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1.  MdmX Promotes Bipolar Mitosis To Suppress Transformation and Tumorigenesis in p53-Deficient Cells and Mice▿ †  
Molecular and Cellular Biology  2007;28(4):1265-1273.
Mdm2 and MdmX are structurally related p53-binding proteins that function as critical negative regulators of p53 activity in embryonic and adult tissue. The overexpression of Mdm2 or MdmX inhibits p53 tumor suppressor functions in vitro, and the amplification of Mdm2 or MdmX is observed in human cancers retaining wild-type p53. We now demonstrate a surprising role for MdmX in suppressing tumorigenesis that is distinct from its oncogenic ability to inhibit p53. The deletion of MdmX induces multipolar mitotic spindle formation and the loss of chromosomes from hyperploid p53-null cells. This reduction in chromosome number, not observed in p53-null cells with Mdm2 deleted, correlates with increased cell proliferation and the spontaneous transformation of MdmX/p53-null mouse embryonic fibroblasts in vitro and with an increased rate of spontaneous tumorigenesis in MdmX/p53-null mice in vivo. These results indicate that MdmX has a p53-independent role in suppressing oncogenic cell transformation, proliferation, and tumorigenesis by promoting centrosome clustering and bipolar mitosis.
PMCID: PMC2258754  PMID: 18039860
2.  Amplification of Mdmx (or Mdm4) Directly Contributes to Tumor Formation by Inhibiting p53 Tumor Suppressor Activity 
Molecular and Cellular Biology  2004;24(13):5835-5843.
Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14ARF or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRasV12. Furthermore, the human Mdmx ortholog, Hdmx, was found to be overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53. Hdmx was also amplified and highly expressed in MCF-7, a breast cancer cell line harboring wild-type p53, and interfering RNA-mediated reduction of Hdmx markedly inhibited the growth potential of these cells in a p53-dependent manner. Together, these results make Hdmx a new putative drug target for cancer therapy.
PMCID: PMC480894  PMID: 15199139
3.  DNA Damage-Induced Phosphorylation of MdmX at Serine 367 Activates p53 by Targeting MdmX for Mdm2-Dependent Degradation†  
Molecular and Cellular Biology  2005;25(21):9608-9620.
Understanding how p53 activity is regulated is crucial in elucidating mechanisms of cellular defense against cancer. Genetic data indicate that Mdmx as well as Mdm2 plays a major role in maintaining p53 activity at low levels in nonstressed cells. However, biochemical mechanisms of how Mdmx regulates p53 activity are not well understood. Through identification of Mdmx-binding proteins, we found that 14-3-3 proteins are associated with Mdmx. Mdmx harbors a consensus sequence for binding of 14-3-3. Serine 367 (S367) is located within the putative binding sequence for 14-3-3, and its substitution with alanine (S367A) abolishes binding of Mdmx to 14-3-3. Transfection assays indicated that the S367A mutation, in cooperation with Mdm2, enhances the ability of Mdmx to repress the transcriptional activity of p53. The S367A mutant is more resistant to Mdm2-dependent ubiquitination and degradation than wild-type Mdmx, and Mdmx phosphorylated at S367 is preferentially degraded by Mdm2. Several types of DNA damage markedly enhance S367 phosphorylation, coinciding with increased binding of Mdmx to 14-3-3 and accelerated Mdmx degradation. Furthermore, promotion of growth of normal human fibroblasts after introduction of Mdmx is enhanced by the S367 mutation. We propose that Mdmx phosphorylation at S367 plays an important role in p53 activation after DNA damage by triggering Mdm2-dependent degradation of Mdmx.
PMCID: PMC1265801  PMID: 16227609
4.  Predicted Functions of MdmX in Fine-Tuning the Response of p53 to DNA Damage 
PLoS Computational Biology  2010;6(2):e1000665.
