p53 (18 page)

Then Evan and his team did the opposite experiment: they kept p53 silenced and out of the picture when they irradiated the mice, and looked to see how they reacted. The scientists were not
surprised initially to find that the animals didn’t get sick, because, with p53 inactivated, there was no mass suicide of cells. But what happened next stopped them in their tracks. When,
after giving the mice time to repair the DNA damage to their cells, they switched p53 back on again, they found that the mice didn’t get sick – again, because there was no mass suicide
of repaired cells – but to their great surprise the animals didn’t get cancer either. In other words, p53 was able to keep cancer in check, even when it was introduced well after the
damage to the DNA had been done.

So what did they make of this extraordinary picture? What this means, explains Evan, is that if you don’t have p53 around when the DNA damage is occurring, most cells will be repaired. In
real life this is a rather hit-and-miss affair, a patch-up process that leads to the kind of ‘mistakes’ that are the driving force behind evolution. But if you then restore p53 after
this repair process has had time to work, the gene will
only
be activated in those cells that have sustained mistakes, or mutations, that make them dangerous (for example, ones that
activate oncogenes) and therefore send out alarm signals to abort. ‘This basically means that you can separate out the DNA damage response from the tumour suppressor response,’ he
comments.

This has enormous implications for treatment of cancer, because it implies that we could devise ways to prevent most of the dreadful side effects of chemo- and radiotherapy – the hair
loss, nausea, exhaustion, immune suppression – that are the direct consequence of hitting all the body’s fast-dividing cells, and clear out just those cells that go on to become
cancerous and that therefore continue to send out alarm signals that activate p53. But how did Evan’s colleagues react to his findings and his theories?

‘You know ideas like this take a long time to percolate through. I mean getting it published . . . I remember one of the reviewers just said, “I refuse to accept that the DNA
response is not the major tumour suppressor pathway.” But this is not a faith-based thing; we’re not a religion! These are the data. And I wasn’t saying, “You’re all
wrong.” I was saying, “These are the data. This is our explanation. This is our hypothesis.” The whole point about publishing in the literature is that you publish the data; you
publish the hypothesis so that it can be tested by the community.

‘That experiment was a very intriguing experiment, and I think a very informative one. And I think the conclusions of it still stand. But the point is that cancers arise in many different
tissues and many different ways, and the issue for me is much less about are these data wrong or are these data right than about getting to understand which set of rules apply in which
case.’

A FINE BALANCE BETWEEN LIFE AND DEATH

Evan had met even stronger resistance to his ideas some years earlier when his research into oncogenes suggested to him that all our cells contain both growth and suicide
programmes that are in constant, hair-trigger competition. Which course of action a cell takes is essentially controlled by its environment and the signals it receives from its neighbours: is it in
the right place and at the right time? Is it behaving normally? If so, it will receive ‘stay alive’ signals; if not, it will be instructed to abort. This is an inbuilt defence mechanism
and one of the reasons cancer is so rare, believes Evan.

Since this story takes us back among the Petri dishes in the lab, it may seem like a diversion from the topic in hand, animal models. But besides describing another pivotal moment in cancer
research, it helps show why it is so important that experiments are performed in living organisms as well as in cell and tissue cultures. It begins in the late 1980s, when Evan, newly recruited to
the ICRF in London, was doing some experiments with the powerful oncogene Myc, looking at how it drives cell proliferation. ‘I made this bizarre observation that when you expressed Myc at
high levels in cells they did indeed proliferate – but when you looked a couple of days later, there were fewer cells than before,’ he explains. ‘I’m a great believer in
personal observation – observing things, you ask questions of what you can actually
see.
So we took these cells and we put them under a microscope and we used time-lapse video to
take one frame every three minutes. Then you speed it up and watch what happens over three or four days in just two or three minutes. And there we saw this amazing phenomenon . . . the cells were
replicating, but also they were dying by apoptosis.’

