p53

by Sue Armstrong

  ‘But it wasn’t that we all sat together and suddenly realised – it was something that had been cooking slowly in our minds and that finally converged in that single two- or three-hour meeting. If you compare one to one, you don’t know what’s right and what’s wrong. You need enough information for all the bits of the Lego to fall into place and to get the feeling of what is the rule and what’s the exception.’

  This was a momentous revelation, but it took a while to sink in. Except among those who were still feeling the intellectual buzz of cloning and testing their creations, interest in p53 had waned as those early experiments showed it wasn’t the novelty everyone had hoped for; it was ‘just another oncogene’, and rather a feeble one at that. This was a time when young scientists looking for jobs and promising paths ahead in the highly competitive world of molecular biology were being warned away from p53 as a dead-end topic. Even some of the old guard who had been in the field since the discovery of the new gene thought of abandoning this line of research and looking for new challenges.

  For nearly a decade p53 research was in the doldrums. But as people began to cotton on to the fact that for several years they had been led up the garden path by mutants, a big question took shape in the collective mind: if p53 was not an oncogene, what else could it be?

  CHAPTER SEVEN

  A New Angle on Cancer

  In which we hear of the revolutionary discovery of tumour suppressors – genes whose job is to protect us from cancer by detecting and eliminating cells with corrupted DNA.

  ***

  Science, my lad, is made up of mistakes, but they are mistakes which it is useful to make, because they lead little by little to the truth.

  Jules Verne

  In retrospect there were many experiments with p53 that raised questions about its true function. Varda Rotter works in a lab two floors down from Moshe Oren at the Weizmann Institute. Now in her sixties, she’s a handsome, stylish, vivacious woman with strong features and short pepper-and-salt hair; she has been involved with p53 since its earliest days, frequently swimming against the tide as her research findings have challenged mainstream thinking. Rotter’s passion for her work is obvious: the gene is almost a character in her life. She uses words like ‘beautiful’ and ‘monster’ and ‘schizophrenic personality’ to describe it. And on the walls of her office in Rehovot – among the framed photos of iconic microscope slides and of Varda with colleagues at various workshops – are pictures, drawn by her granddaughter, of p53 depicted as an angel and a devil. She smiles broadly and with obvious affection as she tells an anecdote about how her obsession with her work has been seen by her family. Her daughters were still little when Rotter joined the lab of the Nobel Prize-winning virologist David Baltimore at MIT as a postdoc and the family moved to the US for a while. ‘On one occasion they were asked at school what their parents did, and my older daughter answered, “Oh, we have a dad and p53!”

  ‘I do my work with all my emotion; that’s just how I am. Even today my head of department said, “Why are you showing me with such enthusiasm such benign data?” I said, “Oh, don’t you see, this is a breakthrough.” And she said, “Every day a breakthrough . . . !”’ Rotter laughs as she mimics her colleague’s exasperated tone. ‘But I think if you do not have this attitude you could not survive,’ she adds theatrically.

  In 1984 Rotter was making her own clones of p53 and running experiments with a virus that infects blood cells and causes leukaemia. History is ruthless with the runners-up or she too might now be credited with discovering p53, for she had stumbled across the gene herself in her research with a mouse virus and its oncogene, called ‘Abelson’ (or Abl for short), while working in Baltimore’s lab in the late 1970s. Like the others, she had found that her efforts to separate Abl protein from everything else in cancerous cells brought along another protein for the ride with a molecular weight close to 53 kilodaltons. But her findings were overshadowed by Lane, Levine and the rest, who published their discoveries just ahead of hers. In her later work with leukaemia cells, Rotter’s findings were very different from most others exploring the function of p53. Far from seeing an over-abundance of the protein, she found none – the gene seemed to have been lost. Researchers in Canada also working with leukaemia viruses found the same thing – most of their blood-cancer cells showed no p53 activity at all.

  The two groups published their research at the height of the debate about what p53 does. So how, I wondered, did the rest of community square these findings with the prevailing dogma that over-expression of the protein is the very hallmark of an oncogene? Rationalisation, replied Moshe Oren. ‘People knew that leukaemias very often have different biology from solid tumours. So you could say, okay, well, maybe leukaemia’s a different story – we don’t understand p53 in leukaemia, but we understand that in the typical types of cancer p53 is behaving just as an oncogene should behave.’

  Not everyone was ready to be convinced by such an argument, however. In 1982 Lionel Crawford, in whose lab the young David Lane had been doing research when he discovered p53, had published a paper describing his work with breast-cancer patients, where he found that almost one in 10 of the afflicted women had antibodies to p53 in her blood serum. This meant that, in effect, the women’s immune systems were preparing to attack one of their bodies’ own proteins as if it were a foreign invader. Crawford’s observations went largely unnoticed because at the time no one knew what to make of them. But they intrigued Thierry Soussi, still working in Paris in the lab of Pierre and Evelyne May.

