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One response that cells make to chemical toxicants is the highly regulated form of cell suicide known as apoptosis. For several years, Sten Orrenius, professor of toxicology in the Institute of Environmental Medicine of Sweden's Karolinska Institute, Stockholm, and his colleagues have been investigating how this process takes place. Many chemicals stimulate apoptosis, Orrenius explained, by triggering involvement of the cell's mitochondria. APOPTOSIS IS a "clean" kind of cell death in which cells shrink, their chromosomes are chopped into small pieces, and the entire cell is eventually packaged into small, membrane-encased packets that can be engulfed either by neighboring cells or by macrophages, immune system cells designed for this task. At the heart of this controlled destruction are a family of protein-chopping enzymes known as cysteine-aspartate proteases (caspases). The entire process is quite different from the other common form of cell death, necrosis, in which cellular regulation seems to break down altogether. In this type of cell death, the cells swell until they burst, spilling their contents into the space around them, often leading to inflammation and other harm for neighboring cells. Orrenius' lab and others have been working to identify the molecular and cellular mechanisms that trigger apoptosis and carry it through until the cell is gone. Several different kinds of chemicals can trigger apoptosis, Orrenius said. These range from anticancer drugs like etoposide and staurosporine to natural cell-signaling molecules such as glucocorticoids to environmental contaminants like tributyltin and dioxins. Mitochondria play an important role in the basic process of chemically triggered apoptosis. Chemical toxicants stimulate the release of several proteins from mitochondria, including cytochrome c, a protein that's normally found in the space between the inner and outer membranes of this organelle, where it plays an important role in respiration. Once released from mitochondria, however, cytochrome c binds to an adaptor molecule called apoptotic protease activating factor-1 and to the inactive form of a caspase known as pro-caspase-9 to form a complex known as an apoptosome. The result is the cleavage and activation of pro-caspase-9, which then activates other pro-caspases that carry out the breakdown steps of apoptosis. What causes mitochondria to release cytochrome c is not well understood. Recent studies suggest that there must be several different ways for this release to happen. One route involves the opening of permeability transition pores in the mitochondrial membrane, a process that involves Ca2+ ions and causes the mitochondria to swell and their outer membrane to rupture, releasing cytochrome c. But cytochrome c can also be released from mitochondria with intact membranes, and experiments in Orrenius' lab find that agents like the anticancer drug etoposide stimulate cytochrome c release even when the formation of permeability transition pores is inhibited. REGULATED PROCESS Many chemicals that kill cells do so in part by interacting with the cell's mitochondria. There they stimulate the release of cytochrome c (Cyt c), apoptosis-inducing factor (AIF), and possibly other proteins from reserves between the organelle's inner and outer membrane as part of a highly regulated process of cell death known as apoptosis. Once released, cytochrome c binds to an adapter molecule, apoptotic protease activating factor (Apaf-1), and the inactive form of a protein-digesting enzyme, pro-caspase-9, to form an apoptosome complex. The complex cleaves the inactive pro-caspase, releasing two subunits that recombine to form caspase-9 in its active form. This caspase, in turn, cleaves and activates caspase 3, starting a chain of events that leads to cell destruction. Many signaling proteins regulate aspects of this process, some of which are also shown. Those in green stimulate apoptosis; those in red inhibit it. The p53 protein "plays this very central role in mediating an external signal to change gene expression, and that's key to a cell's response to a chemical." IT MAY BE that the release of cytochrome c involves permeabilization of the mitochondria's outer membrane by apoptotic signaling proteins of a family known as Bcl-2 proteins, Orrenius suggested. Some members of this family prevent apoptosis, but others help to trigger it. Two proteins of this family, Bax and Bid, for example, are known to induce cytochrome c release, and they do so without altering the structure of the mitochondria. Recent studies show that Bid and another protein from the Bcl-2 family, Bik, can also stimulate cytochrome c release but by a different route. Normally, cytochrome c is anchored to the inner membrane of mitochondria by interaction with the mitochondrial lipid cardiolipin, and studies suggest that this lipid may also be a target of action of Bcl-2 proteins that stimulate apoptosis. Cytochrome c release may be a two-step process, Orrenius suggested, in which the protein is first liberated from cardiolipin and then released through pores or channels that form in the mitochondria's outer membrane. Recent work in Orrenius' lab suggests that two proteins activated in cells under stress may protect the cells from harm by interfering with the mitochondria's role in apoptosis. The proteins are known as heat-shock proteins, although they respond to other types of stress as well. Heat-shock protein 27 (Hsp 27) seems to work by preventing the release of cytochrome c, Orrenius suggested. Hsp 72, by contrast, does not seem to interact with mitochondria directly, but rather to interfere with the activation of caspases by the apoptosome. According to toxicologist James L. Stevens, a research adviser at Lilly Research Labs, Greenfield, Ind., another cellular organelle, the endoplasmic reticulum, also plays a key role in the cell's response to chemical stress. Several years ago, Stevens, then at the W. Alton Jones Cell Science Center in Lake Placid, N.Y., and his colleagues noted that genes that respond to stress in the endoplasmic reticulum are activated by some chemical toxicants. Further, they found, if these genes are activated before cells are exposed to toxicants, the cells are protected from injury. "Before this work, we had no information that the endoplasmic reticulum was a particularly important organ in cell injury," Stevens said. "We tried to probe a number of biochemical responses to see which ones might be most important in this particular endoplasmic reticulum-based stress response," Stevens explained. "By a process of elimination, we arrived at the conclusion that calcium and the ability of the endoplasmic reticulum to control cellular calcium levels is a critical event, both in cell injury and in cell repair and protection." To confirm that processes taking place in the endoplasmic reticulum are important to the cell's response to toxic agents, the authors used an antisense agent to block one of the endoplasmic reticulum response genes, one that produces a protein called glucose-regulated protein 78 (Grp-78). By preventing an increase in this particular protein, the researchers eliminated the protective effect of prior stress. MOST OF Stevens' studies have been with halogenated hydrocarbons that damage kidney epithelial cells. He has also done studies in intact rat kidneys. Halogenated hydrocarbons cause both necrosis and apoptosis in kidney cells, Stevens said. Recent work indicates that preactivating endoplasmic reticulum response genes can protect cells from both kinds of cell death, but the mechanisms of this protection appear to be different for the two types of cell death. In a second set of experiments using an antisense agent to block Grp-78, Stevens found that the cells became resistant to death by apoptosis. The antisense agent, they found, was causing two effects. In addition to blocking production of Grp-78, the agent was also causing an increase in the production of another stress response protein called Grp-94. This second protein, they believe, has a greater role than does Grp-78 in protecting cells against death by apoptosis. "We're not at this point entirely certain how this protection works, because every time we try to perturb one part of the endoplasmic reticulum stress response pathway, other parts tend to upregulate to compensate," Stevens said. "That tells us this is a very plastic system that can adjust to a variety of stresses." Indeed, plasticity and variety of response to chemical stresses seemed a common theme among the speakers at the symposium. As Orrenius put it, "A cell's response to chemical toxicity is complex. It can be modulated by activation of defense mechanisms, and the final outcome is governed by this balance." |
| UPDATE | 10.01 |
| AUTHOR | Karolinska Institute's Institute of Environmental Medicine's Sten Orrenius |
| LITERATURE REF. | This data is not available for free |
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