by Nick Lane
In their 1972 paper, Kerr, Wyllie, and Currie presented evidence that the form of cell death is basically similar in numerous disparate circumstances—in normal embryonic development as well as teratogenesis (malformation of the embryo); in healthy adult tissues, cancers, and tumour regression; and in the shrinkage of tissues with disuse and ageing. Apoptosis is also critical to immune function: immune cells that react against our own body tissues commit apoptosis during development, enabling the immune system to distinguish between ‘self’ and ‘non-self’. Thereafter, immune cells exert many of their own effects by inducing damaged or infected cells to undergo apoptosis themselves. This kind of screening by immune cells eliminates incipient cancer cells before they get a chance to proliferate.
The sequence of events in apoptosis is precisely choreographed. The cell shrinks and begins to develop bubble-like blebs on its surface. The DNA and proteins in the nucleus (the chromatin) condense in the vicinity of the nuclear membrane. Finally, the cell breaks up into small membrane-wrapped structures called apoptotic bodies, which are taken up by immune cells. Effectively, the cell packages itself into bite-sized chunks, which are then cannibalized without fanfare. Consistent with such a choreography, apoptosis requires a source of energy in the form of ATP—if deprived of ATP, a cell cannot undergo apoptosis. So the process is very different from the swelling and rupture characteristic of necrosis, the violent unpremeditated form of cell death. Also unlike necrosis, there is no aftermath to apoptosis, in particular no inflammation: nothing to mark the passing of a cell but its absence. It is a death foretold, but unremembered.
The executioners
For more than a decade, Andrew Wyllie and a handful of others persevered, evangelists of apoptosis, in the face of indifference in the wider biological community. Wyllie began to convert the unbelievers through his discovery that, in apoptosis, the chromosomes break up into segments that exhibit a characteristic laddering pattern on biochemical analysis. This finding enabled apoptosis to be diagnosed in the lab, overcoming the cynical biochemists’ perpetual suspicion of electron-microscope artefact. But the real turning point came in the mid 1980s, when Bob Horvitz, at MIT in Boston, set about identifying the genes responsible for apoptosis in the nematode worm Caenorhabditis elegans, research for which he shared the Nobel Prize in 2002. C. elegans is a tiny, microscopic worm which offered several big advantages—first, it is transparent, so researchers could actually make out the fate of individual cells down the microscope; second, a small, predictable group of cells, 131 of the 1090 somatic cells (body cells, as opposed to germ-line cells) comprising the nematode, die by apoptosis during embryonic development; and third, the mean lifespan of C. elegans is barely 20 days, so its swift development is easily tracked in the lab.
Horvitz and his colleagues discovered several genes that coded for the effectors of cell death in nematodes—the death genes. Their findings were fascinating in their own right, but by far the most unexpected and important discovery was that there were exact equivalents of the death genes in flies, mammals, and even plants. Cancer researchers had already identified some of these genes at the time, but why or how they were involved in cancer was still unknown. The link with nematodes made their function clear, while giving another demonstration of the fundamental unity of life. Not only were the human genes unambiguously related to the nematode genes, but also they could even be genetically engineered to replace the nematode genes in the worms themselves, where they worked equally well! Mutations that disabled any of the death genes prevented the nematodes from losing their 131 cells by apoptosis as usual. The implications for cancer were plain: if the same mutations had a similar effect in people, then incipient cancer cells would likewise fail to commit suicide, and would instead continue to proliferate to form a tumour.
By the early 1990s, researchers realized that a number of oncogenes and tumour suppressor genes, which we discussed earlier as the causes of cancer, did indeed control the fate of the cell through their effect on apoptosis. In other words, cancers arise from cells that have lost the ability to kill themselves by apoptosis, after mutations in the death genes. The death genes are any genes that normally cause a cell to commit apoptosis, and so can potentially include both oncogenes and tumour-suppressor genes, both of which can overrule a cell’s commitment to die in the interest of the body as a whole. As Wyllie put it at the time: ‘The ticket to cancer comes with a ticket to apoptosis built in; the apoptosis ticket has to be cancelled before reaching cancer.’
