Murine Double Minute 2 Expression Research Articles

Identification of functional DNA variants in the constitutive promoter region of MDM2

Although mutations in the oncoprotein murine double minute 2 (MDM2) are rare, MDM2 gene overexpression has been observed in several human tumors. Given that even modest changes in MDM2 levels might influence the p53 tumor suppressor signaling pathway, we postulated that sequence variation in the promoter region of MDM2 could lead to disregulated expression and variation in gene dosage. Two promoters have been reported for MDM2; an internal promoter (P2), which is located near the end of intron 1 and is p53-responsive, and an upstream constitutive promoter (P1), which is p53-independent. Both promoter regions contain DNA variants that could influence the expression levels of MDM2, including the well-studied single nucleotide polymorphism (SNP) SNP309, which is located in the promoter P2; i.e., upstream of exon 2. In this report, we screened the promoter P1 for DNA variants and assessed the functional impact of the corresponding SNPs. Using the dbSNP database and genotyping validation in individuals of European descent, we identified three common SNPs (−1494 G > A; indel 40 bp; and −182 C > G). Three major promoter haplotypes were inferred by using these three promoter SNPs together with rs2279744 (SNP309). Following subcloning into a gene reporter system, we found that two of the haplotypes significantly influenced MDM2 promoter activity in a haplotype-specific manner. Site-directed mutagenesis experiments indicated that the 40 bp insertion/deletion variation is causing the observed allelic promoter activity. This study suggests that part of the variability in the MDM2 expression levels could be explained by allelic p53-independent P1 promoter activity.

Murine double minute‐2 expression is required for capillary maintenance and exercise‐induced angiogenesis in skeletal muscle

Exercise-induced angiogenesis is a key determinant of skeletal muscle function. Here, we investigated whether the E3 ubiquitin ligase murine double minute-2 (Mdm2) exerts a proangiogenic function in exercised skeletal muscle. Mdm2 hypomorphic (Mdm2(Puro/Δ7-9)) mice have a 60% reduction in Mdm2 expression compared with that in wild-type animals. Capillary staining on muscle sections from Mdm2(Puro/Δ7-9) sedentary mice with a wild-type or knockout background for p53 revealed that deficiency in Mdm2 resulted in 20% capillary regression independently of p53 status. In response to one bout of exercise, protein expression of the proangiogenic vascular endothelial growth factor-A (VEGF-A) was increased by 64% in muscle from wild-type animals, and endothelial cell outgrowth from exercised muscle biopsy samples cultured in a 3-dimensional collagen gel was enhanced by 37%. These proangiogenic responses to exercise were impaired in exercised Mdm2(Puro/Δ7-9) mice. Prolonged exercise training resulted in increased Mdm2 protein expression (+49%) and capillarization (+24%) in wild-type muscles. However, exercise training-induced angiogenesis was abolished in Mdm2(Puro/Δ7-9) mice. Finally, exercise training restored Mdm2, VEGF-A, and capillarization levels in skeletal muscles from obese Zucker diabetic fatty rats compared with those in healthy animals. Our results define Mdm2 as a crucial regulator of capillary maintenance and exercise-induced angiogenesis in skeletal muscle.

MDM2 expression is repressed by the RNA-binding protein RNPC1 via mRNA stability

The RNA-binding protein RNPC1 is a target of the p53 family and forms a feedback regulatory loop with the p53 family proteins. The MDM2 oncogene, a key negative regulator of p53, plays a critical role in a variety of fundamental cellular processes. MDM2 expression is found to be regulated via gene amplification, transcription, protein translation, and protein stability. In the current study, we reported a novel regulation of MDM2 by RNPC1 via mRNA stability. Specifically, we found that over-expression of RNPC1 decreases, whereas knockdown or knockout of RNPC1 increases, the level of MDM2 transcript and protein independent of p53. To uncover the underlying mechanism, we found that RNPC1 is able to destabilize the MDM2 transcript via binding to multiple AU-/U-rich elements in MDM2 3′UTR. Consistent with this, we showed that RNPC1 inhibits expression of exogenous MDM2 from a expression vector as long as the vector contains an AU-/U-rich element from MDM2 3′UTR. Finally, we showed that the RNA-binding activity of RNPC1 is required for binding to MDM2 transcript and consequently, for inhibiting MDM2 expression. Together, we uncover a novel regulation of MDM2 by the RNA-binding protein RNPC1 via mRNA stability.

