Search

Background: The CpG island methylator phenotype (CIMP) is a major molecular pathway in colorectal cancer. Approximately 25% to 60% of CIMP tumors are microsatellite unstable (MSI-H) due to DNA hypermethylation of the MLH1 gene promoter. Our aim was to determine if the distributions of clinicopathologic factors in CIMP-positive tumors with MLH1 DNA methylation differed from those in CIMP-positive tumors without DNA methylation of MLH1., Methods: We assessed the associations between age, sex, tumor-site, MSI status BRAF and KRAS mutations, and family colorectal cancer history with MLH1 methylation status in a large population-based sample of CIMP-positive colorectal cancers defined by a 5-marker panel using unconditional logistic regression to assess the odds of MLH1 methylation by study variables., Results: Subjects with CIMP-positive tumors without MLH1 methylation were significantly younger, more likely to be male, and more likely to have distal colon or rectal primaries and the MSI-L phenotype. CIMP-positive MLH1-unmethylated tumors were significantly less likely than CIMP-positive MLH1-methylated tumors to harbor a BRAF V600E mutation and significantly more likely to harbor a KRAS mutation. MLH1 methylation was associated with significantly better overall survival (HR, 0.50; 95% confidence interval, 0.31-0.82)., Conclusions: These data suggest that MLH1 methylation in CIMP-positive tumors is not a completely random event and implies that there are environmental or genetic determinants that modify the probability that MLH1 will become methylated during CIMP pathogenesis., Impact: MLH1 DNA methylation status should be taken into account in etiologic studies., (©2015 American Association for Cancer Research.)

Tumor hypoxia and hypoxia-inducible factor 1 (HIF-1) activation are associated with cancer progression. Here, we demonstrate that the transcription factor TAp73 opposes HIF-1 activity through a nontranscriptional mechanism, thus affecting tumor angiogenesis. TAp73-deficient mice have an increased incidence of spontaneous and chemically induced tumors that also display enhanced vascularization. Mechanistically, TAp73 interacts with the regulatory subunit (α) of HIF-1 and recruits mouse double minute 2 homolog into the protein complex, thus promoting HIF-1α polyubiquitination and consequent proteasomal degradation in an oxygen-independent manner. In human lung cancer datasets, TAp73 strongly predicts good patient prognosis, and its expression is associated with low HIF-1 activation and angiogenesis. Our findings, supported by in vivo and clinical evidence, demonstrate a mechanism for oxygen-independent HIF-1 regulation, which has important implications for individualizing therapies in patients with cancer.

Activation of serine biosynthesis supports growth and proliferation of cancer cells. Human cancers often exhibit overexpression of phosphoglycerate dehydrogenase (PHGDH), the metabolic enzyme that catalyses the reaction that diverts serine biosynthesis from the glycolytic pathway. By refueling serine biosynthetic pathways, cancer cells sustain their metabolic requirements, promoting macromolecule synthesis, anaplerotic flux and ATP. Serine biosynthesis intersects glutaminolysis and together with this pathway provides substrates for production of antioxidant GSH. In human lung adenocarcinomas we identified a correlation between serine biosynthetic pathway and p73 expression. Metabolic profiling of human cancer cell line revealed that TAp73 activates serine biosynthesis, resulting in increased intracellular levels of serine and glycine, associated to accumulation of glutamate, tricarboxylic acid (TCA) anaplerotic intermediates and GSH. However, at molecular level p73 does not directly regulate serine metabolic enzymes, but transcriptionally controls a key enzyme of glutaminolysis, glutaminase-2 (GLS-2). p73, through GLS-2, favors conversion of glutamine in glutamate, which in turn drives the serine biosynthetic pathway. Serine and glutamate can be then employed for GSH synthesis, thus the p73-dependent metabolic switch enables potential response against oxidative stress. In knockdown experiment, indeed, TAp73 depletion completely abrogates cancer cell proliferation capacity in serine/glycine-deprivation, supporting the role of p73 to help cancer cells under metabolic stress. These findings implicate p73 in regulation of cancer metabolism and suggest that TAp73 influences glutamine and serine metabolism, affecting GSH synthesis and determining cancer pathogenesis.

Methylation of the MLH1 gene promoter region is an underlying cause of colorectal cancer (CRC) with high microsatellite instability (MSI-H) diagnosed in persons without a germ line mutation in a mismatch repair (MMR) gene (non-Lynch Syndrome CRC). It is unclear whether relatives of CRC cases with MLH1 methylation have an increased risk of colorectal or other cancers. In this retrospective cohort study, we assessed risk of CRC and other cancers for the first- and second-degree relatives of CRC cases with a methylated MLH1 gene, by comparing observed numbers of cases with those expected on the basis of age-, sex-, and country-specific cancer incidences (standardized incidence ratios). The cohort consisted of 3,128 first- and second-degree relatives of the 233 MLH1-methylated CRC cases with no MMR or MUTYH gene mutations. The standardized incidence ratio (SIR) for CRC was 1.60 [95% confidence interval (CI), 1.22-2.16] for first-degree relatives and 1.08 (0.74-1.60) for second-degree relatives. The SIR for gastric cancer was 2.58 (1.52-4.71) for first-degree relatives and 4.52 (2.23-10.61) for second-degree relatives and, for ovarian cancer, it was 2.16 (1.29-3.86) for first-degree relatives. The risk of liver cancer was also increased significantly in first-degree relatives but the estimate was on the basis of only two cases. These data imply that relatives of CRC cases with MLH1 methylation may be at increased risk of CRC and stomach cancer and possibly ovarian and liver cancer, suggesting that there may be a heritable factor for CRC and other cancers associated with MLH1 methylation in non-Lynch syndrome CRCs., (©2011 AACR.)

Tumor suppressor p53 is crucial for embryonic implantation through transcriptional up-regulation of uterine leukemia inhibitory factor (LIF). This article reports that p53 and estrogen receptor α were activated in endometrial tissues during implantation to coordinately regulate LIF production. By using human p53 knockin (Hupki) mice carrying a single nucleotide polymorphism (SNP) at codon 72 (arginine/proline), the arginine allele was demonstrated to produce higher uterine LIF levels during implantation than the proline allele. In humans, the diversity of haplotypes of the p53 gene has decreased during evolution, because the arginine allele, existing in only a subset of haplotypes, is under positive selection. This observation is consistent with previous results showing that the proline allele is enriched in patients undergoing in vitro fertilization (IVF). Studies with p63- and p73-knockout mice have demonstrated the involvement of p63 and p73 in female reproduction and their roles in egg formation and apoptosis (p63) and spindle checkpoint (p73) in female mice. Here, the role of p63 and p73 in human reproduction was investigated. Selected alleles of SNPs in p63 and p73 genes were enriched in IVF patients. These findings demonstrate that the p53 family members are involved in several steps to regulate female reproduction in mice and humans.