The executioners responsible for carrying out the cell death program are proteins known as caspases (a rather more evocative name than the biochemists’ original ‘cysteine-dependent aspartate-specific proteases’). More than a dozen different caspases have now been discovered in animals, 11 of which also operate in people. All work in essentially the same way: they slice up proteins into bits and pieces, some of which are activated in turn, such that they go on to degrade other components of the cell, like DNA. Interestingly, the caspases are not made to order when needed, but are produced continuously, whereupon they wait in an inactive state for the call to arms: they hang poised over the cell like the sword of Damocles, suspended by a thread above the man who would be king. It is a sobering thought that almost all eukaryotic cells contain within themselves, at all times, this silent apparatus of death.
We can be grateful, we who sit beneath the suspended sword, that the thread is strong. Once the caspases have been activated, there is little hope of turning back the clock; but many checks and balances must be triggered before the ancient machinery grinds into operation. These controls are the subject of nearly two decades of intensive research, and the welter of names and acronyms is enough to confuse all but the most dedicated student. The situation is not helped by the retention of historical names for the same gene in different organisms. I am reminded of Celtic music, in which the same tune goes by several names, and the same title refers to several different tunes: an endless stream of lovely variation, but scarcely conducive to a straightforward understanding. Just to give a genetic example, the gene ced-3 in nematode worms is known as nedd-2 in mice, dcp-1 in Drosophila, and ICE, or interleukin-1 beta-converting enzyme, in humans (as at the time it was known to be involved in the production of the immune messenger, interleukin 1-beta). After discovering its importance in nematode worms, ICE turned out to be the prototype caspase in humans too, and it is now known as caspase-1, although it seems to play a lesser role in human apoptosis. Similar caspase enzymes, and related ones called para-caspases and meta-caspases, have been found in fungi, green plants, algae, protozoa, even sponges: they are virtually universal among the eukaryotes, and so presumably their forerunners were present in some of the earliest eukaryotes, perhaps 1.5 to 2 billion years ago.
There is no need for us to get bogged down in the detail. Suffice to say that the regulation of apoptosis is complex, involving a number of steps in which one caspase sets off the next in a cascade, leading finally to the activation of a small army of executioners, which slice up the cell.1 Virtually all these steps are opposed by other proteins, which are responsible for counteracting the cascade, thereby preventing a false alarm turning into an orgy of death.
Mitochondria, angels of death
This was the state of knowledge a decade ago, in the mid 1990s. None of it has been contradicted. Yet since then, there has been a change of emphasis that amounts to a revolution, overturning the nascent paradigm. The paradigm was that the nucleus is the operations centre of the cell, and controls its fate. In many respects this is of course true, but in the case of apoptosis it is not. Remarkably, cells lacking a nucleus can still commit apoptosis. The radical discovery was that the mitochondria control the fate of the cell: they determine whether a cell shall live or die.
There are two ways in which the apparatus of death is sprung. These originally seemed quite distinct, but more recent work shows that they share some common features. The first mechanism is known as the extrinsic pathway, because death i
s signalled from the outside, via ‘death’ receptors on the outer membrane of cell. For example, activated immune cells produce chemical signals (such as tumour necrosis factor) that bind to the death receptors on incipient cancer cells. The death receptors pass on the message, activating the caspases within the cell, to induce apoptosis. While many details clearly needed filling in, the broad outline seemed plain enough. Not a bit of it!
