Mouse Double Minute 2 Overexpression Research Articles

TRIM47 silencing inhibits the malignant biological behaviors of prostate cancer cells by regulating MDM2/p53 signaling.

Prostate cancer (PCa) is a prevalent male malignancy globally. Tripartite motif 47 (TRIM47) has been reported to be associated with PCa. However, how TRIM47 acts on PCa is still incompletely understood. Here, we explored the biological roles of TRIM47 in PCa cells and investigated its potential regulatory mechanism. TRIM47 expression in PCa cells was detected by qRT-PCR and western blot. After TRIM47 silencing, the viability of PCa cells was measured using CCK-8 method. Flow cytometry was employed to estimate cell cycle. Cell apoptotic level was subjected to appraisement with TUNEL assay. Additionally, wound healing- and transwell assays were adopted for evaluation of migration and invasion of PCa cells. Moreover, the Biogrid database and HDOCK SERVER predicated that TRIM47 could interact with mouse double minute 2 (MDM2), which was detected using the Co-immunoprecipitation (co-IP) assay and glutathione S-transferase (GST) pull-down assay. The expression of proteins in MDM2/p53 signaling was detected by western blot analysis. Results indicated that TRIM47 expression was highly expressed in PCa cells. TRIM47 knockdown inhibited PCa proliferation and cell cycle whereas promoted cell apoptosis. Besides, TRIM47 knockdown significantly inhibited the migration and invasion of PCa cells. In addition, TRIM47 was proved to bind to MDM2 and regulated MDM2/p53 expression. Importantly, MDM2 overexpression counteracted the impacts of TRIM47 knockdown on cell viability, cell cycle, apoptosis, migration and invasion by regulating the MDM2/p53 pathway. Collectively, our results suggested that TRIM47 silencing inhibits the malignant biological behaviors of prostate cancer cells by regulating MDM2/p53 signaling, which may provide a novel therapeutic target for PCa treatment.

Characterizing the protein–protein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumour suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of wild type p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localisation and stability to inhibit its activity. Therefore, small molecule stabilisation of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterisation of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a ‘multivalent’ fashion. Instead, the two phosphorylated MDM2 motifs ‘rock’ between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ/MDM2 interaction is amenable to small molecule stabilisation. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in stablisation of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilisers.

KLF5/MDM2 Axis Modulates Oxidative Stress and Epithelial-Mesenchymal Transition in Human Lens Epithelial Cells: The Role in Diabetic Cataract

Diabetic cataract (DC) is a common cause of visual loss in older diabetic subjects. Krüppel-like factor 5 (KLF5) plays an essential role in migration and the epithelial-mesenchymal transition (EMT) in diverse cells and is involved in oxidative stress. However, the effects of KLF5 on DC remain unknown. This study aimed to examine the biological function of KLF5 in DC and its underlying mechanism. The expression patterns of KLF5 were detected in vivo and in vitro. Then, KLF5 was knocked down in human lens epithelial cells (HLECs) to explore its functional roles and underlying mechanisms. Dual-luciferase reporter assay and chromatin immunoprecipitation analysis were used to detect whether KLF5 could bind the promoter of E3 ubiquitin ligase mouse double minute 2 (MDM2), a key regulator of EMT. Lastly, the regulation of KLF5 in the biological behaviors of HLECs via MDM2 was analyzed. We found a significant increase of KLF5 in the DC lens anterior capsular, diabetic rat lens, and high glucose (HG)-stimulated HLECs. Knockdown of KLF5 inhibited oxidative stress, inflammation, migration, and EMT of HG-stimulated HLECs. KLF5 silencing impeded MDM2 expression and restricted the activation of MARK1/FAK and NF-κB signaling pathways in HLECs under HG condition. Additionally, KLF5 was found to bind the MDM2 promoter and enhance the transcriptional activity of MDM2. The protective effects by silencing KLF5 on HG-cultured HLECs could be offset by MDM2 overexpression. We demonstrated that knockdown of KLF5 alleviated oxidative stress, migration, and EMT of HG-cultured HLECs by regulating MDM2, suggesting a potential therapeutic strategy for DC.

