p53 (19 page)

In fact, the feedback loop – in which the inhibitory
protein is switched on by the protein it then inhibits – did not quite make sense yet. There were still
two vital pieces of the puzzle missing, and Oren’s lab was again at the forefront of the search. They had started to use Mdm2’s ability to restrain p53 as a tool much like the
temperature-sensitive mutant: by adding or removing the gatekeeper from cells in which p53 was present, they could switch the protein between active and inactive states. ‘We were using it
routinely to try to get insights into what p53 does, which is what
doesn’t
happen when Mdm2 inhibits it,’ explained Oren. However, the researchers soon started to notice
something puzzling: that whenever they put the two proteins together, instead of Mdm2 simply attaching itself to p53 and stopping it in its tracks, as they had expected, the p53 protein seemed to
disappear altogether. Initially the scientists didn’t trust their experiments, so they repeated them with slight modifications and extra care. But when they came up with the same results
again and again, they knew they were real: Mdm2 was destroying p53. And it was doing so, they discovered later, by delivering the ‘kiss of death’ – attaching a little chemical tag
to the p53 protein that marked it out for collection and degradation by the cell’s recycling machine, the proteasome.

So, the picture Oren’s team had built up thus far showed that p53 switches on Mdm2, which in turn marks p53 up for degradation in an endless cycle, lasting around 5–20 minutes, that keeps
p53 protein in our cells at almost undetectable levels most of the time. (This incidentally helped to explain why there were such high levels of the protein in cells in those early experiments with
the mutant clones: mutation typically severs the bond between p53 and its controller Mdm2). But how then does the tumour suppressor escape its gatekeeper in order to protect us from cancer in the
normal course of events? This was the final missing jigsaw piece, and it was discovered tucked away in a corner of Carol Prives’ lab in Columbia, where a ‘fantastically talented’
young postdoc called Sheau-Yann Shieh from Taiwan was interested in a process called phosphorylation.

Phosphorylation is one of the most important mechanisms by which proteins are activated and silenced in cells, and it works by attaching small phosphate molecules, as ‘tags’, to some
of the amino acids that make up the protein. Shieh was focusing on p53 and asking the question: does the protein get phosphorylated? If so, how does this affect its function? She found that p53
does indeed become phosphorylated; it changes shape and, incidentally – for she was not paying special attention to the Mdm2 story, which was still evolving – that this weakens the bond
between the p53 and its minder.

‘But we were missing a critical link,’ explained Prives. The pressing questions that remained were: what is the trigger for p53 to become phosphorylated? Does this happen in real
life? (So far they had seen it only in cell cultures in the lab.) And is it related to DNA damage and other stressful events in the cells? With the help of some fancy new tools for singling out
phosphorylated proteins, they found the answer to all three questions. They discovered that phosphorylation of p53 does happen in real life. It is indeed related to DNA damage and the stress
response. And it is the mechanism by which ATM – the gene that makes patients with ataxia telangiectasia so acutely sensitive to radiation – signals distress and triggers the response
from p53.

This piece of the jigsaw completed the picture of the p53/Mdm2 feedback loop and suggested how it might work. In essence, this is how it looked: in the normal course of events, p53 protein is
being made in our cells all the time so that it can give a hair-trigger response to danger signals; and it is being cleared away almost as fast as it is made by Mdm2 lest it lead to unnecessary
death of cells. But danger or stress activates genes such as ATM, which phosphorylate p53, preventing Mdm2 from getting a proper hold and allowing the p53 protein to accumulate in the cells and
perform its job
of orchestrating the response – arresting the cell temporarily and sending in the repair team; condemning it to permanent arrest or senescence; or
forcing it to commit suicide.

Another researcher, Gigi Lozano, working with mouse models at MD Anderson Cancer Center in Houston, confirmed just how important each protein is to the normal functioning of the other in real
life when she created mice with Mdm2 knocked out, and discovered that this was biologically lethal: with no holds barred, p53 caused massive apoptosis. But when Lozano created mice with both p53
and Mdm2 knocked out, they could survive until, eventually, they developed cancer.

Exactly how the stress-response mechanism is turned off again when it is no longer needed, and how the tight regulation of p53 is restored, no one is completely sure as yet. But it could be that
when the stress signals stop coming, any newly produced p53 will not be protected from degradation by phosphorylation, and the protein will resume its normal dance of death with its minder, Mdm2.
This remains an important question since the feedback loop offers tantalising opportunities for tinkering with the regulation of p53 to create new cancer treatments. It’s a topic I will
return to in a later chapter.

***

In the mid-1990s, p53 emerged briefly from the rarefied environment of academia to play a dramatic role in the battle between public-health authorities and the tobacco industry
by decisively nailing the connection between smoking and lung cancer. The connection itself was not news. Nearly half a century earlier, in 1950, the Oxford-based epidemiologist Richard Doll
– a maverick and somewhat controversial figure among the medical establishment because of his membership of the Communist Party – had drawn attention to the association between
cigarettes and lung cancer in a paper for the influential
British Medical Journal.
In it, Doll and his collaborator, Austin Bradford Hill, reported their findings from a research project
aimed at figuring out what was causing the sudden explosive rise in lung-cancer cases in the UK.

In the quarter-century from 1922 to 1947, deaths from lung cancer in England and Wales rose from 612 to 9,287 per annum – ‘one of the most striking changes in the pattern
of mortality recorded by the Registrar-General’, Doll said in his report. Tobacco had been a rare source of comfort to men fighting in the trenches and on the high seas of World
War I and cigarettes were included among a soldier’s or sailor’s rations in both the British and American armed forces. Indeed, General John Pershing, leader of the American
Expeditionary Forces in World War I, is reported as saying, ‘You ask me what we need to win this war. I answer tobacco as much as bullets . . . We must have thousands of tons without
delay.’

