p53

by Sue Armstrong

  Parry ended his letter, ‘Please let me know if your people are interested in supporting the type of work outlined, bearing in mind that we can modify the components in various ways to also fit in with your interests.’

  Curt Harris, original founder of the database and by now editor of the journal Carcinogenesis, had been taking a personal interest in the controversy. Faced with such clear evidence of a conflict of interest, he blew the whistle on Parry, editor of Mutagenesis, with Oxford University Press, which publishes both journals. Although she understood full well the seriousness of the allegation against Parry, Janet Boullin, the editorial director of journals at OUP, pointed out that she had limited scope for action since OUP was simply the publisher, not the owner, of Mutagenesis. But she immediately strengthened the rules about disclosure of interests for both contributors and editors of all OUP journals. Parry was not prepared to comply with the new rules and he resigned as editor of Mutagenesis soon afterwards. But he remained on the journal’s editorial board, whose members were not required to sign competing-interest statements.

  Among the documents on the tobacco industry website, Hainaut found evidence, too, that Philip Morris was gathering intelligence on his own institution, IARC, home of the p53 database. However, satisfied for the time being with what he had learnt from his own sleuthing, he handed the evidence he had gathered over to Stanton Glantz, an anti-tobacco campaigner in the US.

  SCIENTISTS FIGHT BACK

  Stanton Glantz, a heart specialist, is Professor of Medicine at the University of California, San Francisco, director of the Center for Tobacco Control and Education, and a scientist with a long history of standing up to the tobacco industry. Originally, his focus was passive smoking, which kills tens of thousands of non-smokers in America alone every year. But his efforts to have smoking banned in public spaces have seen him hassled constantly by industry supporters. ‘After every barrage of personal attacks against me in the American Smoker’s Journal or the American Smoker’s Alliance [set up by Philip Morris and others] or these other publications from these groups, I get a series of hate mail,’ he told the Public Broadcasting Service (PBS) in an interview for their programme Frontline in 1996. ‘I have had hate mail, hate faxes, hate e-mail, hate phone calls. I mean the police department here intercepts some of my mail now, and it’s a drag.’

  Turning up for work on 12th May, 1994, Glantz found dumped on his desk a large box containing several thousand pages of confidential internal documents from the tobacco company Brown and Williamson, a subsidiary of BAT. There was no clue as to who had sent the parcel, simply the name ‘Mr Butts’ on the ‘return address’ label. The documents covered a period from the early 1950s to the mid-1980s, and related to such issues as the addictiveness of nicotine, cancer and the company’s public relations and legal strategies. Since these were not directly relevant to his own research, Glantz intended passing the documents on to a colleague working in these areas. But after flicking through them for 20 minutes or so he found himself unable to stop reading. ‘The thing that sucked me into them was not their potential political or legal import,’ he told PBS. ‘It was the documents as history, the documents as science. It was just an unbelievable find. As a professor, it would be like an archaeologist finding a new tomb in Egypt or something . . . I mean it’s an amazing, amazing story of what was going on inside these cigarette companies during this crucial period.’

  Glantz hung on to the documents and worked through them systematically with a team of reviewers, writing a number of papers for journals before pulling them together, in collaboration with colleagues, into a book, The Cigarette Papers, published by the University of California Press in 1998. He remained interested in the tobacco industry and its subversive activities, and a few years later began investigating its involvement with p53, adding further detail to what Hainaut had discovered and writing up the story in an explosive paper for The Lancet.

  Glantz’s paper is littered with the names of scientists paid by Big Tobacco to carry out research on its behalf, frequently with the explicit aim of discrediting the evidence of a causal link between smoking and cancer, or to write letters to medical journals or popular newspapers for the same purpose. Sifting through the wealth of information the tobacco industry has been obliged to publish, Glantz found evidence that by the early 1990s, BAT had identified p53 as being especially important, with ‘more papers published on it than any other topic on cancer’. The company monitored research on the gene and pressed its paid scientists for intelligence about what their colleagues were discovering and for advance copies, if possible, of relevant papers submitted for publication. Furthermore, BAT regarded information about the research organisations it supported as confidential and advised individual scientists that they were ‘free to publish their work without further reference’ to the company. BAT and others also appear to have anticipated Pfeifer and Denissenko’s findings about the effects of tobacco tar on p53, and to have worked on a strategy to discredit them in advance.

