Yeast cell cycle; what has this taught us?

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Published: 23 Nov 2016
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Prof Iain Hagan - CRUK Manchester Institute, Manchester, UK

Prof Hagan speaks with ecancertv at NCRI 2016 about the lessons learned in cell biology through yeast models.

He gives historical perspectives on how cell cycle limiters have been instrumental in revealing checkpoints and pathways through which tumourigenesis can be initiated.

CDKs and cell cycle checkpoints were also discussed by Dr Ghadeer Shubassi at an ecancer ESMO symposium on CDK therapy, with video from the session available here.

I’ve been presenting a historical perspective on the contribution of studies in yeast to understanding cancer with a particular focus on cell cycle control. Effectively because yeast is a single cell it lives in a defined media and it just divides and grows and divides and grows and it’s got very strong genetics, it’s got excellent cell biology so it’s been a real driver of our understanding of the control of cell division.

How did you go about this process?

When you don’t understand how a process works you have to try and find clues as to how to do it, how is that process controlled? Using yeast genetics is a totally unprejudiced way, so effectively you can isolate a series of mutations, a series of mutants that cannot grow at one temperature but can grow at another temperature, you have no idea what you’re looking for and then slowly you can build up the characterisation of those mutations, you can define the regulation maps without really knowing the identity of the genes, you can work out the relationship between the genes and then ultimately you can clone the genes, find the identity of the genes and put those functions onto the map of the cell division cycle that you’ve created. So this was done in the 1970s and 1980s by Lee Hartwell, Paul Nurse and all of their colleagues and they defined a series of concepts which have really underpinned our understanding of cell division. So they defined the concept of Start where one takes the decision to commit to the cell cycle and this is a process that happens before the cells have duplicated their DNA. It’s very analogous to the restriction point that was defined around the same time by a guy called Arthur Pardee in some really seminal work. He had used human tissue culture and he had put on serum, taken it off, put on serum, taken it off a bit later and a bit later and he’d discovered there’s a point at which once you remove serum the cells will keep going regardless. So there’s a rate-limiting step in both the yeast cell cycle and mammalian cell cycle and the work in yeast… there are two yeasts, there’s the budding yeast and there’s the fission yeast and this work has been done in both of the yeasts. Their lifestyles are very different, their cell cycles are very different and so we’ve learnt complementary answers from the two different yeasts. Work in the fission yeast showed that there are actually two rate limiting steps and showed that there’s one gene that acts in both of those steps and that it could be mutated to either accelerate the cycle or slow down the cycle. Subsequently it was found that that particular gene was very similar to the gene that regulated Start in the budding yeast and then ultimately the human equivalent of that gene was cloned and that gene was called CDC2, we now know it as CDK1. So that was a really major advance that came really quite rapidly.

It’s almost analogous, if you think about the way that we look in an unprejudiced way for finding cell cycle mutations without knowing what they’re about, it’s the same as sequencing the genome. We don’t know what’s gone wrong in a cancer, let’s open up the bonnet and have a look at the engine underneath and find out what it looks like and then find out what it looks like when the process has gone wrong. It’s effectively the same kind of philosophy however it’s really unlocked understanding of the cell cycle.

There’s one other, perhaps the most important, contribution that has come from yeast in terms of understanding cell cycle control and, in fact, biological processes in general has been the concept of the checkpoint. This came from a really seminal paper from Lee Hartwell and Ted Weinert in 1989 and effectively it’s describing a dependency relationship. So you have the cell cycle, it’s just inevitably going, cells will inevitably divide, but if you damage them they’ve got to stop that division otherwise they won’t be able to do it very well because they’re damaged but actually they will propagate that to the next generation. So it’s essential that you stop that process so there’s a series of pathways, checkpoint pathways, which will sense some damage in DNA or a number of issues and they will feed into the core cell cycle controls and they’ll stop that cell cycle control from happening. Obviously now we know the molecules that are involved in those checkpoint pathways and they are major targets in cancers because one of the first things that happens in a cancer is that the genome gets jumbled up and it accumulates damage and so it becomes more and more dependent upon DNA damage, DNA replication checkpoint controls. Really you can trace all of this approach to the original yeast concept that came from Hartwell and Weinert.

Are there any checkpoint inhibitors that are related to this?

One of the points that I kind of stressed in the talk which is a real yeast cell cycle nerd perspective, to some extent, is that I mentioned that Start is a commitment point, it’s a rate-limiting step so if cells are small they wait until they get to the right size to go through Start and then the second rate-limiting step of going into mitosis is also a rate-limiting step. Then on top of that you have checkpoints and currently there’s a bit of confusion, everything has become a checkpoint, everything has become the G1S checkpoint or the G2M checkpoint. In reality it’s really important to think about what’s a rate-limiting step and what’s a commitment point and what’s a checkpoint. So in terms of pure checkpoint inhibitors one has to think about what a checkpoint is and a checkpoint is the signal that there has been some damage, there’s then a transduction pathway and there’s then a target of that particular checkpoint. So the classic checkpoint, DNA damage checkpoint, you would have DNA PK, protein kinase, that would recognise the DNA damage, that’s drugable and there are drugs being developed to that, that will then signal to Chk kinases, and there’s Chk1 and Chk2 depending upon whether it’s replication or DNA damage, and there are inhibitors to those. The intermediate kinase, ATM, ATR, will be triggering those Chk kinases and so there are inhibitors to those. So those I would regard as classic checkpoint pathways, they then feed in to control a protein kinase called Wee1 and Wee1 controls the activity of the CDK cyclin complex. I didn’t mention the massive contribution from Xenopus where effectively the CDC2 gene that Paul Nurse had found that was really important for controlling the timing of division is partnered by a regulatory subunit called cyclin. That provides the specificity and provides the activity, takes it to its substrates, and the activity of this CDK cyclin complex is regulated by Wee1 kinase. Wee1 shoves on inhibitory phosphates, when the time is right those phosphates are removed by Cdc25 phosphatase and that’s how you enter division.

