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PATHOLOGY Tumor Suppressor Shows Another Way to Get Job Done P53 Prevents Rereplication of DNA, Presents as Protein to Be Restored in Fighting Cancer The early hours of a cell's growth cycle are a time of singular activity. As the cell wakens from its resting state, bevies of proteins congregate and attach themselves to sites along the chromosomes, preparing them for replication. This event must happen only once before a cell divides, otherwise the cell will be thrown into chaos with excess genetic material. P53's latest trick was discovered by Anindya Dutta (front); Sandeep Saxena, Yesu Jeon, Charles Lee (middle, l to r); Kazutaka Murata, Yuichi Machida, and Cyrus Vaziri (back, l to r). (Photo by Graham Ramsay) To enforce this rule, the cell dispatches two proteins, cdk and geminin, to keep the molecular minions from reattaching to the chromosomes before the cell undergoes mitosis. These two enforcers were thought to be enough, but Cyrus Vaziri, Sandeep Saxena, Anindya Dutta, and their colleagues report in the April 25 Molecular Cell that they have discovered a third protein that maintains the no rereplication rule. It is none other than the tumor suppressor protein p53. That a third anti-rereplication agent exists was surprise enough, but that it is the already accomplished p53 was completely unexpected. "P53 is a very cool molecule, but who would have thought that this one factor would have so many tentacles in the way, stopping the cell from going haywire?" said Dutta, HMS associate professor of pathology at Brigham and Women's Hospital. The discovery raises intriguing questions about the workings of the famous molecule. P53's main task is to monitor the cell for DNA damage and to stop the cell from cycling or trigger cell death if damage is detected. Does p53 sense excess genetic material as a form of DNA damage? And what does it do once it detects rereplication? Dutta's study provides some hints. In the Absence of P53 What is clear is that cancer cells have even more to gain than previously thought by having p53 disabled. Not only can their proliferation-enhancing mutations function free of constraints without p53, the protein's absence also means that they are more likely to rereplicate and produce many copies of the deadly oncogenes. "You often find hundreds of copies of an oncogene in cancer cells," Dutta said. "If you can turn p53 on, you will have a handle on rereplication. The hard part is how do you turn it on?" --Anindya Dutta What the study also makes obvious is how much cancer patients could benefit if it were possible to restore p53's activity. "What this basically tells you is that it is still p53; if you can turn p53 on, you will have a handle on rereplication. The hard part is how do you turn it on?" he said. "It is very hard to put inhibitors back into cells. It is much easier to knock them out." Thwarting cancer cells by stopping them from replicating more than once is an idea that only a handful of cancer biologists have considered. In fact, the details of how healthy cells replicate have only recently come to light. The process begins during G1, the first phase of the cell cycle, when clusters of prereplication proteins decorate particular chromosomal sites, dubbed origins. As cdk levels rise, these origins become activated, or fire, causing the two ribbons of DNA to separate. Each strand doubles in the following S phase, setting the scene for cell division during the final phase. "The amazing thing is that for the eight hours of S phase, origins all over the chromosome are replicating, but no region fires more than once," said Dutta. Replication Blockers In the mid-1990s, the Nobel-prize winning researcher Paul Nurse noticed a curious thing: the high levels of cdk in the second half of the cell cycle were actively preventing rereplication. Others went on to show that high cdk achieves this by inhibiting the prereplication proteins from binding to the already replicated chromosome. Then in 1998 Marc Kirschner, the Carl W. Walter professor of cell biology and head of that department at HMS, along with colleagues, discovered that replication could be blocked by adding geminin to Xenopus extracts. Like cdk, geminin appeared to be turned on after the replication-initiating proteins had done their job. Dutta, who had been working on those proteins, was intrigued. "Here was God's gift to my lab--geminin, a natural inhibitor of the very initiation process that we were studying," he said. A flurry of discoveries followed. Dutta and his colleagues identified geminin's target, the protein Cdt1 (see Focus, Jan. 12, 2001). Nurse showed that Cdt1, along with the protein Cdc6, helps load the cluster of prereplication proteins onto the chromosome. It looked like geminin might be inhibiting rereplication by interfering with Cdt1's enzyme-loading function. To see exactly how Cdt1 works and whether geminin might be interfering, Saxena, HMS research fellow in pathology at BWH, and Vaziri, of the Boston University School of Medicine, added the replication-inducing Cdt1 and its Cdc6 partner to a strain of cancer cells. The cells' genetic material underwent multiple duplications. "This was the first time anyone had triggered rereplication in mammalian cells by manipulating the replication initiator proteins," said Dutta. Sure enough, geminin suppressed the rereplication. Until now, the researchers had no idea that they were on the brink of discovering a novel anti-rereplication agent. But it had occurred to them that the strain of cancer cells they were using lacked the p53 tumor suppressor. Thinking that the absence of p53 might be biasing their results, they repeated their manipulations on p53-containing cancer and normal cells. The cells would not rereplicate. Further investigation bore out what they had begun to suspect--that p53 was responsible for stopping rereplication. To begin, they noticed that the addition of Cdt1 and Cdc6 had caused p53's faithful aide-de-camp, p21, to be activated. In addition, p53's upstream partners ATM/ATR and Chk2 also were activated, suggesting p53 was both receiving and sending information along its usual channels. What kind of information p53 is picking up is not clear, though there are clues. Yesu Jeon, a graduate student in Dutta's lab, and Charles Lee, HMS instructor in pathology, tracked the rereplicating chromosomes in the cells without p53 and found that only selected origins were firing, and they were doing so at a faster rate than usual. "So there could be something abnormal about the rereplication process that is perceived by the cell as DNA damage," said Dutta. Restoring the well-known tumor suppressor to lawless cancer cells may not be feasible at the moment, but studying the pathways by which p53 is alerted could lead to alternative strategies to prevent cancer cells from rereplicating. For example, it may be possible to artificially stimulate p53 or its downstream lieutenants. "We know Cdt1 and Cdc6 are activating p53," Dutta said. "Now we want to dissect them. Is one better than the other at turning on the checkpoint pathway that prevents rereplication? And are there any other checkpoint pathways?" --Misia Landau