  The idea to challenge the scientists’ interpretation of data from the IARC database seems to have come from Jim Parry, who published the two critical papers in Mutagenesis. But though Hainaut was relieved when he first discovered the evidence of skulduggery, he found himself unable to dismiss the attacks out of hand. ‘I think, to be fair, the tobacco industry criticism had a point,’ he told me. ‘Our data were based on putting together bits and pieces of our database from studies which were not aimed at demonstrating what we wanted to demonstrate. We had data on never-smokers, for example, that were scattered around something like 20 different papers, none of which had the scope on its own to demonstrate what we wanted to show; it was just by putting them together that they made the point.’

  He and his fellow researchers decided to go back to the drawing board and come up with an analysis that was rigorous and powerful enough to prove their case. ‘It took us two years, but we did it and we published the paper in 2005 in Cancer Research,’ said Hainaut. ‘Nobody now can say the connection is not there; it’s really watertight. I think the whole story is now behind us, but that’s what it took! And maybe in that respect it was a good thing we had this attack, because otherwise we might not have done the paper.’

  Finally nailing Big Tobacco more than half a century after the first warnings of the dangers of smoking was a triumph for p53 and public health, but it’s not the only one. The mutant p53 database is proving a rich resource for the disease detectives, who are finding a growing list of carcinogens that leave their unique fingerprints on the cancers they cause. Besides tobacco, mouldy peanuts on liver cancer and sunlight on skin are just two of the direct relationships the scientists have been able to work out in forensic detail.

  CHAPTER FIFTEEN

  Following the Fingerprints

  In which we learn that besides lung cancer, other types of cancer, including liver and skin, frequently have mutant p53 that carries the unique fingerprint of the agent that caused the disease.

  ***

  The most exciting phrase to hear in science, the one that heralds the most discoveries, is not ‘Eureka!’ (I found it!) but ‘That’s funny . . .’

  Isaac Asimov

  Liver cancer is the seventh most common cancer worldwide, but in South East Asia and sub-Saharan Africa, where the great majority of cases occur, it kills more people every year than any other tumour type. In these regions Hepatitis B, which is a major risk factor for liver cancer everywhere, is extremely widespread – passed between sexually active adults and from mother to child, much like HIV. And like the AIDS virus, too, it can wreak havoc in a person’s body without them being aware of the infection, and become endemic in communities. Hep B generally takes many years to cause liver cancer, but in the high-incidence countries of Asia and Africa people’s risk of getting the disease is compounded by exposure also to aflatoxin, a poison produced by the fungus Aspergillus that flourishes on peanuts and grains stored in warm, damp conditions without adequate ventilation.
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br />   Aflatoxin is a known carcinogen and was one of the chemicals investigated by Curt Harris’s lab in the late 1980s and early ’90s for its mechanism of action in human cells. As with BaP in tobacco tar, Harris knew that aflatoxin is metabolised and transformed in cells into a substance that sticks to DNA and causes mutations. But it was work he did with colleagues in China, analysing the genetic mutations in liver tumours in Qidong county, on the north side of the Yangtze River opposite Shanghai, that showed the poison at work in the real world and pointed the finger at p53 as being the target for mutation. Rates of liver cancer in the county were exceptionally high; so too was people’s exposure to aflatoxin from mouldy grains and beans in their diet, and the researchers were struck by the frequency of an unusual mutation in p53 at codon 249. This resulted in the building blocks of the gene being swapped, a G to a T – the same as with tobacco tar, but in a different hot spot on the gene. Could this be the fingerprint of aflatoxin?