Tumor suppressor protein p53 is regulated by two structurally homologous proteins, Mdm2 and MdmX. In contrast to Mdm2, MdmX lacks ubiquitin ligase activity. Although the essential interactions of MdmX are known, it is not clear how they function to regulate p53. The regulation of tumor suppressor p53 by Mdm2 and MdmX in response to DNA damage was investigated by mathematical modeling of a simplified network. The simplified network model was derived from a detailed molecular interaction map (MIM) that exhibited four coherent DNA damage response pathways. The results suggest that MdmX may amplify or stabilize DNA damage-induced p53 responses via non-enzymatic interactions. Transient effects of MdmX are mediated by reservoirs of p53∶MdmX and Mdm2∶MdmX heterodimers, with MdmX buffering the concentrations of p53 and/or Mdm2. A survey of kinetic parameter space disclosed regions of switch-like behavior stemming from such reservoir-based transients. During an early response to DNA damage, MdmX positively or negatively regulated p53 activity, depending on the level of Mdm2; this led to amplification of p53 activity and switch-like response. During a late response to DNA damage, MdmX could dampen oscillations of p53 activity. A possible role of MdmX may be to dampen such oscillations that otherwise could produce erratic cell behavior. Our study suggests how MdmX may participate in the response of p53 to DNA damage either by increasing dependency of p53 on Mdm2 or by dampening oscillations of p53 activity and presents a model for experimental investigation.
Author Summary
A Molecular Interaction Map (MIM) akin to a circuit diagram of an electric device can give a comprehensive view of cellular processes and help understand complex protein functions in cells. To this end, we generated a MIM focused on the p53-Mdm2-MdmX network proteins and performed computer simulations to help understand how Mdm2 and MdmX may regulate p53. Proper regulation of p53 is important for cell survival: elevated levels of p53 can lead to cell death, and decreased levels of p53 can lead to cancer. Mdm2 and MdmX are structurally homologous proteins that regulate p53. Mdm2 negatively regulates p53 by degradation, but MdmX regulation of p53 is not well understood. Recently, Mdm2 and MdmX have been recognized as potential cancer therapeutic targets. In an effort to better understand how MdmX can alter the p53 activity under various conditions, we used mathematical models based on the MIM network to generate hypotheses that can be tested by experiments. Our simulations suggest that MdmX may increase the dependency of p53 on Mdm2 or dampen p53 oscillations during DNA damage response.
PMCID: PMC2824598  PMID: 20174603
5.  Abnormal MDMX degradation in tumor cells due to ARF deficiency 
Oncogene  2011;31(32):3721-3732.
MDMX is a hetero dimeric partner of MDM2 and a critical regulator of p53. MDMX level is generally elevated in tumors with wild type p53 and contributes to p53 inactivation. MDMX degradation is controlled in part by MDM2-mediated ubiquitination. Here we show that MDMX turnover is highly responsive to changes in MDM2 level in non-transformed cells, but not in tumor cells. We found that loss of ARF expression, which occurs in most tumors with wild type p53, significantly reduces MDMX sensitivity to MDM2. Restoration of ARF expression in tumor cells enables MDM2 to degrade MDMX in a dose-dependent fashion. ARF binds to MDM2 and stimulates a second-site interaction between the central region of MDM2 and MDMX, thus increases MDMX-MDM2 binding and MDMX ubiquitination. These results reveal an important abnormality in the p53 regulatory pathway as a consequence of ARF deficiency. Loss of ARF during tumor development not only prevents p53 stabilization by proliferative stress, but also causes accumulation of MDMX that compromises p53 activity. This phenomenon may reduce the clinical efficacy of MDM2-specific inhibitors by preventing MDMX down regulation.
PMCID: PMC3290737  PMID: 22120712
MDM2; MDMX; ARF; p53; ubiquitination; Nutlin
6.  An observational study on the expression levels of MDM2 and MDMX proteins, and associated effects on P53 in a series of human liposarcomas 
Inactivation of wild type P53 by its main cellular inhibitors (MDM2 and MDMX) is a well recognised feature of tumour formation in liposarcomas. MDM2 over-expression has been detected in approximately 80% of liposarcomas but only limited information is available about MDMX over-expression. To date, we are not aware of any study that has described the patterns of MDM2 and MDMX co-expression in liposarcomas. Such information has become more pertinent as various novel MDM2 and/or MDMX single and dual affinity antagonist compounds are emerging as an alternative approach for potential targeted therapeutic strategies.
We analysed a case series of 61 fully characterized liposarcomas of various sub-types by immunohistochemistry, to assess the expression levels of P53, MDM2 and MDMX, simultaneously. P53 sequencing was performed in all cases that expressed P53 protein in 10% or more of cells to rule out mutation-related over-expression.
