p53

by Sue Armstrong

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.’

  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 eve
nts 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.

  CHAPTER NINE

  Master Switch

  In which we discover that p53 functions by attaching itself to the DNA in a damaged cell and taking control of other genes – switching them on and off as necessary to prevent the cell from multiplying.

  ***

  A true scientist is bored by knowledge; it is the assault on ignorance that motivates him – the mysteries that previous discoveries have revealed.

  Matt Ridley

  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 seve
n 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.