The p53 family of proteins consists of p53, p63 and p73, which are transcription factors that affect both cancer and development. It is now emerging that these proteins also regulate maternal reproduction. Whereas p63 is important for maturation of the egg, p73 ensures normal mitosis in the developing blastocyst. p53 subsequently regulates implantation of the embryo through transcriptional control of leukaemia inhibitory factor. Elucidating the cell biological basis of how these factors regulate female fertility may lead to new approaches to the control of human maternal reproduction.

MDM2, a negative regulator of p53, is elevated in many cancers that retain wild-type p53. A single nucleotide polymorphism (SNP) in the human MDM2 promoter increases the affinity of Sp1 resulting in elevated MDM2 levels. We generated mice carrying either the MDM2(SNP309T) or the MDM2(SNP309G) allele to address the impact of MDM2(SNP309G) on tumorigenesis. Mdm2(SNP309G/G) cells exhibit elevated Mdm2 levels, reduced p53 levels, and decreased apoptosis. Importantly, some Mdm2(SNP309G/G) mice succumbed to tumors before 1 year of age, suggesting that this allele increases tumor risk. Additionally, the Mdm2(SNP309G) allele potentiates the tumor phenotype and alters tumor spectrum in mice inheriting a p53 hot-spot mutation. These data provide causal evidence for increased cancer risk in carriers of the Mdm2(SNP309G) allele., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Murine double minute 4 (MDM4) shares significant structural homology with murine double minute 2 (MDM2) and interacts and regulates transcriptional activity of the tumor suppressor p53. In tumors with wild-type p53, there is often overexpression of MDM2 or MDM4 leading to functional inactivation of p53. A single-nucleotide polymorphism (SNP) in the promoter of human MDM2 (SNP309) was shown to associate with increased MDM2 expression and increased risk of cancer. This study evaluated the association of a SNP in human MDM4 (C>T) with age of onset of breast cancer in two independent cohorts. In cohort 1 of 675 patients, the average age of diagnosis for women with estrogen receptor (ER)-positive and ER-negative breast cancers was 53.2 and 48 years, respectively. In this cohort, homozygous variant (TT) carriers developed ER-negative carcinomas at an earlier age than homozygous wild-type (CC) or heterozygous (TC) such that the age at diagnosis was accelerated by 5.0 years (P = 0.018). This association was validated in a second cohort of breast cancer patients (n = 148), where TT carriers with ER-negative cancer developed the disease 3.8 years earlier than CC carriers (P = 0.006). The effect was more pronounced in Caucasians with ER-negative ductal carcinomas with TT homozygotes developing disease 7.5 years (P = 0.031) and 6.2 years (P = 7 x 10(-5)) earlier than CC carriers in cohorts 1 and 2, respectively. No association was seen in ER-positive ductal cancers suggesting that the SNP in MDM4 only has a functional association in ER-negative breast cancer.

A large body of evidence strongly suggests that the p53 tumor suppressor pathway is central in reducing cancer frequency in vertebrates. The protein product of the haploinsufficient mouse double minute 2 (MDM2) oncogene binds to and inhibits the p53 protein. Recent studies of human genetic variants in p53 and MDM2 have shown that single nucleotide polymorphisms (SNPs) can affect p53 signaling, confer cancer risk, and suggest that the pathway is under evolutionary selective pressure (1-4). In this report, we analyze the haplotype structure of MDM4, a structural homolog of MDM2, in several different human populations. Unusual patterns of linkage disequilibrium (LD) in the haplotype distribution of MDM4 indicate the presence of candidate SNPs that may also modify the efficacy of the p53 pathway. Association studies in 5 different patient populations reveal that these SNPs in MDM4 confer an increased risk for, or early onset of, human breast and ovarian cancers in Ashkenazi Jewish and European cohorts, respectively. This report not only implicates MDM4 as a key regulator of tumorigenesis in the human breast and ovary, but also exploits for the first time evolutionary driven linkage disequilibrium as a means to select SNPs of p53 pathway genes that might be clinically relevant.

Microsatellite instability (MSI) occurs in 10% to 20% of colorectal cancers (CRC) and has been attributed to both MLH1 promoter hypermethylation and germline mutation in the mismatch repair (MMR) genes. We present results from a large population- and clinic-based study of MLH1 methylation, immunohistochemistry, and MMR germline mutations that enabled us to (a) estimate the prevalence of MMR germline mutations and MLH1 methylation among MSI-H cases and help us understand if all MSI-H CRC is explained by these mechanisms and (b) estimate the associations between MLH1 methylation and sex, age, and tumor location within the colon. MLH1 methylation was measured in 1,061 population-based and 172 clinic-based cases of CRC. Overall, we observed MLH1 methylation in 60% of population-based MSI-H cases and in 13% of clinic-based MSI-H cases. Within the population-based cases with MMR mutation screening and conclusive immunohistochemistry results, we identified a molecular event in MMR in 91% of MSI-H cases: 54% had MLH1 methylation, 14% had a germline mutation in a MMR gene, and 23% had immunohistochemistry evidence for loss of a MMR protein. We observed a striking age difference, with the prevalence of a MMR germline mutation more than 4-fold lower and the prevalence of MLH1 methylation more than 4-fold higher in cases diagnosed after the age of 50 years than in cases diagnosed before that age. We also determined that female sex is an independent predictor of MLH1 methylation within the MSI-H subgroup. These results reinforce the importance of distinguishing between the underlying causes of MSI in studies of etiology and prognosis.

The p53 protein is a transcription factor that integrates various cellular stress signals. The accumulation of the mutant huntingtin protein with an expanded polyglutamine tract plays a central role in the pathology of human Huntington's disease. We found that the huntingtin gene contains multiple putative p53-responsive elements and p53 binds to these elements both in vivo and in vitro. p53 activation in cultured human cells, either by a temperature-sensitive mutant p53 protein or by gamma-irradiation (gamma-irradiation), increases huntingtin mRNA and protein expression. Similarly, murine huntingtin also contains multiple putative p53-responsive elements and its expression is induced by p53 activation in cultured cells. Moreover, gamma-irradiation, which activates p53, increases huntingtin gene expression in the striatum and cortex of mouse brain, the major pathological sites for Huntington's disease, in p53+/+ but not the isogenic p53-/- mice. These results demonstrate that p53 protein can regulate huntingtin expression at transcriptional level, and suggest that a p53 stress response could be a modulator of the process of Huntington's disease.

