p53

by Sue Armstrong

  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.

  CHAPTER FOUR

  Unseeable Biology

  In which we peer into the machinery of the cells to see how the genes make the proteins that do virtually all the work in our bodies.

  ***

  Every cell in nature is a thing of wonder. Even the simplest is far beyond the limits of human ingenuity. To build the most basic yeast cell, for example, you would have to miniaturise about the same number of components as are found in a Boeing 777 jetliner and fit them into a sphere just 5 microns across; and then you would have to persuade that sphere to reproduce.

  Bill Bryson

  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.

  Proteins are made of amino acids, of which there are 20 different kinds. The instructions from the gene determine which amino acids are to be used, in what order and what quantities. For this process, the string of simple letters represented by the sequence of base pairs on the gene, the As, Ts, Gs and Cs, need to be translated into more complex ‘words’, so the ribosome protein factory reads the linear information on the strand of mRNA in three-letter chunks. These chunks, or ‘words’, are called codons, and they identify which amino acids should be used to make a specific protein. The amino acids are brought to the assembly point, the ribosome, from other parts of the cell by transfer RNAs (tRNA), which dock on to the appropriate codons and deposit their cargoes of amino acids. When all the amino acids are in place they are strung together as a chain, which detaches itself from the strand of mRNA and goes off to another part of the cell to be folded. This process is vitally important, since a protein’s function is determined not just by its amino acid components but also by the way it is folded.

  The other biological process central to the story of p53 is that of DNA replication, which occurs in every cell that is about to divide. In this process once again the enzyme helicase unwinds the DNA and unzips it – not all the way along, but a fragment at a time. Small molecules called single-strand binding proteins, or SSBs, attach temporarily to each separated strand to stabilise them while they are being copied and to ensure that they stay separate. Then DNA polymerase travels along each separated strand attaching new nucleotides – the nano-scale Lego blocks described earlier – to each of the existing ones, pairing up the bases in the conventional way, A with T and G with C, thus constructing a parallel strand of DNA, block by block. A subunit of the polymerase travels along behind, ‘proofreading’ the new DNA to see that it has been faithfully copied. Then an enzyme ‘glue’ called DNA ligase seals the fragments of copied DNA into a continuous double-sided strand that rewinds itself automatically.

  In the replicated DNA, one strand of the double helix will be from the original (known as the parent strand) and the other will be the new copy (known as the daughter strand). The cell is now ready to divide into two cells with equal shares of identical genetic material. This process, going on ceaselessly in billions of cells in our bodies as we repair and replace tissue and our hair and nails grow, is so efficient that mutations – mistakes that escape the proofreader – occur at the rate of about one in 109 nucleotides per replication.

  It’s interesting to note that this knowledge, this understanding of how the machinery of life works, is built on the foundations of Watson and Crick’s discovery of the structure of DNA. The double helix – the spiral staircase drawn originally for their paper in Nature by Crick’s wife Odile – remains one of the iconic images of 20th-century science. Yet when the paper announcing their discovery came out in 1953, it was noticed by hardly anyone beyond a small group of enormously brainy and ambitious scientists working in the same field – some of whom had been racing to make the discovery themselves first.

  Of the media, only one paper, the British News Chronicle, carried the story about ‘an exciting discovery about what makes YOU the sort of person you are . . .’ And for nearly a decade, only a tiny proportion of scientists writing about DNA in professional journals mentioned the double helix. It was a beautifully elegant model, but many biochemists, intensely preoccupied with working out how we synthesise the proteins in our cells, were sceptical that genes – still rather an abstract notion in the early 1950s – had anything to do with it.

  CHAPTER FIVE

  Cloning the Gene

  In which we hear about the huge technical challenge and the hot competition to clone p53 as the first step to discovering how the gene and its protein work.

  ***

  We stand on the wrong side of the tapestry – a confusion of colours, knots and loose ends. But, be assured, on the other side there is a pattern.

