p53

by Sue Armstrong

  Talking of his move from Sydney to Oxford many years later, Harris told an interviewer, ‘I got a telephone call from Hugh Ward, the Professor of Bacteriology [at Sydney], to say that he had Florey in his office and would I like to meet him. I said, “I must be dreaming, you mean the Florey?” He said, “Yes, come on over.” So I dropped what I was doing and I went over and there was Florey. He looked very much like a moderately successful businessman, but his speech was very laconic, very direct and he said, “Ward tells me that you like doing experiments, Harris, is that right?” I said, “Yes, I quite enjoy myself, break a bit of glassware, make a noise.” He said, “Well, how would you like to come to Oxford?” I said, “That’s like asking a man in the desert whether he would like a drink.”’

  This was the era of the virologists whose flood of insights into the deepest workings of the cell so excited the cancer community that their theories of how tumours start – typically through the acquisition of aggressive new powers in the cell – overshadowed all else. ‘My reaction to this unanimity of opinion was intransigent disbelief,’ wrote Harris in a review for the journal of the Federation of American Societies of Experimental Biology, FASEB. He figured that, with or without the agency of a virus, the rate at which mutations occur naturally in our cells is such that if malign mutations were always dominant – that is, able to override any other operating instructions in the cell – hardly a child would be born without a tumour already forming.

  In experiments run by fellow cell biologists Georges Barski and Francine Cornefert that seemed to confirm the virologists’ theories of the driving forces in cancer, Harris was struck by something the two scientists had dismissed in the interpretation of their results. Their experiment involved fusing malignant mouse cells with normal mouse cells to see which set of instructions prevailed. When, in due course, a tumour developed, they concluded that the genetic material from the malignant cell had dominated that of the normal cell. The fact that the resulting tumour cells had a depleted number of chromosomes they thought was of no consequence. Harris did not agree. Could it be, he wondered, that in becoming malignant the cells had lost genes that might have suppressed cancer, rather than gained genes that encouraged malignancy? It was exactly the question posed a few years later in Texas by Alfred Knudson, looking at the evidence from retinoblastoma cases.

  Over the next few years, Harris and his colleagues at Oxford – in collaboration with a lab in Stockholm that had the best materials to play with – explored this question by fusing malignant cells with normal cells of various different types. They demonstrated conclusively that for the hybrid cells to produce tumours, something in the DNA had to be lost – something that presumably was suppressing the malignant growth while it was still present. They published their findings in Nature in 1969, two years before Knudson’s retinoblastoma studies – and well before it was possible to home in on the individual gene or genes that might be responsible.

  But Harris and Knudson were up against the limits of technology in proving their theories; they were ahead of their time and their ideas caused barely a ripple in the cancer community.

  THE FIRST TUMOUR-SUPPRESSOR GENE IS FOUND

  That began to change in the late 1970s when cytologists – scientists who study the structure and function of cells – noticed that in the tumour cells of children with retinoblastoma, chromosome 13 was unusually short: it seemed to be missing a large chunk of DNA. What is more, in those children with a family history of retinoblastoma, all the cells in their bodies had a truncated chromosome 13. It gave researchers a place to look for the offending gene, and suddenly a hotly competitive race was on to find it and clone it. This promised to be the novelty everyone was seeking – something that might explain the many anomalies that were thrown up by their pursuit of oncogenes.

  But though the discovery had narrowed the field considerably, finding the retinoblastoma gene remained a Herculean task, for chromosome 13 is a mighty bundle of DNA some 60 million base pairs long. Furthermore, scientists weren’t even sure whether they were looking for a single gene or a clutch of genes that normally worked in concert to suppress tumours. They got their answers by an almost impossible stroke of luck. Arriving at Bob Weinberg’s lab in the mid-1980s, a young postdoc named Steven Friend announced he wanted to clone the retinoblastoma gene. As Weinberg tells it, he met this request from his new recruit with frank astonishment: ‘What? How on earth are you going to do that? You don’t know anything about cloning; nobody knows exactly where it is in chromosome 13.’ But Friend was not deterred. ‘Don’t worry. I’ll do it,’ he said.