  Standing back from the jigsaw puzzle to get a better view, Soussi wondered: could the fact that most solid tumours produce an over-abundance of p53 protein, and the fact that in some cases this abundance triggers the immune system to produce antibodies, be related? Might this be an indication that the protein – and the gene that produces it – is mutant and therefore malfunctioning, which is why the immune system sees it as foreign? It was a portentous question because, if this was the case, it would mean going back to the drawing board to discover the true nature of p53.

  ALFRED KNUDSON AND THE ‘TWO-HIT’ HYPOTHESIS

  For clues to what this true nature might be we need to rewind the clock to the late 1960s to meet paediatrician and polymath Alfred Knudson, then working at the MD Anderson Cancer Center in Houston, Texas. Here Knudson developed the ‘two-hit’ hypothesis of tumour formation that was to spark a revolution in cancer research. The two-hit hypothesis is such an important concept in the history of p53, and the information about Knudson the man so sparse, that I decided to seek him out myself to hear his story. I flew into Philadelphia where he lives with his wife, Anna Meadows, a paediatric cancer specialist, on a hot July afternoon in 2012.

  Arriving at their penthouse apartment in the city centre, I was ushered into a large, elegant drawing room with thick cream carpets, a feeling of space and light, and views over Philadelphia’s narrow tree-lined back streets, with their red-brick terraced houses and tiny gardens. The walls of the Knudsons’ apartment are hung with artworks – paintings and collages of such vivid colour and compelling compositions that they immediately draw the eye and invite conversation. But what most caught my attention was a striking, man-sized sculpture in scrap metal of a spindly figure with one huge eye thickly fringed with eyelashes made from nails. The Knudsons had spotted it at one of Philadelphia’s outdoor art shows and, so pertinent was it to Alfred’s seminal work with retinoblastoma, a childhood tumour of the eye that inspired his ‘two-hit hypothesis’, that they bought it. ‘It was made by the wife of one of Al’s scientific colleagues, Zoila Perry,’ explained Anna, a petite, pretty woman who exudes energy and a sense of purpose. It appealed to the Knudsons because, viewed from one side, the eye is blue and from the other it’s green, seeming to symbolise the ‘two hits’. ‘Both of us have worked in retinoblastoma – Al in the genetics and me in the clinical aspects including new therapy to save eyes,’ commented Anna.

  That first evening, the three of us took
a bottle of white wine from the fridge and went out into the warm summer evening for a meal at one of the Knudsons’ favourite Italian restaurants. Next day, Alfred Knudson and I walked together through the city to the College of Physicians. Here we were shown into the library, a hushed space of dark wood and dusty books behind a heavy door, where we sat down to talk. Knudson is now in his nineties, a spare man of medium height with a mop of white hair, a deep, slow voice and eyes that hold yours unblinkingly as he speaks.

  Born in Los Angeles in 1922, he was the first person in his family to go to university, but felt an academic career was his destiny from his earliest days in high school. Then, he mostly imagined himself as a mathematician or physicist, but the current carried him towards medicine instead. He got a place at Caltech (the California Institute of Technology), where he was required to take courses in chemistry and biology as well as physics and maths and says with a deep chuckle, ‘I hate to admit how naïve I was – and you’ll be amused by this . . . I came to the conclusion that they already knew everything in physics and it didn’t seem like a very interesting field to get into!’ Biology, on the other hand, excited him with its possibilities and he decided eventually to study genetics, which seemed to combine biology with his love of maths. ‘It was obvious there was a lot to be discovered in terms of human genetics, because people were almost totally ignorant about heredity and disease at that time.’

  Still at Caltech when America entered World War II in December 1941, Knudson was advised that his best hope of staying in school was to join the forces and apply to study medicine or engineering, which were considered strategic skills and sponsored by the government. ‘I didn’t think I wanted to do engineering – I had the same objections as with physics, only more so!’ He accepted a place at Columbia University Medical School in New York, and left southern California for the first time in his life. He decided to study paediatrics. ‘Children have some interesting genetic diseases, and I’d had a course in embryology and thought, oh, here’s a great field that is way behind the times – there are all kinds of possibilities with this. I thought paediatrics would give me a chance to combine genetics and developmental biology, and it did.’

  Knudson’s first serious encounter with childhood cancer came during his residency at New York Hospital, when he was required to spend a month at Memorial Sloan Kettering Cancer Center just across the street. ‘It had a little unit for children’s cancers. There were about 20 patients there. I had seen a child with Wilms’ tumour before, and somebody with leukaemia – but to see 20 children with cancer just blew me away . . . I never forgot that.’

  Knudson rejoined the army for the Korean War, believing they would draft him anyway, but never saw action: he reckoned they had little use for a paediatrician on the front line. After two years kicking his heels at Fort Raleigh, Kansas, he felt life was passing him by, he told me, and he was anxious to get back into the world of ideas. He returned to Caltech to do a PhD in genetics and biochemistry in 1953, just months after Crick and Watson had cracked the mystery of DNA’s structure. The institute, on the crest of the wave of genetic research, was buzzing with intellectual energy. The molecular-biology revolution was gathering momentum.