In cancer cells, the function of the tumor suppressor protein p53 is usually blocked. Impairment of the p53 pathway results in tumor cells with endogenous overexpression of Mdm2 via a naturally occurring single nucleotide polymorphism (SNP) in the mdm2 gene at position 309. Here we report that in mdm2 SNP309 cells, inactivation of p53 results in a chromatin-associated Mdm2-p53 complex without clearance of p53 by protein degradation. Nuclear accumulation of p53 protein in mdm2 SNP309 cells results after 6 h of camptothecin, etoposide, or mitomycin C treatment, with the p53 protein phosphorylated at Ser15. Chromatin immunoprecipitation demonstrated p53 and Mdm2 bound to p53 responsive elements. Interestingly, although the p53 protein was able to bind to DNA, quantitative PCR showed compromised transcription of endogenous target genes. Additionally, exogenously introduced p53 was incapable of activating transcription from p53 responsive elements in SNP309 cells, confirming the trans-acting nature of the inhibitor. Inhibition of Mdm2 by siRNA resulted in transcriptional activation of these p53 targets. Our data suggest that overproduction of Mdm2, resulting from a naturally occurring SNP, inhibits chromatin-bound p53 from activating the transcription of its target genes.

Twelve years ago, the Mdm2 oncogene was shown to bind to and inhibit the tumor suppressor protein, p53. During the past 12 years, both genetic and biochemical studies have demonstrated that Mdm2 is a key negative regulator of the tumor suppressor p53. Mdm2 and p53 form an oscillating auto-regulatory feedback loop, which is tightly controlled to allow the appropriate response to environmental stresses in order to suppress tumor formation. When Mdm2 activity is inappropriately heightened, as it is in many human tumors, p53 activity is attenuated and tumor susceptibility arises. The p53 gene is mutated in 50% of all human tumors, but in those tumors that retain wild type p53, inhibiting Mdm2 activity could activate p53 tumor suppression and therefore provide a therapeutic strategy for the treatment of cancer.

The tumor suppressor p53 gene is mutated in minimally half of all cancers. It is therefore reasonable to assume that naturally occurring polymorphic genetic variants in the p53 stress response pathway might determine an individual's susceptibility to cancer. A central node in the p53 pathway is the MDM2 protein, a direct negative regulator of p53. In this report, a single nucleotide polymorphism (SNP309) is found in the MDM2 promoter and is shown to increase the affinity of the transcriptional activator Sp1, resulting in higher levels of MDM2 RNA and protein and the subsequent attenuation of the p53 pathway. In humans, SNP309 is shown to associate with accelerated tumor formation in both hereditary and sporadic cancers. A model is proposed whereby SNP309 serves as a rate-limiting event in carcinogenesis.

The tumor suppressor p53, one of the most intensely investigated proteins, is usually studied by experiments that are averaged over cell populations, potentially masking the dynamic behavior in individual cells. We present a system for following, in individual living cells, the dynamics of p53 and its negative regulator Mdm2 (refs. 1,4-7): this system uses functional p53-CFP and Mdm2-YFP fusion proteins and time-lapse fluorescence microscopy. We found that p53 was expressed in a series of discrete pulses after DNA damage. Genetically identical cells had different numbers of pulses: zero, one, two or more. The mean height and duration of each pulse were fixed and did not depend on the amount of DNA damage. The mean number of pulses, however, increased with DNA damage. This approach can be used to study other signaling systems and suggests that the p53-Mdm2 feedback loop generates a 'digital' clock that releases well-timed quanta of p53 until damage is repaired or the cell dies.

The p53 gene is a critical tumor suppressor that is inactivated in a majority of cancers. The central role of p53 in response to stresses such as DNA damage, hypoxia, and oncogene activation underlies this high frequency of negative selection during tumorigenic transformation. Mutations in p53 disrupt checkpoint responses to DNA damage and result in the potential for destabilization of the genome. Consistent with this, p53 mutant cells have been shown to accumulate genomic alterations in cell culture, mouse models, and some human tumors. The relationship between p53 mutation and genomic instability in human osteosarcoma is addressed in this report. Similar to some other primary human tumors, the mutation of p53 correlates significantly with the presence of high levels of genomic instability in osteosarcomas. Surprisingly, osteosarcomas harboring an amplification of the HDM2 oncogene, which inhibits the tumor-suppressive properties of p53, do not display high levels of genomic instability. These results demonstrate that the inactivation of p53 in osteosarcomas directly by mutation versus indirectly by HDM2 amplification may have different cellular consequences with respect to the stability of the genome.

The p53 protein plays a key role in the cellular response to stress by inducing cell growth arrest or apoptosis. The polyproline region of p53 has been shown to be important for its growth suppression activity. p53 protein lacking the polyproline region has impaired apoptotic activity and altered specificity for certain apoptotic target genes. Here we describe the role of this region in the regulation of p53 by its inhibitor Mdm2. p53 lacking the polyproline region was identified to be more susceptible to inhibition by Mdm2. Furthermore, the absence of this region renders p53 more accessible to ubiquitination, nuclear export, and Mdm2-mediated degradation. This increased sensitivity to Mdm2 results from an enhanced affinity of Mdm2 toward p53 lacking the polyproline region. Our results provide a new explanation for the impaired growth suppression activity of p53 lacking this region. The polyproline region is proposed to be important in the modulation of the inhibitory effects of Mdm2 on p53 activities and stability.

The intracellular activity of the p53 tumor suppressor protein is regulated through a feedback loop involving its transcriptional target, mdm2. We present a simple mathematical model suggesting that, under certain circumstances, oscillations in p53 and Mdm2 protein levels can emerge in response to a stress signal. A delay in p53-dependent induction of Mdm2 is predicted to be required, albeit not sufficient, for this oscillatory behavior. In line with the predictions of the model, oscillations of both p53 and Mdm2 indeed occur on exposure of various cell types to ionizing radiation. Such oscillations may allow cells to repair their DNA without risking the irreversible consequences of continuous excessive p53 activation.

p53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro and in vivo with p73 alpha, beta, gamma, and delta. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage.

There is growing evidence that the p53 tumor suppressor protein not only can function to activate gene transcription but also to repress the expression of specific genes. Although recent studies have implicated the transcriptional repression function of p53 in the pathway of apoptosis, the molecular basis of this activity remains poorly understood. This study takes a first step toward elucidating this mechanism. We report that trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs), abrogates the ability of p53 to repress the transcription of two genes that it negatively regulates, Map4 and stathmin. Consistent with this finding, we report that p53 physically associates in vivo with HDACs. This interaction is not direct but, rather, is mediated by the corepressor mSin3a. Both wild-type p53 and mSin3a, but not mutant p53, can be found bound to the Map4 promoter at times when this promoter preferentially associates with deacetylated histones in vivo. Significantly, inhibition of p53-mediated transcriptional repression with TSA markedly inhibits apoptosis induction by p53. These data offer the first mechanistic insights for p53-mediated transcriptional repression and underscore the importance of this activity for apoptosis induction by this protein.

