p53 (12 page)

With the retinoblastoma gene, Rb, they did at least know they were looking for a tumour suppressor, because such an entity had been strongly predicted by the pattern of the disease in children,
explained Oren. But with p53 there was no such firm foundation for predicting a new model for cancer, only a bunch of baffling anomalies in the experiments with those early clones.
‘It’s challenging to be totally innovative conceptually, because you do experiments and
so often they lead you to results that are artefacts; they don’t
lead you anywhere. You really need to have something to grab on to to say, okay, here’s something that makes sense because we’ve already seen something like it before and we know that
it was true.’

Excited by his results, Oren, like Vogelstein and Levine, submitted a paper to a journal –
Nature
in his case. His paper was under review when, in a fluky rerun of Levine’s
experience, Oren attended a small p53 workshop in Gaithersburg, near Washington DC, at which the other two were also scheduled to speak. He had no inkling beforehand of his fellow scientists’
discoveries, and was amazed when they all came up with the same story. It was a moment of revelation, he said, because until he heard the evidence from Vogelstein’s lab, working with human
tumours, Oren had remained sceptical. ‘The results were very impressive, very strong, but I wasn’t sure whether they were clinically relevant or whether this was just some kind of nice
laboratory result without any connection to your cancer,’ he commented. ‘And so when it all came together it was really explosive. We said, wow, this is the greatest thing that has
happened with p53!

‘I came home very excited . . . This was still the age of written communication, and when I got home there was a rejection letter from
Nature
waiting for me in the mailbox.’
There had been a smile in Oren’s voice and his eyes were shining as he recalled the events of the Gaithersburg meeting. Then the rejection. He shook his head and looked down at his desk as
though seeing again in his mind’s eye the letter from the journal lying in his mailbox, feeling the anticipation of opening it and having the value of his discovery endorsed, and not quite
believing what he did see. ‘I was so upset I didn’t keep the letter, but one of the reviewers, I remember, said something like, “Oren is trying to jump on the bandwagon of tumour
suppressors; it’s fashionable, but it’s wrong. It’s very clear that p53 is an oncogene.” I don’t remember who this reviewer was, and I’m sure they’d not
admit to it now.’

The exhilaration Oren had felt at Gaithersburg was on a par with the thrill he had felt at pulling out the first successful clone of p53 five years earlier, and the let-down now was almost
unbearable. ‘I spoke to Vogelstein on the phone and I remember I cried to him about the injustice.’

Oren’s paper was, eventually, published soon after the other two, not in
Nature
but in
PNAS.
Vogelstein’s lab followed up with another paper from Baker’s
fellow graduate student Janice Nigro, to whom the excited Baker had first shown her result. Nigro had looked at a number of other tumour types where one copy of chromosome 17 was also known to be
lost and, sure enough, she had found the same thing – mutant p53 on the remaining chromosome. Clearly this was a general phenomenon, not one relevant only to colon cancer.

The observations from Vogelstein’s lab ‘opened a floodgate’, commented David Lane: suddenly researchers everywhere began going through historic samples of tumour tissue stored
in hospital pathology labs, some dating back to Victorian times. ‘With the tools available one could survey literally thousands of tumours very, very quickly for alterations, and in a pretty
explosive period in 1990–91, we and others showed that p53 alterations were the most common genetic change that occurs in human tumours.’

The papers from this period mark one of the most important milestones in the history of p53 and of cancer research in general. Not only was the new paradigm for the growth of tumours – as
a malfunction of the accelerator and/or failure of the brakes inside cells – strongly endorsed, but p53 turned out to be extraordinary. While under normal circumstances it acts to protect us
from cancer, it is corrupted by mutation in more than half of all human tumours – and a much higher proportion still in some specific types. In lung cancer, for example, p53 is mutated in 70
per cent of cases, while the figure for colon, bladder, ovary, and head and neck cancer is 60 per cent and for non-melanoma skin cancer 80 per cent of cases. What is more, instead of being knocked
out altogether by mutation and rendered non-functional, as happens with all other tumour suppressors (around 30 have been discovered and confirmed to date), p53 can sometimes simply change
character, taking on new and different roles in the machinery of the cell, as I will recount in a later chapter.

