p53 (16 page)

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.

***

In the long history of p53, huge amounts of data have been generated by scientists poring over little scraps of tissue and clusters of cells in test tubes and Petri dishes
– specimens that have been coaxed and manipulated in super-controlled environments. ‘These systems are easy and convenient, but they’re not the real world,’ says David Lane,
sounding a note of caution. ‘The more I look at p53, the more I realise that in the real world it’s operating at a very different level and in a different sort of way.’ Tissue
culture itself puts cells under stress and p53 into a state of alert, he says, and, rather than studying the difference between active and inactive protein, what most researchers are in fact
studying is the difference between very active and moderately active protein. Experiments using animal models tell a story that’s different and a lot more subtle.

Recognition of this fact lies behind one of the legendary stories of p53 research, and it involves David Lane and his friend and colleague Peter Hall, both working at Dundee University at the
time. The year was 1992. The story goes that the two scientists had been sharing a pint in a local pub at the end of a busy day and mulling over the crucial question of whether or not p53 responds
to cellular stress in real life, as it does in tissue culture in the lab. They knew others were asking the same question and that competition to find answers was hot. They knew, too, that they
faced a forest of paperwork to obtain Home Office permission for animal experiments, and their frustration at the prospect of the inevitable delay was intense. Then Hall had an idea: why not
conduct the experiment on themselves? Without hesitation, he volunteered to be the guinea pig, and the two began to make plans. Telling me the story some years later, Hall said with his
characteristic note of defiance that he and Lane knew they risked incurring the wrath of the authorities for not following standard procedure, but they were too fired up at that point to care.

The experiment involved subjecting Hall’s arm to radiation from a sun lamp – ‘equivalent to 20 minutes on a Greek beach’ – and taking a series of time-staggered
skin biopsies to watch the activity of p53. ‘We reckoned that if this gene does respond to stress in living organisms, we should see the accumulation of p53 protein in the cells in my
radiated skin. And that’s exactly what we did see,’ said Hall, rolling up his sleeve to reveal nine neat scars. ‘We did the experiment on me because we wanted quick results . . .
The scars all got infected,’ he laughed, ‘but the experiment worked brilliantly, and it moved the field on considerably.’

Such maverick experiments notwithstanding, yeast, worms and fruit flies have taught us a great deal about how cells work. But for insights into the workings of more complex organisms like
ourselves – with organs and skeletons, circulating blood and immune systems – the animal model of choice is the mouse. Similar to us, mice have around 23,000 genes, almost all of which
have counterparts in our own DNA. Furthermore, mice are cheap to maintain; they breed fast, producing a new litter roughly every nine weeks; and their genomes are relatively easy to manipulate.

For decades, scientists used selective breeding techniques to produce mice with desired genetic traits. Or they blasted their DNA with chemicals known to produce specific mutations: a process
known as ‘chemical mutagenesis’. Then in 1989 came the birth of the first transgenic mouse, created using a sophisticated technology called ‘homologous recombination’. Such
mice provided a new ‘precision tool’ that changed everything, and homologous recombination won its developers, Mario Capecchi and Oliver Smithies, both working in the US, the 2007 Nobel
Prize for Medicine. They shared the prize with a Briton, Martin Evans, who was the first person to isolate the embryonic stem cells from which transgenic mice are created.

The story goes that Evans was on a month’s visit to the US, where he had gone to learn some new technological tricks at the Whitehead Institute in Cambridge, Massachusetts. With so little
time for his mission, he was determined not to be sidetracked into giving lectures or meeting new people. He didn’t even want to speak to anyone outside the lab. Then he got a phone call from
Smithies, a fellow Brit who had left for the US many years earlier. Smithies was eager to learn more about Evans’s embryonic stem cells, which were so vital to his own research goals.
‘I remember to this day, I said to him, “Oliver, you are the only person who I will come and visit . . .”’ Evans told an interviewer for the Nobel Committee. And he turned
up the following weekend at Smithies’ place with a flask of the cells in his pocket.

Homologous recombination – more descriptively known as ‘gene targeting’ – exploits the cell’s natural propensity for repairing breaks in its DNA by stitching in
little pieces of matching DNA taken from another chromosome. In gene targeting, scientists insert into the cell a foreign piece of DNA carrying the desired genes, and they rely on it to find the
appropriate place (where it recognises a matching sequence of genes) to insert itself into the host DNA, in this case kicking out the original sequence.

Over the decades, this method has been used to create many thousands of mice precisely engineered to model human conditions and diseases, from cancer, diabetes and cystic fibrosis to blindness,
obesity and alcoholism. Indeed, creating transgenic mice has become something of a cottage industry, Mario Capecchi told his audience in Stockholm during his Nobel lecture. That is largely thanks
to his own obstinacy when, in 1980, he approached the National Institutes of Health for funding to develop his new technology and was told to forget it; his chances of success in applying it to
mammalian cells were vanishingly small and he should give up. Convinced he was on to a good thing, Capecchi took no notice and soon his whole lab was working on the project. When it became clear in
1984 that their experiments with mammal cells were working, he applied again to the same department at the NIH for funds. This time he was successful, and the NIH had the grace to say, in their
letter of approval, ‘We are glad that you didn’t follow our advice.’

MARIO CAPECCHI AND THE ‘KNOCK-OUT’ MOUSE

As far as p53 research is concerned, one of the most valuable transgenic models has been the so-called ‘knock-out’ mouse, in which a specific gene is deleted from
the mouse’s DNA to see how the animal functions without it. While the three Nobel winners are collectively known as ‘the fathers of transgenic mice’,
⁸
the knock-outs, which are created using a modified version of the gene-targeting technology, are the brainchild of Capecchi, a man whose journey towards the pinnacles of
science no novelist could have made up convincingly. Capecchi lived rough on the streets of war-torn Italy for five years from the age of four, and didn’t go to school until he was nine.

He was born in Verona in 1937 – a time when Fascism, Nazism and Communism were raging throughout the country, he wrote in his autobiographical sketch for the Nobel Committee. ‘My
mother, Lucy Ramberg, was a poet; my father, Luciano Capecchi, an officer in the Italian Air Force. They had a passionate affair, and my mother wisely chose not to marry him.’

Capecchi’s mother studied at the Sorbonne in Paris, where she became politically active, joining the Bohemians, a group of poets who openly opposed Fascism. She returned to Italy in 1937,
giving birth to Mario in October of that year and settling eventually with him in a chalet in the Alpine Tyrol. Fearing that her activism would mark her out, she began saving money to enable her
neighbours, an Italian peasant farming family, to take care of her child if she was taken away.