p53 (6 page)

THE PARIS GROUP

In Paris, a third group of scientists who also independently discovered p53 were equally mystified by the protein they
found sticking close to large T
antigen in their experiments with SV40. The discovery occurred in the lab of Pierre and Evelyne May at the Integrated Cancer Research Institute in Villejuif, where researchers were working
simultaneously with SV40 and another closely related virus called polyoma, which causes multiple tumours in animals. Besides having a large T antigen like SV40, polyoma also has a small T and a
middle T antigen; these are involved in transforming the cells the virus infects, with middle T antigen being especially powerful at causing tumours. Most people working in the field assumed that
SV40 also had a middle T antigen that was likely to be equally potent; this was in the minds of Pierre May’s team when his doctoral student, Michel Kress, found large quantities of a new
protein in his experiment.

Thierry Soussi, who was to become an important figure in the unfolding story of p53, was working in another lab along the corridor from the Mays in 1979, studying SV40 replication. ‘I
vividly remember a postdoc
⁴
bursting into our laboratory to announce that his friend, Michel Kress, had identified the middle T antigen of the SV40
virus: it was a 53 kilodalton protein,’ he wrote in a brief review of p53’s history for a molecular-biology journal in 2010. But when further investigation revealed that the new protein
Kress had discovered in his SV40-infected cells came not from the virus but from the host, no one knew quite what to make of this. He and the Mays published the findings without embellishment in
the
Journal of Virology
, read by few people outside this specialist field.

Pierre May died in 2009, and Evelyne May and Michel Kress are retired, so when I visited Thierry Soussi at the Karolinska Institute in Stockholm where he now works, I asked about the discovery
of p53 in Paris. Ushering me into a bright, modern office opposite his lab, Soussi turned off the opera he likes to listen to while he works, pulled an old file from a high shelf and opened it,
blowing away the dust as the scent of musty old paper wafted from the typewritten pages, yellowed with age. This was a copy of Kress’s original doctoral thesis and his paper about p53. Soussi
leafed through it with a faint air of regret: ‘Michel Kress has been forgotten, which I think is a pity. He has been forgotten for two reasons. First, he is not ambitious at
all
.
Second, he discovered p53 and one year later he had to go for a postdoc, and he went to a very good lab. He wanted to work on p53, and this lab told him “p53 has no future; you are going to
work on something else.” Therefore he had to give up on p53 right at the beginning – which was not too much of a problem because at this time no one was really believing anything about
p53. No one could be excited by something when you don’t know what it is.’

Except, that is, for Pierre May who, according to his widow speaking to me on the phone from Paris, had a hunch from the beginning that this would turn out to be significant. Over the following
decades May won several prestigious prizes for his work on the gene, though at the time of its discovery in 1979 he had the greatest difficulty raising funds to continue the investigation.

Ironically, the lacklustre response to p53 is exactly why Soussi himself chose to study the gene when he joined the Mays’ lab in 1983: he figured it was just the kind of quiet,
uncompetitive backwater that he could cope with alongside the heavy burden of teaching he was expected to fulfil as a university researcher. He smiles at the thought of how wrong he was – but
he wasn’t alone. ‘You know, I was working with a student at this time who was doing her thesis on p53 and she wanted to apply afterwards to INSERM (Institut National de la Santé
et de la Recherche Médicale), which is the French equivalent of the National Institutes of Health. On the board for her thesis we put the director of INSERM, and at the end he told her,
“Okay, I understand that you want a position here. There should be no problem: you have a good application. But just don’t work on this bullshit protein; change your topic.” This
was exactly his word! No one honestly could anticipate in the early 1980s what p53 would become – it was impossible.’

In spite of his own personal conviction that p53 was important – perhaps even key to the development of cancer – David Lane faced the same kind of prejudice in the early days. Soon
after his discovery, he spent a few months at Cold Spring Harbor laboratory on Long Island, New York, where James Watson – just one of many Nobel-winning scientists who had worked there
– was director. Unimpressed by Lane’s recent discovery, one of his new colleagues predicted he would one day be ashamed of the claims he had made for p53’s significance.