The INK4a-ARF locus encodes two distinct tumor suppressors, p16(INK4a) and p19(ARF). Whereas p16(INK4a) restrains cell growth through preventing phosphorylation of the retinoblastoma protein, p19(ARF) acts by attenuating Mdm2-mediated degradation of p53, thereby stabilizing p53. Recent data indicate that Mdm2 shuttles between the nucleus and the cytoplasm and that nucleo-cytoplasmic shuttling of Mdm2 is essential for Mdm2's ability to promote p53 degradation. Therefore, Mdm2 must export p53 from the nucleus to the cytoplasm where it targets p53 for degradation. We show here that coexpression of p19(ARF) blocks the nucleo-cytoplasmic shuttling of Mdm2. Moreover, subnuclear localization of Mdm2 changes from the nucleoplasm to the nucleolus in a shuttling time-dependent manner, whereas p19(ARF) is exclusively located in the nucleolus. In heterokaryons containing Mdm2 and p19(ARF), the longer the Mdm2 shuttling is allowed, the more Mdm2 protein colocalizes with p19(ARF) in the nucleolus, implying that Mdm2 moves from the nucleoplasm to the nucleolus and then associates with p19(ARF) there. Furthermore, whether or not Mdm2 colocalizes with p19(ARF) in the nucleolus, p19(ARF) prevents Mdm2 shuttling. This observation suggests that Mdm2 might be exported through the nucleolus and p19(ARF) could inhibit the nuclear export of Mdm2 by tethering Mdm2 in the nucleolus. Taken together, p19(ARF) could stabilize p53 by inhibiting the nuclear export of Mdm2.

The Hdm2 oncoprotein inhibits p53 functions by two means: (i) it blocks p53's transactivation activity and (ii) it targets p53 for degradation in a proteasome-dependent manner. Recent data indicate that Hdm2 shuttles between the nucleus and the cytoplasm and that the regulation of p53 levels by Hdm2 requires its nuclear export activity. Two different models are consistent with these observations. In the first, Hdm2 binds to p53 in the nucleus and shuttles p53 from the nucleus to the cytoplasm, and then it targets p53 to the cytoplasmic proteasome. Alternatively, Hdm2 and p53 could be exported separately from the nucleus and then associate in the cytoplasm, where Hdm2 promotes the degradation of p53. To distinguish between these two models, several Hdm2 mutants were employed. Hdm2NLS lacks the ability to enter the nucleus, whereas Hdm2NES is deficient in nuclear export. Hdm2NLS, Hdm2NES, or the combination of both mutants were unable to promote p53 degradation in the cotransfected 2KO cells (which were null for both the p53 and mdm2 genes), although wild-type Hdm2 efficiently reduced p53 levels under the same conditions. This observation is not a result of the differences in expression levels or stability between Hdm2 and these mutants. Moreover, coexpression of these mutants had no effect on wild-type Hdm-2-induced p53 destabilization. Thus, Hdm2 must shuttle p53 from the nucleus to the cytoplasm to target it for degradation in the cytoplasm.

Degradation of the p53 tumor suppressor protein has been shown to be regulated by Mdm2. In this study, we identify regions of Mdm2 that are not required for p53 binding but are essential for degradation. Mdm2 mutants lacking these regions function in a dominant negative fashion, stabilizing endogenous p53 in cells by interfering with the degradative function of the endogenous Mdm2. p53 protein stabilized in this way does not strongly enhance the expression of p21(Waf1/Cip1), the product of a p53-responsive gene, supporting the model in which binding of Mdm2 to the NH2-terminal domain of p53 inhibits interaction with other components of the basal transcriptional machinery. Interestingly, COOH-terminal truncations of Mdm2 that retain p53 binding but fail to mediate its degradation are also stabilized themselves. Because Mdm2, like p53, is normally an unstable protein that is degraded through the proteasome, this result suggests a direct link between the regulation of Mdm2 and p53 stability.

The p53 protein is activated in response to physiological stress resulting in either a G1 arrest of cells or apoptosis. As such, p53 must be tightly regulated, and the MDM2 oncoprotein plays a central role in that regulatory process. The transcription of the Mdm2 oncogene is induced by the p53 protein after DNA damage, and the MDM2 protein then binds to p53 and blocks its activities as a tumour suppressor and promotes its degradation. These two proteins thus form an autoregulatory feedback loop in which p53 positively regulates MDM2 levels and MDM2 negatively regulates p53 levels and activity. Immediately after ultraviolet (UV) irradiation MDM2 messenger RNA and protein levels fall in a p53-independent fashion, resulting in increased p53 levels. The p53 protein is then activated as a transcription factor by posttranslational modification permitting p53 to initiate its cell-cycle arrest or apoptotic (programmed cell death) functions. At later times, after the repair of DNA, MDM2 levels increase in a p53-dependent fashion. This induction of MDM2 results in the inhibition of p53 transcriptional activity and the degradation of p53 protein. MDM2-p53 complexes in the nucleus are transported to the cytoplasm via signals present in the MDM2 protein, where p53 is degraded in the proteasome. Thus MDM2 acts as a nuclear-cytoplasmic shuttle for the p53 protein. There are many levels at which this process is regulated, and as such there are many places for chemotherapeutic interventions. The amino-terminal domain of the MDM2 protein is all that is required to bind the p53 protein. The MDM2 protein has additional domains and therefore may have additional functions. Any of these MDM2 domains may contribute to MDM2's activities as an oncogene independent of its inhibition of the tumour suppressor functions of p53. Thus MDM2 itself could be a target for cancer therapeutic intervention.

