From microbes to cancer
This essay was written by Don Williamson and Lee Johnston and was first published in the 2000 Mill Hill Essays.
Studies on the growth and reproduction of micro-organisms have always played an important role in the research activities of the Institute. In the early days attention was largely focussed on the mechanisms of action of anti-bacterial antibiotics, compounds like penicillin which are harmless to humans but effectively kill disease-producing bacteria.
Penicillin had been produced in useful amounts by the middle of the second world war, and to this day remains the basis of much effective therapy of infectious diseases ranging from comparatively trivial ailments like boils and sore throats to life-threatening conditions like scarlet fever, meningitis, pneumonia, syphilis, gonorrhoea and many others. However, the way penicillin works was then completely unknown, and since understanding of this would hopefully lead to development of more and better drugs, its mode of action was high on the list of priorities of the Institute right from the early days of its opening in 1950.
The drug works best on many types of bacteria, all of which possess a thick cell wall, an outer envelope with a particular chemical composition. One of the first results of our research was the discovery that penicillin prevents many bacteria from making this envelope. The result is that as they attempt to grow, they burst open and die, rather like an over-inflated balloon. Armed with this information, the search was on for the bacterial components which were the primary target of penicillin, the so-called penicillin-binding proteins, and many were found, but the real target of the antibiotic was not finally discovered until the 1960s.
Meanwhile, the ability of bacteria to fight back by developing ways of resisting penicillin and other antibacterial agents was increasingly a topic of interest and importance. Several years earlier, it had been realized that amongst populations of bacteria growing in the environment there were often a few that were naturally resistant to compounds like penicillin. With the 20/20 vision of hindsight this is hardly surprising, since several of these useful drugs are themselves produced by micro-organisms normally present in soil, where they are used to deter competitors and boost their own survival in the harsh microbial world. The awful realization then was that treatment of bacteria with antibiotics might allow these few resistant organisms to grow unhindered and come to dominate the scene. This topic has become of the utmost importance in modern day medicine, as we have all become increasingly aware that the development of antibiotic resistance, particularly in the closed environments of hospitals, where antibiotics are in common use, has allowed resistant bacteria to flourish, with serious and sometimes fatal consequences.
The early recognition of this problem had important consequences for the direction of microbiological work in the Institute. The need to find ways of combating resistance stimulated research not only into the details of the way the antibiotics actually work but also into the ways in which some bacteria manage to resist attack. One such way is based on the extraordinary capacity some bacteria have to be economical with their activities. They carry the genetic instructions for many compounds which they only make as required, in response to signals from the outside world. They don’t waste precious energy making them when not needed, and these genes are normally silenced. However, just as you or I might change our clothing when the weather changes, so bacteria faced with a food source they don’t normally use, can “switch on” the genes needed to make the necessary tools, so-called enzyme proteins, to use it.
It turned out that this ability also extended to one of their defence mechanisms against penicillin. Certain bacteria, when exposed to penicillin, would respond by flooding the environment with a penicillin-destroying enzyme. The gene that carried the information for making this enzyme was inactive in the absence of penicillin, but was switched on when penicillin was detected, and studies on the mechanism underlying this type of drug resistance eventually led to great advances in understanding the way in which the genetic apparatus of bacteria functions. It is worth mentioning in passing that these studies on the way genes work in bacteria were of fundamental importance. It turns out that the same broad mechanisms controlling the use of genes apply to all organisms. Consequently much of our present understanding of, for example, the way a foetus develops in humans, the way our immune system responds to infection, and even the mechanisms which allow some human cells to become cancerous, owes a lot to these early studies on bacteria.
Tied in with this early work on gene activity in bacteria was the astonishing finding that the genes involved in resistance to many antibiotics could be transferred from one bacterium to another on tiny particles known as plasmids. This transfer not only happens between organisms of the same species but is also possible between unrelated bacteria. Thus for example, the harmless bacteria that live normally in the human gut might carry an antibiotic-resistance gene which could be transferred to an unrelated disease-producing bacterium, making treatment of a disease much harder. This added a new dimension to the whole field of antibiotic resistance, and so exploration of the nature of these plasmids, the way they are produced and the way they are transferred between bacteria, became another focus of our microbiological work.