Ras homolog gene family, member A promotes p53 degradation and vascular endothelial growth factor‐dependent angiogenesis through an interaction with murine double minute 2 under hypoxic conditions

Tumor neovascularization (TNV) is a common pathologic basis for malignant growth and metastasis. However, the mechanism of TNV pathogenesis is not fully understood. Ras homolog gene family, member A (RhoA), a Rho guanosine triphosphatase (GTPase) family member, may be involved in a hypoxia-induced vascular endothelial growth factor (VEGF) pathway that regulates TNV angiogenesis through an unclear mechanism. The regulation of RhoA on p53, the p53 binding protein homolog murine double minute 2 (MDM2), and VEGF was analyzed in hypoxic MCF-7 cells using Western blot analysis, real-time polymerase chain reaction (PCR) analysis, coimmunoprecipitation, and immunofluorescence staining assays. Changes in proliferation, invasion, migration, stress fiber formation, and tube formation were detected in an MCF-7 human umbilical vein endothelial cell (HUVEC) coculture system. Correlations of RhoA expression with MDM2, wild-type p53 (wt-p53), and VEGF expression in breast cancer tissues and relations between RhoA and breast cancer clinical features were analyzed by immunohistochemistry. Activated RhoA down-regulated p53 protein, which increased VEGF expression in hypoxic MCF-7 cells; whereas p53 messenger RNA levels were not altered. In addition, the ubiquitin-mediated degradation of p53 was enhanced by active RhoA. RhoA and MDM2 colocalized in the cytoplasm of hypoxic MCF-7 cells and interacted with each other physically. Furthermore, nutlin-3, a specific MDM2 inhibitor, was capable of reducing activated RhoA-induced p53 protein stability and attenuating VEGF accumulation. In an MCF-7-HUVEC coculture system, nutlin-3 effectively inhibited HUVEC proliferation, invasion, migration, stress fiber formation, and tube formation mediated by activated RhoA under hypoxic conditions. Data from 129 clinical breast cancer specimens with wt-p53 revealed that high RhoA expression was correlated with high MDM2 expression, low wt-p53 expression, and high VEGF expression. The current data suggested that activated RhoA promotes VEGF expression and hypoxia-induced angiogenesis through the up-regulation of MDM2 to decrease p53 stability.

Study of the Murine Double Minute 2 status in patients with gastric and colorectal carcinomas and its correlation with prognostic factors

Gastric and colorectal cancers are the second and the fourth most common cancers in Iran, respectively. The presence of Murine Double Minute 2 (MDM2) has been identified in many cancers and its relationship with prognosis is under investigation. This study aimed to assess the status of MDM2 and its relationship with prognostic factors in gastric and colorectal carcinoma. This study was performed on 99 paraffin blocks of gastric and colorectal cancers, during the years 2001 to 2007 from Mostafa Khomeini Hospital, Tehran, Iran. Tissue sections were prepared, stained with Hematoxylin and Eosin and immunohistochemistry to evaluate for MDM2 expression. The type of tumor, lymph node involvement and tumor grade was determined. Of the 99 cases, 34.3% and 65.7% cases were diagnosed with gastric and colorectal adenocarcinoma, respectively. The average tumor size was 5.5 cm. MDM2 expression level was 82.4% and 90.8% in gastric and colorectal adenocarcinoma, respectively. No statistical difference was found between MDM2 expression and various prognostic factors; however, significant correlation was observed between gastric (P = 0.03) and colorectal (P = 0.03) tumor size and the percentage of MDM2 immunoreactivity. Considering the role of MDM2 in cell growth and its positive correlation with tumor size (an established prognostic factor), it can be indirectly concluded that MDM2 is also important in prognosis. However, additional investigation is needed.