The hdm2 oncoprotein contains a C-terminal domain that binds RNA and has been suggested to bind zinc(II) in an unusual RING finger domain in which Thr 455 was postulated as a ligand. We have reported experiments to test whether this C-terminal cysteine-rich motif is indeed a RING finger domain. We also tested the affinity of the hdm2 C-terminal peptide for metal binding, metal linkage to the folding of the C-terminal peptide, and the peptide's affinity for RNA. Truncation mutants demonstrate that amino acids 425-491 are necessary and sufficient for RNA binding. However, divalent metal ions do not seem to affect the specific RNA recognition. Metal binding studies suggest that hdm2 indeed binds to two molecules of zinc in an intertwined motif similar to the BRCA1 RING finger peptide. However, there is no similarity in overall tertiary structure, nor is there direct sequence homology with other RING fingers. Fluorescence energy transfer studies give a dissociation constant of (0.22 +/- 0.03) microM for cobalt(II) binding to site 1, while K2 for cobalt(II) binding was estimated to be 15 +/- 5 microM from ultraviolet absorbance. Studies of two mutant peptides confirm the assignment of binding residues in hdm2 and suggest that the coordination of Thr 455 previously proposed by sequence alignments is incorrect. Structural studies of hdm2 in the presence and absence of metal indicate only a small amount of secondary structure by circular dichroic spectroscopy. Metal binding did not seem to nucleate folding as in the case of two other RING finger proteins. However, distance measurement from fluorescence energy transfer indicated that the Tyr 489 residue was only approximately 14 A away from the first metal center, suggesting that the hdm2 protein exists in a compact form, at least in the presence of metal ion. In summary, hdm2 binds metal and RNA, but the RNA binding does not seem to occur in a zinc-dependent manner.

The MDM2 oncoprotein targets the p53 tumor suppressor protein for degradation when the two proteins are expressed in cells. The regulation of p53 levels by MDM2 requires the ability of MDM2 to be exported from the nucleus by utilizing its nuclear export signal (NES). The drug leptomycin B (LMB) blocks the formation of nuclear export complexes consisting of CRM1, RanGTP, and NES-containing proteins. It is predicted that LMB should inhibit nuclear-cytoplasmic shuttling by MDM2 and subsequently stabilize p53. This communication demonstrates that LMB treatment of various cell lines led to an increase in the steady-state levels of the p53 protein as a result of an increase in its stability. The stabilized p53 protein localized to the nucleus and was an active transcription factor. These results indicate that the low steady-state levels of p53 in the absence of DNA damage result from p53's nuclear export for cytoplasmic degradation. LMB also led to p53 stabilization in cell lines that contain human papillomavirus (HPV) DNA and express HPV E6, a protein that targets p53 for degradation. MDM2 is not necessary for E6-dependent degradation of p53, as evidenced by the observation that E6 promoted p53 degradation in cells lacking endogenous MDM2. In addition, LMB reduced E6's ability to degrade p53 in the absence of MDM2, demonstrating that complete degradation of p53 by E6 requires nuclear export and therefore likely occurs in cytoplasmic proteasomes. These data suggest that the nuclear export of p53 to the cytoplasm for degradation is a general mechanism for regulating p53 levels.

p53 is a transcriptional activator that plays a key role in the integration of signals inducing cell division arrest and programmed cell death. Moreover, p53 is a tumor suppressor gene, mutations of which are the most commonly detected mutations in diverse malignancies. In order to better understand the significance of p53 mutations to human cancer, we isolated mutant alleles of p53 that had lost transcription factor activity in yeast. These mutant alleles were evaluated for their precise changes, their activity against three different p53 responsive enhancers and their ability to act in a transdominant fashion to the wild type allele. While many of the mutations isolated in yeast resembled those found in human tumors, consistent with the importance of transcription factor activity for p53 in mammalian cells, the mutational spectrum obtained was dependent upon the p53 enhancer employed for the selection. Some mutations specifically inactivated p53 in yeast for a single enhancer element. Virtually all missense mutations tested had a dominant inhibitory effect on wild type p53 in yeast. Since some of these transdominant mutations are virtually unknown in human tumors we conclude that transdominance, per se, fails to predict which mutations occur frequently in cancer.

The hdm2 gene is overexpressed in a variety of human tumors. Its gene product localizes predominantly to the nucleus, where it acts as an inhibitor of the p53 tumor suppressor gene product. It is shown here that the hdm2 oncoprotein constantly shuttles between the nucleus and the cytoplasm. Shuttling of hdm2 does not depend on its interaction with p53. Nuclear export of hdm2 is mediated by a signal sequence similar to the nuclear export signal of the rev protein from human immunodeficiency virus and other lentiviruses. Mutation of this signal sequence abolishes detectable nucleo-cytoplasmic shuttling. When fused to a carrier protein, the hdm2 signal sequence can mediate nuclear export after intranuclear microinjection into HeLa cells. The export of hdm2 can be blocked by a competitive inhibitor of rev export, arguing that the export pathways for hdm2 and rev are either overlapping or identical. Inhibition of its export modifies the ability of hdm2 to block p53-mediated transcriptional activation, and hdm2's export function is required to accelerate the degradation of p53. Thus the rev nuclear export pathway may be used to regulate an oncogene product's activity and modulate cellular growth.

The mdm2 oncogene encodes a 90-kDa protein that can bind to the p53 tumor suppressor protein and negatively regulate its functions in transcription, cell cycle arrest, and apoptosis. The mdm2 gene is frequently amplified in human sarcomas, which may be responsible for the malignant transformations. We present evidence that the mdm2 oncoprotein is cleaved by an interleukin 1beta-converting enzyme-like protease (caspase) during p53-mediated apoptosis. The protease that cleaves mdm2 has a specificity similar to that of CPP32 (caspase-3), and recombinant caspase-3 is able to cleave mdm2 in vitro. The protease cleavage site has been mapped to between residue 361 and 362 of human mdm2. The proteolytic cleavage removes the COOH-terminal RING finger domain of mdm2, resulting in the loss of RNA binding activity. The p53 binding and inhibition functions of mdm2 are not affected by the cleavage. The cleavage site sequence of mdm2 is evolutionarily conserved, suggesting that regulation by caspase cleavage during apoptosis is an important feature of mdm2.

Background: DNA damage in mammalian cells stabilizes the p53 protein which then functions as a cell cycle checkpoint by leading to growth arrest or apoptosis. p53 is a transcription factor and positively regulates the expression of the p21/WAF-1 gene and the mdm2 gene. After high-dose UV irradiation, p53 increases the expression of the p21/WAF-1 gene immediately (2 to 5 hours after irradiation) while the induction of the mdm2 gene is delayed (8 to 12 hours after irradiation). Experiments presented here explore this differential expression of two different p53-regulated genes., Materials and Methods: IP-Western (protein) and Northern (mRNA) blot experiments are used to follow mdm2 and p21/WAF-1 expression in primary rat or mouse cells after a low-dose (4 J/m2) or a high-dose (20 J/M2) of UV irradiation. Northern blot and nuclear run-on experiments are employed to study mRNA stability as well as transcription rates of selected genes., Results: After high-dose UV irradiation, p53 is rapidly stabilized and the expression of p21/WAF1 is immediately increased. By contrast, both protein and mRNA levels of mdm2 first decrease in a p53-independent manner, and later increase in a p53-dependent manner. The initial decline of mdm2 expression following high-dose UV irradiation is UV-dosage dependent and regulated at the level of transcription., Conclusion: p53 regulates two genes, p21/WAF1 (blocks cell cycle progression) and mdm2 (reverses p53 activity), that mediate opposite actions. This process is regulated in a temporal fashion after high-dose UV irradiation, so that cell cycle progression can be halted while DNA repair continues prior to reversal of p53-mediated arrest by mdm2.

