Cell cycle regulatory functions of the human oncoprotein MDM2

The protein (MDM2) coded by the mouse double minute-2 (mdm2) gene or its human homologue is well known as an oncoprotein. Malignant human tumors particularly breast tumors and soft tissue sarcomas frequently overexpress MDM2. Artificial amplification of mdm2 gene derived from a transformed murine cell line enhances tumorigenic potential of murine cells. Consistent with its tumorigenic property, mouse or human MDM2 can inactivate several functions of the tumor suppressor p53 and can degrade p53. The protein also interacts with other tumor suppressors, and these interactions may contribute to its tumorigenic property. In spite of its oncogenic role, mouse or human MDM2 induces G(1) arrest in normal human or murine cells. Some cell lines bearing known genetic mutations are insensitive to MDM2-mediated growth arrest. This review is aimed to collect available information on the functions of MDM2 that could potentially regulate cell cycle and to discuss how this information may fit in one model that could explain the two apparently opposite G(1) arrest and oncogenic function of MDM2.     

The RING finger domain of MDM2 is essential for MDM2-mediated TGF-beta resistance

In this study, we attempt to gain insights into the molecular mechanism underlying MDM2-mediated TGF-beta resistance. MDM2 renders cells refractory to TGF-beta by overcoming a TGF-beta-induced G1 cell cycle arrest. Because the TGF-beta resistant phenotype is reversible upon removal of MDM2, MDM2 likely confers TGF-beta resistance by directly targeting the cellular machinery involved in the growth inhibition by TGF-beta. Investigation of the structure-function relationship of MDM2 reveals three elements essential for MDM2 to confer TGF-beta resistance in both mink lung epithelial cells and human mammary epithelial cells. One of these elements is the C-terminal half of the p53-binding domain, which at least partially retained p53-binding and inhibitory activity. Second, the ability of MDM2 to mediate TGF-beta resistance is disrupted by mutation of the nuclear localization signal, but is restored upon coexpression of MDMX. Finally, mutations of the zinc coordination residues of the RING finger domain abrogates TGF-beta resistance, but not the ability of MDM2 to inhibit p53 activity or to bind MDMX. These data suggest that RING finger-mediated p53 inhibition and MDMX interaction are not sufficient to cause TGF-beta resistance and imply a crucial role of the E3 ubiquitin ligase activity of this domain in MDM2-mediated TGF-beta resistance.     

Senescence-like changes induced by expression of p21(waf1/Cip1) in NIH3T3 cell line

P21(Waf1/Cip1) is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21(Waf1/Cip1) involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21(Waf1/Cip1) and cellular senescence. While in murine cells, the role of p21(Waf1/Cip1) is indefinite. We explored this issue using NIH3T3 cells with inducible p21(Waf1/Cip1) expression. Induction of p21(Waf1/Cip1) triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21(Waf1/Cip1)-transduced NIH3T3 cells expressed beta-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p2l(Waf1/Cip1) can also induce senescence-like changes in murine cells.     

A role for p53 in maintaining and establishing the quiescence growth arrest in human cells

The p53 tumor suppressor protein induces transient growth arrest or apoptosis in response to genotoxic stress and mediates the irreversible growth arrest of cellular senescence. We present evidence here that p53 also contributes to the reversible, growth factor-dependent arrest of quiescence (G(0)). Microinjection of expression vectors encoding either MDM2 or a pRb-binding mutant of SV40 T antigen, both of which abrogate p53 function, stimulated quiescent normal human fibroblasts to initiate DNA synthesis and were 40-70% as effective as wild-type T antigen. Electrophoretic mobility shift and p53 transactivation assays showed that p53 activity was higher in quiescent and senescent cells compared with proliferating cells. As proliferating cells entered G(0) after growth factor withdrawal, the p53 mRNA level increased, followed by transient accumulation of the protein. Shortly thereafter, the expression (mRNA and protein) of p21, a p53 target gene and effector of cell cycle arrest, increased. Finally, stable expression of the HPV16 E6 oncogene or dominant negative p53 peptide, GSE-22, both of which inhibit p53 function, delayed entry into quiescence following growth factor withdrawal. Our data indicate that p53 is activated during both quiescence and senescence. They further suggest that p53 activity contributes, albeit not exclusively, to the quiescent growth arrest.     