This was exciting, but it didn’t make sense. Then a thought struck Evan that drew on his early training in immunology, where a common theory was that the immune system plays a part in
protecting us from cancer by eliminating rogue cells that it recognises as foreign. ‘I thought what if, instead of the immune system acting as a police service to find aberrantly
proliferating cells, there is, hard-wired into the very warp and weft of how cells proliferate, an abort programme? Every time you pick up the machinery to proliferate, you also pick up the
machinery to kill yourself?’

If that were the case, he reasoned, there must be something that tells the cells whether to live or die, and here he found a clue in the growth medium he was using for his experiments. Most of
the time, he used serum – the colourless liquid the body produces at the site of a wound that makes it ‘weep’ – because serum contains substances that promote clotting of
blood, and survival, growth and proliferation of cells to help in the recovery and regeneration of injured tissue. Myc was killing cells when Evan removed the serum with all its life-enhancing
properties and put the cells in a medium that was more like what they would find under normal conditions in the body.

‘Myc turned out to be, I think, the first example of what
we now know as a generic feature of how growth control is orchestrated within our cells – which is
that everything that makes a cell proliferate (and is potentially therefore a cancer risk if it gets mutated and stuck in the “on” position) comes with something that also suppresses
the expansion and growth of those cells.’

Similar experiments with other oncogenes showed that they too shut down growth programmes one way or another after a short spurt of proliferation. Ras, for example, does it by permanently
arresting, but not killing, the cell – putting it in a state known as ‘replicative senescence’, where it stops dividing but stays alive and active. But this raised a number of
further questions. Oncogenes like Myc and Ras, when not mutated, have regular work to do in promoting growth in cells, but if they also serve to shut down or kill cells after a while, how is new
tissue ever produced? ‘The answer seems to be that if a cell switches on Myc in response to a growth signal and starts to replicate, if that cell is in the right place in the body, and it
stays in its little niche and doesn’t spill out like a cancer, then it will get all the goodies that tell it not to commit suicide, okay?

‘So cell replication is an obligatorily social enterprise. Cells are not autonomous. By taking them out and putting them in a bottle and adding all the things that would stop them dying,
we just completely ignored this fundamental piece of biology. It had always been ignored! Now, the notion that things that drive cell growth also drive cell death and growth arrest is, I think,
completely embedded in the understanding of molecular biology; it’s just generally accepted that this is how things work. But at the time, people literally walked out of my talks!’

In fact, Scott Lowe, then doing his PhD at MIT, and his supervisor Earl Ruley, had observed the same extraordinary phenomenon – oncogenes killing cells or condemning them to replicative
senescence. And they too had had a tough time getting people to listen. ‘If you’d walk down the hall at MIT
Cancer Center and say, “I have an oncogene and
it kills cells”, they’d think you were crazy. Because that’s not what oncogenes do; they make cells grow better,’ laughs Lowe.

The insights he and Evan gained in this work also helped to explain a long-standing mystery:
why
oncogenes are only able to generate tumours in co-operation with one another. Evan
believes that when, for example, you put Myc and Ras together, Myc overcomes the replicative senescence programme of Ras, and Ras overcomes the apoptosis programme of Myc. Thus singly, the growth
spurt fuelled by either oncogene soon fizzles out; together, all hell breaks loose. In time, he and Lowe would discover that the effects they had both witnessed independently and wondered about
– death among their oncogene-driven cells – were caused by the oncogenes switching on tumour suppressors, frequently p53.

The multiple experiments with mouse models – knocking out p53 altogether, or else toggling the gene back and forth between active and passive – made it very clear that this is an
extremely powerful protein. As an arbiter of life-and-death decisions within our cells it must be under strong control. So how does it work?

***

‘Guardian of the genome’, the epithet David Lane gave to p53 in 1992, caught the popular imagination and has helped give this extraordinary gene with the eminently
forgettable name a public profile in the media. But in the 20 years since that phrase was coined, the list of stresses to which p53 responds has expanded way beyond simple damage to the DNA. Arnie
Levine suggests it might be more appropriate to think of the gene as a ‘fidelity factor’ – something that ensures faithful copying of the DNA during cell division. We now know
that the gene responds to heat shock and cold shock (when a cell is subjected to temperatures above or below the ideal body temperature), to lack of oxygen or glucose, to certain poisons, to
natural ageing and to oncogene activity – all things that threaten the fidelity of the DNA, without actually breaking it, as the cells divide.

