p53 (11 page)

Now in his sixties, Vogelstein has an air of restless energy and as he talked about his life in science his conversation was punctuated by an extraordinary high-pitched laugh that sounded like a
cross between mirth and tears. He comes from a long line of rabbis – 13, he believes – but he defied his apparent destiny to study science. He came into cancer research in 1978, at the
height of the oncogene craze, setting up his own lab at Johns Hopkins, where he had qualified as a medical doctor and spent a few years on the wards. In his spare time at medical school he had
worked in the molecular biology lab of Howard Dintzis and loved it. ‘I started doing research with Howard just to learn what research was, and I did it every summer and every chance I could
get during the school year – when I had an elective, or at nights and weekends. And then I also learnt how to take care of patients. I found both of them very satisfying, but I found the
research more intellectually stimulating. It was a tough choice, because the gratification you get from
treating patients is often immediate, whereas the gratification you
get from doing research can sometimes take years – and sometimes never comes. But probably the defining moments were when I started taking care of cancer patients.’

Vogelstein’s first paediatric case was a little girl named Melissa, just three years old, whose parents brought her to hospital because she was pale and had suddenly become prone to
bruising. Vogelstein diagnosed leukaemia. ‘It’s still scary when I think about it, because my own granddaughter is two and a half.’ He broke off for a moment, imagining himself in
the shoes of the little girl’s parents. ‘Out of the blue, bang! One day she’s fine and the next day she’s got cancer. Her father was a mathematician, a young guy about the
same age as I was. He asked me, “Why did this happen to my little girl?” and I had no answer. No one did. Intellectually he was asking, “What’s going on? What’s the
basis for this disease?” And we had no idea, absolutely no idea. I mean there were a hundred different theories, but just no idea . . . Cancer was a total black box! You could throw some
things at the child, and some kids even back then were responding – many of them were. But they were poisons, you know? It was a nightmare.’

The experience, indelibly etched in his mind, helped tip the balance in favour of research; doing something useful for humanity is part of his family tradition, and Vogelstein wanted to have a
go at solving the mighty puzzle of cancer. His motivation to fight the disease was constantly reinforced by the fact that his first lab at Hopkins was directly above the radiotherapy unit; he and
his students had to walk through it to get to work, passing rows of very sick people, many of them in wheelchairs, awaiting treatment. It was impossible not to run up those stairs and start
working, he said.

Vogelstein’s plan was to join the hunt for genes that might be involved in cancer. ‘But I wanted to do it in humans – I think that was part of my medical training. I thought
the only way to really understand what was going on in the
human disease was to actually study humans.’ But his idea was flatly rejected; no one would give him funds
for his research, he told me with a peal of his infectious laughter. ‘I was told that the only way to get insights into the disease process was to use an experimentally amenable system, which
meant mice or worms or fruit flies, because you can manipulate them, or tissue culture or something. That was the paradigm of the time – and to a large extent still is.’

So how did he do his research? ‘Well, I don’t
know
how I did it, okay? I mean I really didn’t have any funding . . . I had to rent my own microscopes and use my
personal money, my salary, to do it. For a couple of years we were really broke!’

Funders eventually came on board as Vogelstein’s lab began to show interesting results. Their initial effort went into ‘fractionating’ tumours – that is, separating the
cancer cells from the normal cells in the lumps of tissue so that they could distinguish clearly between the DNA of the two. ‘Fractionating tumours sounds trivial, but it’s anything
but
trivial and it’s been a major stumbling block for people,’ Vogelstein told me. The common practice was to grind up tumour material to use in experiments, so
cross-contamination of cells was always a problem. ‘We did it just with a razor blade under a microscope. Each tumour would take us four or five hours. Stan Hamilton was the pathologist who
collaborated with us. We would spend hours a day in the pathology lab micro-dissecting these tissues.’

Having developed the tools and perfected the art of isolating and labelling the DNA from human colon cancer – chosen as a research subject because you can almost watch cancer develop from
a growing polyp that starts off benign – Vogelstein and his team were ready to look for the genes responsible for the tumours. This is when they learnt the truth about ‘wild-type’
p53 – by following much the same line of reasoning as Steve Friend and the others pursuing the retinoblastoma gene.

