Monday, January 14, 2013

The human machine: decommissioned components

The previous post in this series can be found here

Happy 2013 from all of us here in the Trenches! We successfully made it one more time around the sun, and if that's not a good excuse for a party I don't know what is! Sadly, however, not all of your cells have been having such a swimmingly good time since the calendar ticked over to January the first - in fact nearly one trillion of them have died in the past fortnight alone, at a rate of roughly 70 billion a day, or 800,000 per second. Don't be alarmed, however, as this has been going on for your whole life and is a vitally important part of being a multicellular organism such as yourself. A human without cell death would be like society without human death - overcrowded, unpleasant, and rife with infirmity. Your body needs a system by which damaged, old, or infected cells can be removed in a controlled manner; this process is known as apoptosis.

In this post I will be discussing what we know about how apoptosis works and how it is a key player in the development of cancer and the fighting of infectious disease. I'll also show how our understanding of how this process works has allowed us to devise targeted therapeutics against a number of debilitating conditions.

Cellular suicide - picking the moment

Your cells are team players - they're willing to do anything to serve you, including laying down their lives. Apoptosis depends on this loyalty because it is actually a form of suicide that your cells perform on themselves. Arguably the most important aspect of this is timing - if your cells are in the habit of committing suicide before it is necessary then you'll waste a lot of energy and resources building replacements that shouldn't be needed. On the other hand, if the cell leaves it too late to kill itself then it may find itself incapable of doing so.

So, how does a cell know when to die? Well the most obvious markers for cell death are simply the various forms of damage that can occur to the components of the cell itself. If a cell's membrane becomes damaged, for example, this can cause excess calcium to leak into the cell and so be sensed by a number of calcium-binding proteins, such as calpain, which in turn signal that apoptosis should begin. Similarly, damage to DNA is sensed by the complex machinery of the DNA repair pathway. For example, PARP is a protein that binds to single-strand breaks in DNA caused by DNA damaging agents such as radiation (think sunburn!) or chemical mutagens like free radicals. PARP and other DNA damage sensors relay their information to a number of signalling proteins, most importantly p53. If p53 is activated in response to DNA damage it signals to stop the usual processes of cell division and begin DNA repair, but if the damage is just too bad it makes the call to start apoptosis and destroy the cell.

These causes of apoptosis are known as 'intrinsic' factors because they emerge from effects sensed within the cell. Other intrinsic factors include heat damage to the cell; sensing viral or bacterial proteins inside the cell (a clear sign that it's been infected and must die asap!); the lack of nutrients in the cell's metabolic processes; and a lack of oxygen (hypoxia). Hypoxia is possibly the most clinically relevant cause of apoptosis because it is responsible for the lasting damage caused by blood flow disorders such as occur during a heart attack or stroke. In these events, blood flow (and so oxygen supply) to either the heart or brain tissue is temporarily cut off, inducing widespread apoptosis in that area and so causing significant damage to the affected organ. Studying the genetic predisposition of individuals to hypoxia-induced apoptosis is an expanding area of research as it will allow better predictions of the severity of stoke or heart attack damage and so allow treatment to be tailored accordingly. 

'Extrinsic' activators of apoptosis are ones that are presented by other cells in order to instruct a cell to die. This is most often the case when a cell is infected with a virus but the viral components have evaded detection within the cell. In this situation, killer T cells (which I've discussed in a previous post - here) detect viral components on the surface of the infected cell and so give it its final orders in the form of a protein called the Fas ligand. The Fas ligand is presented on the surface of the T cell and binds to the Fas receptor on the surface of the infected cell, which kick starts the whole process of apoptosis.

Going from initiation to action

So, our unhappy cell is ready to go, it's had its orders and needs to start the process of packing itself up. What happens now is dependent on whether the initial signal was intrinsic or extrinsic, but both have the same outcome. Ultimately, both pathways cause the activation of a host of enzymes that deconstruct the cellular skeleton, dismantle its organelles, and demolish its DNA. Taking the last of these as an example, DNA in an apoptotic cell is slowly condensed into a single mass (pyknosis) before being released from its usual home of the nucleus and into the rest of the cell (karyorrhexis), and then finally enzymatically chopped up and disposed of by a family of proteins called the DNAses (karyolysis). Similar events happen for every component of the dying cell until the cell begins to break apart into small blobs called apoptotic bodies that are then devoured by roaming macrophages and the cell is finally no more.

The death throes of an apoptotic cell breaking up into apoptotic bodies and being consumed.

As I say, however, how we get to this point depends on where the cell's instructions came from, but both rely heavily on a group of proteins called caspases (short for 'cysteine-dependent aspartate-directed proteases', but which I like to think were named after Casper the friendly ghost). Caspases are enzymes that cleave other proteins, particularly other caspases. Normally, caspases are not active in a cell because their full structure is self-inhibiting, but they can become activated by cleaving off a small part of their structure. This means that an active caspase can activate other caspases, which then activate more caspases and so on in a cascade of caspase activity. This is irreversible - once the caspases are firing a cell's time is up! 

Extrinsic death signals usually cause the formation of the death-inducing signalling complex (DISC), which forms around the aforementioned Fas receptor at the cell membrane. DISC contains two caspases, caspase 2 and caspase 8, which become active within the complex. Their activity has two main effects: the activation of caspase 3, which is the caspase responsible for activating most of the demolition machinery mentioned earlier; and the activation of a protein called tBid. The role of tBid is linked closely to the mechanism of intrinsic apoptosis.

Apoptosis caused by intrinsic or extrinsic signals - different routes, same outcome.