Background: The MDM2 oncoprotein binds to the tumor suppressor p53 and inhibits its anti-oncogenic functions., Materials and Methods: To determine the amino acids of MDM2 that are critical for binding to p53, a modified two-hybrid screen was performed in yeast. Site-directed mutagenesis was then performed to identify MDM2 residues important for p53 interaction. Mutant MDM2 proteins were subsequently tested for their ability to bind to p53 in vitro and for their ability to regulate p53-mediated transcription in vivo., Results: The yeast genetic screen yielded two Mdm2 mutations (G58D and C77Y) which disrupted binding to p53 in vitro without altering the conformation of MDM2 as determined with conformation-sensitive monoclonal antibodies. Site-directed mutagenesis yielded mutations of two additional amino acids of MDM2 (D68 and V75) that prevented binding to p53 in vitro. The mutant MDM2 proteins were unable to inhibit p53-dependent transcription in vivo, which is consistent with prior indications that a physical interaction between the two proteins is required for MDM2's inhibition of p53. Finally, the crystal structure of the MDM2-p53 complex shows that two of the four critical residues identified here contact p53 directly, while the remaining two residues play important structural roles in the MDM2 domain., Conclusions: MDM2 residues G58, D68, V75, and C77 are critical for MDM2's interaction with the p53 protein. Mutation of these residues to alanine prevents MDM2's interaction with p53 in vitro, and MDM2's regulation of p53's transcriptional activity in vivo.

The Mdm2 gene is the best known cellular regulator of p53 tumor suppressor activity. We report here the cloning and characterization of Xdm2, its homolog in Xenopus laevis. Human, mouse and Xenopus MDM2 proteins are more than 65% identical in several regions which are likely to be important for the biological activities of MDM2. Region I is sufficient for binding p53 and inhibiting its G1 arrest and apoptosis functions. Region II contains most of a central acidic region required for interaction with the L5 ribosomal protein and a putative C4 zinc finger. Region III is nearly identical from Xenopus to human and comprises the RING finger domain. We show that this structural conservation is associated with the conservation of three biochemical activities of MDM2; binding to the p53 and L5 proteins and specifically to RNA. Lastly, Xdm2 expression during early development is mainly restricted from the oocyte stage I/II to the blastula stage and is possibly independent of transcriptional activation by p53. These data as well as the utilization of Xenopus laevis to investigate the roles of MDM2 and p53 during early embryogenesis are discussed.

The MDM2 oncoprotein is a cellular inhibitor of the p53 tumor suppressor in that it can bind the transactivation domain of p53 and downregulate its ability to activate transcription. In certain cancers, MDM2 amplification is a common event and contributes to the inactivation of p53. The crystal structure of the 109-residue amino-terminal domain of MDM2 bound to a 15-residue transactivation domain peptide of p53 revealed that MDM2 has a deep hydrophobic cleft on which the p53 peptide binds as an amphipathic alpha helix. The interface relies on the steric complementarity between the MDM2 cleft and the hydrophobic face of the p53 alpha helix and, in particular, on a triad of p53 amino acids-Phe19, Trp23, and Leu26-which insert deep into the MDM2 cleft. These same p53 residues are also involved in transactivation, supporting the hypothesis that MDM2 inactivates p53 by concealing its transactivation domain. The structure also suggests that the amphipathic alpha helix may be a common structural motif in the binding of a diverse family of transactivation factors to the TATA-binding protein-associated factors.

Background: The cellular mdm2 gene has transforming activity when overexpressed and is amplified in a variety of human tumors. At least part of the transforming ability of the MDM2 protein is due to binding and inactivating the p53 tumor suppressor protein. Additionally, this protein forms a complex in vivo with the L5 ribosomal protein and its associated 5S ribosomal RNA and may be part of a ribosomal complex., Materials and Methods: A RNA homopolymer binding assay and a SELEX procedure have been used to characterize the RNA-binding activity of MDM2., Results: The MDM2 protein binds efficiently to the homopolyribonucleotide poly(G) but not to other homopolyribonucleotides. This binding is independent of the interaction of MDM2 with the L5 protein, which occurs through the central acidic domain of MDM2. An RNA SELEX procedure was performed to identify specific RNA ligands that bind with high affinity to the human MDM2 (HDM2) protein. After 10 rounds of selection and amplification, a subset of RNA molecules that bound efficiently to HDM2 was isolated from a randomized pool. Sequencing of these selected ligands revealed that a small number of sequence motifs were selected. The specific RNA binding occurs through the RING finger domain of the protein. Furthermore, a single amino acid substitution in the RING finger domain, G446S, completely abolishes the specific RNA binding., Conclusions: These observations, showing that MDM2 binds the L5/5S ribosomal ribonucleoprotein particle and can also bind to specific RNA sequences or structures, suggest a role for MDM2 in translational regulation in a cell.

The mdm-2 gene encodes a 90-kDa polypeptide that binds specifically to the p53 tumor suppressor protein. This physical interaction results in the inhibition of the transcriptional functions of p53 (J. Chen, J. Lin, and A. J. Levine, Mol. Med. 1:142-152, 1995, and J. Momand, G. P. Zambetti, D. C. Olson, D. George, and A. J. Levine, Cell 69:1237-1245, 1992). Experiments are described that demonstrate the ability of mdm-2 to abrogate both the p53-mediated cell cycle arrest and the apoptosis functions. In addition, the results presented here suggest that mdm-2 binding to p53 and the resultant inhibition of p53 transcription functions are critical for reversing p53-mediated cell cycle arrest. The N-terminal half or domain of the mdm-2 protein is sufficient to regulate these biological activities of p53, consistent with the possibility that the highly conserved central acidic region and the C-terminal putative zinc fingers of mdm-2 may encode other functions.

