In which we discover that another, even more powerful strategy p53 uses to suppress tumours is to drive damaged cells to commit suicide.
***
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.
Kerr’s series of time-staggered electron micrographs published in a journal caught the eye of Alastair Currie, Professor of Pathology at Aberdeen. Currie was seeing something similar in work with a young PhD student, Andrew Wyllie, when they treated the adrenal glands of their lab rats with steroids, causing atrophy. Wyllie, who went on to make important contributions to p53 research, had come under the influence of the energetic and generous-spirited Currie as a medical student and been taken under his wing to study for a PhD. Currie died in 1994, but when I met up with Wyllie on the fringe of a pathology conference in Sheffield, he told me, ‘Alastair was interested in everybody and knew everyone’s names. He took an interest in individuals – and he took an interest in me.’ As we sat drinking coffee in a side room, he recalled with affection the ‘great gladiatorial discussions’ between Currie and his students which the professor, a man with a sharp mind and mischievous sense of humour, clearly loved.
Before accepting the post in Aberdeen – which brought him and his large family back to their beloved Scotland – Currie had spent three years as Head of Pathology at the Imperial Cancer Research Fund in London, and one of the questions that had intrigued him from the start was how tumours shrink. With no obvious signs of death, it must be some kind of cell ‘drop-out’ process, he concluded, and this is what he set his young PhD student to investigate.
‘I began to wonder in a very immature way if the regression was part of a process which had wider significance,’ Wyllie told me. ‘A lot of the things that tumours do are kind of caricatures of things that normal cells do. And if normal cells go through cycles of death and birth, then maybe the regression of tumours has something to do with that. These were ideas that were floating around in the ether, but it was difficult to design experiments to take them much further.’
The experiments with the adrenal gland were set up to test normal physiological processes and it was here that Wyllie and Currie began to see the single scattered cells that had intrigued Kerr. But the two Aberdeen pathologists were working with ordinary light microscopes which were incapable of showing the little round blobs in any detail, and Currie was excited by what he saw in Kerr’s high-resolution images. He managed to combine a visit to his daughter working in Australia with a spell as a visiting professor in Brisbane, where he made a point of meeting Kerr, and he suggested that the Australian spend his upcoming sabbatical in Aberdeen.
‘Before he went, Alastair drew my attention to some beautiful papers of John Kerr’s in the Journal of Pathology, and I have to say that I didn’t catch on initially.’ Wyllie, a slight, bespectacled Scotsman now in his late sixties, who speaks softly, precisely and with his whole body, gave a gleeful laugh as he recalled his first reaction to what turned out to be the start of something truly momentous: collaborative work between the three men that would uncover one of the most fundamental processes in biology and change forever the way cancer was perceived.
On arrival in Aberdeen to start his sabbatical, Kerr looked at the adrenal-gland tissues under his electron microscope and confirmed the presence of ‘shrinkage necrosis’ with identical characteristics to what he had seen in his experiments back home. Wyllie had found the same phenomenon in breast tumours in rats that shrank when the rats’ ovaries were removed, depriving the tumours of the hormones on which they were dependent. Hearing about their work, Allison Crawford, a developmental biologist also doing a PhD in the Pathology Department at Aberdeen, drew their attention to the extensive literature on programmed cell death in the developing embryo. It is a sign of how single-minded and narrowly focused scientific research can be that none of them had been aware of this rich body of knowledge before. But now they knew that what they themselves had seen in a variety of tissues and under a variety of cellular conditions, both normal and pathological, was a natural process with a role to play in many aspects of life – a process essential and complementary to mitosis, or cell division, in regulating the population of cells in an organism by clearing out old, damaged or excess cells as new ones are made.
