The MDM2 RING-finger domain is required to promote p53 nuclear export.

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.

Ubiquitination of p53 and p21 is differentially affected by ionizing and UV radiation.

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 G1 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.

A Drosophila ribosomal protein functions in mammalian cells.

A cDNA expression vector encoding Drosophila ribosomal protein S14 was transfected into cultured Chinese hamster ovary (CHO) cells that harbor a recessive RPS14 emetine resistance mutation. Transformants synthesized the insect mRNA and polypeptide and consequently displayed an emetine-sensitive phenotype. These observations indicate that the insect protein was accurately expressed and correctly assembled into functional mammalian 40S ribosomal subunits.

In vivo ubiquitination and proteasome-mediated degradation of p53(1).

The levels of the tumor suppressor protein p53 are generally quite low in normal cells, due in part to its rapid turnover. Previous studies have implicated ubiquitin-dependent proteolysis in the turnover of wild-type p53 but have not established whether or not p53 is itself a substrate of the ubiquitin system. In this study, inhibitors of the 26S proteasome have been used to further explore the role of ubiquitin proteolysis in regulating p53 turnover. Increased levels of the tumor suppressor protein p53 were observed in normal cells, as well as in cells expressing the human papillomavirus 16 E6 oncoprotein, on exposure of the cells to proteasome inhibitors. Pulse-chase experiments indicated that the increased p53 levels resulted from stabilization of the protein. Furthermore, ubiquitin-p53 conjugates were detected in untreated as well as gamma-irradiated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of p53. Increased levels of the cyclin:cyclin-dependent kinase inhibitor p21, a downstream effector of p53 function, were also observed in proteasome inhibitor-treated cells, and this increase was due in part to an increase in p2l mRNA.

Relationship between radiation-induced G1 phase arrest and p53 function in human tumor cells.

Three widely studied cell lines were used to examine the nature of the G1 arrest induced in human tumor cells by ionizing radiation and its relation to p53 status. Cell lines MCF-7 and RKO express wild-type p53, whereas HT29 expresses mutant p53. Exponentially growing cells were irradiated with 6 Gy, and the progression of G1 cells into S phase was monitored at regular intervals by flow microfluorimetric and continuous labeling autoradiographic techniques. In some experiments, cells were incubated with Colcemid prior to irradiation in order to block them in mitosis and to prevent the accumulation of cells in the second post-irradiation G1 phase. No evidence of a significant arrest at the first post-irradiation G1-S checkpoint was observed in any of the three cell lines. These results suggest that p53 function alone does not control the progression of irradiated human tumor cells from G1 into S during the first post-irradiation cell cycle. In particular, we found no evidence that radiation induced a prolonged G1 arrest in tumor cells expressing wild-type p53 as has been reported by some investigators.

Hypoxia induces p53 accumulation through MDM2 down-regulation and inhibition of E6-mediated degradation.

Hypoxia, a result of DNA-damaging agents such as ionizing radiation, induces the nuclear accumulation of the p53 tumor suppressor protein. However, unlike the effect in ionizing radiation, hypoxia readily induces the nuclear accumulation of p53 in HPV E6-infected cells. In HPV-infected cells, a key regulator of p53 protein levels is the E6 oncoprotein. In association with the endogenous cellular protein E6-associated protein (E6AP), E6 can accelerate the degradation of p53 under aerobic conditions. To better define the mechanism of p53 induction in E6-infected cells by hypoxia, we studied the expression and association of E6 and E6AP with p53 in vivo. We found that hypoxia did not alter the protein levels of E6 or E6AP as compared with those found under aerobic growth conditions, indicating that protein inhibition of E6 or E6AP alone is not sufficient to explain the increased accumulation of p53 under hypoxic conditions. However, p53 did fail to coprecipitate with E6AP under hypoxia, indicating that hypoxia uncouples the interaction of p53 with E6 and E6AP. We also present evidence to indicate that hypoxia decreases the expression of the endogenous cellular regulator of p53 protein, the human MDM2 protein, resulting in an inhibition of p53 export from the nucleus to the cytoplasm for degradation. Taken together, these results suggest that the hypoxic induction of p53 is attributable to the down-regulation of MDM2 protein levels and uncoupling of p53 from its interaction with the E6/E6AP complex.