  Anon

  The cool reception and slow build-up of recognition for the double helix – culminating in the Nobel Prize for James Watson, Francis Crick and the biophysicist Maurice Wilkins in 1962 – are instructive. This is how science works, says Peter Hall. ‘It’s the analogy of the man in the dark warehouse with a small pen-torch. He can only see a tiny part of what’s there. The whole thing only becomes clear when he gets more light.’

  Until they had a clone of p53, the scientists were guddling around in the dark without even a pen-torch, and in the early 1980s the race was on among a handful of individuals in labs around the world, from the UK and US to Russia and Israel, to clone, or make identical copies of, the gene – the first, essential step to finding out what it is and how it works. The first person to succeed was Moshe Oren, who started the process while working as a postdoc in Arnie Levine’s lab in Princeton, but completed it at the Weizmann Institute in Israel, where I went to talk to him on a hot October afternoon some three decades later. Entering his small office on the top floor of the institute, with its views up into the wide sky, I was greeted by the scent of citrus. Oren was seated behind his desk with a little pile of orange peel and pips in front of him – clementines picked from his own garden that morning, he told me, as he offered me a handful. We sat eating the sweet fruit as we talked.

  ‘I was looking for a new project because I needed to change. Here was this interesting protein that people were beginning to look at and it was my chance to clone something,’ he recalled. ‘In those days cloning was a major technical challenge. It was probably two and a half years from me saying, okay, I’m going to clone p53 to actually getting the thing. It took a lot of setting up protocols and testing different approaches, some of which didn’t work. It’s kind of amazing how we’ve progressed: cloning a gene now is trivial; it’s become probably a high-school exercise! But when we did it the tools were very limited; there were very few genes that had been cloned, and each of them was cloned by a variety of improvised tricks . . . It was not easy at all.’

  One of the hardest tasks for the would-be cloner even today is identifying the individual genes on a continuous strand of DNA – where does one gene end and the next begin? Oren’s strategy was to look for the
gene after it had been switched on, when the relevant segment of DNA had been copied, or ‘transcribed’, into ‘messenger’ RNA (mRNA), left the nucleus and gone to the ribosome – the protein-making factory – in the body of the cell. Using antibodies tailored to recognise the p53 protein from among a mass of proteins being made at the same time, he isolated the relevant protein factory and scraps of mRNA. Using the mRNA as a template, he synthesised ‘complementary’ DNA (cDNA) by attaching new nucleotides, block by block, along its length, pairing up the bases as appropriate. This cDNA, he hoped, would give a faithful copy of the p53 gene – or at least that part of the gene responsible for making the protein.

  In order to multiply the little scraps of cDNA, he transferred them to a bacterium – E. coli, which, you will remember from the Asilomar story, is one of the workhorses of biotechnology because it’s easy to manipulate, efficient at taking up new genetic material and can pump out clones at a terrific rate, given a good food supply. In order to transfer the cDNA to the bacteria, Oren had to use a suitable vector – something that would breach the walls of the bacteria and take up residence inside without killing its host – and for this he chose a plasmid.

  Plasmids are tiny rings of DNA between 1,000 and 10,000 base pairs long that some bacteria have floating around in their cells, independent of their regular genomes. When a bacterium dies and its body – a single cell – disintegrates, the plasmids are scattered into the environment and are often absorbed into other bacteria, which then begin to express the new traits encoded by the plasmids. This is how recombination – the phenomenon debated at Asilomar that still excites such public controversy over biotechnology today – happens in nature, and it has provided a marvellous tool for cloners. The scientists put the plasmids in solution with the scraps of cDNA they want copied, having used a ‘snipping’ tool, an enzyme, to cut a gap in the plasmid ring where the new material should be inserted. With a little coaxing, the cDNA moves of its own accord into the gap, where it is glued into place by ‘repair enzymes’ added to the solution. This plasmid is now a recombinant DNA molecule – a mixture of genetic material from different organisms. The cloner then adds it to another solution containing the E. coli, or whatever else he or she has decided to use as a ‘clone factory’, and waits for it to find its way in among the machinery.