According to Wyllie, Alastair Currie was troubled by the name ‘shrinkage necrosis’, which didn’t distinguish it sufficiently from the processes of putrefaction. And ‘programmed cell death’ seemed to suggest it was a developmental programme and nothing more. ‘Without question, the first description of the phenomenon, the first proper analysis of it, was John’s. But I think it was Alastair’s vision to emphasise the stereotypical quality of a process of cell death which was different from necrosis,’ he told me. ‘And then the funny bit of the story . . . This could only happen somewhere like Aberdeen, a small university, an outgoing individual meeting other professors at lunch . . . Alastair met the Professor of Greek and Latin, James Cormack, and he asked him to suggest a term. If the term rhymed with “mitosis” it would be kind of handy as well.’ Cormack proposed ‘apoptosis’. It was a word from ancient Greek poetry describing the dropping of leaves in autumn, and had been used in a medical context already – by the Greek physician Galen almost 2,000 years previously, to describe the sloughing of scabs from wounds.
Kerr, Wyllie and Currie introduced the term to the wider world in August 1972 in a paper in the British Journal of Cancer. ‘Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics’ has clocked up a huge number of citations in the scientific literature over the years, but at the time it was received with stunning indifference. ‘The stone just dropped into the well,’ recalled Wyllie, beginning to laugh. ‘In fact, The Lancet, in their Christmas edition, had a crossword quiz and one of the questions was: what is apoptosis? We were the joke of biology! “These guys are studying death, ho, ho!”’
The trio was not deterred, however, and, together with a handful of others ready to brave being called nuts, they worked away beyond the limelight to uncover the mechanics of apoptosis and to chip away gradually at the single-minded obsession with cell growth that gripped the mainstream cancer community at that time. But it was the discovery of a link between the p53 DNA damage response and apoptosis – or ‘cell suicide’, as some like to call it – many years later that finally convinced the sceptics of the wide significance of this phenomenon and revolutionised the thinking about cancer.
Thus far, the prevailing view had been that cancer was a disease of anarchic growth. Apoptosis suggested a complementary model of tumour formation: one in which cells that grow at a normal rate fail to die at the appropriate time. Filling a bath with water is a good analogy here. You can fill it up by turning on the tap faster than it can run down the plughole (proliferation) or you can leave the tap at the normal speed, but put the plug in to stop the water draining away (blocking cell death). You end up with the same effect by two different routes.
Serendipity played a role in this radical change of perspective about cancer, as it has in so many important scientific discoveries (think penicillin and the little dish of cultured bacteria Alexander Fleming accidentally left uncovered on his lab bench, allowing it to become contaminated with an antibiotic mould). In the case of p53 the serendipitous events occurred in the lab of Moshe Oren in Israel. The year was 1990. Oren and his team were investigating the activity of p53 mutants known to be oncogenic, trying to find out how they contribute to malignancy, when they were asked to move their lab up one floor at the Weizmann Institute. They were a small operation at the time and they soon had their equipment, including their two incubators, up and running in the new setting and were able to carry on with their experiments. But they began to notice that cells in one of the incubators were no longer flourishing as they had downstairs, and as they continued to do in the other incubator, even though both offered an identical environment and were set at 37°C (99°F). What could be wrong?
Cells not growing are bad news in a lab because there is ‘nothing’ to study, and Oren’s group tried all the usual tricks, like changing the culture medium in the dishes, to coax the sluggish cells back to life. ‘But after two months of doing the obvious things and still having problems, we sat together and said, “Okay, what’s going on here?”’ explained Oren, when I visited him at the Weizmann. ‘We realised that only some cells – and only in that incubator – were not growing well. And when we looked more closely, we realised that it was only the cells that had a particular mutant that were affected.’ Clearly something about the incubator was making just the cells with that p53 mutant unhappy, and the team decided eventually to check the temperature. ‘I was kind of ashamed that we didn’t do this earlier. When I was a student I was instructed that you always keep a flask of water in your incubator with a thermometer in, and don’t just trust the digital display. And once we did that we realised that this incubator was about 33.5°C (92°F) instead of 37°C (99°F).’
