p53 (14 page)

‘This is when I got Bert Vogelstein involved,’ he says. ‘You know, I was a clinician who happened to have a very small lab and was doing these cell-cycle studies; Bert had this
big machine . . . Since we were at the same institution I knew they were able to sequence p53. So I got on the phone to him one day in between seeing patients, and I said, “Bert, I think we
know what p53 is doing. Will you sequence these cell lines for us?”’ Kastan described his experiments and his hypothesis to Vogelstein and explained that he wanted to check which of his
cells had wild-type p53 and which had mutants, and to compare their activity. ‘Bert didn’t believe a word of my story!’ laughed Kastan. ‘But he said sure, he’d have
the cells sequenced. And lo and behold, when we tested them, those that had wild-type p53 arrested at the G1 checkpoint after radiation, and those that had mutant p53 didn’t . . . I
immediately had a sense of how important this might be: all of those experiments told us p53 plays a role in DNA damage responses.’

Kastan published his findings in
Cancer Research
in 1991 hot on the heels of news from Vogelstein’s own lab, working in collaboration with Carol Prives, that p53 was a master
switch. All of a sudden, scattered pieces of the jigsaw began to fall into place.

THE JIGSAW BEGINS TO TAKE SHAPE

Another of those jigsaw pieces came from Kastan’s clinical casebook and involved a rare, inherited, neurodegenerative condition called ataxia telangiectasia, or AT. This
devastating disease affects between one in 40,000 and one in 100,000 people worldwide. Children typically start to show signs of AT as toddlers, as it kills brain cells and progressively disrupts
their motor co-ordination, affecting everything from walking and balancing to speaking, swallowing and moving the eyes; it generally sees them wheelchair-bound by the time they are in their teens.
Treating his young patients with the condition, Kastan knew their risk of cancer was exceptionally high – in fact, 37 to 100 times that of the general population. He knew also that they were
especially sensitive to ionising radiation, so medical procedures such as X-rays and CT scans were to be avoided if at all possible. Now, he began to wonder if both phenomena might have something
to do with p53. Were the cells of patients with AT able to halt the cycle at the GI checkpoint and activate a DNA repair programme as they should, or was this mechanism defective?

‘We had cell lines from patients with AT and it became clear very early on that p53 did not get induced normally,’ Kastan says. ‘We had absolutely no idea what the gene was
that was missing in these patients. But whatever it was, we realised it was somehow required for the induction of p53 after radiation.’

At the same time as investigating the AT connection, Kastan was collaborating with a scientist at MIT, Tyler Jacks, who had created experimental mice with no p53. Sure enough, thymocytes –
important components of the immune system – in Jacks’ mice failed to arrest at the G1 checkpoint when bombarded with radiation. Together with Vogelstein, Kastan was also collaborating
with a third group, at the National Cancer Institute near Washington DC, who had discovered a collection of genes called GADDs that are directly responsible for arresting growth of cells with
damaged DNA (indeed, their name is derived, imaginatively, from Growth Arrest and DNA Damage). The three teams found that GADD 45 was controlled by p53, and was one of the genes switched on by the
tumour suppressor to cause arrest at the GI checkpoint that Kastan had first uncovered. Very soon, Vogelstein found another gene, p21, involved in the same event and also controlled directly by
p53.

The picture that emerged of p53 from these disparate bits of research was of a master switch at the hub of a communication network within cells. Its job is to respond to incoming signals
indicating DNA damage by recruiting the relevant genes ‘downstream’ to halt growth of the cell pending future decisions about its fate. In this way cells with scrambled DNA that might
threaten the organism are disabled.

It was this picture that the researchers described in a paper they published together in
Cell
in 1992 and that Kastan says was ‘the most fun thing I ever did in my scientific
career’. Just before the paper came out, he attended his first big p53 meeting, hosted that year by Moshe Oren and Varda Rotter in Israel. Oren had seen Kastan’s original paper on
checkpoint arrest following radiation and been sufficiently excited to invite the American to speak at the plenary session – to the full, august gathering of the p53 community. ‘What
was so much fun was that I was a
total
unknown in the p53 field,’ said Kastan. ‘I go to this meeting; I get up to the podium and give this talk about this whole signal
transduction pathway the day the paper was published in
Cell
.’ No one had seen the data before, and it had a powerful effect on the audience.