Novel Roles for P53 in the Genesis and Targeting of Tetraploid Cancer Cells

Tetraploid (4N) cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. P53 is a tumor suppressor protein and key regulator of tetraploidy. As part of the “tetraploidy checkpoint”, p53 inhibits tetraploid cell proliferation by promoting a G1-arrest in incipient tetraploid cells (referred to as a tetraploid G1 arrest). Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In the current study, Nutlin-3a promoted a p53-dependent tetraploid G1 arrest in two diploid clones of the HCT116 colon cancer cell line. Both clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis compared to their diploid precursors. These findings demonstrate that transient p53 activation by Nutlin can promote tetraploid cell formation from diploid precursors, and the resulting tetraploid cells are therapy (IR/CP) resistant. Importantly, the tetraploid clones selected after Nutlin treatment expressed approximately twice as much P53 and MDM2 mRNA as diploid precursors, expressed approximately twice as many p53-MDM2 protein complexes (by co-immunoprecipitation), and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays novel roles in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher P53 gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin).     

MDM2 protein-mediated ubiquitination of numb protein: identification of a second physiological substrate of MDM2 that employs a dual-site docking mechanism

The E3 ubiquitin ligase, MDM2, uses a dual-site mechanism to ubiquitinate and degrade the tumor suppressor protein p53, involving interactions with the N-terminal hydrophobic pocket and the acidic domain of MDM2. The results presented here demonstrate that MDM2 also uses this same dual-site mechanism to bind to the cell fate determinant NUMB with both the N-terminal hydrophobic pocket and the acidic domain of MDM2 also involved in forming the interaction with NUMB. Furthermore, the acidic domain interactions are crucial for MDM2-mediated ubiquitination of NUMB. Contrary to p53, where two separate domains form the interface with MDM2, only one region within the phosphotyrosine binding domain of NUMB (amino acids 113-148) mediates binding to both these regions of MDM2. By binding to both domains on MDM2, NUMB disrupts the MDM2-p53 complex and MDM2-catalyzed ubiquitination of p53. Therefore, we have identified the mechanism NUMB uses to regulate the steady-state levels of the p53 in cells. By targeting the acidic domain of MDM2 using acid domain-binding ligands we can overcome MDM2-mediated ubiquitination and degradation of NUMB impacting on the stabilization of p53 in cells. Furthermore, delivery of MDM2 acid domain-binding ligands to cancer cells promotes p53-dependent growth arrest and the induction of apoptosis. This highlights the dual-site mechanism of MDM2 on another physiological substrate and identifies the acid domain as well as N terminus as a potential target for small molecules that inhibit MDM2.     

The alternative translated MDMXp60 isoform regulates MDM2 activity

Isoforms derived from alternative splicing, mRNA translation initiation or promoter usage extend the functional repertoire of the p53, p63 and p73 genes family and of their regulators MDM2 and MDMX. Here we show cap-independent translation of an N-terminal truncated isoform of hMDMX, hMDMX^p60, which is initiated at the 7th AUG codon downstream of the initiation site for full length hMDMX^FL at position +384. hMDMX^p60 lacks the p53 binding motif but retains the RING domain and interacts with hMDM2 and hMDMX^FL. hMDMX^p60 shows higher affinity for hMDM2, as compared to hMDMX^FL. In vitro data reveal a positive cooperative interaction between hMDMX^p60 and hMDM2 and in cellulo data show that low levels of hMDMX^p60 promote degradation of hMDM2 whereas higher levels stabilize hMDM2 and prevent hMDM2-mediated degradation of hMDMX^FL. These results describe a novel alternatively translated hMDMX isoform that exhibits unique regulatory activity toward hMDM2 autoubiquitination. The data illustrate how the N-terminus of hMDMX regulates its C-terminal RING domain and the hMDM2 activity.     

The tumor suppressor p53 and the oncoprotein simian virus 40 T antigen bind to overlapping domains on the MDM2 protein.

The oncogene mdm2 has been found to be amplified in human sarcomas, and the gene product binds to the tumor suppressor p53. In this report, we describe the dissection of the MDM2-binding domain on p53 as well as the p53-binding domain on MDM2. We also demonstrate that the oncoprotein simian virus 40 T antigen binds to the product of cellular oncogene mdm2. We have constructed several N- and C-terminal deletion mutants of p53 and MDM2, expressed them in vitro, and assayed their in vitro association capability. The N-terminal boundary of the p53-binding domain on MDM2 is between amino acids 1 and 58, while the C-terminal boundary is between amino acids 221 and 155. T antigen binds to an overlapping domain on the MDM2 protein. On the other hand, the MDM2-binding domain of p53 is defined by amino acids 1 and 159 at the N terminus. At the C terminus, binding is progressively reduced as amino acids 327 to 145 are deleted. We determined the effect of human MDM2 on the transactivation ability of wild-type human p53 in the Saos-2 osteosarcoma cell line, which does not have any endogenous p53. Human MDM2 inhibited the ability of human p53 to transactivate the promoter with p53-binding sites. Thus, human MDM2 protein, like the murine protein, can inactivate the transactivation ability of human p53. Interestingly, both the transactivation domain and the MDM2-binding domain of p53 are situated near the N terminus. We further show that deletion of the N-terminal 58 amino acids of MDM2, which eliminates p53 binding, also abolishes the capability of inactivating p53-mediated transactivation. This finding suggests a correlation of in vitro p53-MDM2 binding with MDM2's ability in vivo to interfere with p53-mediated transactivation.     