MEK-ERK Signaling Dictates DNA-Repair Gene MGMT Expression and Temozolomide Resistance of Stem-Like Glioblastoma Cells via the MDM2-p53 Axis

Overcoming the resistance of glioblastoma cells against temozolomide, the first-line chemotherapeutic agent of choice for newly diagnosed glioblastoma, is a major therapeutic challenge in the management of this deadly brain tumor. The gene encoding O(6) -methylguanine DNA methyltransferase (MGMT), which removes the methyl group attached by temozolomide, is often silenced by promoter methylation in glioblastoma but is nevertheless expressed in a significant fraction of cases and is therefore regarded as one of the most clinically relevant mechanisms of resistance against temozolomide. However, to date, signaling pathways regulating MGMT in MGMT-expressing glioblastoma cells have been poorly delineated. Here in this study, we provide lines of evidence that the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK)-murine double minute 2 (MDM2)-p53 pathway plays a critical role in the regulation of MGMT expression, using stem-like glioblastoma cells directly derived from patient tumor samples and maintained in the absence of serum, which not only possess stem-like properties but are also known to phenocopy the characteristics of the original tumors from which they are derived. We show that, in stem-like glioblastoma cells, MEK inhibition reduced MDM2 expression and that inhibition of either MEK or MDM2 resulted in p53 activation accompanied by p53-dependent downregulation of MGMT expression. MEK inhibition rendered otherwise resistant stem-like glioblastoma cells sensitive to temozolomide, and combination of MEK inhibitor and temozolomide treatments effectively deprived stem-like glioblastoma cells of their tumorigenic potential. Our findings suggest that targeting of the MEK-ERK-MDM2-p53 pathway in combination with temozolomide could be a novel and promising therapeutic strategy in the treatment of glioblastoma.

Plumbagin induces apoptosis via the p53 pathway and generation of reactive oxygen species in human osteosarcoma cells

Osteosarcoma, which is the most common primary bone tumor, occurs most frequently in adolescents. A number of studies have indicated that plumbagin (PL) (5-hydroxy-2-methyl-1, 4-naphthoquinone), a compound found in the plants of the Plumbaginaceae and Droseraceae families, possesses anticancer activity. However, its anticancer effects and mechanisms against osteosarcoma have not been explored. To determine the anticancer effect of PL on osteosarcoma cell lines MG-63 and U2OS, cell viability, apoptosis, cell cycle distribution, caspase-3 and caspase-9 activity and intracellular reactive oxygen species (ROS) generation were measured, and Western blot analyses were performed. PL significantly inhibited the growth of osteosarcoma cells, particularly U2OS cells. PL up-regulated the expression of p53 in U2OS cells and p21 in the two osteosarcoma cell lines causing cell cycle arrest by decreasing the expression of murine double minute 2 (MDM2)/cyclin B1 and cyclin D1. Furthermore, PL altered the ratio of Bax/Bcl-2, and may have triggered the mitochondrial apoptotic pathway, resulting in caspase-3 and caspase-9 activation. We also found that PL induced the generation of ROS in osteosarcoma cell lines. To conclude, PL exerted anticancer activity on osteosarcoma cells by inducing pro-apoptotic signaling and modulating the intracellular ROS that causes induction of apoptosis. These effects may relate to the p53 status.

Immunohistochemical Comparison of the Expression of p53 and MDM2 Proteins in Ameloblastomas and Keratocystic Odontogenic Tumors

Ameloblastoma and keratocystic odontogenic tumor (KOT) is characterized by a benign but locally invasive behavior with a high risk of recurrence. MDM2 (murine double minute 2), an amplifier of cell proliferation, and p53, a tumor suppressor gene, are overexpressed in some odontogenic lesions. The aim of this study was to compare the expression of MDM2 and p53 in ameloblastoma and KOT as 2 lesions with similar biologic behavior, by immunohistochemistry. The expressions of MDM2 and p53 proteins were determined in 39 ameloblastomas (15 follicular types, 15 plexiform types, and 9 unicystic types) and 15 KOTs. P53 protein was expressed in 100% of KOTs and 77.8% of ameloblastomas, and MDM2 was detected in 74.8% of ameloblastomas and 80% of KOTs. There was no statistical difference between MDM2 and p53 expressions in different subtypes of ameloblastomas and also when KOTs were compared with them (P > 0.05). There was a significant difference between immunohistochemical reactivity of MDM2 among subtypes of ameloblastomas (P < 0.05). MDM2 and p53 expressions were positively correlated. Overexpression of p53 and MDM2 is associated with the pathogenesis and oncogenesis of ameloblastomas and KOT. Overexpression of these markers can contribute to similar biologic behavior of these lesions.