The second route to apoptotic death is called the intrinsic pathway. As the name suggests, the impetus to commit suicide comes from within, usually from cell damage. For example, DNA damage from ultraviolet radiation activates the intrinsic pathway, leading to apoptosis of the cell, without any external signal. Hundreds of triggers have now been discovered that activate the intrinsic pathway of apoptosis—they do not operate through ‘death receptors’, but damage the cell directly. The sheer variety of these is breathtaking. Many toxins and pollutants can cause apoptosis, as do some chemotherapeutic drugs used for treating cancer. Viruses and bacteria can provoke it directly, most notoriously in the case of AIDS, where the immune cells themselves die. Many physical stresses cause apoptosis, including heat and cold, inflammation, and oxidative stress. And cells may commit apoptosis in waves of death following a heart attack or stroke, or in a transplanted organ. All these diverse triggers independently bring about the same response, the activation of the caspase cascade, and so produce a similar pattern of cell death by apoptosis in each case. Presumably, the signals had somehow to converge on the same ‘switch’. All somehow had to convert an inactive form of a caspase enzyme into the active form, a biochemical task that is as specific as the turn of a key in a lock. But what on earth could recognize such a diverse array of signals, calibrate their strength, and then integrate them into a single common pathway by turning the caspase key in its lock?
The first part of the answer was supplied in 1995 by Naoufal Zamzami and his colleagues, in Guido Kroemer’s research team at the Centre National de la Recherche Scientifique, in Villejuif, France. Their results were published in two papers in the Journal of Experimental Medicine, which came to be among the most widely cited papers in medical research. A number of factors had already suggested that mitochondria might play a role in apoptosis, but Kroemer’s team proved that mitochondria are in fact key to the process. In particular, they showed that a loss of the membrane potential across the inner mitochondrial membrane—the proton gradient generated by respiration (see Part 2)—was one main trigger for apoptosis. If the inner membrane depolarized for a period, then the cells invariably went on to commit apoptosis. In their second paper, Kroemer’s team showed that the process takes place in two steps. The initial membrane depolarization was followed by a burst of oxygen free-radical generation, which seemed to be required for apoptosis to progress to the next stage.
This mitochondrial two-step—membrane depolarization and free-radical release—takes place in response to virtually all intrinsic triggers. In other words, the mitochondria act as both sensors and transducers of a wide variety of cell damage. Transferring apoptotic mitochondria into a normal cell is enough to cause the nucleus to fragment and the cell to die by apoptosis. Conversely, blocking the mitochondrial two-step can delay or even prevent cells from committing apoptosis. But a question remained: how do apoptotic mitochondria communicate with the rest of the cell? In particular, how do they activate the caspase enzymes?
The answer came from Xiaodong Wang’s group at Emory University in Atlanta, Georgia, in 1996, and was greeted with ‘general stupefaction’, as one expert put it. It was cytochrome c. We met cytochrome c, if you recall, in Part 2. It is a protein component of the respiratory chain, originally discovered by David Keilin in 1930, and is responsible for shuttling electrons from complex III to complex IV of the chain. It is normally tethered to the outside of the inner mitochondrial membrane, adjacent to the inter-membrane space (see Figure 5, page 77). Wang’s group discovered that, in apoptosis, cytochrome c is released from the mitochondria. Once free in the cell, it binds to several other molecules to form a complex (the apoptosome), which in turn activates one of the final executioners, caspase 3. Release of cytochrome c from the mitochondria commits the cell inexorably to die—as indeed does just injecting it into a healthy cell. In other words, an integral component of the respiratory chain (which generates the energy needed for the life of the cell) turns out to be an integral component of apoptosis, responsible for the death of the cell. The link between life and death hinges on the subcellular location of a single molecule. Nothing in biology quite compares with this two-faced Janus: life, looking one way, death the other, the difference between the two but a few millionths of a millimetre.
Cytochrome c is not the only protein released from the mitochondria in this way. A number of other proteins are also released, which play a role in apoptosis too—sometimes a more prominent role than cytochrome c. Some of these additional proteins activate caspase enzymes, while others (such as the apoptosis-inducing factor, or AIF) attack other molecules, like DNA, without the involvement of the caspase enzymes. Like so much in biochemistry, the details often seem endlessly involved, but the underlying principles are simple enough: depolarization of the mitochondrial inner membrane and free-radical generation releases cytochrome c and other proteins into the cytosol, which set in motion the enzymes that slice up the cell.