‘I was a nobody with a no-technology lab,’ he continued, ‘but I just happened to ask an important question because I read the literature carefully. And I asked it at the right
time, with the right techniques and in the right cell type.’

RARE DEGENERATIVE DISEASE HOLDS THE KEY

Not everyone was ready to accept Kastan’s model entirely. The fuzziest part of the picture at that stage, in 1992, was the connection with ataxia telangiectasia. No one
knew what the missing element was in these patients that made them so sensitive to radiation; they knew only that, in normal circumstances, it was essential for signalling to p53 that the DNA was
dangerously damaged and for turning the whole damage-response system on. Things became clearer when, after a Herculean effort by 30 international scientists and hot competition between the labs to
find the gene or genes responsible, a team led by Yossi Shiloh at Tel Aviv University announced success in 1995.

The single-minded search for the AT gene took more than 15 years of his life, Shiloh told me when I spoke to him over the phone from New York, where he was on sabbatical in 2012. It began when
his mentor at university, Professor Maimon Cohen, suggested that the young scientist join him on a field trip to a small village in southern Israel; there they would meet a family of Moroccan
Jewish origin afflicted with ataxia telangiectasia. Shiloh had recently completed his Masters degree and was casting around for a topic for his PhD thesis. ‘Professor Cohen had a hidden
agenda – to interest me in AT,’ he said. ‘It worked very well because when I saw those patients I decided almost on the spot that this was an important problem to work on. First,
because it’s an extreme human tragedy and at that time it was an “orphan disease” – no one cared much about these rare diseases with long names. And second, it was clear
that understanding AT would have broad ramifications in many areas of medicine – neurology, immunology, genetic predisposition to cancer and whatnot – because AT is like a microcosm of
medicine, it involves so many systems in the human body.’

Shiloh had no illusions about how difficult it would be to find a common cause for such diverse symptoms – and for many years the consensus among AT researchers was that there were four
distinct types of the disease and probably at least four different genes responsible. The first breakthrough – what Shiloh identifies as the starting gun for the race to find the genes
– came from Richard Gatti at the University of California in Los Angeles, whose study population was the Amish people of Ohio. In 1988, Gatti had managed to localise the gene responsible for
AT to a region on chromosome 11, homing in on this stretch of DNA through a technique called linkage analysis, which looks for genetic markers – small strips of DNA with unusual
‘spelling’ dotted along the genome that are consistently present in people with a particular genetic disease, and never found in healthy individuals. The researchers then use statistics
to suggest which marker or markers is closest to the target gene. This narrows the search area, but finding the actual gene is still akin to looking for a person’s house when you have only
the name of the city in which they live to go on, and it was another eight years before Shiloh and his team managed to achieve their aim.

‘When I look back I’m surprised yet again that for eight years the
entire
lab was working on that one project . . .’ he said. ‘You know, scientists are very
individual . . . Today we still work on AT, but every student in the lab has his or her own project. At that time the entire lab, several generations of students and postdocs, was focused on just
fishing out genes from that region of chromosome 11, analysing them, cloning them.’

Today, thanks to the Human Genome Project and the wealth of data about genes and sequences available at the click of a computer mouse, such an exercise is relatively straightforward. But in the
mid-1990s it was slow and labour-intensive, and relied on close co-operation with the AT-affected families whose personal DNA was the lifeblood of the research. Among the hundreds of genes
Shiloh’s team cloned was one that specially caught their attention because it was unusually long – so long in fact that they had to repeat the cloning exercise a number of times to
convince themselves it was real. Clearly this was the recipe for a huge protein – and one, they soon discovered, that had the hallmarks of a ‘signalling’ protein responsible for
sending messages within the cell.

Shiloh remembers the day they realised this was what everyone had been looking for. ‘I had been teaching and when I came back to the lab from my class my student was holding a Southern
blot
⁷
in her hand. She said to me, and I remember her words clearly, “There is something odd about this gene in this family.” This was
one of our Palestinian Arab families. I looked at the blot and it was clear that a big portion of that specific gene was deleted in that family. It was a very dramatic result. Of course my heart
skipped several beats, but I said to her as calmly and quietly as possible, “This indeed looks interesting, there might be something here. Why don’t you repeat the experiment with DNA
samples from the entire family and additional controls?”’

