MDM2: life without p53

The MDM2 protein suppresses the ability of p53 to inhibit cellular proliferation or to induce cell death. This property underlies the oncogenic potential of MDM2, which is overexpressed in various human tumours. However, MDM2 also has p53-independent activities, which we focus on here. Similar to other oncogenes, surveillance pathways might counteract the deleterious effects of deregulated MDM2 expression. These pathways need to be inactivated for MDM2 oncogenic activity, which targets p53 but also other proteins.     

Stabilization of the MDM2 oncoprotein by mutant p53

MDM2 is a short-lived protein that regulates p53 degradation. We report here that transient coexpression of MDM2 and several p53 hotspot mutants resulted in stabilization and increased expression of MDM2. Ectopic expression of the mutant p53(175H) allele by recombinant adenovirus infection or stable transfection also stabilized endogenous MDM2 in p53-null cells. A panel of human tumor cell lines expressing different endogenous mutant p53 alleles also contained stabilized nuclear MDM2 at elevated levels when compared with p53-null cells. MDM2 was present in complexes with mutant p53 in tumor cells, and stabilization of MDM2 required direct binding to mutant p53. These results reveal a novel property of mutant p53 and a unique feature of tumors with p53 missense mutations. Accumulation of stable MDM2 may contribute to tumorigenesis through its p53-independent transforming functions.     

MDM2 induces hyperplasia and premalignant lesions when expressed in the basal layer of the epidermis

The MDM2 oncogene is overexpressed in 5-10% of human tumours. Its major physiological role is to inhibit the tumour suppressor p53. However, MDM2 has p53-independent effects on differentiation and does not predispose to tumorigenesis when it is expressed in the granular layer of the epidermis. These unexpected properties of MDM2 could be tissue specific or could depend on the differentiation state of the cells. Strikingly, we found that MDM2 has p53-dependent effects on differentiation, proliferation and apoptosis when it is expressed in the less differentiated basal layer cells. MDM2 inhibits UV induction of p53, the cell cycle inhibitor p21(WAF1/CIP1) and apoptosis ('sunburn cells'). Importantly, MDM2 increases papilloma formation induced by chemical carcinogenesis and predisposes to the appearance of premalignant lesions and squamous cell carcinomas. p53 has a natural role in the protection against UV damage in the basal layer of the epidermis. Our results show that MDM2 predisposes to tumorigenesis when expressed at an early stage of differentiation, and provide a mouse model of MDM2 tumorigenesis relevant to p53's tumour suppressor functions.     

High expression of MDM2 protein and low rate of p21(WAF1/CIP1) expression in SCID mice Epstein Barr virus-induced lymphoproliferation.

To study the prevalence of p53 inactivation and MDM2/p21(WAFI/CIP1) expression in severe combined immunodeficient (SCID) mice Epstein-Barr virus (EBV)-induced lymphoproliferation, 19 samples obtained after ip injection of peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors or lymphoblastoid cell lines (LCL) were analyzed. In all samples tested, overexpression of Ki-67 antigen was shown by immunohistochemistry, indicating a high proliferative index of SCID mice EBV-induced lymphoproliferation. P53 mutations were screened by functional assay in yeast in 14 samples. With this test, a p53-inactivating mutation was found in only one case; the remaining cases exhibited a wild-type p53 pattern. However, an accumulation of p53 protein was detected by immunohistochemistry in six of 19 samples. P21 expression was found in seven of 19 samples but was not correlated with the rate of p53 protein in tumors. In contrast, high levels of nuclear accumulation of MDM2 were found in all samples by immunohistochemistry. These results suggest that a high Ki-67 proliferative index in SCID mice EBV-induced lymphoproliferation is not due to the inactivation of p53 by mutation, but could be associated with an overexpression of MDM2, which would act by a p53-independent mechanism.(J Histochem Cytochem 47:1315-1321, 1999)     

MDM2, an introduction

The murine double minute 2 (mdm2) gene encodes a negative regulator of the p53 tumor suppressor. Amplification of mdm2 or increased expression by unknown mechanisms occurs in many tumors. Thus, increased levels of MDM2 would inactivate the apoptotic and cell cycle arrest functions of p53, as do deletion or mutation of p53, common events in the genesis of many kinds of tumors. MDM2 functions as an E3 ubiquitin ligase to degrade p53. MDM2 also binds another tumor suppressor, ARF. This interaction sequesters MDM2 in the nucleolus away from p53, thus activating p53. Many additional MDM2 interacting proteins have been identified. Functions of MDM2 independent of p53 have also been identified. This article is an introduction to MDM2, its structure and biological functions, as well as its relationship to its binding partners.     