The cellular protein MDM2 can bind to the tumor suppressor gene product p53 and abrogate its transcriptional activity. In addition, p53 can regulate expression of the mdm2 gene. We and others have previously shown that p53 is present at high levels in adenovirus-transformed cells which express the larger E1B protein. In view of these observations the expression of MDM2 in a panel of adenovirus transformed human cell lines has been examined. Two major species (98K and 80K) were detected, together with a number of minor species of higher and lower molecular weight. While there was little variation in levels of 98K protein between cell lines, appreciable differences in the expression of the 80K component were apparent. There was no correlation between MDM2 and p53 expression in any of the adenovirus transformants, nor with the viral proteins expressed. The pattern and level of MDM2 detected was similar to that seen in human tumor cell lines and in human fetal tissue. Northern blot analysis suggested that MDM2 expression was regulated at the transcriptional level. Stable interactions were observed between p53 and MDM2 in the adenovirus-transformed cell lines and in Ad5 E1 HEK 293 cells a ternary complex of p53, MDM2, and the Ad5 E1B 58K protein was demonstrated. In view of the lack of correlation between the level of p53 and MDM2 in adenovirus E1-transformed cells, the capacity of p53 to cause transcriptional activation was assessed using transfected CAT constructs linked to p53 responsive elements. p53 transcriptional activity was similar in all of the cell lines examined and did not correlate with protein expression. It is concluded, on the basis of all of these data, that the high concentrations of p53 found in adenovirus transformants are not transcriptionally active and have no influence on MDM2 expression. However, when expression of p53 was increased following infection with mutant adenoviruses, which do not express the larger E1B proteins, there was an appreciable increase in p53 transcriptional activity and in the levels of all of the MDM2 components.

Inactivation of tumour-suppressor genes leads to deregulated cell proliferation and is a key factor in human tumorigenesis. Both p53 and retinoblastoma genes are frequently mutated in human cancers, and the simultaneous inactivation of RB and p53 is frequently observed in a variety of naturally occurring human tumours. Furthermore, three distinct DNA tumour virus groups--papovaviruses, adenoviruses and human papillomaviruses--transform cells by targeting and inactivating certain functions of both the p53 and retinoblastoma proteins. The cellular oncoprotein, Mdm2, binds to and downmodulates p53 function; its human homologue, MDM2, is amplified in certain human tumours, including sarcomas and gliomas. Overproduction of Mdm2 is both tumorigenic and capable of immortalizing primary rat embryo fibroblasts. Here we show that MDM2 interacts physically and functionally with pRB and, as with p53, inhibits pRB growth regulatory function. Therefore, both pRB and p53 can be subjected to negative regulation by the product of a single cellular protooncogene.

Mutations in the p53 gene are common in many cancers. They have been documented to occur in about 55% of all cancers of 51 different cell and tissue types. These mutations are accompanied by overexpression of the p53 protein in the nucleus of the cell, and this protein has lost its tumor suppressor function. In this study, 25 testicular germ-cell (TGC) tumors were tested for p53 mutations and the level of p53 protein expression. While 67% of the tumors overproduced the p53 protein in the nucleus of 10-60% of their cells, in all cases the DNA sequence of exons 4-9 of the p53 gene was wild type. In this tumor type, there was apparently no selection pressure for p53 mutations. The mdm-2 gene residues on chromosome 12 (12q13-q14), a chromosome often altered in TGC tumors. mdm-2 gene amplification (2.5- to 10-fold) was detected in three (12%) of these TGC tumors. These three tumors, and eight additional TGC tumors, overexpressed mdm-2 mRNA. There was a good correlation between overexpression of p53 protein and overexpression of mdm-2 mRNA (P = 0.01). This may well result from the fact that the level of mdm-2 mRNA is regulated by the p53 level. These studies demonstrate that TGC tumors fail to be selected for p53 mutations but nonetheless frequently expressed high levels of wild-type p53 protein in the cell nucleus. Perhaps this produces the excellent response to radiation and chemotherapy of these tumors, which generally have a good prognosis. Wild-type p53 may mediate apoptosis in these cells in response to the DNA damage.

Background: Mdm-2, a zinc finger protein, negatively regulates the p53 tumor suppressor gene product by binding to it and preventing transcriptional activation (16)., Materials and Methods: Assays for p53 mediated transcription, repression and activation by mutant and wild-type p53 proteins were used to measure the ability of mdm-2 to block each activity., Results: Mdm-2 was able to inhibit all three functions of the wild-type and mutant p53 activities; transcriptional activation by the wild-type protein, transcriptional activation by the mutant p53 protein, and repression by the wild-type protein., Conclusions: The mdm protein binds to the amino terminal portion of the p53 protein and, in so doing, blocks the ability of p53 to interact with the transcriptional machinery of the cell (23). The mdm-2 protein binds to both leucine-tryptophan residues at amino acids 22 and 23, from the amino terminal end of the protein, and in so doing, prevents all p53 functions. The ability of a mutant p53 protein to transactivate a multidrug resistance-1 gene promoter is blocked by mdm-2 and the ability of the wild-type p53 protein to repress transcription of some genes is also blocked by the mdm-2 protein. Thus, all three functions of the p53 protein-transcriptional activation, repression and mutant protein activation-require the p53 amino terminal domain functions and are regulated by the mdm-2 protein in a cell. When mdm-2 is overproduced, resulting in a tumor or transformation of a cell, all of the p53 activities are inactivated.

Throughout the purification of the mdm-2 or mdm-2-p53 protein complexes, a protein with a molecular weight of 34,000 was observed to copurify with these proteins. Several monoclonal antibodies directed against distinct epitopes in the mdm-2 or p53 protein coimmunoprecipitated this 34,000-molecular-weight protein, which did not react to p53 or mdm-2 polyclonal antisera in a Western immunoblot. The N-terminal amino acid sequence of this 34,000-molecular-weight protein demonstrated that the first 40 amino acids were identical to the ribosomal L5 protein, found in the large rRNA subunit and bound to 5S RNA. Partial peptide maps of the authentic L5 protein and the 34,000-molecular-weight protein were identical. mdm-2-L5 and mdm-2-L5-p53 complexes were shown to bind 5S RNA specifically, presumably through the known specificity of L5 protein for 5S RNA. In 5S RNA-L5-mdm-2-p53 ribonucleoprotein complexes, it was also possible to detect the 5.8S RNA which has been suggested to be covalently linked to a percentage of the p53 protein in a cell. These experiments have identified a unique ribonucleoprotein complex composed of 5S RNA, L5 protein, mdm-2 proteins, p53 protein, and possibly the 5.8S RNA. While the function of such a ribonucleoprotein complex is not yet clear, the identity of its component parts suggests a role for these proteins and RNA species in ribosomal biogenesis, ribosomal transport from the nucleus to the cytoplasm, or translational regulation in the cell.