Synergistic Suppression of Prostatic Cancer Cells by Coexpression of BothMurine Double Minute 2Small Interfering RNA and Wild-Typep53Gene In Vitro and In Vivo

Our objective was to evaluate cell growth and death effects by inhibiting Murine Double Minute 2 (MDM2) expression in human prostate cancer cells overexpressing the wild-type (WT) p53 gene. Prostate PC-3 tumor cells were transfected with a plasmid containing either mdm2 small interfering (Si-mdm2) or the WT p53 gene (Pp53) alone, or both (Pmp53), using Lipofectamine in vitro and attenuated Salmonella enterica serovar Typhi vaccine strain Ty21a (Salmonella Typhi Ty21a) in vivo. Cell growth, apoptosis, and the expression of related genes and proteins were examined in vitro and in vivo by flow cytometry and Western blot assays. We demonstrated that human prostate tumors had increased expression of MDM2 and mutant p53 proteins. Transfection of the PC-3 cells with the Pmp53 plasmid in vitro offered significant inhibition of cell growth and an increase in apoptotic cell death compared with that of the Si-mdm2 or Pp53 group. These effects were associated with up-regulation of p21 and down-regulation of hypoxia-inducible factor 1α expression in Pmp53-transfected cells. To validate the in vitro findings, the nude mice implanted with PC-3 cells were treated with attenuated Salmonella Typhi Ty21a carrying the plasmids, which showed that the Pmp53 plasmid significantly inhibited the tumor growth rate in vivo compared with that of the Si-mdm2 or Pp53 plasmid alone. Tumor tissues from mice treated with the Pmp53 plasmid showed increased expression of p21 and decreased expression of hypoxia-inducible factor 1α proteins, with an increased apoptotic effect. These results suggest that knockdown of mdm2 expression by its specific small interfering RNA with overexpression of the WT p53 gene offers synergistic inhibition of prostate cancer cell growth in vitro and in vivo.

Subunit 6 of the COP9 signalosome promotes tumorigenesis in mice through stabilization of MDM2 and is upregulated in human cancers

The mammalian constitutive photomorphogenesis 9 (COP9) signalosome (CSN), a protein complex involved in embryonic development, is implicated in cell cycle regulation and the DNA damage response. Its role in tumor development, however, remains unclear. Here, we have shown that the COP9 subunit 6 (CSN6) gene is amplified in human breast cancer specimens, and the CSN6 protein is upregulated in human breast and thyroid tumors. CSN6 expression positively correlated with expression of murine double minute 2 (MDM2), a potent negative regulator of the p53 tumor suppressor. Expression of CSN6 appeared to prevent MDM2 autoubiquitination at lysine 364, resulting in stabilization of MDM2 and degradation of p53. Mice in which Csn6 was deleted died early in embryogenesis (E7.5). Embryos lacking both Csn6 and p53 survived to later in embryonic development (E10.5), which suggests that loss of p53 could partially rescue the effect of loss of Csn6. Mice heterozygous for Csn6 were sensitized to γ-irradiation-induced, p53-dependent apoptosis in both the thymus and the developing CNS. These mice were also less susceptible than wild-type mice to γ-irradiation-induced tumorigenesis. These results suggest that loss of CSN6 enhances p53-mediated tumor suppression in vivo and that CSN6 plays an important role in regulating DNA damage-associated apoptosis and tumorigenesis through control of the MDM2-p53 signaling pathway.

Potent in vitro and in vivo antitumor effects of MDM2 inhibitor nutlin‐3 in gastric cancer cells

The tumor suppressor gene p53 is the most frequently mutated gene in human cancers. However, its mutation rate is relatively low in gastric cancer compared with other cancers. In this study, we investigated the mechanisms underlying the antitumor effects of nutlin-3, an inhibitor of human homolog of murine double minute 2 (MDM2). MDM2 is a negative regulator of p53. Four gastric cancer cell lines with wild-type p53 (wt p53) and three with mutant-type p53 (mt p53) were analyzed for MDM2 and MDM4 expression by immunoblotting, and for their gene amplification by quantitative real-time PCR. Moreover, the viability of cells exposed to nutlin-3 was examined by WST-8 assay, and the expression of p53 and its downstream genes was analyzed by immunoblotting. Nutlin-3 stabilized p53 and increased the expression of p21(WAF1) and Noxa, and cleaved poly (ADP)-ribose polymerase regardless of the pre-expression levels of MDM2 and MDM4 in gastric cancer cells with wt p53. Flow cytometry revealed that nutlin-3 arrested the cell cycle in G(1) phase and induced apoptosis in the cell lines. These nutlin-3 effects were not observed in the cell lines with mt p53. Nutlin-3 exerted additive or synergistic cytotoxicity in combination with 5-fluorouracil or cisplatin in most cell lines with wt p53. An in vivo antitumor effect of nutlin-3 alone and its additive augmentation by 5-fluorouracil were confirmed in an MDM2 overexpressed xenograft tumor model. Nutlin-3 showed potent antitumor activity against human gastric cancer cells with wt p53 and shows promise as a single agent and in combination with conventional anticancer drugs.