  With his doctorate under his belt, Knudson moved on to the City of Hope Medical Center in Duarte, California, to head a new paediatric unit with a special focus on cancer. But it was at MD Anderson in Houston, which recruited him 10 years later to start a programme in genetics, that he developed his two-hit model of tumour formation. Knudson figured that in order to tease out what was happening at a molecular level in cancer it was best to work with one of the less complicated tumour types. ‘To start out studying something like polyposis was kind of hopeless, you know?’ (Polyps, he explained, are fleshy outgrowths of normal tissue in the wall of the colon that can eventually turn malignant, and the progression to cancer can take years and follow many different paths.) ‘But if a kid can be born with cancer it’s about as simple as it can get. That was my thinking.’ Retinoblastoma met this criterion; it was the ideal topic for research.

  A rare tumour of the retina, or light-detecting cells of the eye, retinoblastoma affects children almost exclusively below the age of five, because it starts in the stem cells of the developing retina that, like the stem cells of all organs of the body, experience an explosion of division and growth during gestation and the early years of life. An early sign of the disease is a milky-white appearance to the pupil of the eye which, left undiagnosed and untreated, as it often is in the developing world, will grow into a grotesque spongy-looking mass of red and white flesh that distorts the child’s whole face and will eventually kill.

  Knudson did not actually see cases of the disease himself. Indeed, he had given up regular paediatrics and treating patients by this stage to concentrate on genetic research, but he struck lucky. He discovered that two people – a paediatrician in England and an ophthalmologist at MD Anderson – had kept detailed records of retinoblastoma cases they had seen. Poring over this rich repository of data, he observed that the disease ran in families as well as occurring sporadically, and that there were distinct differences between the two sorts of patients. Affected children from families with the disease typically developed cancer at a much younger age than those with no family history. And they tended to have multiple tumours in both eyes, while the sporadic form of the disease typically led to a single tumour in only one eye.

  So what did Knudson make of what he was seeing? Something ‘outrageously brilliant’ in its simplicity and far-reaching implications, said Peter Hall, reviewing the pivotal moments in cancer research nearly four decades later. Here in a nutshell is how Knudson himself explained it to me as we sat together in that quiet library in Philadelphia.

  Every gene in the body, except those of the sex cells, ova and sperm, is made up of two copies of itself (or two ‘alleles’), one copy inherited from the father and one from the mother. Knudson realised that in those cases of retinoblastoma with a family history of the disease, the gene responsible was inherited – though in the late 1960s no one was yet able to isolate genes or identify them individually. The overwhelming likelihood, he believed, was that only one copy of the gene, one allele – inherited from either mother or father – would be faulty at birth and the disease would occur only if and when the other allele developed a fault. His reasoning was confirmed by the fact that among the familial cases, not all the siblings of an affected child developed tumours, though they would all have inherited the same genes from their parents and been similarly susceptible.

  In those cases in his data set with no family history of retinoblastoma, faults had obviously developed in both copies of the gene quite by chance over time – explaining why victims tended to be older than those with familial retinoblastoma and typically to have no more than one tumour.

  All this suggested to Knudson that one faulty copy of the gene involved in the development of the retina is not enough on its own to cause tumour growth at that site. The other copy has to develop a fault also. But – and this was the revolutionary suggestion – until the normal copy develops a fault, it seems to keep the already faulty copy in check, so that the cell functions perfectly normally. Only when both copies are faulty do the cells start to behave erratically and to develop into a tumour. Put another way, in all the cases Knudson observed, something seemed to be breaking down and the cells involved to be losing their ability to function properly. He proposed that what had happened in these cases was that some kind of brake on proliferation of cells was lost. It was such a simple suggestion, but it swam against the tide of cancer research which, in 1971 when Knudson published his ‘two-hit hypothesis’, was intensely preoccupied with the driving forces of cancer, the newly discovered oncogenes, which implied a gain of function. He was proposing the existence of an equal and opposite force, an ‘anti-oncogene’ (soon to be renamed a ‘tumour suppressor’) that allows cancer to develop when it is knocked out – implying a loss of function.

  Her
e, the car offers a useful analogy for visualising the forces at work inside cancerous cells, as proposed by Knudson. Think of oncogenes as the accelerator pedals, and tumour suppressors as the brakes: a defective accelerator cable might stick, forcing the car to speed up uncontrollably (a gain of function); brake failure (loss of function) will have a similar effect in that the car won’t be able to stop. But the analogy can be taken still further, since in most cars there are two brake systems. If one system fails, you still have the other and generally both sets of brakes have to fail for catastrophe to occur. Likewise, for tumour-suppressor genes to be involved in cancer, it usually requires both copies of the gene, both alleles, to be disabled. This is the central lesson of retinoblastoma, and the essence of Knudson’s ‘two-hit hypothesis’.

  HENRY HARRIS HAS THE SAME IDEA

  Knudson had arrived at his theory through mathematical modelling of the data before him. There was, however, more direct experimental evidence to back it up. It came from the laboratory of cancer geneticist Henry Harris, an obstinate (by his own admission) and independent-minded Australian who had been recruited in 1952 by fellow countryman Howard Florey – famous for his collaboration with Alexander Fleming in the discovery of penicillin, for which they won a Nobel Prize – to work with him at Oxford University.