‘We had cell lines from patients with AT and it became clear very early on that p53 did not get induced normally,’ Kastan says. ‘We had absolutely no idea what the gene was that was missing in these patients. But whatever it was, we realised it was somehow required for the induction of p53 after radiation.’
At the same time as investigating the AT connection, Kastan was collaborating with a scientist at MIT, Tyler Jacks, who had created experimental mice with no p53. Sure enough, thymocytes – important components of the immune system – in Jacks’ mice failed to arrest at the G1 checkpoint when bombarded with radiation. Together with Vogelstein, Kastan was also collaborating with a third group, at the National Cancer Institute near Washington DC, who had discovered a collection of genes called GADDs that are directly responsible for arresting growth of cells with damaged DNA (indeed, their name is derived, imaginatively, from Growth Arrest and DNA Damage). The three teams found that GADD 45 was controlled by p53, and was one of the genes switched on by the tumour suppressor to cause arrest at the GI checkpoint that Kastan had first uncovered. Very soon, Vogelstein found another gene, p21, involved in the same event and also controlled directly by p53.
The picture that emerged of p53 from these disparate bits of research was of a master switch at the hub of a communication network within cells. Its job is to respond to incoming signals indicating DNA damage by recruiting the relevant genes ‘downstream’ to halt growth of the cell pending future decisions about its fate. In this way cells with scrambled DNA that might threaten the organism are disabled.
It was this picture that the researchers described in a paper they published together in Cell in 1992 and that Kastan says was ‘the most fun thing I ever did in my scientific career’. Just before the paper came out, he attended his first big p53 meeting, hosted that year by Moshe Oren and Varda Rotter in Israel. Oren had seen Kastan’s original paper on checkpoint arrest following radiation and been sufficiently excited to invite the American to speak at the plenary session – to the full, august gathering of the p53 community. ‘What was so much fun was that I was a total unknown in the p53 field,’ said Kastan. ‘I go to this meeting; I get up to the podium and give this talk about this whole signal transduction pathway the day the paper was published in Cell.’ No one had seen the data before, and it had a powerful effect on the audience.
‘I was a nobody with a no-technology lab,’ he continued, ‘but I just happened to ask an important question because I read the literature carefully. And I asked it at the right time, with the right techniques and in the right cell type.’
RARE DEGENERATIVE DISEASE HOLDS THE KEY
Not everyone was ready to accept Kastan’s model entirely. The fuzziest part of the picture at that stage, in 1992, was the connection with ataxia telangiectasia. No one knew what the missing element was in these patients that made them so sensitive to radiation; they knew only that, in normal circumstances, it was essential for signalling to p53 that the DNA was dangerously damaged and for turning the whole damage-response system on. Things became clearer when, after a Herculean effort by 30 international scientists and hot competition between the labs to find the gene or genes responsible, a team led by Yossi Shiloh at Tel Aviv University announced success in 1995.
The single-minded search for the AT gene took more than 15 years of his life, Shiloh told me when I spoke to him over the phone from New York, where he was on sabbatical in 2012. It began when his mentor at university, Professor Maimon Cohen, suggested that the young scientist join him on a field trip to a small village in southern Israel; there they would meet a family of Moroccan Jewish origin afflicted with ataxia telangiectasia. Shiloh had recently completed his Masters degree and was casting around for a topic for his PhD thesis. ‘Professor Cohen had a hidden agenda – to interest me in AT,’ he said. ‘It worked very well because when I saw those patients I decided almost on the spot that this was an important problem to work on. First, because it’s an extreme human tragedy and at that time it was an “orphan disease” – no one cared much about these rare diseases with long names. And second, it was clear that understanding AT would have broad ramifications in many areas of medicine – neurology, immunology, genetic predisposition to cancer and whatnot – because AT is like a microcosm of medicine, it involves so many systems in the human body.’