‘Ever since its evolution in invertebrates, p53 has been a fidelity factor,’ says Levine. Starve a worm of glucose and it won’t produce eggs; radiate a fruit fly and it
won’t produce
sperm or eggs until progenitor cells with healthy genomes are restored. With the evolution of the vertebrates – organisms with much more complex
bodies, including us – the principle of protecting the fidelity of the germ cells, the sperm and eggs that give rise to offspring, was also applied to all other cells of the body for the
first time. ‘And that’s where p53 comes in,’ says Levine. ‘It responds to stress, and it kills. It enforces fidelity by death! So it has a very interesting evolutionary
history.’

Very recently scientists have discovered another fascinating aspect of p53’s role in fidelity assurance. Imagine for a minute what would happen if, in the normal course of events, our
biological clocks could go backwards in time; if our mature cells could revert to their original undifferentiated state as stem cells, complete with the potential to develop afresh into something
new. It’s a nightmare scenario in which your liver cells might morph spontaneously into bone cells, gut into teeth, blood into kidney, and no bodies would be stable. It is p53’s job to
ensure that such de-differentiation doesn’t happen; that biological time moves inexorably forward and that our bodily development cannot unravel (except, that is, in the deranged environment
of cancer). Scientists creating what are known as ‘induced pluripotent stem cells’ (IPSs) – stem cells with the potential to become any kind of specialised cells that have been
engineered in the lab from already differentiated body cells – are frustrating a fundamental law of nature, and they must overcome p53’s defences to do so.

Many people have been involved in uncovering the mechanism controlling this powerful gene, and once again Moshe Oren’s temperature-sensitive mutants provided vital insights. This was the
early 1990s, before the creation of knock-out mice had become a cottage industry. Oren and his team were using his temperature-sensitive mutant p53 as a tool in cell cultures to ask a simple
question: what is different in cells with active p53 compared with those
in which the gene is inactive? They soon observed that a protein appeared hitched to the p53 protein
whenever the tumour suppressor was behaving like wild-type p53, at 33°C (91°F), but never at the higher temperature, when it behaved like a mutant.

Levine had observed the same thing in a different set of experiments designed to explore the workings of wild-type p53, and he had identified the hitchhiking protein as Mdm2, already known to
cancer researchers as a possible oncogene. Levine had also discovered that by attaching itself to p53, Mdm2 restrained the tumour suppressor – more like a policeman handcuffed to a criminal
suspect than a hitchhiker. Tinkering with the temperature in their Petri dishes, Oren’s team discovered that Mdm2 protein appeared only in the cells with normal p53, and was absent altogether
from the mutant cells. What did this mean?

Oren and company soon realised they had in their hands a crucial piece of the jigsaw that would reveal the control mechanism of p53. Timing was all. ‘This was 1993, and a year earlier we
wouldn’t have known what to do except to say that this was very interesting,’ he commented. But just the year before, Carol Prives and Bert Vogelstein had shown that p53 is a
‘transcription factor’ whose job is to switch other genes on and off; Vogelstein had identified the first of its ‘downstream’ targets and it seemed reasonable to suggest
that Mdm2 was another – that it depended on wild-type p53 to switch it on. Subsequent experiments proved their hypothesis right, as Oren explained: ‘Knowing what Mdm2 does to p53 in the
way of acting as an inhibitor, which was Arnie’s discovery, and seeing that p53 itself is activating Mdm2, we discovered this “feedback loop” – which luckily turned out to
be not just interesting but extremely important, because this is the main way in which p53 is regulated in the cells. This Mdm2 feedback loop is perhaps the heart of the p53 regulatory
network.’