ONE MAN’S JOURNEY OF DISCOVERY
For David Lane, who began working with SV40 in the mid-1970s, this was a completely new field. Trained in immunology at University College London, he was still trying to write up his PhD thesis for his charismatic professor, Avrion Mitchison, when he was asked to join the laboratory of Lionel Crawford at the Imperial Cancer Research Fund. Besides offering the excitement of a new intellectual challenge, cancer research had emotional significance for Lane. During his first year at university, his father had died of colorectal cancer within six months of complaining of backache that would not go away. He left a still-young wife and five children ill-prepared to face such loss. ‘It was very fast and very shocking,’ Lane said as we sat talking over coffee in the conservatory of his home in Scotland, the late-summer sun streaming into the room and the occasional ear-splitting roar from a jet fighter streaking across the sky from nearby Leuchars air base. He cared for his father in his last weeks. ‘I saw the disease as it really affects people, and that was a pretty formative experience. I felt it very strongly – we all did as a family.’
As an immunologist Lane had learnt how to use isotopes of iodine to label proteins within cells so that they could be watched in experiments, and it was this skill that Crawford’s lab at ICRF was after. ‘Incorporating the isotope makes proteins very radioactive and screamingly easy to detect,’ he told me. ‘I was good at this; I had to do it for my PhD. But not everyone likes handling these materials. I think it’s a completely safe isotope, but it’s kind of noisy. I mean, the Geiger counter goes crrkk! And you know you’ve done the trick!’
The facts that SV40 can ‘transform’ normal cells into cancer cells and that its effect is dramatic are what made it attractive to researchers at the ICRF. Just before Lane went to work there, the viral gene responsible for transformation, SV40’s oncogene, was identified as something called ‘large T antigen’. To begin to tease out exactly how the virus transforms cells when it infects its host, scientists needed to study the protein it produces when the large T antigen is switched on, and this is what Crawford’s lab was doing. Lane’s task was to develop reagents, or tools, to highlight and extract the large T antigen protein from infected cells. The challenge was to obtain manageable quantities of the stuff that was pure and separated from all the other gunge in the cells. The reagent Lane developed worked on the same principle as the immune system. Just as our bodies design antibodies to seek out and ‘capture’ invading foreign particles for destruction by scavenger cells, so he designed antibodies to recognise specifically large T antigen.
‘It was a tremendously exciting time,’ recalls Lane, a tall, outgoing man with boyish good looks and the kind of good-humoured enthusiasm that says that, no matter what the challenges, the pressures and the politics, science for him has always been fun. ‘I felt we were right at the centre of things. We were on this fantastic floor at the ICRF with the real pioneers in the field. Renato Dulbecco [who had just won the Nobel Prize for Medicine for his work with tumour viruses] was there, and I remember Harold Varmus came over on sabbatical. The atmosphere was very intellectual, very knowledgeable; lots of meetings; lots of critique of your data.’
Working together on the development of their reagents for harvesting purified large T antigen, Lane and Crawford had just begun to make progress when the older man went for a year’s sabbatical in the US, leaving his newest recruit effectively in charge of his lab. Lane, still in his early twenties, was plunged into the office politics, with a number of people angling to increase their lab space and exercise their authority by telling the young scientist what to do while his boss was away. But Lane was not to be diverted. Not only is he naturally resistant to being bossed, but he had spent his formative years under the tutelage of the brilliant, unconventional and irreverent Avrion Mitchison, nephew of the eminent British biologist J B S Haldane and a man whose teaching philosophy was to let students follow their own noses (according to Lane, his professor once set an exam for his students by simply putting out a row of objects on a bench and asking them to comment – a test of their imagination that fazed those who had swotted up on the questions they had expected to be asked). The years with Mitchison had strengthened Lane’s independent spirit; at the ICRF he ignored his senior colleagues’ attempts to redirect his research, and just got on with what he wanted to do.
‘We made what we thought was a terrifically good, specific reagent to large T antigen,’ he recalled of the period leading up to the discovery of p53. ‘We were really happy with it because we’d done all kinds of clever things to make it right. But when we used it, instead of – as we’d hoped – just bringing down the one protein, it always brought down this other protein, with a molecular weight of 53 kilodaltons, as well.’
The process he used to harvest the large T antigen was called electrophoresis, which involves passing an electric current through a gel sandwiched between glass plates to which the protein mixture has been added in little wells. The current causes the protein molecules to migrate through the gel according to their size and electrical charge, thus separating them; the large molecules don’t migrate far, while the little ones go a long way. But no matter how hard Lane tried, he never seemed able to get the large T antigen pure and simple on its own: there was always this nagging ‘shadow’ in the gel.