50 cases over-expressed MDM2 and 42 of these co-expressed MDMX at varying relative levels. The relative expression levels of the two proteins with respect to each other were subtype-dependent. This apparently affected the detected levels of P53 directly in two distinct patterns. Diminished levels of P53 were observed when MDM2 was significantly higher in relation to MDMX, suggesting a dominant role for MDM2 in the degradation of P53. Higher levels of P53 were noted with increasing MDMX levels suggesting an interaction between MDM2 and MDMX that resulted in a reduced efficiency of MDM2 in degrading P53. Of the 26 cases of liposarcoma with elevated P53 expression, 5 were found to have a somatic mutation in the P53 gene.
The results suggest that complex dynamic interactions between MDM2 and MDMX proteins may directly affect the cellular levels of P53. This therefore suggests that careful characterization of both these markers will be necessary in tumours when considering in vivo evaluation of novel blocker compounds for MDM proteins, as a therapeutic strategy to restore wild type P53 function.
PMCID: PMC4028812  PMID: 24330579
Liposarcoma; MDM2; MDMX; P53; Targeted therapy
7.  MdmX Protects p53 from Mdm2-Mediated Degradation 
Molecular and Cellular Biology  2000;20(3):1001-1007.
The p53 tumor suppressor protein is stabilized in response to cellular stress, resulting in activation of genes responsible for either cell cycle arrest or apoptosis. The cellular pathway for releasing normal cells from p53-dependent cell cycle arrest involves the Mdm2 protein. Recently, a p53-binding protein with homology to Mdm2 was identified and called MdmX. Like Mdm2, MdmX is able to bind p53 and inhibit p53 transactivation; however, the ability of MdmX to degrade p53 has yet to be examined. We report here that MdmX is capable of associating with p53 yet is unable to facilitate nuclear export or induce p53 degradation. In addition, expression of MdmX can reverse Mdm2-targeted degradation of p53 while maintaining suppression of p53 transactivation. Using a series of MdmX deletions, we have determined that there are two distinct domains of the MdmX protein that can stabilize p53 in the presence of Mdm2. One domain requires MdmX interaction with p53 and results in the retention of both proteins within the nucleus and repression of p53 transactivation. The second domain involves the MdmX ring finger and results in stabilization of p53 and an increase in p53 transactivation. The potential basis for stabilization and increased p53 transactivation by the MdmX ring finger domain is discussed. Based on these observations, we propose that the MdmX protein may function to maintain a nuclear pool of p53 protein in undamaged cells.
PMCID: PMC85217  PMID: 10629057
8.  p53 inactivation by MDM2 and MDMX negative feedback loops in testicular germ cell tumors 
Cell cycle (Georgetown, Tex.)  2010;9(7):1411-1420.
Testicular germ cell tumors (TGCT) are unique in their excellent response to DNA-damaging chemotherapy. Mutation of p53 is rare in both untreated and relapsed TGCTs, suggesting that p53 fails to respond effectively against malignant transformation in germ cells. Previous studies implicated the presence of a poorly defined TGCT-specific mechanism of p53 inactivation. Here we show that disruption of p53-mdm2 binding using the MDM2-specific inhibitor Nutlin activates p53 in TGCT cells and is sufficient to induce strong apoptosis. Knockdown of MDMX cooperates with Nutlin to activate p53. Surprisingly, we found that p53 activation induced a two-fold increase in MDMX mRNA and protein expression in TGCT cells. A p53-responsive promoter is identified in MDMX intron 1 that contains a functional p53-binding site, suggesting that MDMX also functions as a negative feedback regulator of p53 in a cell line-dependent fashion. These findings suggest that MDM2 and MDMX are responsible for the functional inactivation of p53 in TGCT. Furthermore, TGCT cells are unique in having a strong apoptosis response to p53. Direct activation of p53 by targeting MDM2 and MDMX may provide a backup approach for the treatment of TGCTs resistant to DNA-damaging drugs.
PMCID: PMC3008305  PMID: 20372076
p53; p21; PUMA; MDM2; MDMX; germ cell tumor
9.  Blockade of Hsp90 by 17AAG antagonizes MDMX and synergizes with Nutlin to induce p53-mediated apoptosis in solid tumors 
Cell Death & Disease  2011;2(5):e156-.