LOC100130075 Promotes Cervical Cancer Progression by Activating MDM2 Transcription through E2F1.

Cervical cancer (CC) represents one of the most frequent gynecological tumors worldwide and it takes a big part in cancer-related deaths in women. The mouse double minute 2 (MDM2) gene has been elucidated to be deregulated in cancers and exert its oncogenic activity. Through ENCODE ( https://www.encodeproject.org/ ), LOC100130075 was discovered to be a nearby gene of MDM2. Emerged as a novel long non-coding RNA (lncRNA), LOC100130075 has not been studied in cancers. Therefore, we aim to figure out the function of LOC100130075 and its interaction with MDM2 in CC progression. The high expression pattern of LOC100130075 and MDM2 and a positive correlation between them were firstly verified in CC cells. Then, it was verified that LOC100130075 interference suppressed the proliferation and enhanced the apoptosis of CC cells. Furthermore, we verified through mechanism assays including ChIP, RNA pull-down, as well as luciferase reporter assays that LOC100130075 bound to E2F transcription factor 1 (E2F1) to activate MDM2 transcription. Furthermore, the result of rescue assays manifested that MDM2 overexpression reversed the inhibitory function of LOC100130075 deficiency on CC development. In a word, LOC100130075 promoted CC malignancy by activating MDM2 transcription through E2F1, which may provide a new direction in the advancement of CC treatments.

Expression profile of tumour suppressor protein p53 and its regulator MDM2 in a cohort of breast cancer patients in a Tertiary Hospital in Ghana.

BackgroundInactivation or mutation of the tumour suppressor gene p53 or its regulator mouse double minute 2 (MDM2) is the commonest event in breast cancer. These altered genes usually express abnormally high levels of their proteins in many carcinomas. The phenotypic expression of p53 and MDM2 in breast cancer cases in our setting is not known. This study investigated the expression of the tumour suppressor protein p53 and its regulator MDM2, using immunohistochemistry in a Ghana breast cancer cohort.MethodA 9-year retrospective cross-sectional study on archived tissue blocks–formalin fixed paraffin embedded tissue (FFPE) was carried out. Demographic data were abstracted. Based on complete clinical data and availability of FFPE archived blocks 203 cases were selected for tissue micro array (TMA) construction. The TMA sections were subjected to immunohistochemistry (IHC) (ER, PR, HER2, p53, and MDM2). Expression of p53 and MDM2 were related to grade and molecular subtypes.ResultsThe age ranged from 17 to 92 years (mean = 49.34 ± 13.74). Most of the cases were high grade; grade II (34.9%) and grade III (55.7%). Fifty-four percent of the cases were triple negative. Invasive ductal carcinoma no special type was the commonest histotype (87.1%). Thirty-six percent (36%) of the cases expressed p53. Significant associations were found between p53 overexpression and histological grade (p = 0.034), triple negative (p = 0.0333) and luminal B (p<0.01) tumors. Most cases (93.1%) were negative for MDM2 expression. Significant association was found between MDM2 and HER2 over-expression as well as Ki-67. There was no significant positive correlation between MDM2 and p53 co-expression (p>0.05).ConclusionThe elevated level of p53 expression in the aggressive breast cancer phenotypes (high histological grade and triple negative) in our cohort suggest that P53 elevation may be a poor prognostic marker in our setting. High expression of MDM2 in our cohort with high Ki67; also in cases with Her2/neu overexpression known with predictable poor prognosis in the absence of target therapy suggest MDM2 may be associated with aggressive biological behaviour in our breast cancer cases. The non-significant association of p53 and MDM2 expression in the same cases as also documented by previous studies suggest independent genetic pathway in tumourigenesis.

