The tissue culture facilities of a modern biological science laboratory can easily be distinguished from the rest of the lab by their extreme cleanliness and, relatively speaking, tidiness. Notices abound, exhorting users to put stuff away properly, chuck out old bottles of solutions, soak glassware in the correct buckets, and dispose of plastics in the special bins labeled “autoclave waste.” All of these measures, unusual in the cheerful anarchy of most labs, are designed to ward off the dreaded twin spectres haunting all tissue culture work: that the cells die because they haven’t been looked after properly, or that somebody (never the cells’ owner, naturally) contaminates the precious cultures with bacteria or fungus. Such calamities at best cause a few days’ labour to be consigned to the bin, but sometimes, months of painstaking work are destroyed in a few moments of carelessly insanitary behaviour. Have you been making bread at the weekend? Then don’t go near your cells, or your baking yeast will mysteriously manage to breach all of the defensive barriers and get into your cultures.

The growing cells are taken from their temperature-controlled incubator, bringing with them the tantalisingly fizzy whiff of CO₂-laden air, and looked at under the microscope, to see whether they are “ready to split”; whether they are starting to outgrow their flask. If they are, one of the big tissue culture hoods is fired up. The air flow is switched on, and the fans rumble into life, providing a barrier of constantly rushing air between the user and the enclosed work surface. Then the front panel is removed, and the inside of the hood is thoroughly swabbed with alcohol, together with all of the bottles of medium and miscellaneous bits of equipment. In former days, enthusiastic workers would set the alcohol on fire “just to make sure” that everything was truly sterile, although the side effect of sometimes setting fire to one’s alcohol-soaked hands (gloves are a modern innovation) in a spectacular but (mostly) painless way may have been a more likely enticement.

The work begins. Endless flasks are brought to the hood, and the cells inside are laboriously removed and counted under the microscope. Then, they are divided up into new flasks pre-filled with growth medium and returned to the warmth and darkness of their incubators. Once the mundane husbandry is finished, there are likely to be experiments to tend to as well. Reagents are added to cells, medium is changed or augmented, and cells are harvested and go off to be analysed back in the real world of the main lab. Hours and hours of time can be spent in tissue culture, often to the accompaniment of appalling rock music, mercifully piped straight into the ear nowadays, but formerly blasted out on boom boxes to willing and unwilling listeners. Conflicts between the Radio 3 cricket commentary brigade and the lovers of The Smiths and the Stones were legion before Mr. Sony came to their rescue. And in the background, the fans driving the airflow of the hoods provide a steady rumbling ostinato.

Why this lengthy preamble? Because, in the face of all of the cumulative tissue culture lore, the mountains of dead cells, and the wasted years of scientific time, in one laboratory at the Imperial Cancer Research Fund, someone finally started to wonder whether all of this death might be trying to tell us something biologically meaningful. Was it possible that cell death was not just an awkward obstacle to doing decent experiments, but might matter? Furthermore, might finding the reason for all of the cell death lead to the answer to an enduring problem with our understanding of cancer: why is it that cancer, a disease of madly proliferating cells, is not more common?

Today, Gerard Evan is a major force in cancer research, but in the mid-1980s, he was a distinctly worried junior faculty member in the Ludwig Institute of Cancer Research in Cambridge. Gerard’s career up to then had had the standard upward trajectory of a smart Oxbridge-trained scientist: a PhD at the Medical Research Council (MRC) Laboratory of Molecular Biology in Cambridge, followed by a very successful postdoc in future Nobel laureate Mike Bishop’s lab in San Francisco, and then a return to Cambridge in the early 1980s to run his own lab as a tenure-track group leader, the lowest rung on the lab head career ladder.

Gerard Evan picnicking in France, ca. 1990. (Photograph courtesy of author.)