So how exactly does the gene work normally? And what happens to us when it goes wrong? These were the next big questions for the scientists.

***

How wild-type p53 works as a tumour suppressor – and what goes wrong to cause cancer – were naturally the next big questions for Vogelstein’s lab. But another
question was also nagging at him in the weeks and months following the discovery that normal p53 is a tumour suppressor not an oncogene. Exactly
when
in the development of a tumour does
the p53 mechanism break down? Vogelstein’s team’s special interest was colorectal cancer, of which they had abundant material, and they were able to show quite quickly that the mutation
of p53 occurs at the transition between benign and malignant. In other words, mutation of the gene allows a relatively harmless growth in the bowel, a polyp, to turn nasty and able to spread;
examination of the mass before this point will likely present intact p53. ‘That’s been shown to be generally true of other systems too,’ explained Vogelstein. ‘We showed it,
for instance, with the brain and the breast . . .’

As for
how
the normal gene works, one of the first vital clues came from outside the p53 field, from the lab of Carol Prives, a biochemist working at Columbia University in New York,
who later collaborated with Vogelstein on teasing out the detail, and has since become one of the stars of the p53 community.

Prives was born and raised in Montreal, Canada, the daughter of artists who had grown up poor and were anxious that their children get the opportunities in life they had not had. ‘They
very badly wanted me to go to college, because they hadn’t. But my mother also saw it as a venue to find the proper sort of husband!’ Prives, an engaging woman with a mop of dark curly
hair, permanently smiling eyes behind wire-rimmed specs and a slight lisp when she speaks, laughed at the memory. ‘This is really going to date me, but it was an era when the strongest
pressure on a woman was to get married!’

Prives went to McGill University, Montreal, where she did extremely well in psychology. But she soon realised that the observational and equivocal nature of the discipline didn’t suit her
temperament; she dropped psychology in favour of her second subject, biochemistry, which calls for more deductive reasoning and satisfyingly firm conclusions. For her PhD she went to work with the
British-born biochemist Juda Quastel, renowned for his work in fields as diverse as the bacteria of soil and crop yields, mental illness and cancer. Prives was not sure until she visited Quastel at
his lab whether to go for cancer or neurobiology, and recounted with typical self-deprecating humour how she reached her decision.

‘Juda Quastel was recruited to McGill to head an institute in biological sciences, and what they gave him was an old mansion which was bequeathed by one of the scions of Montreal’s
department-store families,’ she explained when I visited her in New York in the summer of 2012. ‘It was actually a gorgeous old house – the dining room was one lab and the living
room another, and there were still the mouldings in the ceilings, and the chandeliers. It was a very odd situation. When I met him, his office was on the second floor. There were three floors, and
the third floor, honestly, it must have been for the servants originally: there were these tiny little rooms and it was very squirrelly. Prof Quastel said to me, “What would you like to
study?” I said, “Well, I’m interested in cancer and I’m interested in the brain.” And he said, “Take your pick . . . The brain is on the third floor;
cancer’s on the second floor.” So I said, “I’ll do cancer.”’ Prives began to laugh: ‘This is how my wonderful career started off – just because I was
too lazy to walk up three flights to those squirrelly little rooms!’

It was many years later that she got involved with p53 and, as with so many others in this field, it was as a consequence of working with the cancer-causing monkey virus SV40. After a spell in
New York following her doctoral studies, she moved with her family – husband and twin daughters – to her husband’s homeland of Israel, where she spent seven stimulating years in
the lab of Ernest Winocour at the Weizmann Institute. It was Prives’ first real experience of living abroad, and she loved the true foreignness of the country and the intellectual challenge
of her work. ‘The Weizmann Institute was a great scientific environment at the time – particularly in this area, because there were some very strong people in SV40. It was one of the
top labs. Ernest Winocour, actually, was an esteemed virologist and many people went to learn this virus from him.’

