NEWS

Beyond the Nobel Prize: Cell Cycle Research Offers New View of Cancer

Ken Garber

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.



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Dr. Lee Hartwell

 


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Paul Nurse, Ph.D., (left) and Tim Hunt, Ph.D., of the Imperial Cancer Research Fund, London.

 
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.



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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)

 
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."



             
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