Corosolic Acid Inhibits Cancer Progress Through Inactivating YAP in Hepatocellular Carcinoma.

Chemotherapy is critical for the treatment of hepatocellular carcinoma (HCC). Despite the proapoptotic effects of corosolic acid (CA) treatment, its underlying mechanism is not completely clear. The aim of this study was to determine the molecular mechanism of CA in HCC treatment. MTT assay was used to determine the IC50 of CA. Immunoprecipitation and immunofluorescence were used to detect the interaction and subcellular localization of Yes-associated protein (YAP) and mouse double minute 2 (MDM2). In addition, in vivo xenotransplantation was performed to assess the effects of CA, YAP, and MDM2 on tumorigenesis. The IC50 of CA was about 40 μM in different HCC cell lines, and CA decreased YAP expression by reducing its stability and increasing its ubiquitination. CA treatment and MDM2 overexpression significantly decreased the crosstalk between YAP and cAMP-responsive element-binding protein (CREB), TEA domain transcription factor (TEAD), and Runt-related transcription factor 2 (Runx2). CA stimulation promoted the translocation of YAP and MDM2 from the nucleus to the cytoplasm and increased their binding. In addition, CA treatment obviously reduced tumorigenesis, whereas this effect was abolished when cells were transfected with sh-MDM2 or Vector-YAP. The present study uncovered that CA induced cancer progress repression through translocating YAP from the nucleus in HCC, which might provide a new therapeutic target for HCC.

FKBP12 is a predictive biomarker for efficacy of anthracycline-based chemotherapy in breast cancer

BackgroundFK506-binding protein 12 (FKBP12) is abundant, ubiquitously expressed cytoplasmic protein with multiple functions in cell signaling transduction. Recently, we reported a novel function for FKBP12 in oncoprotein mouse double minute 2 (MDM2) self-ubiquitination and degradation, which greatly enhanced the sensitivity of cancer cells to chemotherapy. However, the clinical relevance remains unclear.MethodsAn immunohistochemical analysis of FKBP12 expression was performed in a cohort of 524 patients with invasive breast cancer. The correlations of FKBP12 expression with patient survival and chemoresponse were statistically analyzed. MDA-MB-468 cells were transfected with FKBP12 siRNA or Myc-tagged FKBP12, and then, the tumor cells were treated with doxorubicin followed by western blot, cell viability, and apoptosis assay.ResultsThe expression of FKBP12 was decreased in breast cancer tissues, and there was a significant correlation between FKBP12 loss and MDM2 overexpression. Furthermore, FKBP12 loss was specifically correlated with poor prognosis and increased resistance to anthracycline-based chemotherapy. Kaplan–Meier survival analysis showed that overall survival (OS) and disease-free survival (DFS) were both significantly lower in the low FKBP12 expression group than those in the high FKBP12 expression group. In patients treated with anthracycline-based preoperative chemotherapy, low FKBP12 expression patients had a significant lower rate of pathologic complete response (pCR). Importantly, these results seemed to be driven mainly by MDM2. These observations were especially prominent in the MDM2-positive subgroup. Univariate and multivariate analyses revealed that FKBP12 loss was an independent factor for predicting prognosis and pCR. In in vitro assay, FKBP12 silence led to significant upregulation of MDM2. Accordingly, MDA-MB-468 cells with FKBP12 silence were less responsive to doxorubicin-induced cytotoxic and apoptotic effect. In contrast, in FKBP12-transfected MDA-MB-468 cells, MDM2 was more greatly inhibited by doxorubicin, resulting in greater cytotoxic and apoptotic effect.ConclusionsWe propose that FKBP12 loss, which can be enhanced by MDM2 overexpression, predicts poor prognosis and chemoresistance. Increasing the expression of FKBP12 may be a valuable strategy to add to anthracycline-based chemotherapy, especially in MDM2-overexpressed patients.

