[Abstract unavailable

MDM2 can bind to p53 and promote its ubiquitination and subsequent degradation by the proteasome. Current models propose that nuclear export of p53 is required for MDM2-mediated degradation, although the function of MDM2 in p53 nuclear export has not been clarified. Here we show that MDM2 can promote the nuclear export of p53 in transiently transfected cells. This activity requires the nuclear-export signal (NES) of p53, but not the NES of MDM2. A mutation within the MDM2 RING-finger domain that inhibits p53 ubiquitination also inhibits the ability of MDM2 to promote p53 nuclear export. Finally, inhibition of nuclear export stabilizes wild-type p53 and leads to accumulation of ubiquitinated p53 in the nucleus. Our results indicate that MDM2-mediated ubiquitination, or other activities associated with the RING-finger domain, can stimulate the export of p53 to the cytoplasm through the activity of the p53 NES.

Levels of the tumor suppressor protein p53 are normally quite low due in part to its short half-life. p53 levels increase in cells exposed to DNA-damaging agents, such as radiation, and this increase is thought to be responsible for the radiation-induced G 1 cell cycle arrest or delay. The mechanisms by which radiation causes an increase in p53 are currently unknown. The purpose of this study was to compare the effects of gamma and UV radiation on the stability and ubiquitination of p53 in vivo. Ubiquitin-p53 conjugates could be detected in nonirradiated and gamma-irradiated cells but not in cells which were UV treated, despite the fact that both treatments resulted in the stabilization of the p53 protein. These results demonstrate that UV and gamma radiation have different effects on ubiquitinated p53 and suggest that the UV-induced stabilization of p53 results from a loss of p53 ubiquitination. Ubiquitinated forms of p21, an inhibitor of cyclin-dependent kinases, were detected in vivo, demonstrating that p21 is also a target for degradation by the ubiquitin-dependent proteolytic pathway. However, UV and gamma radiation had no effect on the stability or in vivo ubiquitination of p21, indicating that the radiation effects on p53 are specific.Mutations in the gene encoding the tumor suppressor protein p53 are the most common genetic alterations detected in human cancer (22). A role for p53 in normal cellular proliferation has not been clearly identified. However, there is abundant evidence that wild-type p53 plays a critical role in the cellular response to DNA damage by serving as a cell cycle checkpoint determinant (reviewed in reference 6). p53 levels increase in cells exposed to ionizing radiation (IR) (29,30,55), and the cells undergo a cell cycle arrest or delay in the G 1 phase of the cell cycle (30, 55). This arrest or delay is thought to allow time for the cells to repair the DNA damage incurred during IR treatment before proceeding in S phase and thereby prevent the accumulation of mutations that would result from replicating a damaged genome. Cells which lack wild-type p53 function fail to arrest following radiation treatment (26-28, 30, 55), indicating an essential role for p53 in the arrest response. The p53-dependent G 1 arrest appears to be mediated by p21 (2, 3, 8), a cyclin-dependent kinase inhibitor whose gene is transcriptionally activated by p53 (11,18,57). p53 also plays a role in the signaling of apoptosis (programmed cell death) in certain cell types following irradiation treatment (1, 32). For example, thymocytes from p53 knockout mice were less susceptible to radiation-induced apoptosis than were thymocytes from mice expressing p53 (32). This apoptotic function may involve the ability of p53 to activate transcription of the gene encoding bax, a protein which is involved in an apoptosis signaling pathway (17). There is increasing evidence that p53 carries out a similar checkpoint function during normal cell division. For example, cells which lack wild-type p53 have high levels of genomic ...

Wild-type p53 is a stress-responsive tumor suppressor and potent growth inhibitor. Genotoxic stresses (e.g. ionizing and UV radiation or chemotherapeutic drug treatment) can activate p53, but also induce mutations in the P53 gene and thus select for p53-mutated cells. Nutlin-3a (Nutlin) is pre-clinical drug that activates p53 in a non-genotoxic fashion. Nutlin occupies the p53-binding pocket of MDM2, activating p53 by blocking the p53-MDM2 interaction. Because Nutlin neither binds p53 directly nor introduces DNA damage, we hypothesized Nutlin would not induce P53 mutations and therefore not select for p53-mutated cells. To test this, populations of SJSA-1 (p53 wild-type) cancer cells were expanded that survived repeated Nutlin exposures, and individual clones were isolated. Group 1 clones were resistant to Nutlin-induced apoptosis, but still underwent growth-arrest. Surprisingly, while some Group 1 clones retained wild-type p53, others acquired a heterozygous p53 mutation. Apoptosis resistance in Group 1 clones was associated with decreased PUMA induction and decreased caspase 3/7 activation. Group 2 clones were resistant to both apoptosis and growth-arrest induced by Nutlin. Group 2 clones had acquired mutations in the p53 DNA-binding domain and expressed only mutant p53s that were induced by Nutlin treatment, but were unable to bind the P21 and PUMA gene promoters, and unable to activate transcription. These results demonstrate that non-genotoxic p53 activation (e.g. by Nutlin treatment) can lead to the acquisition of somatic mutations in p53 and select for p53-mutated cells. These findings have implications for the potential clinical use of Nutlin and other small molecule MDM2 antagonists.