Authors: Sue Armstrong
A question that intrigued Milner about biology in general was how cells switch from a dormant, or quiescent, state to a dividing state as tissues grow or repair themselves in the normal course
of life. She was investigating this question in healthy white blood cells, which remain happily quiescent in their nutrient-rich culture medium in the lab until stimulated to enter the cycle of
division. For her experiments she was using antibiotic drugs as biochemical ‘tools’. One of these antibiotics was a toxin derived from the death-cap mushroom that works by inhibiting
the synthesis of essential proteins and cutting off the metabolism in cells so that they grind to a halt and die.
Milner found, however, that if she exposed the cells in her culture only briefly to the toxin, the effect was reversible. She developed a method that enabled her to put the brakes on in her
cells just at the moment they were about to enter division and then, by removing the toxin, to release the brakes and let the cells carry on through the cycle. Using this method, she found clear
evidence of a gene expressing a protein that appeared briefly in her cells and stimulated them to switch from quiescence to division, before it was degraded and disappeared. ‘Obviously the
next question was what that gene might be, and what was the protein involved,’ she commented.
p53 had recently been discovered and, after reading the papers by Lane and Levine, Milner thought it would be interesting, just on the off chance, to check this gene out. She managed to get hold
of two different antibodies that had been tailor-made to recognise and flag up p53 when it was present in cells. Using these antibodies she found that p53 was indeed expressed, and that one of the
antibodies recognised the protein in the quiescent cell and the other recognised it as the cell began to divide. Neither antibody recognised the protein in both states. Here, it seemed, was a good
candidate for the genetic switch. But why was the protein different – so different that it was undetectable by some tailor-made antibodies to p53 – during each stage of the cell cycle?
‘The only thing I could think of,’ said Milner, ‘was that here was a protein that was changing conformation (or structure). So you have one epitope (the face of a molecule to
which an antibody attaches) exposed and another hidden away in one conformation.’ She clenched her fist to demonstrate a folded protein showing its face to the antibody. ‘And then when
you stimulate it, the conformation changes and the other epitope is exposed.’ She opened her fist slightly to show another face as the protein refolded itself.
This was a revolutionary idea, and her results raised the question of whether this was one protein changing shape or two slightly different versions of the protein produced by the same gene in
order to flip the switch in the cell. It’s here that Moshe Oren’s temperature-sensitive mutant comes back into the picture. Just as Milner and her team were asking themselves about the
nature of the p53 protein that was throwing the switch in dividing cells, she and her lab technician attended a p53 conference at which they heard Oren speak of his serendipitous findings, and a
light went on in Milner’s head. ‘I thought: fantastic! We can check his mutant to see if our conformation idea is right,’ she recalled.
Her idea was to look at how a single blob of protein produced by the mutant p53 folded itself at the different temperatures, to see if this was what dictated its changes in behaviour from a
growth suppressor to a growth promoter and back. When she suggested such an experiment to Oren he was doubtful it would work, fearing that the techniques involved, which included a period of
incubation on ice, would upset the mutant’s temperature sensitivity. He had not considered the protein’s conformation, and anyway he had other research ideas in mind for his mutant.
However, Milner still believed the experiment was worth a try and, together with her technician, went ahead as soon as she arrived back in Cambridge. ‘It was just beautiful!’ she
said with a big smile at the memory. ‘That was a real high point, because for a long, long time we’d been trying to get a handle on conformation, juggling with different conditions to
see if we could induce any change. And here we had this mutant that did it perfectly. It was one of the special moments in life . . .’ Its great significance was the revelation that p53 can
switch behaviour – from a suppressor to a promoter of growth and back again – without the need for mutation.
Both
roles, it seems, are part of the protein’s normal
repertoire, and this flexibility of form is what makes it possible. Its flexibility is also an explanation for how p53 is able to play such a varied, subtle and central role in cells, both normal
and diseased. It has turned out that the concept is much broader than just p53, said Oren. ‘But p53, in my mind, set the paradigm for this duality of function.’
THE ‘DOMINANT-NEGATIVE’ EFFECT
The discovery of p53’s intrinsic shape-shifting nature opened the door to all kinds of experiments designed to help us understand the activity of the gene in more detail.
