Cancer Research

Y. Haupt, R. Maya, A. Kazaz, M. Oren, "Mdm2 promotes the rapid degradation of p53," Nature, 387:296-99, 1997. (Cited in more than 230 papers since publication) Comments by Moshe Oren, a professor in the Department of Molecular Cell Biology at the Weizmann Institute of Science, Rehovot, Israel M.H.G. Kubbutat, S.N. Jones, K.H. Vousden, "Regulation of p53 stability by Mdm2," Nature, 387:299-303, 1997. (Cited in more than 215 papers since publication) Comments by Karen H. Vousden, interim director of the Advanced Bioscience Laboratories Basic Research Program at the Frederick Cancer Research and Development Center in Frederick, Md. In trying to publish their findings on a novel aspect of the relationship between the protein Mdm2 (mouse double minute 2, after the first source from which the gene was cloned) and the frequently mutated tumor suppressor protein p53, the authors of these two papers faced a good deal of skepticism. Fortunately, the two labs, already familiar with each other from previous collaborations, were able to divide the somewhat painstaking labors necessary to convince reviewers that their work was worthy of publication. "There was a very long shopping list of things, and some of them were quite time-demanding and very tedious," says senior author Moshe Oren of the required laboratory tasks. "We agreed that we would continue working independently but would stay in close contact and send the papers out together," comments Karen H. Vousden, the senior author of the companion paper. "Each of the labs did part of the work, and in this way we [reached] the finish line for publication much faster," remarks Oren. "It was a very enjoyable and a very productive case of parallel science." The two papers ended up in Nature after first being rejected by Science. Normal cells trigger a tumor-suppressing response in the p53 gene in the face of some types of potential oncogenic stress or DNA damage. By understanding the factors that impinge on p53 protein function, scientists are attempting to find the best way to tinker with the system to devise effective therapies. Prior to these two papers' publication, investigators already knew a few things about the relationship between p53 and Mdm2 and its role in carcinogenesis: They knew that Mdm2 binds to p53, and inhibits its ability to function as a transcription factor. They also knew that Mdm2 itself is a transcriptional target to p53--the two participate in a negative feedback loop in which p53 acts as a transcription factor and activates transcription of the mdm2 gene. The Mdm2 protein then, in turn, binds to the N terminus of p53 and stops it from activating transcription. These two 1997 papers further dissected the Mdm2-p53 interaction by demonstrating that Mdm2 not only binds to p53 but promotes its degradation. The only major difference between the two efforts was the method used to make the initial observation: Western blot analysis in the case of Oren's lab, cell sorting in the case of Vousden's. Both labs found that the normally low level of p53 is maintained through Mdm2-driven degradation: Upon radiation-induced DNA damage, p53 accumulated rapidly. But after the DNA damage, Mdm2 caused p53 to degrade rapidly and things returned to the normal basal level. The findings suggested that the high levels of mutant p53 seen in tumor cells--a common tumor marker in the clinic for many years--result from the p53 protein's inability to turn on the mdm2 gene and activate the feedback loop that ends in the degradation of p53. Tumor cells with mutant p53 cannot turn on the mdm2 gene, don't make the Mdm2 protein, and, as a result, degrade little if any of the mutant p53 protein, allowing for its continued accumulation. Vousden speculates that reviewers had doubts about these characteristics because investigators had been thoroughly studying the Mdm2-p53 relationship since 1992. Any such significant interactions, these researchers reasoned, already would have been uncovered. Says Vousden, "I think then when people actually went back, they did see that they had seen it [but] hadn't really noticed." Once considered legitimate, however, the findings were destined to garner a great deal of attention since p53 is the tumor suppressor gene of choice for a large number of cancer researchers based on its oft-observed role in tumorigenesis.1 Since these 1997 papers, several labs have extended these findings to a variety of other cell lines and, in doing so, have suggested that Mdm2 is, in fact, the major regulator of p53 degradation. By stabilizing p53 levels using peptides or antibodies to stop Mdm2 from binding p53 in normal cells, the lab of David Lane, a professor of biochemistry at the University of Dundee in England, has confirmed the regulatory action of Mdm2 and has introduced a way to activate p53 without causing DNA damage.2 According to Oren, the major effort of the last couple of years has been aimed at understanding the biochemical basis of Mdm2's p53-degrading capability. A Tokyo lab has made perhaps the most significant breakthrough in this regard by showing that Mdm2 possesses an enzymatic activity known as ubiquitin protein ligase.3 In effect, Mdm2 attaches a ubiquitin group to p53, which tags it for degradation. Researchers continue to examine Mdm2's role as a ubiquitin ligase by looking for other proteins that it might target for degradation. Efforts in Oren's lab and others have also focused on the interplay between p53 and Mdm2 in normal cells. They've found that the phosphorylation and other types of biochemical modification affecting both p53 and Mdm2 in response to DNA damage disrupt the interaction between Mdm2 and p53 and prevent p53 degradation. "There are many mechanisms through which you can make p53 immune to Mdm2 degradation," Vousden explains. The best understood, she says, is through expression of a protein called p14ARF (alternative reading frame), which binds directly to Mdm2 and stops it from degrading p53. Having to go through p14ARF protects the organism from the outgrowth of tumor cells by adding another line of defense. "It's a really strong sort of fail-safe mechanism," says Vousden. Recent research in her lab points to a cell cycle protein called E2F1 that activates p14ARF as an important part of that mechanism and of protecting against aberrant cell growth. Such findings suggest one obvious approach to cancer therapy: Block Mdm2's binding to p53 or block Mdm2 from degrading p53, possibly by using an agent that mimics ARF. "The part that we don't know," explains Oren of the ongoing investigation into the Mdm2-p53 interaction, "is whether the changes that prevent the degradation of p53 are necessarily the same ones that allow p53 to avoid biochemical inhibition by Mdm2." Investigators, including Oren and Vousden, would also like to better understand how p53 is regulated at other levels through interaction with other critical proteins, proteins that may have little to do with Mdm2. P. Smaglik, "Taking aim at p53: researchers are targeting the tumor suppressor with vectors, viruses, and small molecules," The Scientist, 13[2]:6, Jan. 18, 1999. A. Bottger et al., "Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo," Current Biology, 7:860-9, 1997. H.R. Honda et al., "Oncoprotein Mdm2 is a ubiquitin ligase E3 for tumor suppressor p53," FEBS Letters, 420:25-7, 1997.

Cancer Research

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