‘We had all these tumours from colon-cancer patients, hundreds of them which we micro-dissected and we looked at alterations. We could see losses of whole
chromosomes and large parts of chromosomes – but we couldn’t find oncogenes that were responsible. So we thought, well, these losses, maybe they represent the losses of
tumour-suppressor genes . . . Tumour-suppressor genes had been
hypothesised
to exist, but they’d never been
shown
to exist at the time – this was the mid-80s. They
were mythical beasts!’

Suzy Baker, who had joined Vogelstein’s lab to study for her PhD, was set the task of seeking out these beasts. It must have looked like a wild-goose chase at the time, but she set to with
the enthusiasm of youth and inexperience. As with the retinoblastoma gene (most often represented simply by ‘Rb’), Baker knew where to start looking for candidates – on a specific
region of chromosome 17 which was missing one copy in at least three-quarters of all colon cancers. Her task was to search through the DNA of the remaining copy for an important gene that was
mutant and malfunctioning, thus signifying that both brakes on the mechanism had failed, one through being lost altogether and the other through mutation – the hallmarks of a tumour
suppressor.

It seemed coincidental that this stretch of chromosome 17 happened to include among its many genes p53, already labelled in all the literature as an oncogene. The fact that it produced lots of
protein in the cells Baker was working with seemed to confirm its designation. But because of the niggling anomalies in recent experiments with the gene, Baker and Vogelstein decided to check it
out so that they could eliminate it decisively from their search. Baker carefully selected a cancer cell that had already lost one copy of p53 along with the chunk of chromosome 17; she then
isolated the gene from the remaining copy and cloned and sequenced it – still at that time a tedious task that took many months. When she finally got the read-out, Baker was fully
expecting the gene to be normal. But to her amazement, she found a mutation.

‘I re-checked the sequence at least 10 times before surreptitiously showing the data to Janice Nigro, a fellow graduate student, to be sure that I had not lost my senses in the escalating
excitement,’ she wrote in a review of the discovery for the journal
Cell Cycle.
‘Knowing that Bert would approach the data with his usual critical rigor and logic
(“What’s the least interesting explanation for your data?”), I tried to be as calm as possible when I walked into his office and announced, “I found something
interesting.”’

Talking of the moment some 23 years later, Vogelstein gave a peal of laughter. ‘This experiment was not meant to show p53 was there, it was meant to
eliminate
it – so we
could look for the
real
tumour suppressor and we wouldn’t have to be bothered by this any more! I remember it quite clearly: we were down on Bond Street, which is a supermarket that
our lab was in back then because we didn’t have any room in the regular hospital. It was actually kinda nice . . . but anyway, it was Friday afternoon about three o’clock and Suzy had
sequenced the whole thing, and she came back and said, “Look, there’s a change.” It was a single change – I think it was a C to T change – and she had sequenced both
the normal and the tumour DNA from the same patient to make sure it was somatic.
⁶
She was excited, but I was concerned, because most interesting
results turn out to be artefacts. And also this particular change is
not
a big change. We expected to see some very pronounced inactivating event. This was the kind of change you could
easily think would do
nothing
to the function of the gene, okay?’

Vogelstein and Baker repeated the sequencing of p53 from the same patient’s tumour over and over again and confirmed it was a genuine new mutation. Then they did the
same thing with tumours from several other colon-cancer patients, once again sequencing the p53 gene from tumours where one copy had already been lost. And they found the same thing
every time: a single small mutation in the gene. ‘When we found the first one I was still doubtful,’ said Vogelstein. ‘When we found two out of two, I was getting pretty excited.
And when we’d done more, then I was sure. It’s just the statistics . . . The chances of you getting this somatic mutation in this many tumours is minuscule – one in a billion or
something.’

