mediated apoptosis, they likely will make the hit list for drug discoveries in this field. This particular branch of apoptosis research is still in its infancy, and according to Nicholls and Murphy, is somewhat contradictory and difficult to interpret-- primarily because some researchers don't appreciate the methodological subtleties for studying mitochondrial physiology. Rather than complain, the two researchers are doing something about this. Last month, they jointly offered an advanced course in mitochondrial physiology to instruct others on how to apply basic bioenergetic principles to mitochondria in situ. Nicholls says, "The field is new, confusing and contradictory. It's different from a mature field where there is total consensus. Here there's total confusion--confusion because there are multiple forms of apoptosis, many models, and some of the techniques [are] not being used appropriately." Laura Defrancesco can be contacted at defrancesco1@earthlink.net. References 1. P. Li et al., "Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade," Cell, 91: 479-89, 1997. 2. R.M. Kluck et al., "Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system," The European Molecular Biology Organization Journal, 16: 4639-49, 1997. 3. N. Rampino et al., "Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype," Science, 275:967-9, 1997. 4. D.C. Wallace, "A mitochondrial paradigm for degenerative diseases and ageing," Novartis Foundation Symposium, 235:247-63, 2001. 5. N.W. Blackstone, D.R. Green, "The evolution of a mechanism of cell suicide," BioEssays, 221:84-8, 1999. 6. R.J. Brennan et al., Oxidative mutations induce intrachromosomal recombination in yeast," Mutation Research, 308:159-67, 1994. 7. D.C Huang, A. Strasser, "BH3-Only proteins-essential initiators of apoptotic cell death," Cell, 103[6]:839-42, 2000.

Death in the Balance

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