Strategies to induce p53 activation in wtp53-retaining tumors carry high potential in cancer therapy. Nutlin, a potent highly selective MDM2 inhibitor, induces non-genotoxic p53 activation. Although Nutlin shows promise in promoting cell death in hematopoietic malignancies, a major roadblock is that most solid cancers do not undergo apoptosis but merely reversible growth arrest. p53 inhibition by unopposed MDMX is one major cause for apoptosis resistance to Nutlin. The Hsp90 chaperone is ubiquitously activated in cancer cells and supports oncogenic survival pathways, many of which antagonize p53. The Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) is known to induce p53-dependent apoptosis. We show here that in multiple difficult-to-kill solid tumor cells 17AAG modulates several critical components that synergize with Nutlin-activated p53 signaling to convert Nutlin's transient cytostatic response into a cytotoxic killing response in vitro and in xenografts. Combined with Nutlin, 17AAG destabilizes MDMX, reduces MDM2, induces PUMA and inhibits oncogenic survival pathways, such as PI3K/AKT, which counteract p53 signaling at multiple levels. Mechanistically, 17AAG interferes with the repressive MDMX–p53 axis by inducing robust MDMX degradation, thereby markedly increasing p53 transcription compared with Nutlin alone. To our knowledge Nutlin+17AAG represents the first effective pharmacologic knockdown of MDMX. Our study identifies 17AAG as a promising synthetic lethal partner for a more efficient Nutlin-based therapy.
PMCID: PMC3122118  PMID: 21562588
Nutlin; 17AAG; wtp53; synergy; cytotoxicity
10.  A Small-Molecule p53 Activator Induces Apoptosis through Inhibiting MDMX Expression in Breast Cancer Cells12 
Neoplasia (New York, N.Y.)  2011;13(7):611-619.
The tumor suppressor p53 is often inactivated in breast cancer cells because the overexpression of its repressors (e.g., MDM2 and MDMX). Restoration of p53 activity by small molecules through counteracting p53 repressors can lead to in vivo tumor regression and is therefore considered a promising strategy for treatments of cancer. Recent efforts in high-throughput drug screening and rational drug design have identified several structurally diverse small-molecule p53 activators, including a pseudourea derivative XI-011 (NSC146109). This small molecule strongly activates p53 while selectively inhibiting growth of transformed cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy. However, the mechanism(s) by which XI-011 activates p53 and the effects of XI-011 on growth of breast cancer cells are currently unknown. Here, we report that XI-011 promoted breast cancer cells to undergo apoptosis through activating p53 and inducing expression of proapoptotic genes. Importantly, we found that activation of p53 by this small molecule was achieved through a novel mechanism, that is, inhibition of MDMX expression. XI-011 repressed the MDMX promoter, resulting in down-regulation of MDMX messenger RNA level in MCF-7 cells. In line with these results, XI-011 decreased the viability of breast cancer cells expressing low levels of MDMX in a less-efficient manner. Interestingly, XI-011 acted additively with the MDM2 antagonist Nutlin-3a to inhibit growth of breast cancer cells. We conclude that XI-011 belongs to a novel class of small-molecule p53 activators that target MDMX and could be of value in treating breast cancer.
PMCID: PMC3132847  PMID: 21750655
11.  MdmX regulates transformation and chromosomal stability in p53-deficient cells 
Cell cycle (Georgetown, Tex.)  2008;7(19):2967-2973.
The cellular homologues Mdm2 and MdmX play critical roles in regulating the activity of the p53 tumor suppressor in damaged and non-damaged cells and during development in mice. Recently, we have utilized genetically defined primary cells and mice to reveal that endogenous levels of MdmX can also suppress multipolar mitosis and transformation in hyperploid p53-deficient cells and tumorigenesis in p53-deficient mice. These MdmX functions are not shared by Mdm2, and are distinct from the well-established ability of MdmX to complex with and inhibit p53 activity. Here we discuss some of the ramifications of MdmX loss in p53-deficient cells and mice, and we explore further the fate of MdmX/p53-double null embryonic fibroblasts undergoing multi-polar cell division using time-lapse video microscopy. We also discuss the relationship between chromosomal loss, cell proliferation, and the tumorigenic potential of p53-deficient cells lacking MdmX.
PMCID: PMC2716213  PMID: 18818521
p53; MdmX; Mdm2; mitosis; transformation
12.  Targeting Mdm2 and Mdmx in cancer therapy 
Genomic and proteomic profiling of human tumor samples and tumor-derived cell lines are essential for the realization of personalized therapy in oncology. Identification of the changes required for tumor initiation or maintenance will likely provide new targets for small molecule and biologic therapeutics. For example, inactivation of the p53 tumor suppressor pathway occurs in most human cancers. While this can be due to frank p53 gene mutation, almost half of all cancers retain the wild type p53 allele, indicating the pathway is disabled by other means. Alternate mechanisms include deletion or epigenetic inactivation of the p53 positive regulator, arf, methylation of the p53 promoter or elevated expression of the p53 regulators Mdm2 and Mdmx. This review discusses current models of p53 regulation by Mdm2 and Mdmx, and presents the rationale for design of future Mdmx-specific therapeutics based on our knowledge of its structure and biological functions. For simplicity, we use Mdmx throughout this review although the protein is also known as Mdm4 in mouse and Hdmx/Hdm4 in humans. A more detailed discussion of Mdm2 as an oncogene and as a target for chemotherapy can be found elsewhere (1-4).