Abstract 309: Effect of histone deacetylase (HDAC) inhibitor on gene expression in MDM2 transfected prostate cancer cells

Abstract Deacetylation of histone gives a tag for epigenetic repression and plays an important role in transcriptional regulation, cell cycle progression, and developmental events. HDACs catalyse the removal of the acetyl moiety from the lysine residues of proteins including the core nucleosomal histones. Through removal of critical acetyl groups from histones, HDACs can create a chromatin conformation that can prevent the transcription of genes that encode for proteins involved in cell cycle regulation. Thus together with histone acetyltransferases (HATs), HDACs regulate the level of acetylation and alter multitude of cellular functions and their characteristics. Several alterations of HDAC and HAT levels and activities have been found to be enacted by translocation, amplification, overexpression, or mutation of the relevant genes in a variety of cancers. In many cancer cell lines, overexpression or activation of the HDAC enzymes result in histone hypo-acetylation and consequent promotion of pro-cancerous mechanisms. Therefore, HDAC inhibitors represent a potential new class of antitumor agents with cytotoxic activity and the ability to regulate gene expression in tumor cells. In this study we evaluated the effects of Vorinostat (suberoylanilide hydroxamic acid), which is a potent inhibitor of HDAC activity, on cell cycle regulation in MDM2 (mouse double minute 2 homolog) overexpressing cells. MDM2 amplification or overexpression is found in many tumors that eventually lead to the inactivation of the cell cycle control and loss of pro-apoptotic functions through both p53 dependent and independent mechanisms. The PCR array, qRT-PCR, and western blot analysis of MDM2 overexpressing prostate cancer cells (LNCaP-MST), after treating with Nutlin-3 (20 µm) and 17-AAG (10 µm), was able to trigger p21 expression and down-regulation of BIRC5 (Baculoviral IAP Repeat Containing 5). Similarly, when we treated the MDM2 transfected LNCaP-MST cells with vorinostat (7.5 µm for 24 hrs), some of the above mentioned changes, similar to Nutlin-3 treatment, were observed. As a result of HDAC inhibition the mRNA levels of p21, p53 and TIMP-1 were significantly elevated, while the levels of BIRC5 was significantly down-regulated. Thus, treatment of MDM2 overexpressing cell lines with HDAC inhibitor resulted in activation of p21 and consequent decrease in cell proliferation due to resumption of cell cycle arrest. Our results with LNCaP-MST cells offer convincing evidence to suggest that the inhibition of HDAC can control cell proliferative signals in MDM2 overexpressing prostate cancer cells. (The generous support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Thiagarajan Venkatesan, Ali Alaseem, Khalid Alhazzani, Thanigaivelan Kanagasabai, Appu Rathinavelu. Effect of histone deacetylase (HDAC) inhibitor on gene expression in MDM2 transfected prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 309. doi:10.1158/1538-7445.AM2017-309

Abstract 1858: Analyzing the impact of MDM2 overexpression on cell cycle pathway-related genes using polymerase chain reaction array