Shiloh had no illusions about how difficult it would be to find a common cause for such diverse symptoms – and for many years the consensus among AT researchers was that there were four distinct types of the disease and probably at least four different genes responsible. The first breakthrough – what Shiloh identifies as the starting gun for the race to find the genes – came from Richard Gatti at the University of California in Los Angeles, whose study population was the Amish people of Ohio. In 1988, Gatti had managed to localise the gene responsible for AT to a region on chromosome 11, homing in on this stretch of DNA through a technique called linkage analysis, which looks for genetic markers – small strips of DNA with unusual ‘spelling’ dotted along the genome that are consistently present in people with a particular genetic disease, and never found in healthy individuals. The researchers then use statistics to suggest which marker or markers is closest to the target gene. This narrows the search area, but finding the actual gene is still akin to looking for a person’s house when you have only the name of the city in which they live to go on, and it was another eight years before Shiloh and his team managed to achieve their aim.
‘When I look back I’m surprised yet again that for eight years the entire lab was working on that one project . . .’ he said. ‘You know, scientists are very individual . . . Today we still work on AT, but every student in the lab has his or her own project. At that time the entire lab, several generations of students and postdocs, was focused on just fishing out genes from that region of chromosome 11, analysing them, cloning them.’
Today, thanks to the Human Genome Project and the wealth of data about genes and sequences available at the click of a computer mouse, such an exercise is relatively straightforward. But in the mid-1990s it was slow and labour-intensive, and relied on close co-operation with the AT-affected families whose personal DNA was the lifeblood of the research. Among the hundreds of genes Shiloh’s team cloned was one that specially caught their attention because it was unusually long – so long in fact that they had to repeat the cloning exercise a number of times to convince themselves it was real. Clearly this was the recipe for a huge protein – and one, they soon discovered, that had the hallmarks of a ‘signalling’ protein responsible for sending messages within the cell.
Shiloh remembers the day they realised this was what everyone had been looking for. ‘I had been teaching and when I came back to the lab from my class my student was holding a Southern blot7 in her hand. She said to me, and I remember her words clearly, “There is something odd about this gene in this family.” This was one of our Palestinian Arab families. I looked at the blot and it was clear that a big portion of that specific gene was deleted in that family. It was a very dramatic result. Of course my heart skipped several beats, but I said to her as calmly and quietly as possible, “This indeed looks interesting, there might be something here. Why don’t you repeat the experiment with DNA samples from the entire family and additional controls?”’
She did so and the conclusion was inescapable: here was the gene whose corruption was the cause of the disease Shiloh’s team were seeing in all their AT patients. It was a time of high tension, recalls Shiloh. The race to find the gene was at its peak, with frequent rumours in the air that someone or other had succeeded, and the temptation to publish his lab’s results immediately was heavy. But he had a hunch that there might in the end be just one gene – not the four that everyone supposed – responsible for the different manifestations of ataxia telangiectasia, and it would take time to prove it. Someone else might get there first, but after intense discussion among themselves everyone in his lab agreed to hold off announcing their results until they had t
ested their hypothesis. It was a nail-biting time, but the gamble paid off: AT is indeed caused by defects in a single gene, which the international consortium named ATM, short for ataxia telangiectasia mutated.
This was the missing detail in Kastan’s picture of the DNA damage response: in time he and others were able to show how the signals are passed down the line from ATM, which first senses the broken strands of DNA, to p53, which then throws the relevant genetic switches to halt the division of the cell. This was biochemical proof of the mechanism, and it finally convinced the doubters that p53’s response to DNA damage is at the heart of its action as a tumour suppressor.
‘You know, you can’t overstate the importance of what Yossi did in cloning the AT gene,’ commented Kastan. ‘He will be somewhat humble in telling it, but people were searching for that gene for 20 years – including him – and it made such an impact . . . It really opened up the whole DNA damage-signalling field. Yossi is a fastidious scientist and it’s because of that fastidious approach that they got to that point.
