p53 (13 page)

‘A footprint is a very elegant kind of experiment that people don’t do very often,’ explained Prives. It involves mixing the purified protein or proteins under investigation
with DNA in a test tube and adding an enzyme to the mixture. The enzyme acts like chemical scissors, chopping the DNA up into fragments. However, if the protein is binding to the DNA, the
‘scissors’ are unable to cut the ribbon of genetic material at that point. Finally the mixture is run through a gel which organises the DNA fragments into a ‘ladder’. If no
protein is binding to the DNA, the ladder is intact; if a protein has bound to the DNA, there will be a gap in the ladder. ‘Looking at the gel will tell you, if you know how to read the
sequence,
exactly
where the protein binds to the DNA,’ said Prives.

Here a bit of explanation is needed in order to understand the significance of what Bargonetti was about to uncover. Generally speaking, the job of a protein that binds to DNA – attaches
itself to a spot on the double-helix ribbon of genes – is to control the expression of genes in that region; to switch them on and off as appropriate. Proteins that do this are called
‘transcription factors’ and are, in effect, the conductors of the orchestra of activities within a cell. As the protein that switches on the DNA replication machinery in cells infected
with SV40, large T antigen was already known to be a transcription factor. Bargonetti’s interest in doing footprint experiments using large T antigen and p53 proteins together in her mixture
was, among other things, to show physically how p53 stops large T from attaching to DNA to switch on the machinery. ‘We told her, “Okay, do it, if you insist!”’ said Prives, who
then left the young scientist to her own devices without further thought.

But Bargonetti found more than she had expected. ‘She came to me one day and she said, “It’s working, but I have this really big problem . . . p53 itself is giving me these
patterns on the gel.”’ Recognising a rare eureka moment in science, Prives responded, ‘That’s not a problem – you are the luckiest person in the world!’ The
clear – and totally unexpected – footprint left by p53 in the DNA ladder was the first clue to how the tumour suppressor works, for it too is a transcription factor. And we now know it
is an extremely powerful one, sitting at the centre of a network that orchestrates life-and-death signals in every single cell in our bodies.

Bert Vogelstein had also sussed out that p53 was a transcription factor, but by another route. His lab had observed that p53 protein is active in the nucleus – the powerhouse of the cell
where the DNA is stored – rather than in the cytoplasm, the body of the cell. Transcription takes place only in the nucleus and many of the proteins active in this site are involved directly
or indirectly in controlling the expression of genes. So Vogelstein’s team did experiments to see whether p53 bound to DNA – a defining feature of a transcription factor – and
they found that it did.

‘The other equally important part of the story,’ explained Vogelstein, ‘is that we weren’t simply looking to see what DNA sequences it binds to – and finding that
it could not only bind to these sequences but also activate “downstream” genes – we were in all cases comparing the wild type to the mutant forms. And what was satisfying and made
us think we were on the right track was that every mutant we looked at was devoid of this binding activity. We had cloned them, right? And this allowed us to test unequivocally whether these
mutants were disrupting this function – and every single one, without exception, prevented its binding to DNA. So that convinced us – and I think the rest of the world, too – that
it was right.’

Jo Bargonetti had also done footprints using protein from Vogelstein’s mutants as a control to her experiments with wild-type p53. ‘Jo said, “You know, it’s only the wild
type that’s making these patterns, not these mutants,”’ continued Prives, putting her hand to her chest to demonstrate her breathless excitement at the revelation.
‘I’m going,
arghh
. . . I’m going to die and go to heaven! I mean, it was just so perfect! . . . Jo’s footprints weren’t great, they were not very clear cut,
but it
was
clear that the wild type had a pattern of recognition of the DNA, and these mutants didn’t. That was
absolutely
clear.’

Prives paused to reflect and then said, ‘You know, I’d been doing science for many, many years, but I don’t think anything I’d done up till then was of anywhere near that
significance.’

Prives’ lab also tested the footprint findings by putting the proteins through their paces in cell cultures in glass dishes in the lab; they found that wild-type p53 did indeed act as a
transcription factor and that mutants did not. Meanwhile, Vogelstein’s lab showed that they did the same in living organisms. The two labs, who were by this time talking regularly, published
their findings as joint authors in the influential journals
Science
and
Oncogene
in 1991.

But what is the connection between p53’s activity as a master switch and its role in protecting us from cancer? How does the tumour suppressor
itself
get switched on? And what
does it actually do when this happens? These were the next things to consider.

***

A critical part of the answer to the question of
how
p53 suppresses tumours came from a fellow medic of Vogelstein’s at Johns Hopkins Hospital, Michael Kastan,
who came to p53 research through his work as a paediatric oncologist. ‘People say it must be depressing to treat kids with cancer,’ commented Kastan when I spoke to him by phone from
North Carolina, where he now lives and works. ‘But first of all, kids do much better than adults; we cure 80 per cent of children with cancer, which is amazing. Also, they deal with it much
better; and you get to know the families really well, so it was socially a more attractive field for me than other aspects of cancer.’

A single case that seemed to epitomise the combined social appeal and scientific challenge of paediatric oncology for Kastan – and convinced him this was the right field for him –
was that of Dora Squires, a little girl with an unusual form of Down’s syndrome. So-called ‘translocation’ Down’s meant that, instead of having a complete extra copy of
chromosome 21, as is most often the case, Dora had an extra scrap of chromosome 21 that had translocated and attached itself to another chromosome. When Kastan, as a young doctor on the wards, met
her she was three years old and already had a long history of cancer.