Her immediate predecessor as a postdoc in Winocour’s lab was Bob Weinberg, and Prives picked up the reins from him. Weinberg had discovered the messenger RNAs – the recipes for
individual proteins – made by SV40. It was Prives’ task to translate those recipes into actual proteins in glass dishes in the lab, as a first step to working out how the monkey virus
causes cancer in its animal hosts. After seven years at the Weizmann, Prives returned to North America to take up a permanent position at New York’s Columbia University, and it was here that
she began to turn her attention towards p53.

‘Those of us who were involved in SV40 research were all obsessed with the large T antigen,’ she explained. ‘I still maintain it’s one of the most amazing proteins people
have ever studied. It’s multi-functional beyond belief – really an extraordinary protein. p53 was this protein discovered by several groups that binds to large T antigen and it was a
very mysterious entity.’ However, this ‘piggyback’ protein did not seem to excite much interest among Prives’ fellow large T antigen fanatics: most of her colleagues were
wholly preoccupied at that time – the mid-1980s – with the fantastic insights large T was offering them into how the DNA copying machinery works in animal cells.

Prives realised with some relief that here was an opportunity for her: p53 offered a relatively empty field in which to play. She had spent 1985–6 on sabbatical at one of the three key
labs working on DNA replication with large T antigen, and says, ‘I left there realising I’d be seriously insane to try to compete with these guys.’ By focusing on p53 she would be
doing her own thing, and she welcomed the new challenge: at this stage, p53 was still baffling scientists as an oncogene that didn’t play by the rules.

INSECT VIRUSES AS FACTORIES FOR FOREIGN PROTEINS

Besides exposing the futility of competing in a field for which she felt ill-equipped, Prives’ sabbatical experience had taught her that, in order to understand a
protein, you need to figure out how to obtain manageable quantities of the stuff to work with. Here she was fortunate to get to know Lois Miller, a specialist in baculoviruses, which are viruses
that exclusively infect arthropods – insects, spiders and crustaceans. Historically, these little scraps of life first appear in ancient Chinese texts describing disease among silkworms,
which the virus liquidates into foul-smelling sludge inside their skins. They played a part in the decline of the European silk industry in the late 19th century, and today they are a threat to the
farmed shrimp industry. But since the 1980s, baculoviruses have been put to beneficial use in the biological control of insect pests in agriculture. Their potential as environmentally friendly
pesticides stimulated intensive study of their molecular biology that revealed another, equally invaluable, property – baculoviruses are able to pump out large quantities of proteins,
including proteins encoded by foreign genes which have been artificially stitched into their DNA.

‘Somehow as a result of our conversations Lois decided to make baculoviruses expressing both SV40 large T antigen
and
p53 proteins. And she very kindly gave us those
viruses,’ said Prives. Working with the two proteins together, Prives soon made an important discovery – that p53 could inhibit the monkey-virus protein from triggering the copying
machinery of the cells. She published her findings not long after the discovery that p53’s normal function is as a tumour suppressor.

‘Next thing, I get a call from this person I’ve never heard of called Bert Vogelstein, who said, “Can you send me some protein, we have some interesting ideas?” So I
said, “Sure.” I didn’t know anything about this guy, but he seemed very friendly.’ Later Vogelstein asked Prives if she would make him some baculoviruses engineered to
provide protein from various p53 mutants taken from the tumours of cancer patients. He wanted to compare the activity of these proteins with that of the normal p53.

FOOTPRINTS REVEAL p53’S FUNCTION

While Vogelstein was busy in his lab in Baltimore, Prives, 270km (170 miles) to the north in New York, was poised to make a momentous discovery about the activity of p53. It
came from the work of a new young postdoc, Jo Bargonetti, who had recently joined her lab. Bargonetti had expressed her interest in figuring out exactly
how
p53 manages to prevent large T
antigen from switching on the machinery of DNA replication in cells infected by the monkey virus. ‘We sort of told her: don’t do that, it’s boring. We’ve already figured
that one out; go do something else,’ said Prives with a laugh. But Bargonetti was insistent: she wanted to do a ‘footprint’, she said.