PMCID: PMC2629357  PMID: 19147532
13.  Controlling the Mdm2-Mdmx-p53 Circuit 
Pharmaceuticals  2010;3(5):1576-1593.
The p53 tumor suppressor is a key protein in maintaining the integrity of the genome by inducing either cell cycle arrest or apoptosis following cellular stress signals. Two human family members, Mdm2 and Mdmx, are primarily responsible for inactivating p53 transcription and targeting p53 protein for ubiquitin-mediated degradation. In response to genotoxic stress, post-translational modifications to p53, Mdm2 and Mdmx stabilize and activate p53. The role that phosphorylation of these molecules plays in the cellular response to genotoxic agents has been extensively studied with respect to cancer biology. In this review, we discuss the main phosphorylation events of p53, Mdm2 and Mdmx in response to DNA damage that are important for p53 stability and activity. In tumors that harbor wild-type p53, reactivation of p53 by modulating both Mdm2 and Mdmx signaling is well suited as a therapeutic strategy. However, the rationale for development of kinase inhibitors that target the Mdm2-Mdmx-p53 axis must be carefully considered since modulation of certain kinase signaling pathways has the potential to destabilize and inactivate p53.
PMCID: PMC2907906  PMID: 20651945
Mdm2; Mdmx; p53; phosphorylation; kinase inhibitor
14.  Controlling the Mdm2-Mdmx-p53 Circuit 
The p53 tumor suppressor is a key protein in maintaining the integrity of the genome by inducing either cell cycle arrest or apoptosis following cellular stress signals. Two human family members, Mdm2 and Mdmx, are primarily responsible for inactivating p53 transcription and targeting p53 protein for ubiquitin-mediated degradation. In response to genotoxic stress, post-translational modifications to p53, Mdm2 and Mdmx stabilize and activate p53. The role that phosphorylation of these molecules plays in the cellular response to genotoxic agents has been extensively studied with respect to cancer biology. In this review, we discuss the main phosphorylation events of p53, Mdm2 and Mdmx in response to DNA damage that are important for p53 stability and activity. In tumors that harbor wild-type p53, reactivation of p53 by modulating both Mdm2 and Mdmx signaling is well suited as a therapeutic strategy. However, the rationale for development of kinase inhibitors that target the Mdm2-Mdmx-p53 axis must be carefully considered since modulation of certain kinase signaling pathways has the potential to destabilize and inactivate p53.
PMCID: PMC2907906  PMID: 20651945
Mdm2; Mdmx; p53; phosphorylation; kinase inhibitor
15.  A Small-molecule Inhibitor of MDMX Activates p53 and Induces Apoptosis 
Molecular cancer therapeutics  2010;10(1):69-79.
P53 inactivation caused by aberrant expression of its major regulators (e.g., MDM2 and MDMX) contributes to the genesis of a large number of human cancers. Recent studies have demonstrated that restoration of p53 activity by counteracting p53 repressors is a promising anti-cancer strategy. Although agents (e.g., Nutlin-3a) that disrupt MDM2-p53 interaction can inhibit tumor growth, they are less effective in cancer cells that express high levels of MDMX. MDMX binds to p53 and can repress the tumor suppressor function of p53 through inhibiting its trans-activation activity and/or destabilizing the protein. Here we report the identification of a benzofuroxan derivative (7-(4-methylpiperazin-1-yl)-4-nitro-1-oxido-2,1,3-benzoxadiazol-1-ium, NSC207895) that could inhibit MDMX expression in cancer cells through a reporter-based drug screening. Treatments of MCF-7 cells with this small-molecule MDMX inhibitor activated p53, resulting in elevated expression of proapoptotic genes (e.g., PUMA, BAX, and PIG3). Importantly, this novel small-molecule p53 activator caused MCF-7 cells to undergo apoptosis, and acted additively with Nutlin-3a to activate p53 and decrease the viability of cancer cells. These results thus demonstrate that small molecules targeting MDMX expression would be of therapeutic benefits.
