Elucidating the DNA Damage Pathway

For this article, Jennifer Fisher Wilson interviewed Thanos Halazonetis, molecular biologist at the Wistar Institute in Philadelphia; Tak Mak, departments of medical biophysics and immunology at University of Toronto; and Carol Prives, department of biological sciences at Columbia University in New York City. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. N.H. Chehab et al., "Chk2/hCds1 functions as a DNA damage checkpoint in G1 by stabilizing p53," Genes & Development, 14:278-88, 2000. (cited in 124 papers) S-Y. Shieh et al., "The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites," Genes & Development, 14:289-300, 2000. (cited in 123 papers) A. Hirao, et al., "DNA damage-induced activation of p53 by the checkpoint kinase Chk2," Science, 287:1824-7, 2000. (cited in 155 papers) Over the last decade, the DNA damage pathway has become a hot topic for scientists who want to know how genomic stability is maintained and lost. The pathway provides key clues to human development, and understanding it could create better treatments for human cancers. The DNA damage pathway becomes inactive in at least half of all human tumors, typically when the tumor suppressor gene p53 is mutated. When functioning normally, p53, which lies at the pathway's center, launches cell cycle arrest or cell apoptosis when it detects damage to the DNA. When mutated or otherwise compromised, p53 doesn't do its job, allowing mutations to accumulate that can lead to cancer. Courtesy of Robert H. ClinkThanos Halazonetis During the 1990s, scientists identified the events downstream of p53 that lead to arrest or apoptosis. Little was known, however, about upstream signaling events that follow DNA damage leading to p53's activation and stabilization. This research led these authors to find the kinase that activates p53. Until this work, "we knew very little about the upstream p53 regulators, only that ATM [Ataxia telangiectasia-mutated] was involved," says Thanos Halazonetis, a molecular biologist at the Wistar Institute in Philadelphia and lead author of one of these papers. The Game's Afoot Clues leading to upstream signaling events emerged in 1995 when geneticist Yosef Shiloh at Tel Aviv University's Sackler School of Medicine cloned the ATM gene, which is responsible for ataxia telangiectasia, a rare autosomal recessive disorder.1 Three years later, Shiloh and cancer researcher Michael Kastan, now at St. Jude Children's Research Hospital, in Memphis, Tenn., published back-to-back papers proving that ATM functions upstream of p53.2,3 Soon afterward, researcher Robert Abraham at Duke University Medical Center, Durham, NC, showed that another DNA damage sensor, ATR (ATM and Rad-3 related), also functions upstream of p53.4 But the findings suggested that some other kinase, activated by ATM and ATR, must stabilize p53 by phosphorylating on the gene's serine-20 (Ser-20) location. Meanwhile, biochemist Steve Elledge, Baylor College of Medicine, Houston, cloned a kinase called Chk1 (checkpoint gene 1), the human homolog of the Chk1 protein kinase in fission yeast.5 Subsequently, Elledge and other research groups cloned Chk2 (checkpoint gene 2, also known as hCds1), the human homolog to cds1 in the yeast model. Known to be located downstream of ATM and ATR, the clonings set off a flurry of activity to discover their roles in human cells.6 It turned out, as the papers featured here indicate, that Chk2 is the missing link between ATM and p53. Two years ago, within a few weeks of each other, three groups published reports demonstrating that Chk2 directly activates p53. In reporting essentially the same findings about Chk2, using different experimental approaches, the reports confirm one another. The DNA repair pathway upstream of p53 remains mostly a mystery—for example, the roles of Chk1 and ATR remain largely unclear—but by determining the role of Chk2, these key papers began to crack the code. Chk2 Stabilizes P53 in the G1 Phase Once Chk2 was cloned, Halazonetis and his group decided to find out whether it was involved in p53 stabilization. Initially, he says, the kinase was thought to be important only in the arrest of the cell cycle's G2 phase because that is where the yeast homolog (cds1) is involved. To determine if Chk2 is active in other phases, such as G1, Halazonetis' group ran biochemical experiments and found that indeed it does. Halazonetis says, "we also think that it functions in G2 and [cell cycle] S-phase as well," although the team has yet to conduct these experiments. In vivo tests showed that Chk2 phosphorylated p53 on Ser-20 after gamma irradiation induced DNA damage. Also, after DNA damage, in vivo ectopic expression experiments of wild-type Chk2 led to increased p53 stabilization while expression of a dominant negative Chk2 mutant nullified both p53 phosphorylation on Ser-20 and p53 stabilization. Furthermore, the paper connected Chk2's action on p53 with increased cell cycle arrest. In vitro experiments showed how Chk2 phosphorylation on p53 led to dissociation of p53 from Mdm2, a protein that regulates p53 levels. As a result, this disconnection leads to increased p53 protein levels and cell cycle arrest or apoptosis. Since publishing these findings, Halazonetis' group has continued to study the DNA repair pathway to find out if other proteins are involved upstream of p53. "We don't know what activates ATM, or if there is a scaffold protein that helps ATM bind with Chk2 and Chk2 with p53. It's still a completely unknown area," he says. Chk1's Role Still in the Dark A second paper