One lasting contribution to current medical practice was the work done at the Institute on an antibiotic known as vancomycin. Originally isolated from a soil micro-organism, this substance offered a new route to fighting bacterial infection, since although its mode of action and effects on sensitive bacteria are superficially similar to those of penicillin, it is a totally unrelated compound, kills penicillin-resistant bacteria and is rarely met by resistance to itself. Our work unravelled much detail of the mode of action of this compound and was instrumental in establishing it as one of the important weapons in our anti-bacterial armoury. Currently vancomycin is regarded as our final defence in treating patients infected with the now infamous “MRSA” strain of bacterium – the “superbug” which is resistant to practically all other known antibacterial agents, and which has surfaced in many hospital wards.
With the passage of time, most infectious diseases of bacterial origin have dwindled in importance in western society, compared with the massive killer that is cancer. Our interest and microbiological skills have reflected these changes, and much of our current microbiology is centred around the use of micro-organisms as models for studying the roots of the biological changes that can give rise to cancer.
Cancer is a desperately complex condition which calls for study at many different levels if we are ever to find effective ways of combating it. In adults most human cells are prevented from dividing by a complicated regulatory network which depends on interactions between the protein products of many different genes. Cells become cancerous when this control mechanism breaks down and they begin to grow and divide unchecked. There are many ways in which this breakdown can occur, but almost invariably the root cause involves damage or alteration to some part of the cell’s genetic apparatus, its DNA. This can result from exposure to toxic chemicals, as found in cigarette smoke, for example, or radiation from the sun’s ultraviolet light, X-rays, cosmic rays or radioactive minerals. It is a hazard that is constantly monitored by living organisms. Not surprisingly, cells of all animals, plants, fungi and bacteria possess elaborate mechanisms to prevent and repair genetic damage from these sources, and in recent years we have contributed a lot of experimental work to understanding the molecular basis of mechanisms involved in both the regulation and maintenance of DNA.
Many different micro-organisms, some very “simple”, have found value as experimental models for this type of work. The E. coli bacterium for example, which normally inhabits our gut and, with rare notable exceptions, is totally harmless to humans, is much used in laboratories world-wide since it grows very fast and is easily manipulated genetically. Shadowing our earlier studies on the way bacteria can respond to environmental changes by making new enzymes “a la carte”, we discovered that E. coli can respond to exposure to noxious chemicals by making, as required, a complex collection of enzymes to repair the damage that they cause to the bacterial DNA. Once the job is done, the enzymes are simply discarded. We later discovered a similar “inducible” repair mechanism in a simple fungus, the parasite which causes “smut” in corn. This was important because although it is a fairly humble organism, at the molecular level this fungus has many of the attributes normally associated with cells of higher organisms. Using it as a model, we also carried out a number of studies on the way genes are inherited, especially in sexual reproduction, an area of particular importance in the biology of humans; failure of proper inheritance of genes in humans leads to spontaneous abortion and birth of babies with defects like Down’s syndrome.
However, our major workhorse for this kind of biology soon became the baker’s yeast cell. As you might buy it from a bakery, a lump of yeast consists of billions of completely harmless single-celled micro-organisms which can be grown easily and cheaply in laboratory cultures, and which, like the smut fungus, reproduce by a form of cell division. Despite external appearances, the functional organisation of yeast cells is amazingly similar to that of the cells of our own body. Many of their biochemical activities are carried out in the same way, using enzymes virtually identical to those in our own body. Some human genes can even be introduced into yeast cells, where they substitute for their yeast counterparts and function perfectly well. This is even true of many of the genes involved in regulating cell division, and this is where the real value of yeast cells lies. They grow and divide at an astonishing rate, an average division cycle of an individual cell taking only an hour or two. This makes them an ideal model system for studying human cancer cells, the more so because their genetic apparatus is remarkably easy to study, so that changes in genes that might lead to cancer in humans can be quickly evaluated and understood.
We started working with yeast in the late 1960s, specifically with the aim of using this model system to explore the mechanism of cell division. Individual cells are too small for easy use, so for this purpose we pioneered the use of procedures in which large numbers of yeast cells are all induced to go through a division cycle together, a process known as division synchronisation. This enables scientists to harvest useful-sized samples from particular stages of the division cycle and know that any results they find on analysis represent the behaviour of individual cells. Initially we focused on what one might call the “natural history” of the division cycle, changes in the chemical composition and structure of the dividing cell. We already knew that the DNA in the cell’s nucleus is duplicated quite abruptly, very early in the cycle, copies of the genes made at that time being distributed with great precision between daughter cells a little later on, as the nucleus itself divides. We concentrated at first just on finding out what enzymes were involved in these stages, but this very soon led us to take a more genetic stance, exploring in depth the activities of the genes controlling these particular enzymes. We rapidly made the interesting observation that a whole group of genes which make enzymes involved in duplicating the DNA in the nucleus are all switched on at the same time, just before duplication starts, and are switched off after use, eerily reminiscent of the behaviour of the bacteria studied before. This led us naturally to ask what sort of mechanism could control the activation of a large number of genes at the same time, and exploration of this and similar conundrums at the genetic level, largely exploiting the synchronisation technique, soon became a major focus of our activities.