Transcription factor Nrf2 maintains the basal expression of Mdm2: An implication of the regulation of p53 signaling by Nrf2

Co-operated regulation of oxidative stress–response transcription factors would be an important issue for animals to determine the cell fate under environmental stress. This notion raises a possibility that NF-E2-related factor 2 (Nrf2), which confers cytoprotection against oxidative stress, and p53 can have a direct co-regulation network. In the current study, we have indentified that the expression of murine double minute 2 (Mdm2) is repressed in nrf2-deleted murine embryonic fibroblasts (MEFs). This was confirmed by microarray, RT-PCR, and immunoblot analyses, and further promoter analysis showed that Nrf2 is directly involved in the basal expression of Mdm2 through the antioxidant response element, which is located in the first intron of this gene. This linkage between Nrf2 and Mdm2 appears to cause the accumulation of p53 protein in nrf2-deficent MEFs. In addition, we show that ovarian carcinoma A2780 cells with Nrf2 shRNA expression displayed higher levels of p53 activation in response to hydrogen peroxide treatment, leading to increased cell death. Collectively, our results suggest novel evidence that the inhibition of Nrf2 can suppress Mdm2 expression, which may result in p53 signaling modulation. In addition, this observation supports the concept that Nrf2 inhibition in cancer cells can facilitate apoptotic response upon environmental stress.

Inhibition of NRF2 in cancer cells enhances apoptotic response and suppresses cell growth:a potential involvement of the p53 signaling

The integrated regulation of oxidative stress‐response transcription factors could be critical for animals to promote the survival of normal cells and to facilitate the removal of abnormal cells with inherited genetic damage. In the current study, we have investigated the effect of NRF2, a cell survival‐promoting factor, on the p53 signaling. In NRF2 inhibited ovarian cancer cells, the growth arrest in G0/G1 phase and the loss of tumorigenecity in mice were accompanied by the increase in p53 transcription activity as well as its target gene expression such as p21 and Gadd45a. In NRF2‐deleted murine embryonic fibroblasts (MEFs), the accumulation of p53 protein together with the suppression of murine double minute 2 (MDM2) expression was observed although accumulated p53 protein might not be functional. Our further promoter analysis showed that NRF2 can be involved in the regulation of basal expression of MDM2 through the proximal ARE in its promoter. Collectively, our results suggest that a novel evidence that the inhibition of NRF2 may stimulate the p53 signaling, which can result in cell growth limitation and facilitation of apoptotic response upon oxidative stress.

P53 and MDM2 in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common type of kidney cancer and follows an unpredictable disease course. To improve prognostication, a better understanding of critical genes associated with disease progression is required. The objective of this review was to focus attention on 2 such genes, p53 and murine double minute 2 (MDM2), and to provide a comprehensive summary and critical analysis of the literature regarding these genes in RCC. Information was compiled by searching the PubMed database for articles that were published or e-published up to April 1, 2009. Search terms included renal cancer, renal cell carcinoma, p53, and MDM2. Full articles and any supplementary data were examined; and, when appropriate, references were checked for additional material. All studies that described assessment of p53 and/or MDM2 in renal cancer were included. The authors concluded that increased p53 expression, but not p53 mutation, is associated with reduced overall survival/more rapid disease progression in RCC. There also was evidence that MDM2 up-regulation is associated with decreased disease-specific survival. Two features of RCC stood out as unusual and will require further investigation. First, increased p53 expression is tightly linked with increased MDM2 expression; and, second, patients who have tumors that display increased p53 and MDM2 expression may have the poorest overall survival. Because there was no evidence to support the conclusion that p53 mutation is associated with poorer survival, it seemed clear that increased p53 expression in RCC occurs independent of mutation. Further investigation of the mechanisms leading to increased p53/MDM2 expression in RCC may lead to improved prognostication and to the identification of novel therapeutic interventions.