MDM2--master regulator of the p53 tumor suppressor protein

MDM2 is an oncogene that mainly functions to modulate p53 tumor suppressor activity. In normal cells the MDM2 protein binds to the p53 protein and maintains p53 at low levels by increasing its susceptibility to proteolysis by the 26S proteosome. Immediately after the application of cellular stress, the ability of MDM2 to bind to p53 is blocked or altered in a fashion that prevents MDM2-mediated degradation. As a result, p53 levels rise, causing cell cycle arrest or apoptosis. In this review, we present evidence for the existence of three highly conserved regions (CRs) shared by MDM2 proteins and MDMX proteins of different species. These highly conserved regions encompass residues 42-94 (CR1), 301-329 (CR2), and 444-483 (CR3) on human MDM2. These three domains are respectively important for binding p53, for binding the retinoblastoma protein, and for transferring ubiquitin to p53. This review discusses the major milestones uncovered in MDM2 research during the past 12 years and potential uses of this knowledge in the fight against cancer.     

The alternative translated MDMXp60 isoform regulates MDM2 activity

Isoforms derived from alternative splicing, mRNA translation initiation or promoter usage extend the functional repertoire of the p53, p63 and p73 genes family and of their regulators MDM2 and MDMX. Here we show cap-independent translation of an N-terminal truncated isoform of hMDMX, hMDMX^p60, which is initiated at the 7th AUG codon downstream of the initiation site for full length hMDMX^FL at position +384. hMDMX^p60 lacks the p53 binding motif but retains the RING domain and interacts with hMDM2 and hMDMX^FL. hMDMX^p60 shows higher affinity for hMDM2, as compared to hMDMX^FL. In vitro data reveal a positive cooperative interaction between hMDMX^p60 and hMDM2 and in cellulo data show that low levels of hMDMX^p60 promote degradation of hMDM2 whereas higher levels stabilize hMDM2 and prevent hMDM2-mediated degradation of hMDMX^FL. These results describe a novel alternatively translated hMDMX isoform that exhibits unique regulatory activity toward hMDM2 autoubiquitination. The data illustrate how the N-terminus of hMDMX regulates its C-terminal RING domain and the hMDM2 activity.     

Identification of a Catalytic Active but Non-Aggregating MDM2 RING Domain Variant

As a key regulator of the tumour suppressor protein p53, MDM2 is involved in various types of cancer and has thus been an attractive drug target. So far, small molecule design has primarily focussed on the N-terminal p53-binding domain although on-target toxicity effects have been reported. Targeting the catalytic RING domain of MDM2 resembles an alternative approach to drug MDM2 with the idea to prevent MDM2-mediated ubiquitination of p53 while retaining MDM2′s ability to bind p53. The design of RING inhibitors has been limited by the extensive aggregation tendency of the RING domain, making it challenging to undertake co-crystallization attempts with potential inhibitors. Here we compare the purification profiles of the MDM2 RING domain from several species and show that the MDM2 RING domain of other species than human is much less prone to aggregate although the overall structure of the RING domain is conserved. Through sequence comparison and mutagenesis analyses, we identify a single point mutation, G443T, which greatly enhances the dimeric fraction of human MDM2 RING domain during purification. Neither does the mutation alter the structure of the RING domain, nor does it affect E2(UbcH5B)–Ub binding and activity. Hence, MDM2-G443T facilitates studies involving binding partners that would be hampered by the low solubility of the wild-type RING domain. Furthermore, it will be valuable for the development of MDM2 RING inhibitors.     

Aberrant expression of nucleostemin activates p53 and induces cell cycle arrest via inhibition of MDM2

The nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis. Both depletion and overexpression of NS reduce cell proliferation. However, the mechanisms underlying this regulation are still unclear. Here, we show that NS regulates p53 activity through the inhibition of MDM2. NS binds to the central acidic domain of MDM2 and inhibits MDM2-mediated p53 ubiquitylation and degradation. Consequently, ectopic overexpression of NS activates p53, induces G(1) cell cycle arrest, and inhibits cell proliferation. Interestingly, the knockdown of NS by small interfering RNA also activates p53 and induces G(1) arrest. These effects require the ribosomal proteins L5 and L11, since the depletion of NS enhanced their interactions with MDM2 and the knockdown of L5 or L11 abrogated the NS depletion-induced p53 activation and cell cycle arrest. These results suggest that a p53-dependent cell cycle checkpoint monitors changes of cellular NS levels via the impediment of MDM2 function.     