MDM2-dependent ubiquitination of nuclear and cytoplasmic P53.

Wild-type p53 is stabilized and accumulates in the nucleus of DNA damaged cells. The effect of stabilizing p53 is to inhibit cell growth, either through a G1 cell cycle arrest or apoptotic cell death. MDM2 can inhibit p53 activity, in part, by promoting its rapid degradation through the ubiquitin proteolysis pathway. In the current study, MDM2-mediated degradation of p53 was partially inhibited in cells treated with leptomycin B (LMB), a specific inhibitor of nuclear export. In contrast, levels of ubiquitinated p53 increased in LMB-treated cells, indicating that nuclear export is not required for p53 ubiquitination. To investigate this further, p53 mutants were generated which localize to either the nucleus or cytoplasm, and their susceptibility to MDM2-mediated ubiquitination was assessed. p53 mutants that localized to either the nucleus or the cytoplasm were efficiently ubiquitinated, and their steady-state levels decreased, when coexpressed with MDM2. In addition, an MDM2-mutant that localized to the cytoplasm was able to ubiquitinate and degrade a p53 mutant which was similarly localized in the cytoplasm. Our results indicate that nuclear export is not required for p53 ubiquitination, and that p53 proteins that localize to either the nucleus or cytoplasm can be ubiquitinated and degraded by MDM2.

Transcriptional activation of bovine mimecan by p53 through an intronic DNA-binding site.

Mimecan is a small leucine-rich proteoglycan that can occur as either keratan sulfate proteoglycan in the cornea or as glycoprotein in many connective tissues. As yet, there is no information on its transcriptional regulation. Recently we demonstrated the presence of eight mimecan mRNA transcripts generated by alternative transcription initiation, alternative polyadenylation, and differential splicing, all of which encode an identical protein. Here we report a conserved consensus p53-binding DNA sequence in the first intron of bovine and human mimecan genes and show that wild-type p53 binds to this sequence in vitro. Co-transfections of Saos-2, HeLa, NIH 3T3, and primary bovine corneal keratocytes with bovine mimecan promoter/luciferase reporter constructs in combination with p53 expression vectors activate the second mimecan promoter through the p53-binding sequence. In addition, we show absence of mimecan expression in different tumors and cancer cell lines, where p53 frequently is inactivated/mutated. Thus, this work provides novel information that links mimecan to the p53 network.

A cDNA encoding human ribosomal protein S24.

We describe the isolation and nucleotide sequence of a cDNA encoding the human 40S ribosomal subunit protein (r-protein) S24 (Mr 15,425). Human S24 is virtually identical to the r-protein encoded by a cloned Xenopus laevis cDNA previously identified as S19 [Amaldi et al., Gene 17 (1982) 311-316].

Downregulation of MDM2 stabilizes p53 by inhibiting p53 ubiquitination in response to specific alkylating agents.

p53 is stabilized in response to DNA damaging stress. This stabilization is thought to result from phosphorylation in the N-terminus of p53, which inhibits p53:MDM2 binding, and prevents MDM2 from promoting p53 ubiquitination. In this report, the DNA alkylating agents mitomycin C (MMC) and methylmethane sulfonate (MMS), as well as UV radiation, stabilized p53 in a manner independent of phosphorylation in p53 N-terminus. This stabilization coincided with decreased levels of MDM2 mRNA and protein, and a corresponding decrease in p53 ubiquitination. Importantly, MDM2 overexpression inhibited the stabilization of p53 and decrease in ubiquitination following MMC, MMS, and UV treatment. This indicates that downregulation of MDM2 contributes to the stabilization of p53 in response to these agents.

Acquisition of p53 mutations in response to the non-genotoxic p53 activator Nutlin-3.

Wild-type p53 is a stress-responsive tumor suppressor and potent growth inhibitor. Genotoxic stresses (for example, ionizing and ultraviolet 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 manner. Nutlin occupies the p53-binding pocket of murine double minute 2 (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 (for example, 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.