To those of us unfamiliar with the microscopic world, it’s hard to imagine getting excited by two small, dark smudges on a plate of gel; or that such a mundane image might suggest the beginning of something momentous. But like beauty, scientific discovery is in the eye of the beholder: it depends on the scientist himself or herself seeing something singular in what to other eyes may seem commonplace or dull, and then having the wit to know what it might mean. To draw an analogy from the macroscopic world, the Laetoli footprints would have been nothing but patches of displaced dust on the floor of a remote African canyon to the untrained eye; but to the palaeoanthropologist Mary Leakey, who discovered them in 1976, they were a window into the fathomless past and thrilling revelations about the origins of humankind.
Those tiny dark smudges in his laboratory gel certainly excited David Lane, even though he could not, at that stage, have known what he had found, for p53 turned out in the fullness of time to be a type of protein never before seen. Others in Lane’s lab were unimpressed: it’s a contaminant, they told him; his tools were not as good as he imagined, some suggested; or perhaps the ‘rogue’ protein was a breakdown product of the large T antigen which was being chopped up into little pieces in the infected mouse cells he was using for his experiments. But Lane trusted his tools, antibodies designed to recognise and attach themselves to large T antigen and to no other protein; he had been extremely careful to avoid contamination of his experiments; and he remained convinced that what he saw repeatedly in those gels was important. ‘I was very sure in my mind that it was something to do with how the virus transformed cells, because I was kind of primed for that. I mean, Joe Sambrook (a highly respected tumour-virus specialist) had written an article saying, “Look, there can’t be many ways large T antigen works. It’s a single protein that goes into a cell, it transforms the cell into a cancer cell; it must be interacting with some part of the host machinery.” So I was looking for exactly that.’
Here again Lane’s background in immunology came in useful, for it told him that if, in the infected cell, the two proteins were sticking to each other physically, an antibody that recognised one would automatically bring down the other – ‘a sort of piggyback idea’. It strengthened his conviction that the interaction between the two was central to the way large T antigen turned the cells cancerous. He was able to get enough evidence to support that view and to excite the imagination of Lionel Crawford, newly returned from sabbatical, who repeated his experiments. The two published their findings in Nature, which was sufficiently impressed to make this the cover story of the journal of 26th April 1979.
OTHERS ON THE SAME TRACK
Meanwhile, across the A
tlantic, in a lab surrounded by quiet leafy parkland at Princeton University, Arnie Levine was also studying the monkey virus SV40 in an attempt to uncover the mechanism by which cells turn cancerous. ‘Ever since I was a kid I’ve been fascinated by viruses,’ Levine told me when I visited him in New Jersey, taking the train from Penn Station out through New York’s scruffy post-industrial suburbs towards the prosperous, sedate university town of Princeton. ‘What caught my imagination was that for a hundred years the effects of viruses were known, but no one had ever seen them. They’re the smallest of all living organisms, and they’re completely degenerate!’3
Levine, a genial man now in his sixties, grew up in New York City. His father owned movie theatres, and from time to time the young Levine would earn pocket money working as an usher and clipping tickets. ‘I must have seen James Cagney in Yankee Doodle Dandy about 19 times,’ he chuckles. ‘I grew up in a wonderful neighbourhood in Brooklyn, a typical American neighbourhood with Italians, Scandinavians, Jewish people – just a real mixture. My grandparents came from Poland and Lithuania. My father actually came from Lithuania as a young boy, but he never went to school; he went right to work, as was the case with most immigrants because they needed to earn money to survive.’
His parents believed fervently in educating their children, and the young Levine went to Harpur College (now Binghamton University) in New York City, where he was taught microbiology by Mildred Shellig, a retired medical doctor whom he credits with inspiring in him his passion for science. ‘Dr Shellig’s enthusiasm was infectious, and what I specially didn’t expect was that I loved the laboratory. We got to repeat people’s experiments from the literature and even try some new things. People stayed in the lab late at night; she had us over to her house to talk, and what did we talk about? We talked about science! It was terrifically infectious for me. This was probably 1959 – only six years after Watson and Crick and their discovery of DNA structure. So the molecular biology revolution was just starting.’
Levine began his career studying how viruses replicate themselves – essentially by taking over the machinery of the host cell to do so, because they are parasites that cannot function outside of other living things, be they plant or animal. He was working with bacteriophages – viruses that infect bacteria, described earlier. They were exciting model systems because everything happens so fast: an infected bacterium can spew out a new generation of virus roughly every 20–60 minutes, compared with an animal cell where the process might take 48 hours. But as the story of oncogenes and animal tumour viruses took off, Levine switched his focus. His imagination was fired. ‘How is that possible?’ he wondered of these minuscule scraps of life. ‘How can one or two simple genetic elements which encode the information for proteins cause a cancer? It seemed to me to be the way into understanding, for the first time, the origins of cancer in humans. So my movement into this field was really based on the use of the simplest system to get at one of the most complex of questions: what is the origin of cancer in us?’