We can illustrate one way we tend to approach these questions with an example taken from one of our earliest notebooks. Nearly twenty years ago, we chose to examine a mutant yeast which was sensitive to temperature in a very specific way; when its growth temperature was raised by a few degrees it stopped dividing at a particular stage of its division cycle. We soon found this mutant had a specific defect in a key gene that is vital for cell division and indeed for the life of the cell. Its actual role is to “stitch” together stretches of DNA that are produced either during DNA synthesis, or following repair of molecules damaged, for instance by X-rays or cancer-inducing chemicals. The enzyme produced by this gene turns out to be a key player in maintaining the essential integrity of DNA. It is one example of hundreds of genes that are shared amongst all living creatures, plants as well as animals, and inherited defects in it and related enzymes can contribute to some forms of leukaemia and certain severe life-threatening conditions which make patients highly sensitive to sunlight and prone to skin cancer.
One notable achievement, which nicely illustrates the value of the yeast cell as a model, was that we were able to use yeast actually to identify this gene, for the first time ever, in human cells. This came about because we were able to cure the defect of the temperature-sensitive mutant cells, simply by inserting the human gene into their nuclei, where it compensated for the activity of the damaged yeast gene. Our ability to follow the activities of this enzyme and the way its controlling gene is regulated in yeast cells helped establish its role in the human diseases as well as in the complex processes involved in the development of cancer.
A more recent illustration of the direction our work now takes is provided by studies on what are known as “checkpoints” in the yeast division cycle. These are specific points in the cycle at which the dividing cell assesses if it is safe to proceed with cell division. Cell division is a critical step for a cell. If its DNA is damaged or incomplete, to go ahead with division would be fatal. Death would also result if the apparatus for apportioning its genes precisely between offspring was faulty. There are in fact at least two major checkpoints in the division cycle of all cells. One of these monitors the quality of the newly made DNA, while another reviews the DNA transmitting-apparatus, known as the “spindle”. Both these checkpoints are complex and involve the action of a host of different genes. If the cell senses damage to its spindle, it immediately switches on a cascade of these genes which prevent division from proceeding. This buys it time to repair the damage, after which it can go ahead. We have recently worked out a novel pathway of the interactions between the genes involved in the spindle checkpoint and we now know in great detail nearly all the gene products involved.
Well, you may say, that’s fine for the yeast cell; what about us? And this is where the value of the yeast cell lies. Nearly all the genes involved in this checkpoint in the yeast cell also occur in human cells. The knowledge from our laboratory experiments on yeast is directly applicable to human cells, and there is simply no way we could have gained this detailed understanding by directly studying humans or even isolated human cells. The significance of this of course brings us back to cancer. Cancerous cells may sometimes result from the checkpoint failing to do its job properly, and once a daughter cell with a defective genetic content is formed, more defects in the regulation of all sorts of genes can occur, and a complete breakdown of all the regulatory processes which hold normal body cells in check allows unlimited growth to occur, with the development of fully-blown cancer. Knowing the genes involved in this checkpoint raises at least a possibility that we could find ways of switching on the checkpoint in cancerous cells and preventing them from proliferating before too much damage is done.
Having said all this, we know that we have only scratched the surface of our understanding of the way cells divide, and the mechanisms which can sometimes go wrong and cause serious or life-threatening conditions like cancer. However, all the time the basic information at our fingertips is increasing rapidly. Workers with yeast have recently unravelled the complete yeast genome – the entire catalogue of genes that control the life of the cell. We have all been humbled by the finding that about half the genes that have been found have as yet no known function, and we bravely assert that it is only a matter of time before that “little local difficulty” is overcome. But to put this into perspective, it is worth pointing out that the yeast cell has only about six thousand genes, while the human genome has over ten times that number, and about the same proportion with unknown functions. Against this background, there is obviously good reason to continue exploration of our humble but beautiful and complex little friend, the baker’s yeast.