[Show abstract][Hide abstract] ABSTRACT: MDM2-A is a common splice variant of murine double minute 2 (MDM2) that is frequently detected in many tumor types. Our previous work has characterized MDM2-A as an activator of p53, and therefore, in a wild-type p53 background, this splice variant would be predicted to confer p53-dependent tumor protection. To test this hypothesis, we used Mdm2-a transgenic mice to assess transformation and tumorigenesis in tumor susceptible murine models. A MDM2-A-dependent decrease in transformation was observed in Arf-null mouse embryonic fibroblasts (MEF) or when wild-type MEFs were exposed to the carcinogen ethylnitrosourea. However, this reduced transformation did not confer tumor protection in vivo; Mdm2-a/Arf-null mice and ethylnitrosourea-treated MDM2-expressing mice developed similar tumor types with equivalent latency compared with their respective controls. Interestingly, when p53 was deleted, MDM2-A expression enhanced transformation of p53-null MEFs and altered tumor spectrum in vivo. In addition, p53-heterozygous mice that expressed MDM2-A developed aggressive mammary tumors that were not observed in p53-heterozygous controls. In conclusion, we found that although MDM2-A expression enhances p53 activity and decreases transformation in vitro, it cannot confer tumor protection. In contrast, MDM2-A seems to exhibit a novel transforming potential in cells where p53 function is compromised. These data show that MDM2 splice variants, such as MDM2-A, may provide protection against transformation of normal tissues having intact p53. However, when such splice variants are expressed in tumors that have defects in the p53 pathway, these isoforms may contribute to tumor progression, which could explain why their expression is often associated with aggressive tumor types.
Full-text · Article · Jul 2009 · Molecular Cancer Research
[Show abstract][Hide abstract] ABSTRACT: MDM2 is the predominant negative regulator of p53 that functions to maintain the appropriate level of expression and activity of this central tumor suppressor. Mdm2-a is a commonly identified splice variant of Mdm2; however, its physiological function is unclear. To gain insight into the activity of MDM2-A and its potential impact on p53, an Mdm2-a transgenic mouse model was generated. Mdm2-a transgenic mice displayed a homozygous-lethal phenotype that could be rescued by a reduction in p53 expression, demonstrating a dependence upon p53. Mdm2-a hemizygous mice exhibited reduced longevity, and enhanced senescence was observed in their salivary glands. In addition, the transgenic mice lacked typical, accelerated aging phenotypes. Growth of transgenic mouse embryonic fibroblasts (MEFs) was inhibited relative to wild-type MEFs, and MDM2-A was shown to bind to full-length MDM2 in an interaction that could increase p53 activity via reduced MDM2 inhibition. Evidence of p53 activation was shown in the Mdm2-a transgenic MEFs, including p53-dependent growth inhibition and elevated expression of the p53 target protein p21. In addition, MDM2-A increased senescence in a p21-independent manner. In conclusion, unexpected roles for MDM2-A in longevity and senescence were identified in a transgenic mouse model, suggesting that Mdm2 splice variants might be determinants of these phenotypes in vivo.
Preview · Article · Feb 2009 · Disease Models and Mechanisms
[Show abstract][Hide abstract] ABSTRACT: The generation of transgenic mouse models to study in vivo functions of specific proteins has become common practice. In addition, PCR technology allows efficient and rapid identification of founder mice by the analysis of tail tip DNA. Whilst the DNA construct used in the microinjection of one-cell-stage embryos is usually sequenced it is not common practice to sequence the PCR product once the transgene has been inserted into the mouse genome. In this report we describe why sequencing of inserted transgenes is important. Upon generation of transgenic mice expressing a splice variant of MDM2, MDM2-A, three of four founders contained mutations within the Mdm2-a cDNA sequence. The observation that selection against expression of wild-type MDM2-A resulted in the generation of mice expressing mutant transgenes highlights the importance of sequencing the transgenes of founder mice.
No preview · Article · Sep 2007 · Transgenic Research
[Show abstract][Hide abstract] ABSTRACT: Of the >40 alternative and aberrant splice variants of MDM2 that have been described to date, the majority has lost both the well-characterized nuclear localization signal (NLS1) and the nuclear export signal (NES) sequence. Because cellular localization of proteins provides insight regarding their potential function, we determined the localization of three different MDM2 splice variants. The splice variants chosen were the common variants MDM2-A and MDM2-B. In addition, MDM2-FB26 was chosen because it is one of the few variants described that contains the complete p53-binding site. All three splice variants predominantly localized to the nucleus. Nuclear localization of MDM2-A and MDM2-B was controlled by a previously uncharacterized nuclear localization signal (NLS2), whereas nucleoplasmic localization of MDM2-FB26 was mediated by NLS1. p53 and full-length MDM2 colocalized with the splice variants in the nucleus. MDM2-A and MDM2-B both contain a COOH-terminal RING finger domain, and interaction with full-length MDM2 through this domain was confirmed. MDM2-FB26 was the only splice variant evaluated that contained a p53-binding domain; however, interaction between MDM2-FB26 and p53 could not be shown. p14(ARF) did not colocalize with the splice variants and was predominantly expressed within the nucleoli. In summary, nuclear localization signals responsible for the nucleoplasmic distribution of MDM2 splice variants have been characterized. Colocalization and interaction of MDM2-A and MDM2-B with full-length MDM2 in the nucleus have important physiologic consequences, for example, deregulation of p53 activity.
Preview · Article · Apr 2007 · Molecular Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Deregulated expression of oncogenes such as MYC and PAX3-FKHR often occurs in rhabdomyosarcomas. MYC can enhance cell proliferation and apoptosis under specific conditions, whereas PAX3-FKHR has only been described as anti-apoptotic.