Tetraploid state induces p53-dependent arrest of nontransformed mammalian cells in G1

A "spindle assembly" checkpoint has been described that arrests cells in G1 following inappropriate exit from mitosis in the presence of microtubule inhibitors. We have here addressed the question of whether the resulting tetraploid state itself, rather than failure of spindle function or induction of spindle damage, acts as a checkpoint to arrest cells in G1. Dihydrocytochalasin B induces cleavage failure in cells where spindle function and chromatid segregation are both normal. Notably, we show here that nontransformed REF-52 cells arrest indefinitely in tetraploid G1 following cleavage failure. The spindle assembly checkpoint and the tetraploidization checkpoint that we describe here are likely to be equivalent. Both involve arrest in G1 with inactive cdk2 kinase, hypophosphorylated retinoblastoma protein, and elevated levels of p21(WAF1) and cyclin E. Furthermore, both require p53. We show that failure to arrest in G1 following tetraploidization rapidly results in aneuploidy. Similar tetraploid G1 arrest results have been obtained with mouse NIH3T3 and human IMR-90 cells. Thus, we propose that a general checkpoint control acts in G1 to recognize tetraploid cells and induce their arrest and thereby prevents the propagation of errors of late mitosis and the generation of aneuploidy. As such, the tetraploidy checkpoint may be a critical activity of p53 in its role of ensuring genomic integrity.     

Mutant p53 can substitute for human papillomavirus type 16 E6 in immortalization of human keratinocytes but does not have E6-associated trans-activation or transforming activity.

Human papillomavirus type 16 (HPV16) E6 and E7 are selectively retained and expressed in HPV16-associated human genital tumors. E6 is active in several cell culture assays, including transformation of NIH 3T3 cells, trans activation of the adenovirus E2 promoter, and cooperation with E7 to immortalize normal human keratinocytes. Biochemically, the HPV16 E6 protein has been shown to bind to tumor suppressor protein p53 in vitro and induce its degradation in a rabbit reticulocyte lysate. To examine the relationship between the various biological activities of E6 and inactivation of p53, we tested the abilities of dominant negative mutants of p53 to substitute functionally for E6 in the three cell culture assays. While wild-type p53 inhibited keratinocyte proliferation, both mouse and human mutant p53s, in conjunction with E7, increased proliferation of the keratinocytes, resulting in generation of immortalized lines. However, in contrast to E6, mutant p53 was unable to induce transformation or trans activate the adenovirus E2 promoter in NIH 3T3 cells. These results suggest that inactivation of wild-type p53 is necessary for HPV-induced immortalization of human keratinocytes and that different or additional activities are required for E6-dependent transformation and trans activation of NIH 3T3 cells.     

Syndecan-2 mediates adhesion and proliferation of colon carcinoma cells

Syndecan-2 is a transmembrane heparan sulfate proteoglycan whose function at the cell surface is unclear. In this study, we examined the function of syndecan-2 in colon cancer cell lines. In several colon cancer cell lines, syndecan-2 was highly expressed compared with normal cell lines. In contrast, syndecan-1 and -4 were decreased. Cell biological studies using the extracellular domain of recombinant syndecan-2 (2E) or spreading assay with syndecan-2 antibody-coated plates showed that syndecan-2 mediated adhesion and cytoskeletal organization of colon cancer cells. This interaction was critical for the proliferation of colon carcinoma cells. Blocking with 2E or antisense syndecan-2 cDNA induced G(0)/G(1) cell cycle arrest with concomitantly increased expression of p21, p27, and p53. Furthermore, blocking of syndecan-2 through antisense syndecan-2 cDNA significantly reduced tumorigenic activity in colon carcinoma cells. Therefore, increased syndecan-2 expression appears to be a critical for colon carcinoma cell behavior, and syndecan-2 regulates tumorigenic activity through regulation of adhesion and proliferation in colon carcinoma cells.     

RYBP stabilizes p53 by modulating MDM2

The mouse double minute 2 (MDM2)–p53 interaction regulates the activity of p53 and is a potential target for human cancer therapy. Here, we report that RYBP (RING1‐ and YY1‐binding protein), a member of the polycomb group (PcG), interacts with MDM2 and decreases MDM2‐mediated p53 ubiquitination, leading to stabilization of p53 and an increase in p53 activity. RYBP induces cell‐cycle arrest and is involved in the p53 response to DNA damage. Expression of RYBP is decreased in human cancer tissues compared with adjacent normal tissues. These results show that RYBP is a new regulator of the MDM2–p53 loop and that it has tumour suppressor activity.