Dora had been born with leukaemia – a not unusual occurrence in Down’s children – which resolved itself without treatment, Kastan told me. She did fine until she was
two-and-a-half years old, when she developed a tumour on her face, a rapidly growing sarcoma that responded well and melted away with radiation therapy. But the little girl was soon diagnosed with
acute myelogenous leukaemia, the condition for which she was being treated when Kastan appeared on the wards.

‘So I come on service and I hear this story, and I say, “Here is a girl with a known chromosomal translocation that in the space of three years has had three different tumours, and
no one is saving her blood to try to figure out why.” Now this is before the era of knowing about oncogenes, but I said, “We don’t know what question to ask now, but if we save
her blood samples, some day we’ll be able to ask questions about what it is with this translocation that led to this story.” Scientifically I found it extraordinarily interesting, and I
felt it was a field ripe for discovery.

‘But the other piece of the puzzle was that Dora was a typical Down’s child: she was very very happy, and she always loved to see us when we came on our rounds. Her father weighed
275kg (600lb) and was too large to sleep on the parent bed in the ward, so he used to sleep on the floor – and she would sleep on his belly. So when we came on our rounds in the morning
we’d open the door, she would see us and she would sit up on his belly and put out her arms for hugs from everyone on the team. You can’t not melt . . . So that’s how I decided to
go into paediatric oncology – it was because of Dora Squires.’

Kastan had sandwiched his medical training around a PhD in molecular biology, for which he studied how cells respond when their DNA is damaged. And when, after completing his specialist training
in paediatrics at Johns Hopkins, he started his own lab – a modest set-up consisting of himself and an inexperienced young assistant – to do research on the side, this was the topic he
was intent on pursuing. His daily experience on the wards had convinced him it was central to cancer biology, he says, and nothing has happened since to change that view.

‘We know DNA damage causes cancer, right? We know this from animal models where you can take an animal, treat it with a carcinogen or radiation and cause tumours. We know it from human
experience – Hiroshima and Nagasaki, for example, showed us how radiation causes cancer. We know it from exposure to carcinogens in the environment, which is why we have laws about what
chemical companies can put in the water. And we know it from familial cancer syndromes, most of which are due to mutations in DNA repair genes.

‘So we know DNA damage
causes
cancer. But we also use DNA damage to cure cancer: radiotherapy and most of our chemotherapeutic agents target DNA. And most of the side effects of
treating cancer – the hair loss, the bone-marrow suppression, the nausea and vomiting – are because DNA damage is killing normal cells. So from a clinical perspective, or a
cancer-biology perspective, DNA damage causes the disease; DNA damage is used to treat the disease; DNA damage is responsible for the side effects of treating the disease . . . It makes it a pretty
important phenomenon! As an oncologist, understanding and characterising DNA damage signals is important in every aspect of cancer.’

Kastan’s research had always focused on blood cells and what goes wrong with them to cause leukaemia. It was his preference for this cell type over the ones that cause solid tumours, or
carcinomas, that enabled him, serendipitously, to discover the key mechanism by which wild-type p53 protects us from cancer – simply because, unlike carcinoma cells, leukaemia cells almost
never have mutant p53. Thus what he observed in his experiments was the activity of normal p53 under a variety of circumstances, whereas he would not have seen anything – because nothing
would have happened – if the cells had contained mutant p53. It was serendipity also because p53 was far from his mind, with no place in his research agenda, when he began his experiments to
look at DNA damage and repair in cells that are dividing.

The cell cycle, as this dividing process is called, has several phases, and Kastan had been intrigued by a paper he had read from researchers studying yeast – one of nature’s
simplest, most pared-down organisms, consisting of a single cell – that described how, if its DNA was damaged by radiation, yeast would stop at a ‘checkpoint’ in its cycle while
the DNA was repaired, before carrying on through the cycle. The two researchers had found the gene responsible for this exquisite control of the cell cycle after damage, and this set up a challenge
to Kastan: could he identify the genes and proteins that might be doing a similar job in us?

The first thing he did with his leukaemia cells was to bombard them with ionising radiation – which typically causes extreme damage to the DNA by breaking both strands of the double helix
– and to take note of the changes in the cell cycle as a consequence. He found that his damaged cells arrested at checkpoints, demonstrating for the first time that what happened to yeast was
a general phenomenon, applicable to the human body too. Now he could start asking the questions that really interested him: were there proteins whose level in the cells increased as a result of the
damage, indicating that a particular gene or genes had been activated by the event? And if so, which of these genes were responsible for arresting the cells at the checkpoints?

Kastan was pinning his bets on the known oncogenes, and he was surprised to see no changes in these. But he had developed especially sensitive tools for measuring protein levels, and he noticed
that the reading for p53 was slightly elevated. This was unusual since, for reasons that will soon become clear, p53 protein is normally present at levels that are barely detectable in cells. Could
this slight increase be significant, he wondered?

Indeed it could. As we shall see, Kastan had begun to uncover the mechanism by which p53 suppresses tumours – by halting defective cells in their tracks so that they cannot divide. He was
on the brink of a momentous discovery, but it would take time and hard work to tease it out.

FOLLOWING THE CLUES

While Kastan was busy with his initial experiments, a paper came out from Steve Friend – the scientist, you will recall, who had discovered the first-ever tumour
suppressor, the retinoblastoma gene, Rb. Friend’s paper showed that if you pushed a dividing cell into producing an over-abundance of p53, it came to a temporary halt at a checkpoint named
G1. It was a simple observation; Friend did not know whether this ever happened in real life, nor what might trigger an over-abundance of p53, but it made Kastan sit up. Could this be part of the
same picture he had observed in his damaged leukaemia cells? Could the damaging event, the ionising radiation, be what activated p53? And could p53 therefore be the protein that was
responsible
for the checkpoint arrest?