Cell Cycle Research Papers
‘Dividing cells pass through a regular sequence of cell growth and division, known as the cell cycle’, according to a college textbook of biology published in 1983 , 5 years before the underlying principles of control were first laid bare during 1988, the annus mirabilis of cell cycle research [2,3]. One of the key architects of that revolution, Paul Nurse, was elected as President of the Royal Society in 2010, and this volume is intended in part as a tribute and in part as a reflection of what we now know, and what remains still to be found out about cell proliferation. Lest we forget that cells have fates other than their own reproduction, Pat O'Farrell  reminds us that many cells in our bodies survive for long periods in a quiescent state. He considers quiescence from the perspective of the developmental biologist, and sees growth factors as surrogates for nutritional signals. He surveys the complexity and the richness of growth control in higher eukaryotes, rightly pointing out what an important topic for future research this remains. This problem is also attacked from the perspective of the single starving cell by Yanagidai, who have been recently studying the effects of nitrogen or glucose deprivation on fission yeast . It turns out that the results are daunting. Wild-type yeast undergoes two rapid divisions to generate quasi-spherical cells if they are suddenly deprived of nitrogen, and undergoes startling changes in intracellular morphology and metabolism that remain difficult to comprehend. Interestingly, defects in these adaptions are accompanied by cell death and hundreds of different genes, in many distant pathways, are required to respond to starvation or to ‘wake up’ when better times come along.
Long-term survival, we must remind ourselves, requires not only cell division but also sex. Dan Mazia, who was the guru of cell division of the 1950s and 1960s, put it thus: ‘More often than not, questions beginning with ‘Why’ are inane and of no service in scientific discourse. In biology, they sometimes make sense. If we ask why cells must divide, the answer can be given in terms of what happens if they do not. The answer is that they die, no matter what criterion of death we apply’ [6, pp. 82–83]. This applies to organisms as well as cells, of course, and it is still somewhat mysterious that we humans can all trace our ancestry back several billion years, yet we are all mortal. The continuity of the germ cells is something that successfully evolved, but is hard to explain and rarely examined. Yet, formation of gametes is amenable to genetic analysis, and van Werven & Amon  present an accessible and wide-ranging survey of the process in budding yeast, fission yeast and higher eukaryotes that makes clear what a delicately regulated process it is. Certain features seem to be common: the existence of a ‘master regulator’ whose activity depends critically on a very particular combination of nutritional or developmental signals, as well as downstream regulatory protein kinase cascades, in particular the TOR pathway and the cyclic AMP-dependent protein kinase. For some reason, functioning mitochondria are apparently also required for successful gamete formation in yeast and mice. Presumably, that has only been true since oxygen entered Earth's atmosphere two and a half billion years ago.
In ‘normal’ cell cycles, there is a gap between the end of mitosis and the start of DNA replication, and control of the G1 to S transition is an important point of no return in the cell cycle. Cross et al.  discuss the evolution of control networks at this stage of the cell cycle, comparing yeast with plants and animals. They find that many individual regulators have either undergone huge sequence divergence from the last common ancestor or have evolved from different origins. Despite this, the topology and dynamic properties of networks have striking similarities. Diffley  looks at the control of initiation of DNA replication and again notes the variety as well as the redundancy of mechanisms that ensure the genome is replicated once and only once each time a cell divides. Arguing from simple assumptions, he points out that suppression of re-replication must be close to 100 per cent efficiency and that a combination of mechanisms, each with a small but finite failure rate, is necessary to reduce the overall failure rate to acceptable levels.