Geldanamycin promotes premature mitotic entry and micronucleation in irradiated p53/p21 deficient colon carcinoma cells.

P53 wild-type and p53-null or mutant cells undergo a G(2)-phase cell-cycle arrest in response to ionizing radiation (IR). In this study we examined the effect of heat-shock protein 90 (HSP90) inhibitor, geldanamycin (GA), on IR-induced G(2) arrest in human colon adenocarcinoma cells with different p53 status. We show that GA treatment abrogates IR-induced G(2)-phase arrest in cells null or mutant for p53. Specifically, GA treatment pushed irradiated p53 signaling-defective cells into a premature mitosis characterized by aberrant mitotic figures, increased gammaH2AX expression and formation of micronucleated cells. Cells expressing wild-type p53 were resistant to GA-induced G(2) checkpoint abrogation. Notably, GA treatment decreased levels of G(2) regulatory proteins Wee1 and Chk1, and inhibitory phosphorylation of Cdc2, independent of p53 status. Further investigation identified p21 as the potential downstream effector of p53 that mediates resistance to G(2) checkpoint abrogation. Clonogenic survival studies demonstrated higher sensitivity to GA alone or combination IR plus GA treatment in p53 and p21-null cells. Collectively, these data demonstrate potential mechanisms through which HSP90 inhibition can enhance the effects of ionizing radiation in p53-compromised cancer cells. Combination IR plus HSP90 inhibitor therapies may be particularly useful in treating cancers that lack wild-type p53.

Oligomerization is required for p53 to be efficiently ubiquitinated by MDM2.

Wild-type p53 is degraded in part through the ubiquitin proteolysis pathway. Recent studies indicate that MDM2 can bind p53 and promote its rapid degradation although the molecular basis for this degradation has not been clarified. This report demonstrates that MDM2 can promote the ubiquitination of wild-type p53 and cancer-derived p53 mutants in transiently transfected cells. Deletion mutants that disrupted the oligomerization domain of p53 displayed low binding affinity for MDM2 and were poor substrates for ubiquitination. However, efficient MDM2 binding and ubiquitination were restored when an oligomerization-deficient p53 mutant was fused to the dimerization domain from another protein. These results indicate that oligomerization is required for p53 to efficiently bind and be ubiquitinated by MDM2. p53 ubiquitination was inhibited in cells exposed to UV radiation, and this inhibition coincided with a decrease in MDM2 protein levels and p53.MDM2 complex formation. In contrast, p53 dimerization was unaffected following UV treatment. These results suggest that UV radiation may stabilize p53 by blocking the ubiquitination and degradation of p53 mediated by MDM2.

MDM2 can promote the ubiquitination, nuclear export, and degradation of p53 in the absence of direct binding.

MDM2 can bind the N terminus of p53 and promote its ubiquitination and export from the nucleus to the cytoplasm, where p53 can then be degraded by cytoplasmic proteasomes. Several studies have reported that an intact MDM2 binding domain is necessary for p53 to be targeted for ubiquitination, nuclear export, and degradation by MDM2. In the current study, we examined whether the MDM2 binding domain of p53 could be provided in trans through oligomerization between two p53 molecules. p53 proteins mutated in their MDM2 binding domains were unable to bind MDM2 directly and were resistant to MDM2-mediated ubiquitination, nuclear export, and degradation when expressed with MDM2 alone. However, these same p53 mutants formed a complex with MDM2 and were efficiently ubiquitinated, exported from the nucleus, and degraded when co-expressed with MDM2 and wild-type p53. Moreover, this effect required MDM2 binding by wild-type p53 as well as oligomerization between wild-type p53 and the MDM2 binding-deficient p53 mutants. Taken together, these results support a model whereby MDM2 binding-deficient forms of p53 can bind MDM2 indirectly through oligomerization with wild-type p53 and are subsequently targeted for ubiquitination, nuclear export, and degradation. These findings may have important implications regarding the DNA damage response of p53.

Role and regulation of p53 during an ultraviolet radiation-induced G1 cell cycle arrest.

p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wildtype p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.