The intrinsic pathway for apoptosis is closely linked to the energy-releasing organelles of the cell, mitochondria (whose role I've discussed before - here). Within mitochondria are a range of proteins of the Bcl-2 family. Some of these a pro-apoptotic (e.g. Bax, Bad, Box etc.) whereas others are anti-apoptotic (Bcl-2, Bcl-xl, Bcl-w etc.). Whether apoptosis occurs due to intrinsic signals depends on the balance of the pro- and anti-apoptotic proteins in the mitochondria. The intrinsic causes of apoptosis that I mentioned above cause the activation of a number of pro-apoptotic proteins and the inactivation of the anti-apoptotic ones, primarily though signals relayed through the aforementioned p53 protein. tBid, activated by extrinsic signals, also promotes the pro-apoptotic cause in mitochondria by inhibiting the anti-apoptotic proteins. Going through exactly what all these various proteins do would take me a lot longer than I have here, but it is the outcome that we're interested in anyway! A mitochondrion in which the pro-apoptotic proteins are winning becomes extremely porous, spewing much of its internal components into the rest of the cell. These include a number of proteins that bind to IAP (inhibitor of apoptosis) proteins and block their activity. IAPs usually bind to and inactivate caspases, so their inhibition by mitochondrial proteins causes caspase activation - thereby starting the whole business of packing up the cell.

One very interesting example of a mitochondrial protein promoting apoptosis is cytochrome c. As discussed previously, cytochrome c is usually involved in the mitochondrial electron transport chain, where is acts as an electron-carrier in cellular energy processing. However, once it is free of the mitochondria, cytochrome c is able to bind to a protein called Apaf1. This binding event alters the structure of Apaf1 such that it starts to cluster together into a wheel-like structure containing 7 copies of Apaf1 and 7 or cytochrome c. This structure is colourfully referred to as the 'wheel of death' (or more boringly, the apoptosome) because, once formed, it is able to activate caspase 9, which in turn activates caspase 3 and voila we have apoptotic lift off! 

The wheel of death! The apoptosome is a key player in cell suicide  (structure from Yuan et al. 2010, Structure of an apoptosome-procaspase-9 CARD complex).

Fine tuning the machine

Apoptosis is, as I say, a process vital to the health of the body as a whole. When it goes wrong, things can get nasty. Cancerous cells have almost always developed partial or complete resistance to apoptosis such that they never die and so are free to divide perpetually and beyond their physiological role. Cells can develop such resistance by accumulating mutations in the proteins that regulate apoptosis such that the death signals are no longer effective. These are grouped into two categories: tumour-suppressor (TS) genes, and oncogenes. A TS gene is one for which a mutation that knocks out its activity can promote cancer. For example, the gene that encodes p53 (TP53) is a TS gene, as without p53 intrinsic death signals cannot be relayed from their initial signals to the mitochondria and so are ineffective. Oncogenes, on the other hand, are genes whose over-stimulation can promote cancer. One such gene is XIAP (X-linked inhibitor of apoptosis protein), which normally regulates apoptosis by binding to and inactivating caspases 3, 7 and 9. Deregulation (i.e. overactivity) of XIAP activity is a factor in a number of lung, prostate, and bowel cancers.

A cruel fact of apoptotic failure is that cancerous cells that are resistant to apoptosis are therefore resistant to the signals that halt cell division upon DNA damage. This means that the cell can continue to accumulate more and more mutations without undergoing the repair pathways that would become active in a non-cancerous cells. Thanks to this, the rate of mutation in cancerous cells can be up to 100 times higher than in non-cancerous cells and so tumours can deteriorate rapidly.

Thanks to our understanding of apoptosis, however, we are now at the stage where we are starting to fight back against death-resistant cells. A number of widely-used anti-cancer drugs, such as Herceptin, Iressa, or Gleevac, work in part through the induction of apoptosis by blocking cellular survival signals, thereby circumventing some of the blocks to the apoptotic machinery that have accumulated due to mutation. Some other treatments target tumour suppressor genes, such as p53, and attempt to reactivate them in the cancerous cells. Future targetted gene therapies may be capable of replacing damaged TS genes in those cells, thereby reconnecting the wiring within cellular signalling that was blocking apoptosis.

Knowledge of apoptosis has also allowed us to connect the dots between some cancers and specific viral infections. A virally infected cell must die as quickly as possible to prevent the virus from spreading. This, needless to say, is bad news for the virus, which will try to stop it at all costs. Some viruses are so effective at this that a by-product of their efforts is a fully cancerous cell. Epstein-Barr virus, for example, has its own version of the Bcl-2 proteins that inhibit apoptotic activation of mitochondria, and so can cause various lymphomas and leukaemias. Similarly, Human Papillomavirus (HPV) is now vaccinated against in young women in many countries because it was discovered its inactivation of p53 and pRb (another TS gene) lead to nearly half a million cases of cervical cancer annually worldwide and 270,000 deaths. Without an understanding of how apoptosis works, such translational treatments would not be possible.

The future

Apoptosis research is still a fairly young field and there is much left to be learned about how apoptosis is regulated in different tissues. One hurdle to such research is that the cell lines molecular biologists usually work with are derived from cancers so that they grow nicely, but these do not display usual apoptotic behaviour and so aren't suitable for work on cell death. As our understanding of apoptosis improves, however, we are better able to say what is and isn't usual in these cell lines and so accommodate the abnormalities accordingly, thereby making research more efficient. Future work promises to undercover in great depth the complex regulatory processes that govern apoptosis, and present more and more targets for treating cancer and other disorders of cell division.

The next post in this series can be found here.

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