The p53 tumor suppressor gene is the most commonly mutated gene in human cancers and, although mutation leads to loss of p53 suppression of cell growth, it can also result in new functions which enhance tumorigenicity. These mutations often lead to loss of p53 transcriptional activation activity, which is likely to account for the loss of growth control. The p53 protein induces expression of the GADD45 and mdm-2 genes, and the respective roles of the products of these two genes in the regulation of growth control, apoptosis, amplification, and the response to DNA damage remain to be determined. Although some facts are clear at this juncture, we can surely expect some surprises in the future, including additional functions for the p53 tumor suppressor gene product and a constant reevaluation of our present concepts. The study of the p53 regulatory pathway, however, should lead us to additional important genes and proteins central to an explanation of the origins of cancer.

The p53 tumor suppressor gene product is a transcriptional activator that may be associated with its ability to suppress tumor cell growth. The acidic amino terminus of the p53 protein has been shown to contain this trans-activation activity as well as the domains for mdm-2 and adenovirus 5 E1B 55-kD protein binding. An extensive genetic analysis of this amino-terminal p53 domain has been undertaken using site-specific mutagenesis. The results demonstrate that the acidic residues in the amino terminus of p53 may contribute to, but are not critical for, this trans-activation activity. Rather, the hydrophobic amino acid residues Leu-22 and Trp-23 of human p53 are both required for trans-activation activity, binding to the adenovirus E1B 55-kD protein and the human mdm-2-p53 protein in vitro. In addition, hydrophobic residues Leu-14 and Phe-19 are crucial for the interactions between p53 and human mdm-2 (hdm-2). Hydrophobic residues Trp-23 and Pro-27 are also important for binding to the adenovirus 5 (Ad5) E1B 55-kD protein in vitro. These mutations have no impact on the ability of the p53 protein to bind to a p53-specific DNA element. These results suggest that 2-4 critical hydrophobic residues in the amino-terminal domain of the p53 protein interact with the transcriptional machinery of the cell resulting in transcriptional activation. These very same hydrophobic residues contact the hdm-2 and Ad5 E1B 55-kD oncogene products.

Genetic alterations in the p53 and mdm2 genes have been reported to occur in soft tissue sarcomas. This study was designed to determine the prevalence and potential clinical value of detected molecular abnormalities and altered patterns of expression of mdm2 and p53 genes in adult soft tissue sarcomas. A cohort of 211 soft tissue sarcomas from adults that were both clinically and pathologically well characterized was analyzed. Monoclonal antibodies directed against mdm2 and p53 proteins were used to measure overexpression of these proteins in the nuclei of cells from sections of these tumors. Seventy-six of 207 tumors had abnormally high levels of mdm2 proteins and 56 of 211 tumors overexpressed p53 protein. Twenty-two cases had abnormally high levels of both mdm2 and p53 proteins based upon immunoreactivity with these antibodies. There was a striking statistically significant correlation between the overexpression of p53 and mdm2 proteins in the same tumor and poor survival (P < 0.05) of the patients. A group of 73 soft tissue sarcomas was chosen for analysis using Southern blots, single strand conformation polymorphisms, and direct DNA sequencing to confirm mdm2 gene amplifications and p53 mutations and correlate these with the results of the immunoreactivities. The overexpression of p53 and mdm2 proteins in the nuclei of tumor cells did not always correlate well with gene amplification at the mdm2 locus or mutation at the p53 gene. The possible reasons for these discrepancies are discussed.

Irradiation of mammalian cells with UV light results in a dose-dependent accumulation of the p53 tumor-suppressor gene product that is evident within 2 hr. UV treatment causes a dramatic increase in p53-specific transcriptional transactivation activity and an increase in expression of the p53-responsive gene mdm-2. UV-stimulated mdm-2 expression is not directly correlated with the level of p53 protein in a cell because mdm-2 induction is delayed at high UV doses even though p53 levels rise almost immediately. Cells lacking p53 protein do not respond to UV by increasing their expression of mdm-2. The delayed induction of mdm-2 at high UV doses suggests that, in addition to p53 protein levels, other factors contribute to the regulation of mdm-2 expression following UV treatment. The time of induction of mdm-2 in cells treated with UV light correlates with recovery of normal rates of DNA synthesis, presumably after DNA repair. These data indicate a possible role for mdm-2 in cell cycle progression.

A protein product of the mdm-2 oncogene (p90) has been recently shown to associate with the protein encoded by the tumor-suppressor gene p53. The mdm-2 gene was originally identified as a gene amplified in a spontaneously transformed Balb/c 3T3 cell line (3T3DM). This report describes the characterization of mdm-2 gene products and their interactions with the p53 protein. Polyclonal and monoclonal antibodies were generated against murine and human mdm-2 protein. These antibodies detected the mdm-2 p90 protein and at least four additional polypeptides (p85, p76, p74, p58-p57) in cultured cells. These additional proteins may arise from different spliced mRNA forms of the mdm-2 gene or post-translational modifications of the mdm-2 protein. The monoclonal antibodies distinguished at least three sets of mdm-2 proteins with distinct combinations of epitopes (p90 and p85; p76 and p74; p58-57). One or two of these proteins forms a complex with the p53 protein (p90, p58). These mdm-2 proteins were found to be overexpressed in 3T3DM cells and a subset of these proteins were complexed with p53. In 3T3DM cells, p90, like p53, had a short half-life of approximately 20 min and was localized to the cell nucleus. In resting cells stimulated with serum p90 levels and p90/p53 complex levels increased in the late G1 phase of the cell cycle. The p90 mdm-2 protein could regulate p53 activity in the late G1 phase of the cell cycle.

The 90-kDa cellular protein encoded by the mouse mdm-2 oncogene binds to the p53 protein in vivo and inhibits its transactivation function (J. Momand, G. P. Zambetti, D. C. Olson, D. George, and A. J. Levine, Cell 69:1237-1245, 1992). cDNA clones encoding the human homolog of the mdm-2 protein (also called hdm-2) were isolated from a HeLa cell cDNA library. A series of monoclonal antibodies have been generated against human mdm-2 protein, and the epitopes recognized by these antibodies have been mapped. By construction of a series of deletion mutants, the region of the mdm-2 protein that is critical for complex formation with the p53 protein has been mapped to the N-terminal portion of the human mdm-2 protein. Interestingly, a monoclonal antibody with an epitope located in this same region failed to immunoprecipitate the mdm-2-p53 complex and appeared to recognize only free mdm-2 protein. The domain of the p53 protein that is sufficient for interaction with human mdm-2 protein has been mapped to the N-terminal 52 amino acid residues of the p53 protein. This region contains the transactivation domain of p53, suggesting that mdm-2 may inhibit p53 function by disrupting its interaction with the general transcription machinery.