In order to evaluate how MYC and PAX3-FKHR oncogenes influenced p53-mediated apoptosis, rhabdomyosarcoma cells were developed to independently express MYC and PAX3-FKHR cDNAs. Exogenous wild-type p53 expression in MYC transfected cells resulted in apoptosis, whereas there was only a slight effect in those transfected with PAX3-FKHR. Both oncoproteins induced BAX, but BAX induction alone without expression of wild-type p53 was insufficient to induce apoptosis. Data generated from genetically modified MEFs suggested that expression of all three proteins; MYC, BAX and p53, was required for maximal cell death to occur.
We conclude that cooperation between p53 and oncoproteins to induce apoptosis is dependent upon the specific oncoprotein expressed and that oncogene-mediated induction of BAX is necessary but insufficient to enhance p53-mediated apoptosis. These data demonstrate a novel relationship between MYC and p53-dependent apoptosis, independent of the ability of MYC to induce p53 that may be important in transformed cells other than rhabdomyosarcoma.
[Show abstract][Hide abstract] ABSTRACT: Renal cancer consists of a heterogeneous tumor group which is characterized by complex cytogenetic and molecular genetic abnormalities. In this study, three different techniques were applied to screen renal cancers for genetic alterations. We studied 99 primary, sporadic renal cancers (96 renal cell carcinomas and three other renal cancers) by a comparative evaluation of different microsatellite markers, comparative genomic hybridization (CGH) and AP-PCR. The AP-PCR produces a genomic fingerprint after an AluEI DNA restriction digest of tumor DNA samples. Microsatellite alterations were investigated using nine microsatellite markers spanning well-known regions of FhiT and VHL (3p14.2, 3p26) but also of oncogenes and tumor suppressor genes like Myc-L1 and TP53Alu (1p32, 17p13.1). To receive a genomic fingerprint, AP-PCR was carried out for all patient samples. Performing AP-PCR, only one case out of 99 displayed genomic imbalance. Seven of 99 investigated primary renal cancers showed alterations in up to four microsatellite loci (TP53Alu, Myc-L1, D3S1300, D3S1560, D3S1317, D3S4260). Three markers (Bat25, Bat26, REN) did not reveal any aberrations within the tested tumor samples. Six cases with microsatellite alterations and four without were examined by CGH. Five samples yielded aberrations, four of them were positive for microsatellite alterations. Only one tumor sample displayed microsatellite alterations, shift patterns in AP-PCR and alterations analyzed by CGH. Our data suggest that genomic aberrations found by microsatellite analysis are also detectable by CGH with the restriction of a minimum of alterated DNA of >10 Mb. Based on this study of RCC and in contrast to other reports for solid tumors, we conclude that AP-PCR is far less informative in investigation of renal cancers.
No preview · Article · Jul 2004 · International Journal of Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: Few studies have investigated the loss of heterozygosity and microsatellite instability in soft tissue sarcomas. Therefore, we analyzed samples of human soft tissue sarcomas to determine the status of the chromosomal region 12q14-15, which contains the MDM2 gene encoding the well-known counterpart of the tumor suppressor p53. In addition, we determined whether an amplified MDM2 gene was present in the samples. Of the 88 soft tissue sarcoma samples, 24 (27%) showed evidence of loss of heterozygosity of markers representing 12q14-15, and 12 (14%) showed evidence of microsatellite instability. Of the 72 samples analyzed by semiquantitative polymerase chain reaction, 15 (21%) possessed an amplified MDM2 gene. Loss of heterozygosity (P =.008) and microsatellite instability (P =.035) were significantly more common in Stage I tumors than in higher stage tumors. This result indicated that these alterations occur early in soft tissue sarcoma progression and possibly define a subgroup of soft tissue sarcoma. Surprisingly, MDM2 amplification in soft tissue sarcoma patients was associated with a prognosis better than that of patients without the amplification; however, this difference was not statistically significant (P =.6). Furthermore, of the tumors with an MDM2 amplification, 40% (6/15) also experienced loss of heterozygosity at 12q14-15; in contrast, only 16% of tumors without an MDM2 amplification (9/57) underwent a loss of heterozygosity. A concomitant occurrence of deletions and amplifications resulting from deficiencies in the nonhomologous end-joining pathway could in part explain this finding.
[Show abstract][Hide abstract] ABSTRACT: Deregulation of DNA mismatch repair is a common mechanism for the development of hereditary nonpolyposis colon carcinoma and related familiar cancers, but it also plays a role in the tumorigenesis of sporadic cancers. Although the protein expression of the two main components of DNA mismatch repair, hMSH2 and hMLH1, has been described in soft tissue sarcoma (STS) patients, its prognostic impact is yet to be determined.
The authors investigated the expression of the DNA repair proteins hMSH2 and hMLH1 by Western blot analysis in tumor samples of 57 STS patients. The correlation between the expression of hMSH2/hMLH1 and survival was studied in a Cox proportional hazards regression model, which was adjusted for the prognostic effects of staging, tumor entity, and radicality of tumor resection.
Nine of 57 STS (16%) showed reduced expression of hMSH2 and reduced expression of hMLH1 was detected in seven STS patients (12%). In a Kaplan-Meier analysis, the median survival for patients with reduced expression of the hMSH2 protein was 18 months, whereas the patients with a normal expression of hMSH2 survived for an average of 68 months. A multivariate Cox proportional hazards regression model revealed a significant correlation between the reduced expression of the hMSH2 protein and poor survival (relative risk = 4.7; 95% confidence interval [CI]: 1.3-17.2; P = 0.019).
Reduced expression of the hMSH2 protein is an independent negative prognostic factor for STS patients.