High fidelity is also a major consideration for the control of key cell cycle transitions. The penalty for failure is high, the difference between success and failure is tiny, and mechanisms for assuring accuracy are numerous and robust. Having evolved over billions of years they may also be rather complicated and difficult to understand, as is the case with the so-called S-phase checkpoints, discussed in detail by Labib & De Piccoli . As soon as people realized the importance of DNA and DNA replication for cells, in the early 1950s, they tested the effects of ionizing radiation and discovered that normal cells quickly stopped synthesizing DNA after X-ray damage (apart from very rare mutant individuals, who were extremely sensitive to X-irradiation and turned out—many years later—to carry mutations in the ATM protein kinase). These irradiated cells did not enter mitosis. After intense study, largely by geneticists, because biochemical analysis for such complex systems is for the most part too difficult, we are now aware of many if not most components of the S-phase checkpoint, but it is still difficult to appreciate how the system really works. Replication forks and collapsed replication forks are complicated structures and the details of how damage is sensed, signalled and repaired are complicated and only gradually being worked out in mechanistic detail. The virtue of Labib's account lies in its historical approach and his attention to describing the experiments that underlie our present understanding. Langerak & Russell  also discuss the effects of DNA damage on cell cycle progression and vice versa, concentrating on the mechanisms that repair double-strand breaks in DNA. These are largely twofold, non-homologous end joining (NHEJ), which tends to occur when DNA is broken during the G1 phase of the cell cycle, and homologous recombination (HR), a largely error-free repair process that uses sister chromatids to reconstruct lost DNA sequences. The latter requires production of long stretches of single-stranded DNA that search for neighbouring homologous DNA sequences and subsequently invade them. The abundance and the activity of a large cast of cofactors are regulated in such a way as to promote NHEJ during G1, when sister DNAs are absent, and HR during S and G2, when they are present.
A key issue in cell cycle studies has been the nature of the triggers for the onset of DNA replication and mitosis. Much to everyone's surprise, both turn out to be triggered by similar molecules, namely S- and M-phase-specific cyclin-dependent kinases (CDKs), and almost every article in this issue refers to these key cell cycle regulators. An important question about these enzymes, apart from their regulation, is their substrate specificity. In particular, and this was confusing in the early days of the modern era of cell cycle studies, how can it be that the same kinase initiates both S and M phase? Why, for example, do cells undergo DNA replication and not attempt to enter mitosis at the first appearance of CDK activity? Moreover, why do not cells re-replicate their chromosomes when a second rise in CDK activity triggers mitosis? Two explanations seemed possible at first: one was that different cyclins imbued Cdks with different properties, so S-phase cyclins promote S phase and M-phase cyclins catalyse mitosis. But an alternative, originally suggested by Paul Nurse and co-workers , is that it takes only a little Cdk activity to initiate S phase, but more to enter M phase. The available evidence suggests that the level of activity is indeed part of the story, as Uhlmann et al.  discuss in their article. However, this is only part of the story. Whether a cell enters to undergo DNA replication or mitosis in response to a rise in Cdk activity is as much determined by the presence or absence of substrates or structures for these kinases to work on. Thus, the reason why Cdks do not trigger S during G2 is that the pre-replication complexes required to initiate DNA replication are absent from this stage of the cell cycle. Likewise, G1 cells that have not yet replicated their DNA do not possess a pair of sister chromatids nor even the cohesion that will hold these together, and cannot undergo anything resembling a physiological mitosis until these have been produced.
Uhlmann et al.  take some trouble to examine whether ‘checkpoints’ impose order on the cell cycle, and conclude that, on the whole, they do not. We note, however, that the concept of checkpoint, while highly popular and therefore much abused in the literature, is often inappropriate in the context of the cell cycle as well as being rather fuzzy on close inspection. In an earlier generation, before yeast genetics was applied to cell cycle control, people like Dan Mazia used to talk about ‘Points of no return’ rather than ‘Checkpoints’. The idea of the checkpoint is that you may not proceed to the next process or event until the one in which you are presently engaged is complete: a quality control check. But there is something else as well—once you have finished a task and been allowed to pass on, you cannot go back. This applies equally to the G1–S, the G2–M and the metaphase to anaphase transitions. Uhlmann et al.  argue that even if the level of Cdks activity has an important role in determining entry into S or M, regulation of phosphatase activity plays an equally important part. The various thresholds for cell cycle transitions are set by ratios of kinase activity to phosphatase activity and not by kinase activity per se. This theme continues in the contribution from Domingo-Sasanes et al. , who restrict their discussion to the control of mitosis, but focus on the recently discovered role of greatwall kinase as a controller of protein phosphatases that both regulate and antagonize Cdks at the G2–M transition.