  With what Weinberg calls ‘irrational enthusiasm – totally irrational and illogical’, Friend went ahead. He struck up a collaboration with a doctor working at the nearby Massachusetts Eye and Ear Infirmary, Ted Dryja, whose caseload at the hospital included children with retinoblastoma. Driven by concern for his small patients as well as by intellectual curiosity and the desire to learn something about DNA, Dryja, who had no formal training in molecular biology, had started to do some lab research to try to find out what lay at the root of this dreadful affliction. Focusing on chromosome 13, he had chopped out and cloned small fragments of the DNA. For him, this was just a means of acquiring some basic skills, but these clones created useful probes for investigating the chromosome further. Dryja shared his new tools with Steve Friend and, soon after the young scientist began his search, ‘Lo and behold, one of these probes landed right in the middle of the retinoblastoma gene and allowed it to be cloned out,’ Weinberg told his audience in the lecture hall at MIT. Spreading his arms wide to emphasise the length of the DNA strand, then stabbing with his finger to indicate the extraordinary landing site of the probe, right on target, Weinberg spoke with a voice slow and deliberate with amazement. ‘Now you know how many mega-bases each human chromosome is long; and you know how astronomically unlikely this stroke of luck was – or is. But it happened . . . This is what’s termed an “unearned run”. It was a terrific finding.’

  Steve Friend and Ted Dryja published their story in Nature in October 1986 and now the world was listening. A scientist called Webster Cavanee, then a postdoc at the University of Utah, had earlier narrowed the field of search down even further, to a specific region on chromosome 13, and his 1983 paper was the first to confirm that Knudson’s two-hit hypothesis was right. On hearing the news that the actual gene had been found and cloned, Cavenee commented, ‘I take my hat off to these guys. You can call it luck, but they did the right experiment, an elegant experiment, and it worked. What more do you have to do before they stop calling you lucky and start calling you a good scientist?’

  Alfred Knudson, too, was excited. ‘I’m delighted this has happened,’ he said. ‘Before, we could only concoct theories about what the retinoblastoma gene does. Now that we have the gene, we can get to work on the facts.’

  This was nothing less than a paradigm shift – a whole new way of looking at tumour formation as a battle of competing forces between oncogenes and tumour suppressors, the accelerators and brakes of our car analogy above. It led also to the recognition, finally, that cancer is altogether an aberration. Oncogenes are not there primarily to drive cancer, and tumour suppressors are not there primarily to suppress cancer; all these genes have regular work to do, including promoting or controlling the growth of cells as part of the endless cycle of building and maintaining our bodies. Only when these vital genes become corrupted and start to malfunction do they acquire the ability to cause cancer.

  When, very soon afterwards, p53 was finally revealed as being the same kind of gene as the retinoblastoma gene – a tumour suppressor, and a powerful one at that – it had an electrifying effect on the field. Researchers reacted like a flock of starlings over a winter field, wheeling around to fly in a new direction, and those who had begun to lose faith in p53 and to consider moving on to other things returned to their work with renewed enthusiasm.

  CHAPTER EIGHT

  p53 Reveals its True Colours
<
br />   In which we hear of the brilliant work and strokes of luck that showed normal p53 to be a tumour suppressor not an oncogene – its job being to press on the brake rather than the accelerator pedal in cells with damaged DNA.

  ***

  People don’t realise that not only can data be wrong in science, it can be misleading. There isn’t such a thing as a hard fact when you’re trying to discover something. It’s only afterwards that the facts become hard.

  Francis Crick

  The evidence that p53 had been miscast as an oncogene and was in fact a tumour suppressor had been accumulating in parallel with the work on the retinoblastoma gene. At the forefront of this challenge to received wisdom was Bert Vogelstein, a legendary figure in the p53 story, whose lab has been involved in many of the most important discoveries relating to the gene. On a stunningly hot afternoon in July 2012, I travelled to Baltimore to meet him, climbing the stairs of an elegant office block, all glass and sunlight and potted greenery, overlooking the original old hospital of Johns Hopkins.

  Vogelstein’s lab is famed almost as much for its fun as for its hard work. For years he headed a rock band, a bunch of musicians from his lab who called themselves Wild Type and who played at scientific conferences and other venues. The band broke up when the drummer’s wife died of leukaemia and, with children to care for alone, he had to drop out.

  ‘We started the band mainly to develop esprit de corps in the lab. Everybody liked it, scientists liked it – we used to play for scientists. And it was fun!’ Vogelstein told me later. ‘I think it’s important to have outside activities. I certainly encourage everyone who works here to do so. Most people who have looked at creativity have recognised that inspiration often comes in the off moments when you’re not focusing on exactly what you’re doing.’

  Arriving a little early for our meeting, I waited in the lobby and leafed through a photo album I found lying on a low coffee table – pictures of Vogelstein’s lab ‘family’ at conferences and social gatherings, and of Wild Type in their heyday playing gigs. Beside the albums on the coffee table was a copy of Grant Making for Dummies. When he emerged from his office, hand outstretched, I was struck by how slight a figure Vogelstein is and by the expression of impish fun on his face as he led the way into his large, cool office and motioned towards a swivel chair. Against the back wall I noticed the keyboard which, he told me, he likes to tinkle on from time to time during the day.

  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.”’