So obviously when you have DNA damage or you have any of the replication checkpoints they will feed into both Wee1 and Cdc25, they will enhance the activity of Wee1 which will keep you from dividing and they’ll reduce the activity of Cdc25 which will keep you from dividing even further. So something that’s showing real promise and great excitement at the moment is inhibiting Wee1. One can think of it as a checkpoint inhibitor but actually, from my perspective, I would say yes it’s the target of the checkpoint that comes in but actually it’s also a major rate-limiting step. So that one has a particular appeal because whatever the damage that is being done, whatever checkpoints are happening, and so when you have a cancer cell it’s gone through oncogenic stress, it is reliant on multiple checkpoint pathways, ultimately they all feed into Wee1, it’s like the bottleneck and so people are using Wee1 inhibitors now to great effect. Effectively what you do is you advance the timing of commitment to division and that just sends you into mitosis, so into the physical process of division with a huge amount of damage and you trigger all kinds of checkpoint pathways within mitosis. So you trigger apoptosis from within mitosis or in the subsequent next cell cycle because you’ve really done things way before you should have done. Normal tissues haven’t accumulated so much damage, they’re not reliant upon these checkpoint pathways and so there’s a real therapeutic window between the transformed cell and the neighbouring normal tissue because the transformed cell has effectively become addicted to checkpoint control in order to survive.

What’s next for your work?

There’s a huge amount that we can still learn. One of the great features of yeast is that it’s a single celled organism that does virtually everything that we do, that our single cells will do, in isolation. It’s got fantastic genetics we can do a huge amount with it. Consequently in the last fifteen years there have been four Nobel prizes, there’s the cell cycle one that I was talking about, there’s telomeres, Jack Szostak, there’s secretion, Randy Schekman, there is now Ohsumi has just got the Nobel for autophagy this year. The reason is because basically fundamental processes can be taken apart in the yeast but one of the processes by which we take things apart is we have a hypothesis, we make a mutant and then we can cross it into numerous genetic backgrounds and invariably we find the process doesn’t exactly work how we think it does but we’re in a new place, we do many more crosses, introduce more genetic backgrounds. So we can very rapidly move through to define signalling networks and signalling pathways and the dawn of CRISPR and genome editing is fantastic in humans, it’s amazing because it allows us to really go in with some precision questions. The challenge is everything has to be done sequentially so the real power of the yeast still persists because we can define the questions, we can use yeast as the test tube in which to define exactly what you should do when you go into humans.

So an example of that is the work we’re doing in our laboratory. There has been a lot of excitement in many signalling transduction cascades and in the cell cycle of the contribution of protein kinases and protein kinases put phosphate onto molecules to change their function. So in order to send you into division CDK cyclins, CDK1 cyclin B, will put phosphate on lots of proteins, the protein now accumulates phosphate and changes. Obviously when you leave mitosis you’ve got to take all those off again otherwise you’ll be stuck in mitosis for ever. The study of phosphatases that do that removal of the phosphates has really lagged behind kinases and the reason for this is that protein kinases are easy to assay, you can pull down your kinase by immunoprecipitation, you can mix it with casein, histone H1, histone H3, myelin basic protein and a bit of ATP and in a range of buffers you’ve got an assay. With the phosphatases you really need the specific target phosphorylated in your assay in order to be able to determine what the specific phosphatase activity is. So consequently the understanding of phosphatases in virtually all fields in cancer biology apart from the signal transduction of tyrosine phosphatases, the core [?? 12:00] phosphatase we really know very little about. So in our laboratory we are using the fact that we can run the yeast cell on one, two, sometimes no phosphatases and just establish core rules by which protein phosphatases are regulated. Just to put some perspective on that, with the protein phosphatase 2A family there are regulatory structural and catalytic subunits, so it’s a trimer, there are multiple genes encoding the catalytic, multiple genes encoding the structural, then there are four types of regulatory, there are multiple genes encoding each of the regulatory and then all of those genes, every gene is subject to differential splicing. So the PP2A-B55 family in humans can be around 280, we don’t know, B56 is the same, whereas in fish and yeast we can run the yeast on one PP2A-B55 or none. So we have described an unexpected relationship whereby one of the major protein phosphatases, protein phosphatase 1, regulates protein phosphatase 2A and we have worked in collaboration with Jon Pines to show that this is conserved through to humans. Now we are using yeast as a test tube in which to define the next level of regulation – how is that relationship controlled, what’s the impact, what’s the target on the PP2A - we’ll define that in yeast and then we move to humans. So that’s just one example, there are many areas where the yeast stuff will still hold its head up and guide the way for human cell cycle research.