with similar findings was published in the same issue of Genes & Development that included Halazonetis' report. This investigation, led by molecular biologist Carol Prives, Columbia University, New York, also set out to determine which kinases phosphorylate p53 on Ser-20; they too found that Chk2 does. But the report focused more on Chk1, another checkpoint kinase active upstream of p53 that also phosphorylates p53 on Ser-20. Prives' team used fractionated nuclear extracts from human HeLa cells and found that Chk1 phosphorylates not only Ser-20 but also on other inducible amino- and carboxyl-terminal sites on p53, suggesting that it is a highly versatile kinase. Further experiments showed that expression of kinase-defective Chk1 or antisense Chk1 led to reduced levels of p53, while overexpression of wild-type Chk1 resulted in increased levels of p53. In vitro experiments with Chk2 found that it, too, phosphorylated Ser-20 as well as the other Chk1 sites on p53. "By showing that these kinases can directly modify p53 at key regulatory sites, the work led to the hypothesis that p53 might be a direct target for one or both of these kinases in vivo," she says. Prior to these observations, she notes that the cdc25C phosphatase was the only suspected target of Chk1 and Chk2. In 1997, Prives' postdoctoral fellow Sheau-Yann Shieh had been the first to show that p53's ability to bind to Mdm2 is significantly reduced when phosphorylated at N-terminal sites. Prives and Shieh proposed that in a resting cell, p53 is underphosphorylated and bound to Mdm2. When DNA damage occurs, p53 is freed from interacting with Mdm2 and thereby becomes stable and active. Prives' group, along with Yoichi Taya at the National Cancer Research Center in Tokyo, showed that on irradiation of cells, p53 becomes phosphorylated at N-terminal sites including Ser-20, and that this reduces its interaction with Mdm2. Since publication, it has become clear that Chk2 is involved as an upstream regulator of p53, Prives says, but the role of Chk1 is murkier. "While we were able to show that modulation of Chk1 levels in cells can result in corresponding changes in the levels of p53; nevertheless, it is yet to be proven whether or when Chk2 is an upstream regulator of p53," she says. Her group is currently studying Chk1 and Chk2 regulation in cells and how it relates to p53 stabilization after various forms of cellular stress. Chk2 Knockout Models' Supporting Role Courtesy of Tak MakTak Mak Just six weeks after the publication of these studies, Science published a report of knockout experiments that demonstrated the same findings as the Halazonetis and Prives groups regarding Chk2's role in the DNA repair pathway. Tak W. Mak's group from the Amgen Institute at the Ontario Cancer Institute in Toronto reported on genetics experiments with Chk2-deficient Rag cells (recombination activating gene). Cells with damaged DNA could not induce p53 activation and these cells could not maintain gamma-irradiation arrest in the cell cycle's G2 phase. The Chk2-deficient cells also resisted DNA-damage-induced apoptosis. Reintroducing Chk2 to these cells restored p53 protein activation after irradiation. As expected in normal functioning cells, the reintroduced Chk2 phosphorylated p53 on Ser-20, inducing cell cycle arrest. "We were quite aware that to do the knockout takes much longer than to do the dominant-negative biochemical studies, and we knew that others were working on those studies," Mak says. "So we rushed the paper and limited our analysis to Chk2-deficient, embryonic fibroblasts, and thymocytes with the Rag reconstitution." Mak's group is currently screening the Drosophila fly for other genes upstream of p53. In addition to its p53 protein activator role, Chk2 might play a key role as a tumor suppressor; its mutation may contribute to a range of cancers, Mak says. He cites a study in patients with a syndrome similar to Li-Fraumeni—a cancer predisposition syndrome in which p53 is mutated at the germline level—who have mutations in Chk2 but not p53.7 Study results from the Chk2 knock-out thymocyte cells provide a mechanistic link, says the author of the Science article, between Chk2 and p53 that "might explain the phenotypic similarity of the two genetically distinct Li-Fraumeni syndrome families." Like Halazonetis and Prives, Mak believes that it will take at least another five years before the DNA repair pathway is wholly elucidated. "It will take a long time to figure out, but it's essential to [do so] because p53 ultimately controls genomic stability," he says. Jennifer Fisher Wilson (jfwilson@snip.net) is a contributing editor. References 1. K. Savitsky et al., "A single ataxia-telangiectasia gene with a product similar to PI-3 kinase," Science, 268:1749-53, 1995. 2. S. Banin et al., "Enhanced phosphorylation of p53 by ATM in response to DNA damage," Science, 281:1674-7, 1998. 3. C.E. Canman et al., "Activation of the ATM kinase by ionizing radiation and phosphorylation of p53," Science, 281:1677-9, 1998. 4. R.S. Tibbetts et al., "A role for ATR in the DNA damage-induced phosphorylation of p53," Genes & Development, 13:152-7, 1999. 5. Y. Sanchez et al., "Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25," Science, 277:1497-501, 1997. 6. S. Matsuoka, et al., "Linkage of ATM to cell cycle regulation by the Chk2 protein kinase," Science, 282:1893-7, 1998. 7. D.W. Bell et al., "Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome," Science, 286:2528-31, 1999.

Elucidating the DNA Damage Pathway

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