  As the paper describing Harris and his colleagues’ findings and suggesting such a possibility was about to go to press, a visiting scientist to Harris’s lab at the National Cancer Institute mentioned casually that another group, working in South Africa, had also discovered an unusual p53 mutation in liver tumours, but were unsure of its significance or what to do with their findings. Realising that this strengthened their case for a direct link between aflatoxin and p53 in liver cancer, Harris pushed the other group, led by Mehmet Ozturk, to write up their research in double-quick time so that the two papers could be published together, and they came out back to back in Nature in April 1991.

  The coincidence of aflatoxin exposure and a distinctive p53 mutation in liver-cancer patients soon became apparent in many other warm, humid places with poor storage for crops. But what was going on in the machinery of their cells? Pierre Hainaut joined the quest to find out. Despite his initial unease at being drawn into the tobacco and cancer controversy, Hainaut is a natural-born sleuth, never happier than when he is doing research on the front line, where faulty tumour-suppressor genes are affecting the lives of real people. He has followed the fingerprints of p53 from China to Brazil, Iran to West Africa and South East Asia, and many other countries. With aflatoxin, his research has focused mainly on Mali, The Gambia and Thailand – three countries where liver cancer is a huge problem. Over the years he and others working on this issue, including Harris and Pfeifer, have revealed a devilish relationship between aflatoxin, the ubiquitous Hep B virus and p53, as they co-operate to cause liver cancer.

  First the scientists worked out how Hep B virus on its own can lead to liver cancer. A viral gene – known simply as ‘x’ because for a long time no one had a clue what it did – codes for a protein that has a dual function: one end of the protein encourages proliferation of the liver cells it’s infecting; the other end promotes apoptosis, cell death. In this way the virus tries to maintain some kind of balance in the population of infected liver cells. But in so doing, it causes cycles of inflammation, damage and repair to the liver that result in cirrhosis – a liver greatly enlarged and distorted by scar tissue and lumps, or nodules, of regenerated cells.

  ‘These cycles of destruction and regeneration can go on for a while, and sometimes they can kill people – they can die from cirrhosis of the liver without getting cancer,’ explained Hainaut. ‘But at some point, in the absence of mutant p53, what happens to people with chronic liver disease and cirrhosis is that HBx becomes accidentally integrated into the genome of the liver cells. At that point it loses it pro-apoptotic part, and what remains is just the part that activates proliferation: the cells then escape destruction and are on their way to cancer. This is why cancer develops as a sequel to cirrhosis in the context of wild-type p53.’

  Harris’s group found also that HBx protein sticks to p53 protein, forming a complex in much the same way as SV40 does with p53. They assumed that in so doing HBx had a similar effect of crippling the tumour-suppressor function of p53, and that this was one of the driving forces towards cancer. However, very recent research by Hainaut and his colleagues in West Africa suggests this assumption is wrong; it has the relationship between the two proteins back to front, for what really seems to be happening is that p53 is blocking the ability of the virus protein, HBx, to trigger apoptosis, while leaving its ability to drive proliferation of cells intact. The crucial point here is that, in real life, the bond between p53 and HBx that transforms the viral protein is only really strong when p53 has the aflatoxin-induced mutation, at codon 249. Then the brakes are off and the liver is especially vulnerable to cancer. ‘The risk of having liver cancer for someone who is a chronic carrier of Hep B is about 5-7 times compared to a non-chronic carrier,’ Hainaut told me. ‘The risk of getting liver cancer with just aflatoxin is very difficult to measure, but is probably no more than twofold. However, the risk of having liver cancer if you have the two is at least 20 times, and some measures suggest it is up to 60 times greater than usual. So it’s truly multiplicative.’