One of the theories around at the time Levine discovered p53 was that cancer cells become reprogrammed to resemble embryonic or fetal cells. In other words, the evolutionary clock is turned back to a stage where the cells’ natural behaviour is to grow and divide rapidly, as they would in a developing embryo. Researchers had found evidence for this in the presence of regressive fetal proteins in liver and colon cancers, and had developed blood tests to detect the antibodies that are created by the adult immune system recognising them as aberrations – out of time and out of place – and arming itself to attack them.
The ‘re-embryonisation of cancer cells’ was an attractive concept because of the obvious behavioural similarities between the two cell types, embryonic and cancer, and the hunt was on in a number of labs to identify proteins that were present in both normal embryo cells and tumour cells, but not in healthy, fully developed adult cells. These proteins, the researchers figured, while obviously good news for developing embryos, could be the driving force behind cancer. In his lab, Levine’s team was looking for evidence of a fetal protein or proteins expressed in response to infection with SV40, and when p53 appeared Levine believed at first that was exactly what they had found. What specially excited him when his graduate student Daniel Linzer, who did the original experiments, showed him his results was that the rogue protein occurred in large quantities in the SV40-infected cells, suggesting it must be doing something important, and that it was interacting specifically with the viral oncogene, large T antigen. What’s more, his team had found exactly the same protein also in uninfected fetal cells.
By the time they published their paper in Cell, Levine and Linzer knew from further experimentation that p53 was not the fetal protein they had been looking for, and over time the theory itself fell from favour. But apart from the fact that it was obviously important because of its regular appearance in cancer cells, Levine did not know what to make of the new discovery. ‘We had no idea at the time where this would go compared to where it went – that’s for sure!’ he chuckles, pausing to reflect. ‘I don’t think any of us thought this was going to be the single most important gene in cancer based on the frequency with which it mutated . . . However, I would say we thought we had found the path into how SV40 causes tumours.’
THE PARIS GROUP
In Paris, a third group of scientists who also independently discovered p53 were equally mystified by the protein they found sticking close to large T antigen in their experiments with SV40. The discovery occurred in the lab of Pierre and Evelyne May at the Integrated Cancer Research Institute in Villejuif, where researchers were working simultaneously with SV40 and another closely related virus called polyoma, which causes multiple tumours in animals. Besides having a large T antigen like SV40, polyoma also has a small T and a middle T antigen; these are involved in transforming the cells the virus infects, with middle T antigen being especially powerful at causing tumours. Most people working in the field assumed that SV40 also had a middle T antigen that was likely to be equally potent; this was in the minds of Pierre May’s team when his doctoral student, Michel Kress, found large quantities of a new protein in his experiment.
Thierry Soussi, who was to become an important figure in the unfolding story of p53, was working in another lab along the corridor from the Mays in 1979, studying SV40 replication. ‘I vividly remember a postdoc4 bursting into our laboratory to announce that his friend, Michel Kress, had identified the middle T antigen of the SV40 virus: it was a 53 kilodalton protein,’ he wrote in a brief review of p53’s history for a molecular-biology journal in 2010. But when further investigation revealed that the new protein Kress had discovered in his SV40-infected cells came not from the virus but from the host, no one knew quite what to make of this. He and the Mays published the findings without embellishment in the Journal of Virology, read by few people outside this specialist field.
Pierre May died in 2009, and Evelyne May and Michel Kress are retired, so when I visited Thierry Soussi at the Karolinska Institute in Stockholm where he now works, I asked about the discovery of p53 in Paris. Ushering me into a bright, modern office opposite his lab, Soussi turned off the opera he likes to listen to while he works, pulled an old file from a high shelf and opened it, blowing away the dust as the scent of musty old paper wafted from the typewritten pages, yellowed with age. This was a copy of Kress’s original doctoral thesis and his paper about p53. Soussi leafed through it with a faint air of regret: ‘Michel Kress has been forgotten, which I think is a pity. He has been forgotten for two reasons. First, he is not ambitious at all. Second, he discovered p53 and one year later he had to go for a postdoc, and he went to a very good lab. He wanted to work on p53, and this lab told him “p53 has no future; you are going to work on something else.” Therefore he had to give up on p53 right at the beginning – which was not too much of a problem because at this time no one was really believing anything about p53. No one could be excited by something when you don’t know what it is.’
Ironically, the lacklustre response to p53 is exactly why Soussi himself chose to study the gene when he joined the Mays’ lab in 1983: he figured it was just the kind of quiet, uncompetitive backwater that he could cope with alongside the heavy burden of teaching he was expected to fulfil as a university researcher. He smiles at the thought of how wrong he was – but he wasn’t alone. ‘You know, I was working with a student at this time who was doing her thesis on p53 and she wanted to apply afterwards to INSERM (Institut National de la Santé et de la Recherche Médicale), which is the French equivalent of the National Institutes of Health. On the board for her thesis we put the director of INSERM, and at the end he told her, “Okay, I understand that you want a position here. There should be no problem: you have a good application. But just don’t work on this bullshit protein; change your topic.” This was exactly his word! No one honestly could anticipate in the early 1980s what p53 would become – it was impossible.’
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