‘He flew to Baltimore to tell me he had the gene clone, and I remember very distinctly, he was sitting in my living room, saying, “Okay, we got the gene, and we’re calling it ATM for ‘ataxia telangiectasia mutated’.” I looked at him and I said, “Well, that sounds great, but you know ATM has another meaning in the US?” And I explained to him about these new automated teller machines. His face dropped, and I said, “Don’t worry, Yossi, people will know that’s where the money’s at in the signalling pathway!” And it’s been true. The field just exploded at that point.’
MULTIPLE STRESSES, MULTIPLE RESPONSES
In labs everywhere, researchers began testing the model, and evidence soon mounted that many more insults to the DNA – as well as more subtle stresses on the cellular machinery – can trigger the p53 response to halt the cell cycle. The increasingly long list includes UV radiation from sunlight, chemicals in the environment, and activated oncogenes, as well as natural ageing and dangerously low levels of oxygen and essential nutrients like glucose in the cell. Importantly, each stressor has its own characteristic pathway – from the protein that sends out the first alarm signal that all is not ideal for the division of the cell, thus triggering the response, to the range of genes that p53 switches on. But they all have the same effect of preventing potentially harmful mutations from being passed on from one generation of cells to the next.
The frenzy of activity among researchers was fuelled also by revelations that, just as there are many different stressors that can trigger p53, there is also a variety of outcomes to the response. Besides inducing a temporary halt in a dividing cell while DNA damage is repaired, p53 can induce a state of permanent arrest, called senescence. And under certain circumstances, it will instruct a seriously damaged cell to commit suicide – a process that many people feel is the most important weapon in its armoury.
In July 1992, David Lane, p53’s co-discoverer, pulled all the information together from widely scattered publications in a review for Nature in which he dubbed p53 ‘the guardian of the genome’ – essentially, the policeman in our cells taking action to clear dangerous individuals from the scene. As a reflection of what many people were thinking, it was neat; but as a statement from a scientist it was unusually bold. ‘In a sense it was sticking my neck on the block,’ said Lane with a mischievous chuckle. ‘You write a scientific paper and you say: it’s not unreasonable to speculate . . . But in this I said: this is how it works! Then everyone thinks, well there’s a challenge! But is it true? Is it not true? Not everyone believes it even now, but it provoked debate, which is what it was intended to do. That’s very important to the progression of science.’
AHEAD OF HIS TIME
There is a poignant footnote to this story. It involves a young scientist called Warren Maltzman, who worked briefly as a postdoc in Arnie Levine’s lab in the early 1980s, before moving on to Rutgers, the State University of New Jersey. Maltzman’s doctoral research at Stanford had focused on how cells repair damaged DNA, and when he joined Levine’s team he became involved, naturally, in p53. At Rutgers the two fields came together when Maltzman observed that in normal, non-cancerous cells subjected to UV radiation (as in sunlight), the levels of p53 shot up. He published his findings in the journal Molecular and Cell Biology in 1984. ‘At that stage,’ says Levine, ‘we didn’t know p53 was a tumour suppressor; we didn’t know what it meant that the level went up, and so his paper was roundly ignored. Had everybody picked it up, we’d have known p53 was involved in DNA damage and repair responses right away; we might have found that it transcribes genes . . . But . . . the time was not ready for anybody to make sense of it.’ Despite a good reference from Levine when he applied subsequently for a research post, Maltzman’s academic career faltered and he went into industry. ‘I feel badly about that because this man made a contribution whose time had not come . . . In many ways it’s the human story of science,’ mused Levine.
CHAPTER ELEVEN
Of Autumn Leaves and Cell Death
In which we discover that another, even more powerful strategy p53 uses to suppress tumours is to drive damaged cells to commit suicide.
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In science it happens every few years that something till then held to be in error suddenly revolutionises the field, or that some dim and disdained idea becomes the ruler of a new realm of thought.