Thus a generation of young men returned to civilian life addicted to tobacco, entrenching a habit that had been growing since the invention in the late 1880s of a machine for rolling cigarettes
that enabled mass production. By the time Doll and Hill were doing their studies in the late 1940s, approximately 80 per cent of British men were regular smokers and the death rate from lung cancer
in England and Wales had increased more than sixfold among adult men and approximately threefold among women in less than 20 years.

The two scientists did not, however, suspect tobacco of being the cause of the premature deaths. Their initial hunch was that it was atmospheric pollution – dust from the new road-building
material, tarmac; exhaust fumes from cars, power stations and the coal fires burning in everyone’s front rooms. But the picture of smoking habits that emerged from the questionnaires
conducted with 1,732 cancer patients and 743 controls in 20 London hospitals pointed compellingly to tobacco exposure as the culprit. Doll and Hill’s
BMJ
paper concludes with the
statement that ‘above the age of 45, the risk of developing [lung cancer] increases in simple proportion with the amount smoked, and that it may be approximately 50 times as great among those
who smoke 25 or more cigarettes a day as among non-smokers’.

Though he had no idea what the carcinogenic substance in tobacco could be, Doll himself gave up smoking on
the strength of the epidemiology. As the evidence of its deadly
effects accumulated, governments in many countries adopted measures aimed at curbing the habit. But as long as the link between smoking and lung cancer remained circumstantial, without a physical
mechanism to back it up, cigarette companies had ample wriggle room to fight the public-health case against them. They continued to promote their product aggressively, particularly in developing
countries with potentially massive markets and poor controls. Even when scientists, following on from Doll and Hill’s work, showed that painting tar – the sticky black residue of
tobacco that coats smokers’ lungs – on to the skin of laboratory mice causes tumours to grow, Big Tobacco was wont to scoff that the experiments were irrelevant: these were mice, not
people.

In fact, the first person to do such an experiment was Angel Honorio Roffo, an Argentinian oncologist who flagged up the link between smoking and cancer nearly two decades before Doll and
Hill’s paper in the
BMJ
. By painting rabbits’ ears and the skin of mice repeatedly with either nicotine or tobacco tar, Roffo identified the latter as being the carcinogenic
substance – nicotine alone had no effect on the animals’ skin no matter how long he left it. But the results of his extensive research into the disease that afflicted his patients went
almost unnoticed by anyone except the tobacco industry. This was at least partly because his most important scientific papers were published only in German and partly because he was way ahead of
his time in his understanding of cancer biology. So far ahead, in fact, that a New York physician who had heard of Roffo’s studies and written to the American Tobacco Company (AT) in May 1939
to ask if his findings were valid was able to be reassured by the cigarette manufacturer’s own research director. Hiram Hanmer replied to the doctor that AT had been following Roffo’s
work for some time and felt that it had left the literature on tobacco ‘in a very beclouded condition’. He assured his correspondent
that ‘the use of
tobacco is not remotely associated with the incidence of cancer’.

Rubbishing the science has always been the tobacco industry’s
modus operandi,
but it became infinitely more difficult – and finally impossible – once the molecular
biologists were on the case. Curt Harris is a bear of a man with a shaggy grey beard and a deep bass voice as rich as a pint of brown ale; he heads the Laboratory for Human Carcinogenesis at the
National Cancer Institute in Bethesda. He is also a pioneer of the relatively young field of molecular epidemiology, the science that traced the origins of the AIDS pandemic back to chimpanzees and
green monkeys in the forests of Africa by following the genetic footprint of the virus that became HIV. It’s the science that uncovers the sources and assesses the virulence of regular flu
outbreaks; and it’s a science used widely today to try to pin down the multiple causes of cancer.

In the 1980s, Harris’s lab was involved in studying the activity of carcinogenic substances, including components of tobacco tar, when they get into cells. They found that a number of
these substances stick themselves firmly to the DNA, and Harris knew from the literature and from his own studies that this would lead to mutations in the genes, the first step along the road to
cancer. He became especially fascinated by Bert Vogelstein’s work with p53, and the two men decided to collaborate on a research project to assess how commonly p53 might be mutated in cancer.
This was 1989, just after Suzy Baker in Vogelstein’s lab had made her sensational discovery that p53 was a tumour suppressor, not an oncogene. Harris and Vogelstein found p53 mutations in
many of the common tumour types they saw in their clinics, including breast, lung, brain and colon. They found too that the mutations showed a pattern and were clustered in four particular
positions along the length of the gene, which they dubbed ‘hot spots’.

The two researchers published their findings in
Nature
in
1989. Soon afterwards Harris, who remained intrigued by the pattern he and Vogelstein had revealed,
decided to collect systematically the information on the different mutations mentioned in the steady stream of papers that appeared on p53. In 1990, in collaboration with Monica Hollstein, then
working at the World Health Organization’s International Agency for Research on Cancer (IARC) in Lyon, France, he formalised his collection into the p53 database, a rare resource for
scientists and clinicians that can provide clues to the identity of carcinogens in the environment, as well as to the likely course of a patient’s disease or the best options for treating
certain tumours.

In 1994, Hollstein left IARC and her place at the helm of the database was taken by Pierre Hainaut, who expanded it, increased the level of detail recorded for each mutation and managed it until
mid-2012. Today the database contains information on tens of thousands of mutations, together with the tumours in which they occur and as much information as possible about the lifestyles and
personal characteristics of cancer patients, including their response to treatment and the final clinical outcome if available.