This brings us to the end of the cell cycle, or the beginning of the next, the metaphase to anaphase transition. Musacchio  entitles his piece ‘Spindle assembly checkpoint: the third decade’, inviting the query, what's taking you so long? The answer is that this is a very complicated piece of machinery involving both hardware (the kinetochore itself, and its connection with spindle microtubules) and software—the error correction mechanisms and surveillance mechanisms that constitute the spindle assembly checkpoint (SAC). Interestingly, this regulatory system, unlike other the so-called checkpoints, has little or no role in repairing the damage sensed and appears solely concerned with regulating cell cycle progression. At least three or four protein kinases (and presumably their counterpart phosphatases) are involved as well as specific regulatory proteins such as Mad2. Working out how the SAC functions will require greater understanding of kintetochore structure as well as further structural work on its target, the anaphase-promoting complex/cyclsome (APC/C). Nevertheless, it is clear that the structural approach has already been extremely illuminating. At the moment, it looks as if the mechanical basis for the tension sensor may be nothing more complicated than a substrate being pulled beyond the reach of a tethered kinase. We may hope that some of the other seemingly complex features of the mitotic checkpoint will proved to have a similarly simple basis, once we understand them.
David Barford's  magisterial review of the APC/C depends even more so on structural determination, but his recent impressive advances required him to work out ways of making large quantities of this enormous, complicated multi-subunit complex. As he describes, we can begin to see how the thing works, although mysteries still remain, particularly in its control. This is connected with the previous paper, of course, because somehow the SAC can reliably amplify a signal from a single kinetochore to inhibit millions of APC/Cs, and somehow (as anyone who has ever watched cells enter anaphase can testify) the inhibition is lifted when all the chromosomes are properly aligned on the metaphase plate, such that it looks as though someone fired a starting pistol (figure 1) to signal chromosome separation.
Mazia's diagram of mitosis with the starting pistol (‘Trigger?’). Adapted from Mazia .
Pines & Hagan  revisit many of the points raised in the preceding articles from a wide-ranging perspective, but they go on to stress the importance of intensely local conditions controlling physically distant processes. A particular concern of theirs is the spindle pole in fission yeast and its role as the place where a commitment to mitosis is made first, and the centrosome in animal cells, which they argue plays a central role in the control of mitotic entry. They make a plea for more quantitative studies in cell biology, and urge the development of reporters that can monitor the local activity of protein kinases and phosphatases in living cells.
Hyman's article  takes up the same theme from a ‘systems biology’ perspective. Actually, one of his main concerns is people's understanding of exactly what is systems biology. Tony makes the important point that both time and space cover vast ranges of scale in biology. Molecular movements in proteins occur on the microsecond timescale, yet it takes years for a human to reach sexual maturity. Or look at Barford's beautiful pictures of the APC/C elsewhere in this issue and remind yourself that a human on the same scale would be roughly twice as big as the Earth. Hyman makes the same plea as Pines and Hagan: more emphasis on quantitative data is necessary to begin to make sense of biological models. He also provides a useful definition for systems biology: ‘It is the approach of collecting quantitative biological information at one level of complexity, and using it to build models that describe the next level of complexity’. Thus, he claims that ‘systems biology is an approach, not a field’. The more we learn about the complexity of the regulatory network controlling cell proliferation the more useful are the systems biology approaches in the cell cycle field.