PMCID: PMC3058295  PMID: 21075910
MDMX; small-molecule inhibitor; p53; targeted therapy; apoptosis
16.  Regulation of MDMX Expression by Mitogenic Signaling▿  
Molecular and Cellular Biology  2008;28(6):1999-2010.
MDMX is an important regulator of p53 transcriptional activity and stress response. MDMX overexpression and gene amplification are implicated in p53 inactivation and tumor development. Unlike MDM2, MDMX is not inducible by p53, and little is known about its regulation at the transcriptional level. We found that MDMX levels in tumor cell lines closely correlate with promoter activity and mRNA level. Activated K-Ras and insulin-like growth factor 1 induce MDMX expression at the transcriptional level through mechanisms that involve the mitogen-activated protein kinase and c-Ets-1 transcription factors. Pharmacological inhibition of MEK results in down-regulation of MDMX in tumor cell lines. MDMX overexpression was detected in ∼50% of human colon tumors and showed strong correlation with increased extracellular signal-regulated kinase phosphorylation. Therefore, MDMX expression is regulated by mitogenic signaling pathways. This mechanism may protect normal proliferating cells from p53 but also hamper p53 response during tumor development.
PMCID: PMC2268405  PMID: 18172009
17.  A critical role for the non-coding 5S rRNA in regulating Mdmx stability 
Molecular cell  2011;43(6):1023-1032.
Both p53 and Mdmx are ubiquitinated and degraded by the same E3 ligase Mdm2; interestingly, however, while p53 is rapidly degraded by Mdm2, Mdmx is a stable protein in most of cancer cells. Thus, the mechanism by which Mdmx is degraded by Mdm2 needs further elucidation. Here, we identified the noncoding 5S rRNA as a major component of Mdmx-associated complexes from human cells. We show that 5S rRNA acts as a natural inhibitor of Mdmx degradation by Mdm2. RNAi-mediated knockdown of endogenous 5S rRNA, while not affecting p53 levels, significantly induces Mdmx degradation and subsequently, activates p53-dependent growth arrest. Notably, 5S rRNA binds the RING domain of Mdmx and blocks its ubiquitination by Mdm2 whereas Mdm2-mediated p53 ubiquitination remains intact. These results provide insights into the differential effects on p53 and Mdmx by Mdm2 in vivo and reveal an critical role of noncoding 5S rRNA in modulating the p53-Mdmx axis.
PMCID: PMC3627499  PMID: 21925390
5S rRNA; Non-coding small RNA; Mdm2; Mdmx; p53; HAUSP; Ubiquitination; Deubiquitination
18.  Increased radio-resistance and accelerated B-cell lymphomas in mice with Mdmx mutations that prevent modifications by DNA damage-activated kinases 
Cancer cell  2009;16(1):33-43.
Mdmx is a critical negative regulator of the p53 pathway that is stoichiometrically limiting in some tissues. Post-translational modification and degradation of Mdmx after DNA damage have been proposed to be essential for p53 activation. We tested this model in vivo, where critical stoichiometric relationships are preserved. We generated an Mdmx mutant mouse in which three conserved serines (S341, S367, S402) targeted by DNA damage-activated kinases were replaced by alanines to investigate whether modifications of these residues are important for Mdmx degradation and p53 activation. The mutant mice were remarkably resistant to radiation, and very susceptible to Myc-induced lymphomagenesis. These data demonstrate that Mdmx down-regulation is crucial for effective p53-mediated radiation responses and tumor suppression in vivo.
P53 function is sensitive to the levels of its negative regulators, Mdm2 and Mdmx. Cell culture studies have suggested the importance of post-translational modifications in Mdm2 and Mdmx for p53 activation, but this has not been rigorously tested in vivo. This work shows that DNA damage and activated c-Myc both require phosphorylation of Mdmx in residues targeted by the damage kinases ATM and Chk2 for robust p53 activation. Preventing Mdmx post-translational modification stabilizes this negative regulator, which mitigates p53 activation, and presumably enables c-Myc to drive tumor cells with defective genomes into cycle in vivo. The data also stress the relevance of Mdmx as a potential therapeutic target.
PMCID: PMC2758524  PMID: 19573810
19.  Stress-Induced Alternative Splice Forms of MDM2 and MDMX Modulate the p53-Pathway in Distinct Ways 
PLoS ONE  2014;9(8):e104444.
MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.