Robert Musil
Paradoxically, one of the most important and dynamic topics today in biology – the science of life and living organisms – is death. Programmed cell death, or apoptosis, to be more precise, which rivals p53 for the number of scientific papers it has generated. But this is not death as most of us know it – a process of decay and putrefaction as the cells in our tissues rupture and spill their contents, to be colonised by bacteria that release bad smells. It is not the death that produces pus in wounds. Necrosis is typically caused by random, traumatic injury and the spilled contents of ruptured cells can cause damage to surrounding tissues, seen as inflammation. Apoptosis, on the other hand, is an integral part of the programme of life – a recycling process in which the cell membrane remains intact while the contents are systematically chopped up and repackaged before being engulfed by phagocytes, the scavenger cells of the immune system, or swallowed by neighbouring cells.
Apoptosis is a process of shrinkage and quiet dispatch. It is unmessy and unseen. For decades it was the territory of embryologists and entomologists, for this is what sculpts our bodies in the womb, removing the web of skin between fingers and toes, hollowing out tubes, shaping organs and building our brains. It is what makes the tadpole’s tail shrink as it grows into a frog. And it is part of the process of metamorphosis, whereby a caterpillar turns into a butterfly or moth in the chrysalis, or a nymph into a dragonfly. Indeed it was an entomologist, Richard Lockshin, who coined the term ‘programmed cell death’ – a decade before it was given the alternative name of apoptosis – to underline the fact that here was a process controlled by the genes, with a beneficial role in biology, not the result of accidental or destructive forces.
Rick Lockshin was one of the earliest biologists to study the phenomenon and became a founder member of the International Cell Death Society and a leading light in the community. He came to the topic as a result of an interest in metamorphosis that developed during his undergraduate years as a biology student at Harvard, when he was given the opportunity to ‘hang out’ in the lab of entomologist Carroll Williams, and work as a dishwasher and lab technician. ‘Williams was one of the world’s experts on insect hormones, and he had brought the custom of afternoon tea back from a sabbatical in England. I therefore spent many afternoons listening in fascination to discussions about the mechanisms of insect metamorphosis,’ he told an interviewer for the journal Cell Death and Differentiation on the occasion of his 70th birthday in 2008.
Williams subsequently became Lockshin’s PhD supervisor and programmed cell death the topic of Lockshin’s thesis. His research was
given a huge boost when Williams, on a trip to Japan, found that moth pupae were selling for a very good price and ordered 20,000 to be shipped to his lab at Harvard. ‘When they arrived, he was horrified to realise that they had all initiated metamorphosis during the voyage,’ said Lockshin. ‘They were going to be nearly useless to almost everybody but me, as long as I was willing to work non-stop, and I was. For a brief time I had more material than I could have ever dreamed of having.’
Programmed cell death began to emerge into the mainstream of biology with the work of three pathologists, John Kerr, Andrew Wyllie and Alastair Currie, who came together in 1971 at Aberdeen University in Scotland, where Kerr was spending a sabbatical year away from his home town of Brisbane, Australia. Kerr had long been intrigued by cell death, having first noticed the phenomenon in London in 1962 while doing research for his PhD, which involved examining the effect on rat livers of cutting off the main blood supply. He could see clear evidence of necrosis in large patches of the livers, which showed all the characteristics of degeneration under the microscope. But gazing down the eyepiece at the wafer-thin slivers of liver, he saw something else, too – single cells scattered sparsely through the living tissue; small round blobs of cytoplasm speckled with fragments of DNA. This was death, too, but without the degeneration, or the inflammation of the surrounding tissue. Unaware of the literature in the insect and developmental-biology fields, he called what he saw ‘shrinkage necrosis’ because of its apparent role in atrophy of the damaged livers.
Back in Brisbane, in the Pathology Department of the University of Queensland, Kerr began studying the process more closely under an electron microscope. Soon he was examining tissues other than liver and finding the same thing – notably in sections of skin cancer and other tumour types. He and his colleagues concluded that programmed cell death must be responsible for the shrinkage of tumours after treatment, and sometimes spontaneous shrinkage too.
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