Gerard had spent his postdoc working on an oncogene called MYC (pronounced Mick), identified in the Bishop lab in 1978 as the gene in the chicken retrovirus MC29 responsible for its cancer-causing activities. v-myc was soon shown to have a cellular counterpart, c-MYC (known as MYC hereafter), which could be converted into an oncogene when overexpressed. By 1984, when Gerard returned to the United Kingdom, MYC was a well-established oncoprotein and had been shown to be able to drive uncontrolled growth when introduced into cells in tissue culture. (A quick note about gene nomenclature: names in italics signify genes, names in normal font signify their protein products. Hence, the MYC gene encodes the MYC protein. Strictly speaking, there is another level of hierarchy depending on what species the gene in question comes from, but I am ignoring that here for reasons of clarity.)

Like many nascent lab heads, Gerard had a dowry from his ex-boss comprising unfinished postdoctoral projects, and he simply continued to work on them. He was allotted a minute amount of lab space in the Ludwig Institute and hired a young research assistant, Dave Hancock, who’d previously been working across the road in the Department of Pathology and Virology but fancied a change. Dave was soon joined part-time by Trevor Littlewood, who was actually the laboratory manager for the Ludwig Institute but was pining for the bench; in fact, he moved into a full-time position with Gerard shortly afterwards, leaving the world of laboratory administration behind forever.

Dave Hancock in the laboratory, 1980s. (Photograph courtesy of Trevor Littlewood.)

The lab’s publication output over the next couple of years was good, but not mind-blowingly so, and Gerard’s growing feeling that, scientifically, he was drifting without purpose, was compounded by his disappointment with the Ludwig Institute, which was not proving to be the vibrant and well-connected place that he had hoped it would be. In 1987, therefore, he started looking around elsewhere for jobs. The ICRF was advertising for junior tenure-track group leaders, the same career rung that Gerard was currently on, and he applied for and was offered a job there. However, his bosses at the Ludwig Institute conducted a charm offensive to persuade him to stay put, and in the end he turned down the offer from London.

Dave Hancock and Trevor Littlewood, Evan laboratory dinner, 1980s.

(Photograph courtesy of Trevor Littlewood.)

The Dominion House laboratories had opened in 1983, as part of Bodmer’s drive to expand the ICRF’s links with clinically relevant cancer research. Under the initial leadership of John Wyke, they were small, comprising just four labs, and from the outset, suffered from being a rather isolated satellite of the main laboratories in Lincoln’s Inn Fields, without the focus that was to make Clare Hall so successful. By 1988, John Wyke had moved on to become Director of the Beatson Institute in Glasgow, and the Dominion House lab heads comprised Mike Owen, an immunologist, and Gordon Peters and Roger Watson, both interested in the molecular biology of oncogenes. Life there was a bit quiet, with occasional excursions to Lincoln’s Inn Fields, a half-hour walk away, being the only excitement; thus, the arrival of the Evan lab was a major social and scientific improvement. Because science is rather incestuous, Gerard and his three new colleagues already knew and liked each other pretty well. Having Gerard bouncing about the corridors galvanised the place, and the Evan laboratory also had a sense of a new beginning.

The world of molecular biology at that time was still in a state of great excitement about the cell cycle, following Lee and Nurse’s 1987 publication showing that the cell cycle control mechanisms were pretty much universal in eukaryotic organisms. Legions of postdocs and graduate students were occupied worldwide in working out how their protein of interest could be bolted onto the cell cycle: Did it regulate the cycle? Did it help cells get into cycle? Did it stop them getting out of cycle? Did cells cycle faster in its presence, and so on, and so on. Naturally, cancer biologists were very much part of this buzz, given the central role of cell growth in their particular obsession, and what many of them had focused on was how entry into the cell cycle was regulated. Clearly, if an oncogene could push cells into cycle when they were supposed to be resting, this was a property that could cause inappropriate growth, and maybe lead to cancer.

Using Hucky’s MYC-expressing stable cell lines, and subsequently, in cell lines they’d made themselves, the Evan lab began to look more closely at MYC’s role in the cell cycle. They soon noticed that MYC was not a typical Immediate Early gene. Most Immediate Early genes come on rapidly after serum stimulation and then switch off equally rapidly, but MYC came on fast and didn’t switch off. Instead, although the amount of MYC protein reduced, it lingered on well into subsequent stages of the cell cycle. Was MYC perhaps doing something different to regulate the cycle?