The final contribution to this collection by Kronja & Orr-Weaver  covers one of the less familiar areas of cell cycle control, namely the control of expression of mRNA at the level of translation. It turns out that there is quite a lot to say, not only in the authors' favourite model system, the Drosophila egg, zygote and early embryo, but also in yeast, frogs and even human cells. It has been known for some time that translation of normal capped mRNAs declines during mitosis, whereas internal ribosome entry sites (IRES) are preferentially used, and some of the important examples of this switch have recently come to light; their underproduction causes faults in cytokinesis. The majority of well-established and better worked-out examples do come from eggs and early embryos, however, which is probably not surprising considering that transcriptional control of gene expression is largely absent in these (typically) enormous cells. The regulatory networks are quite complicated, as can be seen from figure 2 of this review. One suspects that there is rather a lot to be learned still in this area.
Altogether, this collection of articles provides a kind of partial snapshot of the current state of understanding in some of the most active areas of enquiry in the broad field of the cell cycle. For the most part, the general principles are reasonably well defined, but the precise details of molecular mechanism in many cases prove harder to pin down than perhaps one might have expected 25 years ago. Moreover, there remain considerable tracts of uncharted territory, that is, areas where even the basic principles are difficult to comprehend. First and foremost among these is the problem of the regulation and coordination of cell growth, and the relationship between growth control and division control, which was arguably the starting point when Paul Nurse decided to work on fission yeast with the late Murdoch Mitchison. It is perhaps appropriate to end with an acknowledgement of Murdoch's contribution to this field. Apart from definitively defining the field in the title of his 1971 monograph ‘The Biology of the Cell Cycle’  (the term was not in common currency before this, surprisingly enough), Murdoch served as a stimulating, quizzical, generous mentor to Paul Nurse as well as two of the editors of this issue, Kim Nasmyth and Bela Novak. Murdoch took a keen interest in the cell cycle field until the very end, and his passing marks the end of an exciting and heroic era.
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This year’s Nobel Prize for physiology or medicine brought welcome recognition to the cell cycle. Awarded to Lee Hartwell, Ph.D., Paul Nurse, Ph.D., and Tim Hunt, Ph.D., for working out cell cycle regulation, the prize gave official stamp to the revolution that has transformed this once-shunned area of research over the last 30 years. The implications for cancer research and treatment are far-reaching.
In 1970, the cell cycle was a black box. Scientists, through microscopes, had long observed the exquisitely choreographed sequence of events culminating in mitosis: Cells grew, replicated their DNA, segregated their chromosomes into two identical sets, and divided. But which molecules drove cell division, and which abnormalities set off uncontrolled cell division and cancer, remained completely unknown—to the extent that few scientists were willing to tackle the problem. “It was a real needle in the haystack,” recalled Nurse, who is the director general of the Imperial Cancer Research Fund, London.
Most biologists felt that the cell cycle could not be dissected without first understanding its obvious features, like DNA replication. Their thinking was, “If you couldn’t understand the processes that were controlled, you couldn’t possibly understand the control circuitry,” said Andrew Murray, Ph.D., of Harvard University.
Hartwell, who is now the president and director of the Fred Hutchinson Cancer Research Center, Seattle, was the first to show otherwise. While he was studying temperature-sensitive budding yeast mutants in 1970, serendipity intervened. An undergraduate in Hartwell’s laboratory noticed that some mutants, at high temperature, all stopped growing buds at exactly the same point during cell division.
“Hartwell immediately realized that these mutants would be fantastic for dissecting how the cell cycle worked,” said Murray. Dozens of cell cycle genes soon were isolated. “They fell out very quickly,” Hartwell recalled. One—cdc28—blocked all the early cell cycle events and identified a point in the cell cycle where cells are irrevocably committed to replicating their DNA. Hartwell called this crucial transition point Start.
Hartwell’s work in California directly inspired Nurse, a British graduate student. “I was doing some very tedious experiments in the middle of the night,” Nurse recalled. “I read a couple of Lee’s papers [and] I thought, ‘This is great stuff.’”