Someone had obviously bumped the thermostat during the removal, and in so doing had revealed an invaluable property of the mutant: it was temperature sensitive. Though the full implications of this took some time to sink in and to test, what Oren eventually discovered was that the mutant behaved like a regular oncogene at 37°C (99°F), the temperature typically set for lab experiments, and like a tumour suppressor – that is, like wild-type, non-mutant p53 – below 34°C (93°F).
Oren was familiar with temperature-sensitive mutants in virology and knew that what he had stumbled across here – in mammalian genes, not viruses, this time – was a very valuable tool; a potential gold mine for p53 research. It meant that scientists could put the mutant into a variety of cell types and watch its activity, first as a regular oncogene helping to drive malignancy; then they could reduce the temperature in the incubator to see how the cancerous cells reacted to the presence of wild-type p53 as the leopard changed its spots. What is more, they could track the reaction over time from Minute Zero.
Oren and his team tested their switchable mutant in a wide variety of cells, and found that at low temperatures, when it was behaving as wild-type p53, it inhibited cell division in those that were damaged – not a new insight by that time, but a confirming one. But they were especially interested to know what would happen in mouse leukaemia cells, which typically have no active p53 at all, if they switched on the wild-type behaviour. So they introduced their temperature-sensitive mutant to a dish of cells given them by Leo Sachs, a leukaemia expert in another lab at the Weizmann, and dropped the temperature in the incubator to 32°C (90°F).
The postdoc given the experiment to do was hoping to see something interesting, but when she returned to check her cells she found to her dismay that they were all dead. ‘Usually when you see cells that are all dead you don’t think about interesting possibilities,’ commented Oren. ‘You think you’ve done something wrong and it just means you have to do the experiment again – which she did, and again it repeated. After about two weeks of repeating the experiment, it was clear there was nothing wrong with the way the experiment was done, the cells were just dying.’
Oren was quick to realise this was interesting and important, though he didn’t know how to interpret it. So he took his data along the corridor to Leo Sachs for comment. Sachs suggested he consider apoptosis – a process Oren had never heard of – and directed him to the still sparse literature on the subject, including the papers by Kerr, Wyllie and Currie. Intrigued, Oren stained the dead cells in his dishes to make them stand out and put them under the microscope. They looked exactly like the textbook images of apoptosis, and further experiments to confirm their findings all pointed to the same thing: ‘p53 was killing cancer cells by apoptosis, and we were very excited about that.’
Oren was particularly keen to share his discovery with his old friend and mentor, Arnie Levine, who chuckled as he told me the story of their discussion when I visited him at Princeton. It was on the fringe of a meeting in Vienna in April 1991, where Oren first presented his findings in public and, full of anticipation, approached his former teacher in one of the coffee breaks. ‘I have to admit to making an error of judgement!’ said Levine. ‘Moshe and I are very close because he was my postdoc. Moshe shows me the data about apoptosis, and he says, “Well what do you think? Nice story!” I said, “I don’t know if this is going to go anywhere.” Moshe looked at me like, oh, that’s not good! But that’s okay, he’s a brilliant guy and he goes on and shows that it is important, and that it’s central and so forth. But I’m always amused by the fact that my first response was, “I can’t figure out why this would be important to anybody!”
‘It goes to show, you know, that you get a mindset about something. You hope that as a scientist you have a completely open mind about things, but of course you get committed to an idea, and you’re willing to run with that idea, and that’s what makes you work hard on it. But it starts to exclude other ideas, right? And that’s just life! That’s the way science works.’ (It also goes to show just how strong was the prejudice against death as a relevant topic for biologists that, 20 years after Kerr, Wyllie and Currie’s paper about apoptosis, some of the most eminent scientists were still so ready to dismiss it.)
Oren’s team was the first to demonstrate that p53 can promote apoptosis, but the setting of their experiments and the way they activated p53 in their cultured cells were artificial. The big question was where and when does this happen in real life? It was a question already being explored by scientists working with transgenic mice on both sides of the Atlantic.