Milner’s next step was to investigate a phenomenon known as the ‘dominant-negative’ effect, in which the behaviour of cells that have a wild type and a mutant copy of the same
gene, both active, is dominated by the mutant. This had never been seen with a tumour-suppressor gene: all those discovered thus far conformed to Knudson’s model in which a cell that still
has one wild-type allele will function as normal until that allele is knocked out by some event. In other words, the wild type dominates over a mutant.
However, many of the people who remained sceptical of the gain-of-function theory – that mutant p53 produces a protein with new and abnormal functions – suggested that the
dominant-negative effect might well be what people had observed with p53 and mistaken for gain of function. After all, they were used to this tumour suppressor breaking the rules. But if this were
the case, it was not true gain of function people were seeing at all, but
loss
of function by an unusual route – by the wild-type p53 being, as it were, overwhelmed by the mutant
which crippled its brakes.
No one knew how this might occur, however, and the object of Milner’s next experiments was to try to understand the relationship between different versions of p53 operating in the same
cell. Do they work as separate agents, or stick together to form a co-operative unit? To make things clear, she used combinations of mouse and human p53 in her test tube because, being slightly
different-sized molecules, the two proteins could be easily followed in her experiments. As with two different-coloured but similar blocks of Lego, you could see how they fitted together, if they
did, rather than being confronted with an amorphous blob.
Milner already knew that p53 protein molecules can clump together in groups of two or four to form co-operative units. Now she showed that this assembly was restricted to proteins of the same
conformation. Thus she found that mixing the pre-formed proteins in her test tubes gave either suppressor-suppressor complexes or promoter-promoter complexes, but not suppressor-promoter complexes.
Clearly, the affinity between the p53 building blocks was determined by their shape.
However, in real life the dominant-negative effect occurs when wild-type and mutant proteins are co-expressed – that is, produced together and simultaneously in the same cell by the two
different alleles, or copies, of a single gene. So Milner and her colleague simulated this scenario in the lab, co-expressing the suppressor (wild-type) and promoter (mutant) forms of p53 side by
side and simultaneously in the same mixture. This was the acid test, and the results were spectacular: not only did the two co-expressed proteins form a complex, but the only antibody that
recognised the new unit was the one tailored to the promoter (mutant) form. The dominant-negative effect people had speculated about was a real possibility, and here was a novel mechanism to
explain it – a clear demonstration that one misfolded p53 protein in a co-operative unit of proteins can force the others to change shape in domino-like fashion.
Milner’s research, conducted under artificial conditions in the lab, was proof of principle; no one knew if this is what happens in real life. But her findings, published in
Cell
in 1991, quickly caught the attention of Stanley Prusiner, a scientist with an equally original mind working in a very different field – that of the so-called ‘spongiform
encephalopathies’ that include mad cow disease and its human equivalent, Creutzfeldt-Jakob disease or CJD, as well as scrapie in sheep. His ideas about how these diseases might arise had been
widely scorned as heretical and he was looking for just such a mechanism as Milner described to strengthen his case.
In 1972, Prusiner, then working as a neurologist at the University of California, San Francisco, had admitted a female patient to his ward suffering from CJD, which kills nerve cells in the
brain, leaving holes that give it the characteristic sponge-like texture. His patient was progressively losing her memory and her ability to perform routine tasks, and Prusiner was told she was
dying of a ‘slow virus’ infection. However, in years of research, no one had been able to pin down this slow virus, so-called because of the long incubation period between exposure to
the agent and the appearance of symptoms.
‘The amazing properties of the presumed causative “slow virus” captivated my imagination, and I began to think that defining the molecular structure of this elusive agent might
be a wonderful research project,’ Prusiner wrote some years later in an autobiographical sketch. His efforts gradually convinced him that he was dealing not with a virus, nor with any other
known infectious agent such as a bacterium or a fungus, but with a misfolded protein – and he named his novel pathogen a ‘prion’. But how could a substance with no DNA to carry
the instructions of replication transmit a disease? This was the heresy that caused the firestorm when Prusiner published his prion hypothesis in 1982. ‘Virologists were generally incredulous
and some investigators working on scrapie and CJD were irate,’ he wrote. ‘The term prion, derived from “protein” and “infectious”, provided a challenge to find
the nucleic acid of the putative “scrapie virus”. Should such a nucleic acid be found, then the word prion would disappear!’