Correlation between expression of p53, p21/WAF1, and MDM2 proteins and their prognostic significance in primary hepatocellular carcinoma

BackgroundTumor Protein p53 (p53), cyclin-dependent kinase inhibitor 1A (p21/WAF1), and murine double minute 2 (MDM2) participate in the regulation of cell growth. Altered expression of these gene products has been found in malignant tumors and has been associated with poor prognosis. Our aim was to investigate the expression of the 3 proteins in hepatocellular carcinoma (HCC) and their prognostic significance.MethodsWe examined p53, p21/WAF1, and MDM2 expression in 181 pairs of HCC tissues and the adjacent hepatic tissues by performing immunohistochemistry and examined the expression of the 3 proteins in 7 pairs of HCC tissues and the adjacent hepatic tissues by using western blot analysis.ResultsThe expression of p53, p21/WAF1, and MDM2 in the HCC tissues was significantly higher than those in the adjacent hepatic tissues (P < 0.05). A statistical correlation was observed between p53 and p21/WAF1 expression in HCC tissues (R = 0.195, P = 0.008). A statistical correlation was observed between expression of p53 and p21/WAF1 (R = 0.380, P = 0.000), p53 and MDM2 (R = 0.299, P = 0.000), p21/WAF1 and MDM2 (R = 0.285, P = 0.000) in 181 liver tissues adjacent to the tumor. Patients with a low pathologic grade HCC (I+II) had a higher tendency to express p53 on tumor cells than the patients with high pathologic grade HCC (III+IV) (P = 0.007). Survival analysis showed that positive p21/WAF1 expression or/and negative MDM2 expression in HCC was a predictor of better survival of patients after tumor resection (P < 0.05).ConclusionsThe proteins p53, p21/WAF1, and MDM2 were overexpressed in all the HCC cases in this study, and p53 and p21/WAF1 overexpression were positively correlated. The expression of p21/WAF1 and MDM2 can be considered as 2 useful indicators for predicting the prognosis of HCC.

A polymorphic variant in human MDM4 associates with accelerated age of onset of estrogen receptor negative breast cancer

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.

Expression and significance of p53-related proteins and LMP-1 in nasal NK/T-cell lymphoma

To investigate the expression of murine double minute 2 (mdm2), p53, p21 and latent membrane protein 1 (LMP-1) in nasal NK/T-cell lymphoma (NKTL) and analyze their relationship with the clinical stage and prognosis. The clinicopathological and follow-up data of 62 patients with NKTL proven by pathology were collected. Paraffin-embedded tissue sections were examined for mdm2, p53, p21 and LMP-1 proteins by tissue microarray technique and immunohistochemistry. In situ hybridization was used to detect EBER1/2. The positive expression rates of mdm2, p53, p21 and LMP-1 proteins in NKTL were 61.3%, 79.0%, 58.1% and 48.4%, respectively, and EBER1/2 was 90.3%. The positive expression rates of mdm2 in Ann Arbor stage I, II, III and IV NKTL were 43.5%, 62.5%, 73.3% and 87.5%, respectively; p53 were 69.6%, 75.0%, 86.7% and 100.0%; p21 were 47.8%, 56.3%, 60.0% and 87.5%; while those of LMP-1 were 60.9%, 50.0%, 26.7% and 50.0%, respectively. With the progression of tumor, the positive expression rates of mdm2, p53 and p21 proteins gradually increased. There were statistically significant differences between them (P<0.05). There were statistically positive correlations among those three genes (P<0.05). The prognosis of the positive expression group of these three genes was worse than that of negative expression group (P<0.05). No statistically significant difference was observed between the expression of LMP-1 protein and the clinical stage or prognosis (P>0.05). The expression of p53 protein was an independent prognostic factor. The expression of mdm2, p53 and p21 proteins is closely related with the pathogenesis and progression of NKTL. They are good markers for judgement of the biological behavior of NKTL. The expression of p53 protein is an independent prognostic factor. Though no significant relationship was found between the expression of LMP-1 protein and the clinical stage or prognosis, it may play some role in tumor progression.