She did so and the conclusion was inescapable: here was the gene whose corruption was the cause of the disease Shiloh’s team were seeing in all their AT patients. It was a time of high
tension, recalls Shiloh. The race to find the gene was at its peak, with frequent rumours in the air that someone or other had succeeded, and the temptation to publish his lab’s results
immediately was heavy. But he had a hunch that there might in the end be just one gene – not the four that everyone supposed – responsible for the different manifestations of ataxia
telangiectasia, and it would take time to prove it. Someone else might get there first, but after intense discussion among themselves everyone in his lab agreed to hold off announcing their results
until they had tested their hypothesis. It was a nail-biting time, but the gamble paid off: AT is indeed caused by defects in a single gene, which the international consortium named ATM, short for
ataxia telangiectasia mutated.

This was the missing detail in Kastan’s picture of the DNA damage response: in time he and others were able to show how the signals are passed down the line from ATM, which first senses
the broken strands of DNA, to p53, which then throws the relevant genetic switches to halt the division of the cell. This was biochemical proof of the mechanism, and it finally convinced the
doubters that p53’s response to DNA damage is at the heart of its action as a tumour suppressor.

‘You know, you can’t overstate the importance of what Yossi did in cloning the AT gene,’ commented Kastan. ‘He will be somewhat humble in telling it, but people were
searching for that gene for 20 years – including him – and it made such an impact . . . It really opened up the whole DNA damage-signalling field. Yossi is a fastidious scientist and
it’s because of that fastidious approach that they got to that point.

‘He flew to Baltimore to tell me he had the gene clone, and I remember very distinctly, he was sitting in my living room, saying, “Okay, we got the gene, and we’re calling it
ATM for ‘ataxia telangiectasia mutated’.” I looked at him and I said, “Well, that sounds great, but you know ATM has another meaning in the US?” And I explained to him
about these new automated teller machines. His face dropped, and I said, “Don’t worry, Yossi, people will know that’s where the money’s at in the signalling pathway!”
And it’s been true. The field just exploded at that point.’

MULTIPLE STRESSES
,
MULTIPLE RESPONSES

In labs everywhere, researchers began testing the model, and evidence soon mounted that many more insults to the DNA – as well as more subtle stresses on the cellular
machinery – can trigger the p53 response to halt the cell cycle. The increasingly long list includes UV radiation from sunlight, chemicals in the environment, and activated oncogenes, as well
as natural ageing and dangerously low levels of oxygen and essential nutrients like glucose in the cell. Importantly, each stressor has its own characteristic pathway – from the protein that
sends out the first alarm signal that all is not ideal for the division of the cell, thus triggering the response, to the range of genes that p53 switches on. But they all have the same effect of
preventing potentially harmful mutations from being passed on from one generation of cells to the next.

The frenzy of activity among researchers was fuelled also by revelations that, just as there are many different stressors that can trigger p53, there is also a variety of outcomes to the
response. Besides inducing a temporary halt in a dividing cell while DNA damage is repaired, p53 can induce a state of permanent arrest, called senescence. And under certain circumstances, it will
instruct a seriously damaged cell to commit suicide – a process that many people feel is the most important weapon in its armoury.

In July 1992, David Lane, p53’s co-discoverer, pulled all the information together from widely scattered publications in a review for
Nature
in which he dubbed p53 ‘the
guardian of the genome’ – essentially, the policeman in our cells taking action to clear dangerous individuals from the scene. As a reflection of what many people were thinking, it was
neat; but as a statement from a scientist it was unusually bold. ‘In a sense it was sticking my neck on the block,’ said Lane with a mischievous chuckle. ‘You write a scientific
paper and you say: it’s not unreasonable to speculate . . . But in this I said: this is how it works! Then everyone thinks, well there’s a challenge! But is it true? Is it not true? Not
everyone believes it even now, but it provoked debate, which is what it was intended to do. That’s very important to the progression of science.’