A DIFFERENT ROUTE TO THE SAME DISCOVERY

The fourth person to discover p53 in 1979 was Lloyd Old, who died of prostate cancer in 2011. Born in San Francisco, California, in 1933, Old started his working life as a
professional violinist, studying the instrument in Paris and at the University of California, Berkeley, before his fascination with science overtook his musical ambitions and he switched to
medicine. Old was a pioneer of tumour immunology, which studies the interaction between our bodies’ immune system and cancer cells. It was while he was trying to identify what it is about
certain tumour cells, but not others, that alerts the immune system and causes it to develop antibodies tailored specifically to recognise only those cells – one of the central conundrums in
tumour immunology and infernally hard to crack with the tools available at the time – that he discovered p53.

As with the virus studies, the rogue protein seemed to piggyback on other proteins that Old and his team at Memorial Sloan Kettering in New York were trying to isolate by using specially
designed antibodies as tools, rather as you would use a magnet to pick out scraps of metal from a bunch of other materials. This was intriguing and, working with laboratory mice, they looked for
the protein in all kinds of cell types, both normal and cancerous. The researchers found p53 in none of the normal cells, but in all of the cancerous ones, and concluded it must be playing a part
in the cancer process.

Old and his colleagues published their findings in the
Proceedings of the National Academy of Sciences
(
PNAS
), but because there was – and still is to a lamentable degree
– so little communication between different fields of cancer research, it was over a year before the immunologists and virologists realised they were all talking about the same thing. But
what exactly was it that had caught the attention of such a disparate bunch of scientists, all following different leads in their widely scattered labs, at about the same time? This was the burning
question now for the cancer community as they began to realise that p53 could not be dismissed as a contaminant or an irrelevance, but may well be key to the transformation of cells. To find out,
they needed to clone the gene.

***

The early 1980s were an incredibly exciting time in biological research, with the increasing ability to clone and sequence genes providing a tool of huge importance. Here a bit
of basic biology is needed to make the next step in the p53 story intelligible. Virtually all the activity in our bodies is performed by proteins, and these are produced, or ‘encoded’,
by genes, which are, in effect, recipes for all the different proteins. The proteins are made only when and where they’re needed, at which time the relevant gene is switched on. And there are
mechanisms for removing the proteins when they have finished their tasks. When its protein is not needed, the gene sits there in the cells, quietly doing nothing.

Sequencing a gene gives, as it were, the exact recipe for the protein it encodes, and provides vital clues to its purpose and function in the cell. Moreover, having the clone of a gene –
that is, endless copies of the same thing – to work with means that all sorts of experiments can be done in cultured cells in the lab to answer questions about how the gene might work. Today,
cloning is a relatively simple process that can be accomplished in a day or two, but in the early 1980s it was a big challenge taking months – even years – and was made especially
difficult by the fact that it involved working with recombinant DNA. This technology remained somewhat controversial for years after the Asilomar Conference, which, you will remember, brought
concerned scientists together in a California hide-out to confront the spectre of Frankenstein species.

It’s worth pausing at this point to ponder what it is that molecular biologists – working away with their pipettes and dishes, test tubes, gels and incubators – are handling.
What can they actually see? What can they feel and smell? And what faculties are they using beyond the basic senses to understand what’s going on? ‘The wonderful thing about molecular
biology is that it’s based upon faith, ultimately,’ says Peter Hall. ‘All the data fit with a model; you build it and you test it, but you can’t
see
it. Oh no. No,
no. You actually infer it from all the experiments that you do, the interpretation of which is this, that or the other.’