Gerard and Trevor, together with a postdoc, Cathy Waters, decided to set up some experiments looking at what happened if MYC was removed at different points in the cell cycle, not just in G₀. To do this, the protocol was to serum-starve control cells and matching stable cell lines carrying extra MYC for a day or so (over the weekend was a handy time), put the cells back into cycle by adding serum, and then do their assays. They got some interesting results, showing that MYC was indeed needed at a different point in the cell cycle from the rest of the Immediate Early gang, but the experiments were severely hampered by a very awkward technical problem: although the control cells without any extra MYC in them seemed to be fine without serum, the MYC-containing cells were completely unreliable, often dropping dead in such numbers that there weren’t enough to analyse at the end of the experiment.

Up to this point, the cells had only ever been looked at under a conventional light microscope, where they could be counted, but where little else in the way of observation was possible. To see exactly how the cells were dying, something far more sophisticated was required. Fortunately for Gerard, it was available at the Lincoln’s Inn Fields laboratories: time-lapse cinemicroscopy.

Cinemicroscopy was a very recent arrival at ICRF and, indeed, in the science world, coming into existence at roughly the same time as Gerard’s relocation to Dominion House. It revolutionised the way people could monitor cells growing in culture, by allowing them to watch the behaviour of individual cells over time, rather than just look at two time points, the start and the end of an experiment. It was the forerunner of today’s incredibly sophisticated time-lapse digitally recording microscopes, but was a very simple setup, consisting of a microscope, a flask full of cells, and an attached cinefilm camera. Every two min or so, the camera was rigged to wind on a frame and take another photo down the microscope. At the end of an experiment, the film was sent off to be developed. Upon its return, it could then be played backwards and forwards endlessly whilst the positions and fates of cells in the field of view were examined and mapped manually.

The immediate response of a scientist when faced with a new fact to research is to hit the library big time. Therefore, Gerard descended on the dusty stacks of the ICRF library and did some frantic self-education on apoptosis. What follows here is a brief summary of what he found.

The difficulties that death had had in establishing itself as a proper biological event worthy of study began to be surmounted after 1972, following the publication of work by an Australian pathologist John Kerr, and his collaborators in Aberdeen, Andrew Wyllie and Alastair Currie. Kerr had rediscovered Flemming’s hallmarks of apoptosis in injured liver tissue, calling it “shrinkage necrosis,” and whilst on sabbatical in Aberdeen, he, Wyllie, and Currie published a paper characterising, in multiple tissue types and cells both during normal development and in disease, the stages and morphology of this form of death. James Cormack, Professor of Greek at Aberdeen, suggested the term apoptosis, meaning the dropping of petals from flowers, or leaves from trees, for Kerr, Wyllie, and Currie’s paper. As with Lockshin’s earlier terminology of programmed cell death, the name was chosen to imply that the process was part of a cycle of life and death and was regulated, rather than haphazard. (Unfortunately for Professor Cormack, his proper Greek pronunciation, “Appo-TOE-sis,” has morphed in the mouths of most of the scientific community into “Appop-TOE-sis,” with only a few die-hards remaining true to the original.)

On the genetics side of things, Bob Horvitz and John Sulston, working in Cambridge on tracking the developmental origins of every cell in the adult nematode worm Caenorhabditis elegans, realised that of the 1090 cells generated during development of an adult hermaphrodite worm, the same 131 cells always died, and moreover, they died by apoptosis. C. elegans is a geneticist’s dream because it grows rapidly and can be mutated easily, and Horvitz and Sulston’s discovery meant that it was now possible to start looking for mutant worms in which cells meant to die stayed alive, or vice versa. The underlying genetic mutations causing “undead” phenotypes would be in genes controlling and executing apoptosis, and these genes could be isolated, cloned, and their DNA sequences determined. The importance of this work was recognised when Horvitz, Sulston, and Sydney Brenner (whose foresight had driven the development of C. elegans as a model organism) won the 2002 Nobel Prize for their discoveries concerning the genetic regulation of organ development and programmed cell death.

