Polymorphisms in the 5′-UTR of <Emphasis Type="Italic">PTEN</Emphasis> and other gene polymorphisms in normal Japanese individuals

Polymorphisms are distributed differently in populations, including those of regions, ethnic groups, and diseased patients. In order to investigate variation in nucleotide sequences in normal individuals, we isolated genomic DNA from the blood of healthy Japanese individuals and sequenced the 5′-untranslated region (5′-UTR) of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene and the gene promoter, intron, and exon nucleotides of p53, p14 ^ARF , murine double minute 2 (MDM2), and the β₂- and β₃-adrenoceptor (?AR). We found polymorphisms in these regions, including a deletion at positions ?465 to ?463 and a substitution at position ?404 in PTEN and a substitution at position ?4924 in p14 ^ARF , in normal individuals. Individuals with or without the PTEN polymorphism harbored a different distribution of polymorphisms, including simultaneous alterations in nucleotides of p53, MDM2, and β₃-AR, and also harbored some polymorphic nucleotides located in the same set of associatively altered nucleotides. Our results show that multiple nucleotides, including the PTEN nucleotides, are altered in normal Japanese individuals and provide useful information for genotyping studies in individuals and populations.     

Hypermethylation of 5'CpG island of p14ARF flanking exon 1 Beta in human colorectal cancers displaying a restricted pattern of p53 overexpression concomitant with increased MDM2 expression

ABSTRACT: BACKGROUND: It has been suggested that inactivation of p14ARF, a tumor suppressor central to regulating p53 protein stability through interaction with the MDM2 oncoprotein, abrogates p53 activity in human tumors retaining the wild-type TP53 gene. Differences in expression of tumor suppressor genes are frequently associated with cancer. We previously reported on a pattern of restricted p53 immunohistochemical overexpression significantly associated with microsatellite instability (MSI), low TP53 mutation frequency, and MDM2 overexpression in colorectal cancers (CRCs). In this study, we investigated whether p14ARF alterations could be a mechanism for disabling the p53 pathway in this subgroup of CRCs. RESULTS: Detailed maps of the alterations in the p14ARF gene were determined in a cohort of 98 CRCs to detect both nucleotide and copy-number changes. Methylation-specific PCR combined with bisulfite sequencing was used to evaluate the prevalence and distribution of p14ARF methylation. p14ARF alterations were then correlated with MSI status, TP53 mutations, and immunohistochemical expression of p53 and MDM2. The frequency of p14ARF mutations was extremely low (1/98; 1%), whereas coexistence of methylated and unmethylated alleles in both tumors and normal colon mucosa was common (91/98; 93%). Only seven of nine tumors (7%) had a distinct pattern of methylation compared with normal colon mucosa. Evaluation of the prevalence and distribution of p14ARF promoter methylation in a region containing 27 CpG sites in 35 patients showed a range of methylated CpG sites in tumors (0 to 25 (95% CI 1 to 13) versus 0 to 17 (95% CI 0 to 2)) in adjacent colon mucosa (P = 0.004). Hypermethylation of the p14ARF promoter was significantly correlated with the restricted p53 overexpression pattern (P = 0.03), and MDM2 overexpression (P = 0.02), independently of MSI phenotype. Although no significant correlation between p14ARF methylation and TP53 mutational status was seen (P = 0.23), methylation involving the proximal CpG sites within the 5' CpG flanking exon 1beta was present more frequently in tumors with restricted p53 overexpression than in those with diffuse p53 overexpression (range of methylated clones 17 to 36% (95% CI 24 to 36%) versus range 0 to 3% (95% CI 0 to 3%), P = 0. 0003). CONCLUSION: p14ARF epigenetic silencing may represent an important deregulating mechanism of the p53- MDM2-p14ARF pathway in CRCs exhibiting a restricted p53 overexpression pattern.     