  The revelation that mutant p53 transforms the function of the virus rather than the other way round has also helped to explain an abiding mystery in African liver-cancer patients. When someone infected with Hep B virus finally succumbs to liver cancer, he or she generally has signs of advanced cirrhosis from years of damage and repair. ‘This is the rule in the Western world,’ said Hainaut. ‘The patient who doesn’t develop cirrhosis before liver cancer is really the exception.’ But this is not what they have found among patients with liver cancer in Africa, despite chronic infection with Hep B. ‘I would say that maybe 15 per cent have cirrhosis beforehand, and then a number of them develop cirrhosis during the proliferation of cancer, as a sort of secondary response of the liver to the inflammatory state, but it does not precede cancer.’

  Hainaut’s theory is that by blocking the virus’s killer function, the mutant p53 prevents the regular cycles of inflammation, damage and repair that cause the scars and nodules of cirrhosis. Thus, paradoxically, aflatoxin exposure can be protective of people with chronic Hep B infection, often for years, until other events in the ordinary course of living render them vulnerable to cancer. ‘We could never understand why we have so little liver cirrhosis in these populations. It’s something I observed about 15 years ago – we had very few patients with cirrhosis. The common response was, “Ah, you’re not looking for them . . . They’re not reporting to doctors, so detection is not good . . . The diagnosis is not accurate,” and so on. But since then we’ve done a few cohort studies (which follow a group of people who share common characteristics and lifestyles) and still we find most patients presenting with liver cancer without any trace of cirrhosis beforehand.’

  In their studies among liver-cancer patients in Thailand, Hainaut and his team found the same phenomenon – those who had the aflatoxin mutation as well as Hep B infection had no signs of cirrhosis. But though aflatoxin-mutated p53 may be protective of livers in the short term, the case for controlling the offending fungus to reduce the burden of liver cancer is overwhelming, as events in Mali have shown serendipitously. Poring over the cancer register in the capital city, Bamako, very recently, Hainaut and colleagues faced another mystery – liver-cancer cases seemed to be plummeting. In the 15 years since the late 1990s, the rate of new cases had declined by about 75 per cent. They looked for flaws and biases in the records, but could find nothing obvious to explain away the dramatic figures.

  On further investigation they discovered that in the mid-1990s, the agriculture ministry had started a programme to prevent aflatoxin contamination of the country’s crops. The primary motivation was not public health but economics: Mali wanted to export its crops for animal feed and needed to comply with international regulations. But this has had far-reaching consequences for the man and woman in the street, said Hainaut. ‘The first thing is that the contamination of food has decreased; and second, most of the crop production has been diverted towards export, so the diet has changed.’ Today, the exposure to aflatoxin of people in Mali is
only a tiny fraction of the exposure of people in The Gambia, where rates of liver cancer remain as high as ever.

  But there is a downside to this story: as Hainaut and his colleagues predicted, doctors are beginning to see more people with liver cirrhosis in Mali than ever before, as Hep B is still widespread but the factor that keeps the offending viral gene under control – aflatoxin-mutated p53 – is no longer so common.

  The great appeal of molecular epidemiology to those involved is that it is often swiftly and directly applicable to real life, and this is the case with liver cancer and mouldy grains. Having worked out the relationship between aflatoxin, p53 and Hep B virus, the scientists find they can read much of what is going on in a person’s liver with a blood test. ‘The point is that when material is being cleared from the liver it goes either into the bile or the blood. It can’t go anywhere else – there’s no direct route to the outside world like in the digestive tract or the lungs,’ explained Hainaut. ‘That means that every bit of DNA from liver cells ends up in the bloodstream. And the liver is such a massive organ that a large part of the free-circulating DNA that you find in the blood comes from the liver.’

  The scientists have worked out a method for retrieving that DNA and screening it for aflatoxin-mutated p53. They are also able to monitor the components of the viral genome and to look at what’s happening with HBx. Unfortunately, however, no such simple test exists for skin cancer, where the carcinogen, sunlight, leaves an equally clear fingerprint on p53. People just have to be on the lookout themselves for the signs and symptoms of disease.