The Expression of Murine Double Minute 2 (MDM2) on Helicobacter pylori-Infected Intestinal Metaplasia and Gastric Cancer

The overexpression of murine double minute 2 (MDM2) is found in several human tumors, and increased expression of MDM2 inactivates the apoptotic and cell cycle arrest function of p53. Interleukin-16 (IL-16) is a pleiotrophic cytokine and the properties of IL-16 suggest that it involve in the pathophysiological process of chronic inflammatory diseases. In this study, we investigated the expression of MDM2 in intestinal metaplasia and gastric cancer as well as the effect of H. pylori infection and IL-16 on epithelial cell proliferation and MDM2 expression in gastric cells in vitro. The expression of MDM2 on gastric biopsies was studied immunohistochemistry. AGS cells were incubated with a combination of IL-16 and Helicobacter pylori (H. pylori). Gastric epithelial cell proliferation was studied by BrdU uptake and the expressions of MDM2 were studied by ELISA. There was no significant difference on the expression of MDM2 between with and without H. pylori infected chronic gastritis. In H. pylori infected gastric mucosa; the MDM2 expression was higher on intestinal metaplasia and gastric cancer than chronic gastritis. IL-16 administration was increased MDM2 expression and cell proliferation on AGS cells, which was decreased by H. pylori infection. In conclusion, the expression of MDM2 in long-term H. pylori infected gastric mucosa may indicate a risk for carcinogenesis. IL-16 secretion in H. pylori infected mucosa is one of the factors for gastric cancer. The expression of MDM2 on mucosa can be a mediator for gastric cancer.

Immunohistochemical localization of mdm-2, p27Kip1 and bcl-2 in Warthin's tumor of the parotid gland

BackgroundWarthin's tumor is a benign monomorphic adenoma with unclear origin that almost occur exclusively in the parotid gland. Etiology of Warthin's tumor as well as its malignant potential are still unclear. Therefore immunohistochemical assessment of Warthin's tumor may be useful to detect its origin or its malignant transformation potential.Aims and objectivesThe present study aims to investigate the immunohistochemical expression of murine double minute-2 (mdm-2), p27Kip1 and B cell lymphoma-2 (bcl-2) in Warthin's tumor of parotid gland and also to clarify the role of these proteins in the behavior of that tumor.MethodsTwenty paraffin blocks of cases previously diagnosed as Warthin's tumor were collected for immunohistochemical staining with primary antibodies against mdm-2, p27Kip1 and bcl-2 using streptavidin-biotin immunoperoxidase staining system.ResultsAll cases showed immunopositivity for mdm-2 and p27Kip1 while 18/20 showed bcl-2 immunopositivity. Both layers of the neoplastic epithelial cells that line the cystic spaces showed immunopositivity with all antibodies used. Goblet cells were mdm-2 immunonegative while myoepithelial cells were p27Kip-1 immunonegative. Areas of epithelial proliferation that formed buds were p27Kip-1 and bcl-2 immunopositive.ConclusionMdm-2 played a tumor-suppressor role that might be implicated with the benign behavior of Warthin's tumor. The mutual expression of both p27Kip1 and bcl-2 suggested a protective role of these slowly proliferating cells from apoptosis to maintain their survival and elevated bcl-2 expression offers a significant protection against p27Kip1-mediated apoptosis.

Regulation of Peroxisome Proliferator–Activated Receptor-α by MDM2

Peroxisome proliferator-activated receptor-alpha (PPARalpha) belongs to the nuclear receptor (NR) family of transcription factors and regulates lipid and glucose metabolism. Like other NRs, the regulation of gene expression by PPARalpha depends on cofactor recruitment to the transcription complex and multiple protein-protein interactions. In this study, Murine Double Minute 2 (MDM2), an E3 ubiquitin ligase, is identified as a PPARalpha-interacting protein that regulates PPARalpha transcriptional activity. MDM2 modulated the transcriptional activity of PPARalpha and PPARbeta/delta, but not PPARgamma in reporter assays. Knockdown of MDM2 by small interfering RNA in rat hepatoma cells inhibited ligand-induced mRNA levels of several PPARalpha target genes involved in lipid metabolism. MDM2 associated with PPARalpha on target gene promoters, and this association increased in response to Wy14,643 treatment. MDM2 interacted with PPARalpha and this interaction occurred with the A/B domain of PPARalpha. Coexpression of MDM2 increased PPARalpha ubiquitination and the E3 ubiquitin ligase activity of MDM2 affected PPARalpha protein expression and transcriptional activity. MDM2 expression was decreased in response to clofibrate in wild-type (WT), but not in PPARalpha null mice, indicating a PPARalpha-dependent regulation. These studies identify a role for MDM2 in regulating PPARalpha-mediated pathways of lipid metabolism.