By the time Gerard had his momentous conversation with Mike Owen, a total of around 500 papers had been published about apoptosis and programmed cell death, mostly in the fields of developmental biology and immunology, with a few also looking at the behaviour of cancer cells. Many were still descriptive, but the most recent crop of papers was starting to identify genes involved in the death process and to try to put these in the context of overgrowth, as occurred in cancer. One of these genes, BCL2, caused Gerard’s scientific radar to start beeping madly because it was already very familiar to him, but in a different context: BCL2 had been suggested to be an oncogene.

The story at this point might just have petered out into another publication in a worthy, second-rank journal, with some dutiful observations, albeit using a cute new technique, about the mechanism by which MYC caused the cell death that so many researchers in the field had already encountered. Indeed, the atmosphere in the burgeoning field of molecular biology at that time was exactly conducive to such an outcome. Almost all of those working on deciphering the ways in which proteins and genes functioned were basic researchers, doing bottom-up, technically challenging science, in many cases still at the dawn of knowledge in their particular fields, where feverish excitement could be generated merely by finding out how protein A might connect to protein B, and how A and B interacted with C. All was unknown, even at the lowest, nuts-and-bolts level. Furthermore, there was a deep and snobbish divide between such research and what in today’s parlance would be called translational research: Taking basic science observations and considering whether they would be useful in a clinical context was widely considered to be the easy option, for those who could not generate new ideas or data but spent their careers recycling the observations of others.

With his science brain configured in this slightly unusual way, Gerard made a conceptual leap that to him seemed blindingly obvious, linking his discovery that MYC could cause apoptosis to that of David Vaux showing that MYC could cooperate with BCL2. Death caused by MYC was a programmed part of the cell’s lifestyle, not a consequence of a mitotic car crash; an executed decision, not an accident. This meant that MYC was constituted to have dual, seemingly opposite functions, able to push cells into cycle, but also forcing them to die under certain circumstances. BCL2, on the other hand, actively stopped cells from cycling and protected them from death. Might not this mean that the whole idea of oncogene cooperation, originally proposed by Parada, Land, and Weinberg to be a simple alliance between one oncogene driving one aspect of tumour formation, such as too much cell cycling, and another oncogene driving a different aspect, such as stopping death, was far too simplistic? Better, surely, was a theory in which cooperating oncogenes not only had complementary cancer-causing functions but were able to override the other’s inbuilt safety mechanisms, delivering a far more potent double whammy to the normal cell? Normally, if a cell accidentally made too much BCL2 it wouldn’t matter because it wouldn’t be able to cycle, and if it made too much MYC, that wouldn’t matter either because it would just die. Only in a situation in which the two oncogenes were wrongly switched on together would MYC override BCL2’s cell cycle block, pushing cells to cycle, and BCL2 stop the apoptotic signals being sent by MYC from being executed, and only then would there be a possibility for a cancer to initiate.

The simplicity of the experiments, and the carefully discussed conclusions, belie the frantic activity that had ensued following the first observations the lab had made. Almost the first step that Gerard took was to make an appointment to go and visit Andrew Wyllie, one of the fathers of apoptosis, who had moved from Aberdeen to be Professor of Experimental Pathology at Edinburgh University. Gerard’s initial conviction that he had stumbled onto something important was confirmed by Wyllie, who could see the enormous implications of apoptosis being a mainstream suicide mechanism rather than an occasional habit of white blood cells and developing embryos. Wyllie became a valued colleague, expert in the arcane lore of the apoptosis field, and was responsible for suggesting to Gerard that the work was so interesting that it should be submitted to Cell, for maximum visibility. Back in the lab, Trevor, Dave, and Cathy were working all hours doing endless tissue culture, sometimes staying all night. Everyone was excited because they all realised that, finally, after all of the awful things that had happened to the lab in the last few years, they had got their big break. If MYC causing apoptosis was as big a deal as they thought, they were set for stardom, or at least temporarily released from the fear of failure that haunts the hearts of all scientists.