PMCID: PMC4126728  PMID: 25105592
20.  Full-length hdmX transcripts decrease following genotoxic stress 
Oncogene  2008;27(52):6657-6666.
Previous studies have suggested that the mdmX gene is constitutively transcribed, and that MdmX protein activity is instead controlled by cellular localization and DNA damage induced Mdm2-mediated ubiquitination leading to proteasomal degradation. In these studies, we report that the human mdmX (hdmX) mRNA is reproducibly decreased in various human cell lines following treatment with various DNA-damaging agents. Repression of hdmX transcripts is observed in DNAdamaged HCT116 colon cancer cells and in isogenic p53−/− cells, suggesting that this effect is p53-independent. Reduction in the amount of hdmX transcript occurs in both human tumor cell lines and primary human diploid fibroblasts, and results in a significant reduction of HdmX protein. Examination of hdmX promoter activity suggests that damage-induced repression of hdmX mRNA is not significantly impacted by transcription initiation. In contrast, changes in hdmX mRNA splicing appear to partly explain the reduction in full-length hdmX mRNA levels in tumor cell lines with the destabilization of full-length hdmX transcripts, potentially through microRNA miR-34a regulation, also impacting transcript levels. Taken together, this study uncovers previously unrecognized cellular mechanisms by which hdmX mRNA levels are kept low following genotoxic stress.
PMCID: PMC2610866  PMID: 18711402
HdmX; p53; transcription; splicing; micro-RNAs
21.  MDMX Promotes Proteasomal Turnover of p21 at G1 and Early S Phases Independently of, but in Cooperation with, MDM2▿ †  
Molecular and Cellular Biology  2007;28(4):1218-1229.
We have shown previously that MDM2 promotes the degradation of the cyclin-dependent kinase inhibitor p21 through a ubiquitin-independent proteolytic pathway. Here we report that the MDM2 analog, MDMX, also displays a similar activity. MDMX directly bound to p21 and mediated its proteasomal degradation. Although the MDMX effect was independent of MDM2, they synergistically promoted p21 degradation when coexpressed in cells. This degradation appears to be mediated by the 26S proteasome, as MDMX and p21 bound to S2, one of the subunits of the 19S component of the 26S proteasome, in vivo. Conversely, knockdown of MDMX induced the level of endogenous p21 proteins that no longer cofractionated with 26S proteasome, resulting in G1 arrest. The level of p21 was low at early S phase but markedly induced by knocking down either MDMX or MDM2 in human cells. Ablation of p21 rescued the G1 arrest caused by double depletion of MDM2 and MDMX in p53-null cells. These results demonstrate that MDMX and MDM2 independently and cooperatively regulate the proteasome-mediated degradation of p21 at the G1 and early S phases.
PMCID: PMC2258738  PMID: 18086887
22.  Stochastic Modeling and Simulation of the p53-MDM2/MDMX Loop 
Journal of Computational Biology  2009;16(7):917-933.
The p53 gene is crucial for effective tumor suppression in humans as supported by its universal inactivation in cancer cells either through mutations affecting the p53 locus directly or through aberration of its normal regulation. The p53 tumor repressor is regulated through a negative feedback loop involving its transcriptional target MDM2. MDMX is also an essential negative regulator of p53. Several computational models have been proposed to simulate the dynamics of the p53-MDM2 loop, but they do not include MDMX, only account for some basic interactions between p53 and MDM2 and cannot capture the intrinsic noise in the loop. In this article, we present a comprehensive model for the p53-MDM2/MDMX loop that accounts for most known interactions among p53, MDM2 and MDMX. Our model is characterized by a set of molecular reactions, which enables us to employ stochastic simulation to investigate the dynamics of the loop. In agreement with experiments, our results show that p53 and MDM2 undergo oscillations after DNA damage in the presence of noise, and the variation in oscillation amplitudes is much higher than that in oscillation periods. Our simulations predict that intrinsic noise contributes to 60%–70% of the total variation in oscillation amplitudes and periods. The protein levels of p53, MDM2, and MDMX after treatment with Nutlin in our simulations are also consistent with experimental results. Our simulation results further predict that p53 levels increase dramatically after MDM2 is knocked out, but increase with a much less amount after MDMX is knocked out. This may partially explain why MDM2-null and MDMX-null mouse embryos die in different developmental stages. Our stochastic model and simulation provide insights into the variability of the behavior of the p53 pathway and can be used to predict the dynamics of the pathway after certain interventions.