Of course no DNA was ever found, and as evidence mounted for his novel theory of infection, the sometimes vicious personal attacks from his critics gradually died down. But still Prusiner needed
an explanation of how a misfolded protein might corrupt the normal protein we all have in our brains. ‘Our paper was the very first evidence that such a thing could happen,’ commented
Milner. ‘Prusiner was visiting a colleague in Germany at the time and contacted me to arrange a meeting. We met in my office in Cambridge and talked for three hours before I drove him back to
the station. It was so exciting to exchange ideas – just lovely!’ Six years later, in 1997, Prusiner was awarded the Nobel Prize for Medicine for his prion hypothesis which, though it
still has its critics, is now widely accepted as the explanation for the deadly spongiform brain diseases.
A LIFE OF ITS OWN?
Not all p53 mutations produce so-called ‘conformational’ (or ‘structural’) mutants that behave this way. ‘Contact’ mutants, which produce a
protein unable to attach to DNA and switch on other genes, are the type most commonly found in human tumours; in these cases the wild-type protein will win the day, keeping the mutant in check
until that good copy of the gene is lost in the course of living. As the unusual variability of p53 mutants became apparent, the debate about ‘loss of function’ versus ‘gain of
function’ became ever more intense and, in the late 1990s and early 2000s, a number of research groups created transgenic mice to try to resolve it and to find out what happens in real
life.
Guillermina (‘Gigi’) Lozano, whom we met working with mouse models at MD Anderson in Houston in
Chapter 13
, headed one such group. Lozano’s family had immigrated to the US from
Mexico in search of a better life, and Gigi was the first among them to go to college. In 1986 she earned a doctorate in biochemistry from Rutgers University in New Jersey, but as a postdoc she
chose to join Arnie Levine’s molecular biology lab at Princeton, attracted by the fact that he was working on one of the very first mouse-tumour models. ‘When I realised you can
manipulate the mouse genome to mimic the kinds of tumours you find in human cancer, I was fascinated. There was no going back for me,’ she told me with a grin when I met her at a mutant p53
conference in Toronto in 2013.
Trained by Levine, Lozano got a job in molecular genetics at MD Anderson, where she is now Professor and Head of the Department of Cancer Genetics. Much of her research involves mouse models and
in the early 2000s she set about creating one that mimics the human Li-Fraumeni syndrome, in which the p53 gene has one wild-type allele and one allele with a ‘point’ mutation, meaning
that just a single letter in its code is changed. People had become adept at creating knock-out mice, with a whole gene or one of the two alleles ‘deleted’ from the DNA, but a knock-in
mouse – one with a point mutation – was an altogether trickier proposition that took time, skill and patience. Lozano and her group chose a mutation that corresponds to the R175H
mutation in human cancers ‘because it’s the worst mutant you can possibly have,’ she explained. ‘And if you’re going to generate a mouse for the first time you
don’t want a mutant that’s kind of wimpy.’
Meanwhile, at MIT in Boston, Tyler Jacks – renowned for creating one of the two first p53 knock-out mice in 1992 – was on the same track. His lab was busy generating two different
mouse models that mimicked LFS – one with the same point mutation as Lozano’s mice, corresponding to human R175H, and another corresponding to R273H. The two groups published their
findings in the same edition of
Cell
in December 2004. What distinguished their mouse models from others designed to test the activity of mutant p53 was that here the gene was being
switched on
naturally
in response to signals from the cell’s environment. In most other models, the gene was switched on artificially by the researchers – and herein lay the
big sticking point. The sceptics argued that in all experiments that appeared to show gain of function, the gene had been over-stimulated by the researchers, leading to an over-abundance of the
protein. The artificial manipulation by researchers was bound to upset the delicate machinery of the cell, they said, and they were not convinced this pooling of protein – and hence gain of
function – is what happens in real life.