To begin with, many cancer biologists reacted with extreme scepticism to the idea that an oncogene, especially one so powerful as MYC, could be hard-wired to cause a cell to kill itself. Gerard recalls the first talk he ever gave about it, at a Gordon conference, traditionally a venue for the frank exchange of views and a very prestigious gig for an up-and-coming scientist. Leaving aside the several people who walked out, whether from disbelief or preferring the competing charms of an early coffee break, many prominent researchers in the field were reluctant to let go of the idea that this property of MYC’s was no more than a by-product of its forcing cells into cycle. The consensus view was that an individual cell was able to sense its health or otherwise, and if it detected that something bad was happening, such as excessive cycling, it would fall sick and die, by a theoretical mechanism snappily named “mitotic catastrophe.” Therefore, the phenomenon “Sick of MYC” was just that: a terminal illness, a set of conflicting signals whereby Myc said one thing to the cell, the rest of the cell thought something else, and the two viewpoints were irreconcilable.

The problem with such anthropomorphic thinking was that, viewed from an evolutionary perspective, it made no sense. Firstly, the notion that an oncogene could have a main function, from which all its properties flowed, was nomenclature-based determinism, a belief that the first thing a gene was discovered to do had to be the most important. Just because MYC was first noticed in the context of excess cycling did not mean that that was the main thing it did. More importantly, evolutionary theory now held that the organism was the fundamental unit of evolution, with its genes just a vast collection of biological meccano that could be assembled into as many different networks as were needed for the body to function and survive; genes had a function, but that function could be used in a whole organism for multiple purposes. Viewed like that, it was perfectly reasonable for MYC to be involved in any number of processes and that these processes might dictate conflicting outcomes was irrelevant: The downstream readout was a function of the pathway as a whole, not its individual components.

In the autumn of 1992, Gerard published a paper in Nature about oncogenic cooperation between MYC and BCL2. As he’d surmised, the two oncogenes were able to work together because they could compensate for each other’s weaknesses: BCL2 protected cells from MYC’s murderous tendencies, whereas MYC chivvied cells into cycle, overcoming BCL2’s cell cycle block. However, the paper was more than just a vindication of Gerard’s original thoughts; the Evan lab had been caught up, and their publication was back-to-back with another saying the same, from the lab that had shown the link between MYC and apoptosis in T cells—that of Doug Green in La Jolla. Events could have taken a very nasty turn, given the highly competitive nature of the subject, but fortunately for both labs, Doug and Gerard soon met, at a conference in the windswept wastes of the University of East Anglia campus. Doug, heading back into the conference hall following a quick trip to the lavatory, met Gerard heading out on the same mission (an awful lot of coffee is consumed at meetings). The two men realised pretty quickly that despite being competitors, they were so similar in temperament and outlook that they couldn’t help but be friends first. Happily, they’ve been best friends ever since: mutual repositories of scurrilous stories, enthusiastic occupants of numerous conference bars, and scientifically distinguished collaborators and competitors.

In 1994, in a paper in the EMBO Journal, the Evan lab proved the most important tenet of Gerard’s theory, that of MYC’s duality. In normal conditions, a cell exists within a network of social survival signals, which suppress MYC’s signals to the apoptotic programme but allow its proliferation signals to make it through, so that cells grow. If the survival signals are removed, MYC’s apoptotic signal is no longer suppressed and cells die. However, if the survival signals are maintained artificially, MYC cannot kill the cells but drives them into cycle, causing tumours. By the time this paper came out, however, the battle for acceptance was long won, and the field had settled down to the long slog of working out the exact mechanisms involved in triggering and then executing apoptosis.