Battle of life and death
If the life or death of the cell depends on the location of cytochrome c and its companions of doom, it’s not surprising that medical research has focused on the specific mechanism that releases these molecules from the mitochondria. Again the answer is complicated, but helps clarify the link between the intrinsic and extrinsic pathways of apoptosis. Overlooking a few exceptions, likely to be refinements, these findings place the mitochondria at the centre of both forms of cell death. In almost all cases, the basic apparatus of death is controlled by the mitochondria. When enough mitochondria in a cell spill out their death proteins—probably beyond a threshold point of no return—then the cell inexorably goes on to kill itself.
The release of cytochrome c takes place in two steps, according to recent research from Sten Orrenius and his colleagues at the Karolinska Institutet in Stockholm. In the first step, the protein is mobilized from the membrane itself. Cytochrome c is normally bound loosely to lipids (especially cardiolipin) in the inner mitochondrial membrane, and is released only upon the oxidation of these lipids. This explains the requirement for free radicals in apoptosis: they oxidize the lipids in the inner membrane to release cytochrome c from its shackles. But this is still only half the story. Cytochrome c is mobilized into the inter-membrane space, and it can’t escape from the mitochondria altogether until the outer membrane has become more permeable. This is because cytochrome c is a protein, and so is too large to cross the membrane in normal circumstances. If it is to escape the mitochondrion, some sort of pore must breach the outer membrane.
The nature of the pore that opens in the outer mitochondrial membrane has foxed researchers for a decade or more. It seems probable that several distinct mechanisms can operate under different circumstances, giving rise to at least two different types of pore. One mechanism apparently involves metabolic stress to the mitochondria themselves, which leads to excess free-radical generation. The rising stress opens up a pore in the outer membrane, known as the permeability transition pore, leading to swelling and rupture of the outer membrane, coupled with the release of proteins.
Another pore, which may be of more general significance, involves a large family of proteins known rather dryly as the bcl-2 family. The name is now largely irrelevant, and stands for ‘B cell lymphoma/leukaemia-2’, which refers to the oncogene discovered by cancer researchers in the 1980s. At least 21 related genes have since been discovered, which code for proteins in the family. These proteins fall into two broad groups, which battle among themselves in ways that are complex and still largely obscure. One group protects against apoptosis. They are found in the outer mitochondrial
membrane and seem to prevent the formation of pores, thus blocking the release of proteins like cytochrome c into the cytosol. The other group are diametrically opposed: they act to form pores, apparently large enough to allow the escape of proteins from the mitochondria directly. This group thereby promotes apoptosis. They are normally found elsewhere in the cell, and migrate to the mitochondria upon receiving some sort of signal. The final outcome—whether or not the cell commits apoptosis—depends on the numerical balance of the feuding family members in the mitochondrial membrane, and the number of mitochondria embroiled in the battle. If the pro-apoptosis members outnumber their protective cousins in a sufficient number of mitochondria, the pores open, the death proteins are spilled out from the mitochondria, and the cell goes on to kill itself.
The existence of the feuding bcl-2 family helps explain the links between the two different forms of apoptosis, the intrinsic and extrinsic pathways. Many different signals alter the balance of the feud in the mitochondria, either in favour of or against apoptosis. For example, both the ‘death’ signals from outside the cell (the extrinsic pathway) and the ‘damage’ signals from inside the cell (the intrinsic pathway) alter the feuding family balance in favour of apoptosis.2 Thus the bcl-2 proteins integrate a diverse array of signals from both outside and inside the cell, and calibrate their strength in the mitochondria. If the balance favours death, pores form in the outer membrane, cytochrome c and other proteins spill out, and the caspase cascade is activated. Thus the final events are the same in most cases.