Around 1975, working in fission yeast, Nurse found temperature-sensitive yeast mutants that divided while abnormally small and reasoned that they expressed genes responsible for driving cell division only when cells grew to a certain size. Nurse had already guessed that just a few such events might govern the cell cycle.
“A key issue was how to identify those events that were rate-limiting,” he recalled. “And I actually hadn’t solved that problem, as to how to do it, until I saw under the microscope fission yeast cells growing, dividing, at a reduced size. And then it clicked into my brain.” Nurse soon identified a key gene, cdc2, that drove mitosis.
Using mutants that make the cell cycle go faster to find such genes was “an extremely clever idea,” commented Murray. “[Nurse] was very single-minded in following that idea through.”
The advent of DNA cloning made possible the next stunning advance. In 1982, Nurse set out to find a budding yeast homologue for fission yeast cdc2. Introducing plasmids with budding yeast genomic DNA into fission yeast mutants, he found one that restored mitotic control. To his (and everyone’s) complete surprise, it turned out to be Hartwell’s cdc28. Thus, the same protein controlled both Start and mitosis, in organisms separated by more than a billion years of evolution. “That was pretty amazing, almost spooky,” recalled Nurse. “Suddenly all this locked together.”
Until then, few scientists believed that cell cycle control in yeast would apply to higher organisms, but Nurse’s experiment changed that view. “It said these regulatory proteins have been enormously conserved during evolution,” said Murray. “That was really revolutionary.” Later, in 1987, Nurse would clone the human homologue of cdc2, removing all doubt.
Meanwhile, another current was developing, based on another serendipitous discovery. In 1983, Hunt, who is now the head of Cell Cycle Control at the Imperial Cancer Research Fund, was studying the fertilization of sea urchin eggs at the Woods Hole Marine Biological Laboratory in Massachusetts. Measuring the level of proteins in newly fertilized eggs, he unexpectedly found one protein that abruptly disappeared at the end of cell division and then gradually appeared again as eggs began the next round of division. Hunt concluded that this protein, which he called cyclin, was driving the cell cycle.
Hunt’s 1983 report in Cell was “a tremendously brave paper,” said Murray. “Because all he saw really was [that] the protein goes up and down and up and down with the cell cycle.” Only later would Hunt and others prove that making and destroying cyclin were essential for cell division.
Hunt’s discovery of cyclin helped pave the way to resolving the field’s two competing world views. On the one hand, Hartwell convincingly portrayed the cell cycle as a genetically controlled set of dependent events—a “domino” model where each stage commenced if, and only if, the previous one was complete. But scientists working with frog eggs offered a completely incompatible model, featuring a central cytoplasmic clock controlling the cell cycle, setting off later events whether or not earlier ones were complete.
Evidence for this model was just as compelling. Yale University’s Yoshio Masui, Ph.D., in 1971, showed that a cytoplasmic substance from frog oocytes (egg precursor cells) induced egg maturation and called the substance maturation promoting factor (MPF). Then, in 1975, Marc Kirschner, Ph.D., and John Gerhart, Ph.D., cut a frog egg in half; the half without the nucleus went through periodic contractions at the same time that the nucleated half went through division, demonstrating that some kind of timing mechanism was at work in the cytoplasm. Hunt’s cyclin was probably the key ingredient. So embryonic and mature cells seemed to be regulated completely differently, a contradiction that puzzled the whole field and stalled progress.
In 1988, the two models came together, spurred by the purification of MPF by James Maller, Ph.D. MPF proved to consist of two protein subunits, one cyclin B and the other cdc2. So both were necessary to generate a “cyclin-dependent kinase” driving the cell cycle, in both developing and mature cells.
The stunning demonstration that changes in a single molecule drove cell division in all cells, from yeast to humans, made the field instantly red-hot. “All of a sudden everything just took off,” said Murray. The following year Hartwell fully reconciled the clock and domino models with his second great contribution: checkpoints.