Today, we know an awful lot about apoptosis—mechanistically, it is probably one of the best understood cellular processes, thanks to the combination of biochemistry and genetics that proved so important in the genesis of the field and have continued to have a vital role. From an average of about 40 papers a year pre-1992, there are now close to 10,000 articles published about apoptosis annually, more than one every hour. However, despite roughly one in 50 of these publications having some mention of MYC in them, we are still not quite sure exactly how MYC works to cause apoptosis. What is known then?

MYC’s ability to cause apoptosis is partially understood, because it can induce the p53 tumour-suppressor protein, causing cells to kill themselves. However, there are clearly MYC-driven apoptotic pathways independent of p53, about which we currently know very little. Rather than being a sole regulator of apoptosis, as it was in the artificially created tissue culture milieu of Gerard’s original experiments, in normal life MYC functions to sensitise cells to a huge range of pro-apoptotic insults, including starvation, oxygen deprivation, DNA damage, and the activation of death receptors—proteins able to transmit apoptotic signals coming from outside the cell. In this duality, it shares a common function with many proto-oncogenes, acting as a driver of proliferation, but also as a sentinel detecting unnatural changes to a cell, nearly always brought about by tumour-specific growth.

Anti-cancer therapies aimed at reawakening a cancer cell’s lost ability to apoptose have been pursued with much energy over the past decade. A 2002 review, written for the inaugural issue of the journal Cancer Cell by Gerard and Doug Green (they concocted it over a shared Thanksgiving weekend, although thankfully, no traces of turkey-based thinking seeped into the final product), pitched the idea that cancers are built on a precarious platform of two mutations, one causing overgrowth and the other causing apoptosis suppression, and that if one or the other of these mutations were to be counteracted, the cancer would die. The hypothesis flows directly from the original insights gained from the interaction of MYC and BCL2, and to general excitement, it has, indeed, proved possible to topple a MYC-driven cancer by knocking out its founding MYC mutation. Gerard’s lab, the lab of his old mentor Mike Bishop, and that of another ex-Bishop postdoc, Dean Felsher, have engineered mice with a switchable MYC gene embedded in their genomes. When the mice are treated with the switch compound, MYC comes on, causing tumours, but these tumours regress rapidly as soon as MYC is switched off again.

And what of Gerard? After all the years of striving to be successful and shaking off the traces of his Cambridge experience at the Ludwig Institute, he realised that, actually, it wasn’t that much fun being a world authority on something, because many people stopped arguing with him, assuming that he knew what he was on about. It was also no fun being part of the furniture of an institution, and so, after a very productive decade at the ICRF, Gerard left to go back to San Francisco, to the new UCSF Cancer Center, where he started again at the bottom of the heap, working on mouse models of cancer and driving a rather snazzy little red sports car across the Golden Gate Bridge to the lab every day. Having worked his way up to become a major player in both the MYC and p53 mouse worlds, he moved back across the Atlantic in 2009, to become Head of the Cambridge Department of Biochemistry and Sir William Dunn Professor. Gerard’s current research is focused on the processes responsible for the genesis and maintenance of cancers, in particular, cancers of the pancreas, colon, brain, skin, and liver. Understanding the molecular mechanisms that underlie the cell suicide machinery and how it can be manipulated therapeutically is still the overarching aim of his laboratory, and he continues to be one of the most productive and thought-provoking cancer biologists working today.

For his work on apoptosis and cancer, Gerard was elected to the UK Academy of Medical Sciences in 1999 and became a fellow of the Royal Society in 2004. His 1992 Cell paper is an acknowledged classic, having been cited more than 2600 times, and remains the most highly cited research paper ever to have been published by scientists working at the London Research Institute.

Web Resources

Bishop JM. 2003. How to win the Nobel Prize. Harvard University Press.

A wonderful autobiography by Gerard’s postdoctoral mentor Mike Bishop, the 1989 Nobel laureate in Physiology or Medicine.

Green DR. 2011. Means to an end: Apoptosis and other cell death mechanisms. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.