Contribution of two independent MDM2-binding domains in p14(ARF) to p53 stabilization

The MDM2 protein targets the p53 tumor suppressor for ubiquitin-dependent degradation [1], and can function both as an E3 ubiquitin ligase [2] and as a regulator of the subcellular localization of p53 [3]. Oncogene activation stabilizes p53 through expression of the ARF protein (p14(ARF) in humans, p19(ARF) in the mouse) [4], and loss of ARF allows tumor development without loss of wild-type p53 [5] [6]. ARF binds directly to MDM2, and prevents MDM2 from targeting p53 for degradation [6] [7] [8] [9] by inhibiting the E3 ligase activity of MDM2 [2] and preventing nuclear export of MDM2 and p53 [10] [11]. Interaction between ARF and MDM2 results in the localization of both proteins to the nucleolus [12] [13] [14] through nucleolar localization signals (NoLS) in ARF and MDM2 [11] [12] [13] [14]. Here, we report a new NoLS within the highly conserved amino-terminal 22 amino acids of p14(ARF), a region that we found could interact with MDM2, relocalize MDM2 to the nucleolus and inhibit the ability of MDM2 to degrade p53. In contrast, the carboxy-terminal fragment of p14(ARF), which contains the previously described NoLS [11], did not drive nucleolar localization of MDM2, although this region could bind MDM2 and weakly inhibit its ability to degrade p53. Our results support the importance of nucleolar sequestration for the efficient inactivation of MDM2. The inhibition of MDM2 by a small peptide from the amino terminus of p14(ARF) might be exploited to restore p53 function in tumors.     

MDM2 promotes ubiquitination and degradation of MDMX

The p53 tumor suppressor is regulated by MDM2-mediated ubiquitination and degradation. Mitogenic signals activate p53 by induction of ARF expression, which inhibits p53 ubiquitination by MDM2. Recent studies showed that the MDM2 homolog MDMX is also an important regulator of p53. We present evidence that MDM2 promotes MDMX ubiquitination and degradation by the proteasomes. This effect is stimulated by ARF and correlates with the ability of ARF to bind MDM2. Promotion of MDM2-mediated MDMX ubiquitination requires the N-terminal domain of ARF, which normally inhibits MDM2 ubiquitination of p53. An intact RING domain of MDM2 is also required, both to interact with MDMX and to provide E3 ligase function. Increase of MDM2 and ARF levels by DNA damage, recombinant ARF adenovirus infection, or inducible MDM2 expression leads to proteasome-mediated down-regulation of MDMX levels. Therefore, MDMX and MDM2 are coordinately regulated by stress signals. The ARF tumor suppressor differentially regulates the ability of MDM2 to promote p53 and MDMX ubiquitination and activates p53 by targeting both members of the MDM2 family.     

Phosphorylation of human p53 at serine 46 determines promoter selection and whether apoptosis is attenuated or amplified

The capacity of DNA damaging agents to induce apoptosis is regulated by target gene induction by p53. We found that p53 targeted MDM2 in cells in which DNA repair was occurring, but persistent DNA damage induced by chemotherapy led p53 to selectively target PTEN. High dose chemotherapy induced the phosphorylation of p53 on serine 46, whereas low dose chemotherapy did not. A nonphosphorylatable serine 46 to alanine p53 mutant (S46A) targeted the MDM2 promoter in preference to that for PTEN. A serine 46 to aspartate mutant (S46D, a phosphorylation mimic) targeted PTEN in preference to MDM2. These observations show that phosphorylation of serine 46 in p53 is sufficient for it to induce the PTEN (phosphatase and tensin homolog deleted on chromosome ten) tumor suppressor protein in preference to MDM2. S46A induced significantly less cell death than the S46D in cells. The phosphorylation-induced change of p53 promoter targeting suppresses the induction of MDM2 and the formation of the autoregulatory feedback loop. Induction of PTEN by p53 followed by expression of PTEN inhibits AKT-induced translocation of MDM2 into the nucleus and sustains p53 function. The protection of p53 from MDM2 by PTEN and the damage-induced activation of PTEN by phosphorylated p53 leads to the formation of an apoptotic amplification cycle in which p53 and PTEN coordinately increase cellular apoptosis.     

Stabilization of p53 by p14ARF without relocation of MDM2 to the nucleolus

The alternative product of the human INK4a/ARF locus, p14ARF, has the potential to act as a tumour suppressor by binding to and inhibiting the p53 antagonist MDM2. Current models propose that ARF function depends on its ability to sequester MDM2 in the nucleolus. Here we describe situations in which stabilization of MDM2 and p53 occur without relocalization of endogenous MDM2 from the nucleoplasm. Conversely, forms of ARF that do not accumulate in the nucleolus retain the capacity to stabilize MDM2 and p53. We therefore propose that nucleolar localization is not essential for ARF function but may enhance the availability of ARF to inhibit MDM2.     