Checkpoints made instant sense. A cell that pauses to check for proper DNA replication before resuming division seemed an evolutionary necessity. It was an alarm clock with a stop button—a stop button missing in the early embryo.
Hartwell recalled that a single conversation with postdoc Ted Weinert, Ph.D., led to the checkpoint breakthrough. “Ted said, ‘I want to look at regulation,’” Hartwell said. “And I remembered that cells regulated in response to radiation.”
Hartwell gave Weinert some radiation-sensitive yeast mutants to look at. “Right away, within a few days ... he discovered mutants that no longer arrested in the cell cycle when they were radiated.” Weinert’s mutants divided immediately after irradiation and then died. The conclusion: They must be defective in some feedback mechanism or checkpoint; otherwise they would not divide at all or would delay division to repair DNA.
This simple experiment changed everything. “One of Hartwell’s great strengths is to do things that, in principle, would have been possible for a fairly long period of time,” said Murray. “After they’re done you go, ‘Oh my lord, that’s so simple, why didn’t anyone else do that?’”
Checkpoints also suggested new ways to target cancer. Since many cancers feature defective checkpoints (leading to uncontrolled growth), it may be possible to find checkpoint-inhibiting drugs that kill tumor cells but not normal cells, or it may be possible to develop drugs that enhance tumor sensitivity to radiation by blocking their weakened checkpoints so they cannot repair their lethally damaged DNA, leading to cell death.
For example, ATM (ataxia-telangiectasia, mutated), part of a family of checkpoint-related kinases, is involved in DNA repair, and several drug companies are targeting it for cancer. “We know if we inhibit ATM, then we make cells very sensitive to ionizing radiation,” said Michael Kastan, M.D., Ph.D., chairman of hematology/oncology at St. Jude Children’s Research Center, Memphis.
Other checkpoint targets, including the Chk1, Chk2 and ATR kinases, are also drug targets. “It’s an important strategy that needs to be tested,” said Nurse. “We really need to push and see if it works.”
Drugs directly targeting cyclin and CDKs are also in development. “I’m less impressed by that,” commented Nurse. “Obviously CDKs are required for cell cycle progression. [But] I see no compelling reason why that’s thought to be much better than inhibiting DNA polymerase or something. But, having said that, such empirical approaches can be useful.”
Another strategy is to use checkpoint mutant cells to find new chemotherapy drugs. Drugs like paclitaxel and 5-FU were once thought to work selectively because cancer cells replicated more quickly than normal cells, but a new view holds that it is because tumors have defective checkpoints. In 1995 Hartwell, in collaboration with the National Cancer Institute, launched the Seattle Project at the Fred Hutchinson Cancer Research Center. The goal: use yeast mutants defective in checkpoint or DNA repair genes to screen compounds for activity.
Hartwell later left the Seattle Project to become director of Fred Hutchinson, but the project is still active. “[Cancer] cells lose some controls over DNA repair,” said Hartwell. “What they gain by that is the ability to evolve rapidly. But they also incur a vulnerability, and if we really could match the vulnerability with the particular treatment, I think that’s still a very viable strategy.”
What’s next? In the dozen years since Hartwell unified the field of cell cycle regulation with the checkpoint concept, scores of proteins have been identified along the signaling pathways involved in checkpoints. But we don’t yet understand how the signals interact with the cell cycle machinery. Exactly how do DNA damage or other defects lead to cell cycle arrest?
“We have the signaling,” said Kastan. “And we know what cell cycle machinery is involved in those steps. But we don’t have the link between them yet. Those are the major breakthroughs, in my mind, waiting to occur.”
Oxford University Press
The winners of this year’s Nobel Prize for physiology or medicine discovered mechanisms that control the cell cycle. (Credit: Courtesy of the Nobel Assembly at the Karolinska Institute)
The winners of this year’s Nobel Prize for physiology or medicine discovered mechanisms that control the cell cycle. (Credit: Courtesy of the Nobel Assembly at the Karolinska Institute)