It’s the ‘unseeable’ nature of molecular biology – because most molecules are smaller than the wavelength of light – that makes it so difficult to grasp and
therefore so intimidating to the layperson. But if, like a deep-sea diver, you’re prepared to learn how to operate in an alien environment, there’s a fabulous world to be explored. So,
in the best tradition of scientists themselves, let’s translate what has been deduced from experimentation about the basic working of cells into mental images and concepts that will make it
more comprehensible.

First of all, it’s not strictly true that everything in molecular biology is invisible. With an electron microscope, under certain conditions, you can actually see DNA, which is a
‘mega-molecule’. It appears sometimes as a fine strand in cells, like a piece of cotton thread. But it is when the thread is compacted, spooled tightly on to structures called histones
and organised into paired chromosomes as a cell prepares to divide that it is most easy to see, if a stain is used to highlight it in the cell. However, no microscope currently available to most
labs can show DNA in enough detail for scientists to be able to determine the order of the ‘bases’ making up the molecule. Thus the genes which are carried on the chromosomes –
not as discrete chunks of DNA but as segments along the continuous stand of genetic material – remain unseeable, and it is these scraps of information that carry the recipes for the proteins
that the scientists are after.

The famous corkscrew structure of DNA – the double helix discovered by James Watson and Francis Crick in 1953 – is made up of components called nucleotides, which stack one on top of
the other like nano-sized blocks of Lego to form long chains. Each nucleotide, or block, has three components: a sugar molecule called a deoxyribose (the D in DNA), a phosphate group and a nitrogen
‘base’. These bases come in four different types: adenine (represented by A), thymine (T), guanine (G) and cytosine (C). The long spiral ribbons of DNA are double-stranded, and the
bases on one strand reach across to pair up with bases on the other, holding the two strands together like the rungs of a chain ladder. No matter what the organism, the bases of their DNA always
pair up in the same way: A with T; G with C.

DNA is measured in base pairs. Human DNA is around 3 billion base pairs long and we have about 1.8 metres (6ft) of it in every one of the trillions of cells in our bodies, apart from the mature
red blood cells, which are unique. You get an idea of the scale of the landscape in which molecular biologists are working when you consider that, if all the DNA in your body was uncoiled and laid
end to end, it would stretch to the moon and back more than 3,000 times. This unimaginably long gossamer thread is the instruction manual for building and operating the machinery that is you or me,
so scientists working on the Human Genome Project, which aimed to decipher the manual’s code, were staggered, and not a little mystified, to find that the genes, the working units of DNA,
account for only around 2–3 per cent of the stuff – that is, 24,000 genes, each averaging 10,000–15,000 base pairs in length. Having no clue to the purpose of the rest, they
labelled it initially ‘junk DNA’.

That was in 2000. Since then researchers have worked out the function of approximately 80 per cent of what is now more respectfully called ‘non-coding DNA’. That function is to
regulate the expression of the genes – when and where they are turned on, and at what volume. Since every cell has an identical complement of DNA and a complete set of genes, these switches
are vital to the way cells can be so different – a liver cell from a heart cell, a brain cell from a skin or bone cell, for example. The ‘switches’ of the non-coding DNA ensure
that only the genes relevant to each organ are activated in the cells of that organ and the other genes left silent.

When a gene is switched on, its recipe is read out to produce a protein. For this to happen, the two strands of DNA need to be separated so that the genetic information, which is on the inside,
is exposed. Here I’m going to switch the analogy for DNA from a spiral chain ladder with rungs to a zip-fastener with teeth. A little machine called a helicase unwinds the DNA on the
chromosome carrying the relevant gene, and then unzips it so that the recipe can be read. The DNA is housed in the nucleus of the cell, but the proteins are made in the body of the cell, the
cytoplasm outside the nucleus. Because the DNA molecule is too big to pass through the pores of the nuclear membrane into the cytoplasm, the genetic information is copied, by means of another
little machine, an enzyme called polymerase, on to a smaller molecule called messenger RNA (mRNA). This forms a single complementary strand to the DNA. The mRNA leaves the nucleus and heads for the
protein assembly factory, the ribosome, in the body of the cell.