PMCID: PMC3148126  PMID: 19580521
algorithms; gene chips; gene networks; genetics; machine learning
23.  Protecting the Genome from Mdm2 and Mdmx 
Genes & Cancer  2012;3(3-4):283-290.
The contribution of Mdm2, and its recently identified family member Mdmx (Mdm4), to tumorigenesis has primarily focused on their negative regulation of the p53 tumor suppressor. Although Mdm2 and Mdmx clearly inhibit p53, which can lead to tumor development, both have also been shown to affect tumorigenesis independent of p53. Given that Mdm2 and/or Mdmx overexpression is common and likely underestimated in human cancers, understanding the functions of these proteins beyond p53 control is critical. In recent years, new functions of Mdm2 and Mdmx that lead to genome instability, a hallmark of malignancy, have emerged. Specifically, roles in the DNA damage response that are distinct from their regulation of p53 have been identified. Inhibition of p53 as well as other components of the DNA damage response by Mdm2 and Mdmx can result in delayed DNA repair and increased genome instability, making Mdm2 and Mdmx a danger to the genome when aberrantly expressed. However, the genome instability caused by altered levels of Mdm2 and Mdmx could be used therapeutically for the treatment of cancer. Specifically, drugs/small molecules that target the interaction between Mdm2 and p53 can stabilize Mdm2, resulting in negative consequences on the genome that could be exploited for cancer treatment, particularly malignancies lacking functional p53.
PMCID: PMC3494371  PMID: 23150761
Mdm2; Mdmx; p53; Nbs1; genome instability
24.  Phosphorylation and Degradation of MdmX is Inhibited by Wip1 Phosphatase in the DNA Damage Response 
Cancer research  2009;69(20):7960-7968.
MdmX and Mdm2 regulate p53 tumor suppressor functions by controlling p53 transcriptional activity and/or stability in cells exposed to DNA damage. Accumulating evidence indicates that ATM-mediated phosphorylation and degradation of Mdm2 and MdmX may be the initial driving force that induces p53 activity during the early phase of the DNA damage response. We have recently determined that a novel protein phosphatase, Wip1 (or PPM1D), contributes to p53 regulation by dephosphorylating Mdm2 to close the p53 activation loop initiated by the ATM/ATR kinases. In the present study, we determine that Wip1 directly dephosphorylates MdmX at the ATM-targeted Ser403 and indirectly suppresses phosphorylation of MdmX at Ser342 and Ser367. Wip1 inhibits the DNA damage-induced ubiquitination and degradation of MdmX, leading to the stabilization of MdmX and reduction of p53 activities. Our data suggest that Wip1 is an important component in the ATM-p53-MdmX regulatory loop.
PMCID: PMC2763051  PMID: 19808970
Wip1; MdmX; Stabilization; Dephosphorylation
25.  The Hsp90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin increases cisplatin antitumor activity by inducing p53-mediated apoptosis in head and neck cancer 
Cell Death & Disease  2013;4(12):e956-.
The tumor suppressor p53 is often inactivated in head and neck cancer (HNC) through TP53 mutations or overexpression of mouse double minute 2 or mouse double minute X. Restoration of p53 function by counteracting these p53 repressors is a promising strategy for cancer treatment. The present study assessed the ability of a heat shock protein 90 (Hsp90) inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17AAG), to induce apoptosis in HNC by restoring p53 function. The effect of 17AAG, alone or in combination with Nutlin-3a or cisplatin, was assessed in HNC cells using growth and apoptosis, immunoblotting, quantitative reverse transcription-polymerase chain reaction, and preclinical tumor xenograft models. 17AAG activated and stabilized p53 in HNC cells bearing wild-type TP53 by disrupting the p53–MDMX interaction. 17AAG upregulated p21 and proapoptotic gene expression, and promoted apoptosis in a concentration-dependent manner. Growth inhibition by 17AAG was highest in tumor cells with MDMX overexpression. The apoptotic response was blocked by inhibition of p53 expression, demonstrating that the effect of 17AAG depended on p53 and MDMX. 17AAG synergized in vitro with Nutlin-3a and in vitro and in vivo with cisplatin to induce p53-mediated apoptosis. 17AAG effectively induced p53-mediated apoptosis in HNC cells through MDMX inhibition and increased the antitumor activity of cisplatin synergistically, suggesting a promising strategy for treating HNC.
PMCID: PMC3877559  PMID: 24336076
17AAG; p53; MDMX; head and neck cancer; apoptosis

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