Mutations in human ARF exon 2 disrupt its nucleolar localization and impair its ability to block nuclear export of MDM2 and p53.

The mammalian ARF-INK4a locus uniquely encodes two cell cycle inhibitors by using separate promoters and alternative reading frames. p16INK4a maintains the retinoblastoma protein in its growth suppressive state while ARF stabilizes p53. We report that human ARF protein predominantly localizes to the nucleolus via a sequence within the exon 2-encoded C-terminal domain and is induced to leave the nucleolus by MDM2. ARF forms nuclear bodies with MDM2 and p53 and blocks p53 and MDM2 nuclear export. Tumor-associated mutations in ARF exon 2 disrupt ARF's nucleolus localization and reduce ARF's ability to block p53 nuclear export and to stabilize p53. Our results suggest an ARF-regulated MDM2-dependent p53 stabilization and link the human tumor-associated mutations in ARF with a functional alteration.     

Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction

MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.     

MDM2 promotes SUMO-2/3 modification of p53 to modulate transcriptional activity

The tumor suppressor p53 is extensively regulated by post-translational modification, including modification by the small ubiquitin-related modifier SUMO. We show here that MDM2, previously shown to promote ubiquitin, Nedd8 and SUMO-1 modification of p53, can also enhance conjugation of endogenous SUMO-2/3 to p53. Sumoylation activity requires p53-MDM2 binding but does not depend on an intact RING finger. Both ARF and L11 can promote SUMO-2/3 conjugation of p53. However, unlike the previously described SUMO-1 conjugation of p53 by an MDM2-ARF complex, this activity does not depend on the ability of MDM2 to relocalize to the nucleolus. Interestingly, the SUMO consensus is not conserved in mouse p53, which is therefore not modified by SUMO-2/3. Finally, we show that conjugation of SUMO-2/3 to p53 correlates with a reduction of both activation and repression of a subset of p53-target genes.Key words: p53, SUMO-2/3, sumoylation, MDM2, ARF, L11     

CARF is a novel protein that cooperates with mouse p19ARF (human p14ARF) in activating p53

The INK4a locus on chromosome 9p21 encodes two structurally distinct tumor suppressor proteins, p16(INK4a) and the alternative reading frame protein, ARF (p19(ARF) in mouse and p14(ARF) in human). Each of these proteins has a role in senescence of primary cells and activates pathways for cell cycle control and tumor suppression. The current prevailing model proposes that p19(ARF) activates p53 function by antagonizing its degradation by MDM2. It was, however, recently shown that stabilization of p53 by p14(ARF) occurs independent of the relocalization of MDM2 to the nucleolus. We have identified a novel collaborator of ARF, CARF. It co-localizes and interacts with ARF in the nucleolus. We demonstrate that CARF is co-regulated with ARF, cooperates with it in activating p53, and thus acts as a novel component of the ARF-p53-p21 pathway.     

Inhibition of MDM2 by hsp90 contributes to mutant p53 stabilization

Stabilization and overexpression are hallmarks of mutant p53 found in nearly 50% of human tumors. Mutations in the conformation-sensitive core domain of p53 often lead to association with molecular chaperones such as hsp70 and hsp90. Inhibition of hsp90 function accelerates mutant p53 degradation. We recently found that expression of p53 core domain mutants inhibits MDM2 degradation, suggesting that mutant p53 can modulate MDM2 functions. In this report, we show that mutant p53 mediates formation of MDM2-p53-hsp90 complexes. Release of MDM2 from the p53-hsp90 complex after DNA damage restores MDM2 but not p53 turnover, whereas dissociation of hsp90 by geldanamycin increases the degradation of both MDM2 and mutant p53. Mutant p53 degradation after hsp90 inhibition requires MDM2 expression. The interaction between MDM2 and hsp90 is disrupted by the 2A10 antibody, which recognizes a site on MDM2 important for binding to alternative reading frame (ARF). Expression of mutant p53 prevents MDM2 from binding ARF and accumulating in the nucleolus in an hsp90-dependent fashion. These results suggest that hsp90 recruited by mutant p53 conceals the ARF-binding site on MDM2 and inhibits its ubiquitin-protein isopeptide ligase function, resulting in the stabilization of both mutant p53 and MDM2.