Anti-tumor efficacy of a novel antisense anti-MDM2 mixed-backbone oligonucleotide in human colon cancer models: p53-dependent and p53-independent mechanisms

BACKGROUND: The MDM2 oncogene is amplified or overexpressed in many human cancers and MDM2 levels are associated with poor prognosis. MDM2 not only serves as a negative regulator of p53 but also has p53-independent activities. This study investigates the functions of the MDM2 oncogene in colon cancer growth and the potential value of MDM2 as a drug target for cancer therapy, by inhibiting MDM2 expression with an antisense anti-human-MDM2 oligonucleotide. MATERIALS AND METHODS: The selected antisense mixed-backbone oligonucleotide was evaluated for its in vitro and in vivo antitumor activity in human colon cancer models: LS174T cell line containing wild-type p53 and DLD-1 cell line containing mutant p53. The levels of MDM2, p53 and p21 proteins were quantified by Western blot analysis. RESULTS: In vitro antitumor activity was found in both cell lines, resulting from specific inhibition of MDM2 expression. In vivo antitumor activity of the oligonucleotide occurred in a dose-dependent manner in both models and synergistically or additive therapeutic effects of MDM2 inhibition and the cancer chemotherapeutic agents 10-hydroxycamptothecin and 5-fluorouracil were also observed. CONCLUSIONS: These results suggest that MDM2 have a role in tumor growth through both p53-dependent and p53- independent mechanisms. We speculate that MDM2 inhibitors have a broad spectrum of antitumor activities in human cancers regardless of p53 status. This study should provide a basis for future development of anti-MDM2 antisense oligonucleotides as cancer therapeutic agents used alone or in combination with conventional chemotherapeutics.     

C-Terminal Ubiquitination of p53 Contributes to Nuclear Export

The growth inhibitory functions of p53 are controlled in unstressed cells by rapid degradation of the p53 protein. One of the principal regulators of p53 stability is MDM2, a RING finger protein that functions as an E3 ligase to ubiquitinate p53. MDM2 promotes p53 nuclear export, and in this study, we show that ubiquitination of the C terminus of p53 by MDM2 contributes to the efficient export of p53 from the nucleus to the cytoplasm. In contrast, MDM2 did not promote nuclear export of the p53-related protein, p73. p53 nuclear export was enhanced by overexpression of the export receptor CRM1, although no significant relocalization of MDM2 was seen in response to CRM1. However, nuclear export driven by CRM1 overexpression did not result in the degradation of p53, and nuclear export was not essential for p53 degradation. These results indicate that MDM2 mediated ubiquitination of p53 contributes to both nuclear export and degradation of p53 but that these activities are not absolutely dependent on each other.     

MDM4 enhances p53 stability by promoting an active conformation of the protein upon DNA damage

Stabilization of p53 protein is an important step in the activation of its function. p53 levels are regulated by ubiquitin-dependent and -independent degradation pathways. MDM4 (MDMX) is an important regulator of p53, able to both stimulate and antagonize p53 degradation. Both of these activities have been attributed to the ability of MDM4 to potentiate or antagonize the function of MDM2, the main ubiquitin ligase of p53, depending on their relative levels. Here, we have investigated the stabilizing function of endogenous MDM4 using genetic models of knockout MEFs and RNA interference in human non-transformed cell lines. Our data demonstrate that MDM4 is able to stabilize p53, protecting it from proteasome-mediated degradation in a MDM2- and ubiquitin-independent manner. Upon DNA damage, MDM4 is associated to p53 independently of MDM2 and promotes a conformational change of the protein toward an active form. This correlates with a decreased association of p53 to the proteasome and increased protein levels. The association between MDM4 and p53 is evidenced in the cytoplasmic compartment, supporting the role of cytoplasmic stabilization of p53 during its activation. This work demonstrates that the ability of MDM4 to enhance p53 stability is actually a specific property of MDM4 accomplished upon DNA damage. In addition, these data support the hypothesis of distinct functions of MDM4 under different growth conditions.     

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.