For Baker, a young scientist just starting out on her career, this was an extraordinary coup. ‘The eureka moment of a discovery is usually drawn out over time as the hypothesis is retested
and confirmed, and a scientist slowly becomes more convinced by the accumulating evidence that they have made a meaningful discovery. But as a naïve and enthusiastic graduate student, I truly
believed that I had identified the critical tumour-suppressor gene in colorectal cancer at the moment that I found the first mutation. Incredibly, it was correct.’

HOT COMPETITION

Unbeknown to Vogelstein, Arnie Levine and Moshe Oren, working independently in New Jersey and Israel, were hot on the same track. You will remember that after a period of
mighty frustration when his lab was scoring blanks with their clone of p53 while others were successfully creating tumours, Levine had realised that his was the only clone of the normal, wild-type
p53 and that everyone else had mutants. His relief at explaining the presumed failure of his clone quickly gave way to excited curiosity. The very recent discovery of the retinoblastoma gene (Rb)
– the first tumour suppressor – set him wondering if wild-type p53 could be the same thing. He repeated the experiments with his wild-type clone that he had initially read as failures
– mixing it with an even wider selection of known oncogenes, including the two powerful ones found so commonly in tumours, Myc and Ras – and in every case he
found that the wild-type p53 stopped transformation. ‘It trumped everything,’ he told me when I visited him in Princeton, clearly relishing the memory all these years later.
‘Every time we got a transformed cell it killed it. That was our first clue that we had a tumour suppressor; we didn’t in fact have an oncogene, though the mutant was behaving that
way.’

Levine’s lab submitted their paper to
Cell
in 1989, around the same time as Vogelstein and Baker submitted theirs to
Science.
But before either set of findings had seen
the light of publication, both teams found themselves attending the same conference, at Cold Spring Harbor on the outskirts of New York City, at which both were scheduled to give presentations.
‘The speaker before me was Bert Vogelstein. I’d never met him before,’ continued Levine. ‘And he gets up and he says the following: “We’ve been sequencing p53 in
human tumours and our results with colon cancers suggest it’s a tumour-suppressor gene, not an oncogene.” I almost fell off my chair, because my talk was coming next and I was going to
say the same thing.’

Did you feel cruelly upstaged, I asked Levine? He shook his head, ‘No, no, I
loved
it! The reason I loved it was two things: because I knew when I published the fact that it was a
tumour-suppressor gene everyone in the field would attack that. Why wouldn’t they? There were 10 years’ worth of oncogene papers, right? I mean 10 years . . . Everyone is committed to
an oncogene. You say it’s not an oncogene and you better prove it really isn’t! Well suddenly I had Vogelstein on my side. I had a second observation, a confirmation. That was the first
thing that made me feel very good about this – because you’re always worried that you’ve made a mistake.

‘But secondly – and this is what made me smile and never stop smiling – it was in
humans.
That’s what Vogelstein
found. We were working on
mice all the time. I mean, Moshe had cloned the human gene, but there was
very
little evidence that humans were going to have p53 mutations. I think throughout the 1980s most people
thought this was a curiosity: SV40 didn’t cause tumours in humans; if this was the way it forms tumours in animals it’s a curiosity that is intellectually very satisfying, but its
application to humans is probably nil.’

Since the meeting in Levine’s office in 1987 at which the light had dawned about the true nature of their clones – that Levine was the only one with a wild-type clone, while all the
others were mutants – Oren too had been keen to discover the function of wild-type p53. And he too had decided to repeat the experiments with other known oncogenes, teaming them up with the
normal p53 clone and looking more closely at the results, for he knew now that ‘nothing’ meant ‘something’ after all. It did not occur to Oren that Levine would have had the
same idea and be doing exactly the same experiments in Princeton, so he was unaware at the time of just how hot was the competition.

‘We got with wild-type p53 exactly the same kind of results that Bob Weinberg’s lab was getting with Rb: you suppress transformation; you inhibit the growth of transformed cells. So
against this background it was easy to conclude that normal p53 was behaving like a tumour suppressor. But had it been three years earlier – before the discovery of Rb – I must say
frankly that I doubt if we would have interpreted it correctly.’