Abstract The human homologue of the mouse double minute 2 (MDM2) oncogene has been well characterized as a negative regulator of the tumor suppressor p53. It has been established that p53 regulates genes that are involved in cell cycle arrest, apoptosis, DNA repair and senescence. MDM2 interacts primarily with p53 in an autoregulatory negative feedback loop and targets it for ubiquitylation and proteasomal degradation there by attenuating p53 cell cycle arrest and apoptosis functions. Studies have shown that MDM2 also exerts its effects via p53 independent pathways. These p53-independent functions of MDM2 may have a role in cancer etiology and progression, yet the exact function and significance of these interactions are not fully understood. MDM2 is overexpressed in more than 40 different types of malignancies, including wide varieties of carcinomas and sarcomas, which is believed to be associated with worse clinical prognosis and increased likelihood of distant metastases. Hence, MDM2 is proving to be a key player in human cancer and thus an important target for therapeutic intervention. In this study, we aimed to investigate the effects of MDM2 overexpression on cell cycle pathways by using LNCaP prostate cancer cells and MDM2 transfected LNCaP-MST. The LNCaP-MST cells expressed at least 10-folds higher levels of MDM2 mRNA due to transfection. We used the Human Cell Cycle PCR array to compare the gene expression profiles of LNCaP and LNCaP-MST cells. Our experimental results clearly indicated that MDM2 modulates the expression of cell cycle related genes. The following genes were found to be significantly down-regulated in LNCaP-MST cells compared to LNCaP: CCND2, GADD45A, RB1, RBBP8, CDKN1A and TP53. Genes such as BIRC5, CDK1 were found to be significantly up-regulated. Heat map and scatter plot analysis further confirmed the alterations in the expression levels of these genes following the MDM2 overexpression. In addition, cluster analysis further denoted that the gene expression patterns positively correlated with cell proliferation and tumor growth. Our experimental results offer conclusive evidence that MDM2 can impact the cancer growth and metastatic potential by altering the gene expressions in the cell cycle pathway. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale is gratefully acknowledged). Citation Format: Priya Dondapati, Thiagarajan Venkatesan, Appu Rathinavelu. Analyzing the impact of MDM2 overexpression on cell cycle pathway-related genes using polymerase chain reaction array. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1858.

The Role of MDM2 Amplification and Overexpression in Tumorigenesis.

Mouse double minute 2 (MDM2) is a critical negative regulator of the tumor suppressor p53, playing a key role in controlling its transcriptional activity, protein stability, and nuclear localization. MDM2 expression is up-regulated in numerous cancers, resulting in a loss of p53-dependent activities, such as apoptosis and cell-cycle arrest. Genetic amplification and inheritance of MDM2 promoter single-nucleotide polymorphisms (SNPs) are the two best-studied mechanisms for up-regulating MDM2 activity. This article provides an overview of these events in human cancer, highlighting the frequent occurrence of MDM2 amplification in sarcoma and the role of SNP309 and SNP285 in regulating MDM2 expression and cancer risk. The availability of large-scale genomic profiling datasets, like those from The Cancer Genome Atlas Research Network, have provided the opportunity to evaluate the consequences of MDM2 amplification and SNP inheritance across high-quality tumor samples from diverse cancer indications.

MDM2 Promotes Invasion and Metastasis in Invasive Ductal Breast Carcinoma by Inducing Matrix Metalloproteinase-9

The molecular mechanisms that underpin invasive ductal breast cancer (IDC) invasion and metastasis are incompletely understood. The oncogene, mouse double minute 2 (MDM2), has been implicated in the pathogenesis of numerous cancers, where it stimulates the expression of matrix metalloproteinase 9 (MMP9), an important enzyme in the breakdown of the extracellular matrix. However, its role in breast cancer remains poorly understood. This study assessed the clinical significance of MDM2 expression in IDC and used in vitro expression assays to determine the molecular roles of MDM2. Immunohistochemical staining for MMP9 and MDM2 was performed using archived tumor blocks from 321 women who underwent surgical resection for IDC at the First Affiliated Hospital of Nanjing Medical University, China between January 2002 and December 2003. MCF-7 and MDA-MD-231 cell lines were transfected with siRNA targeted against MDM2, or MDM2 was overexpressed using transiently expressed vectors. The invasion, cell migration and proteolytic capabilities of cells that over- or underexpressed MDM2 was then assessed and compared against control cells, in addition to the consequent effects on MMP9 expression using RT-PCR. In vivo, 54.9% and 49.6% of samples were positive for MMP9 and MDM2 expression, respectively, and their expression was significantly correlated (r2 = 0.171, P = 0.012). Moreover, MDM2 expression was markedly correlated with disease-free survival (HR 2.56, 95% CI 1.02–6.40, P = 0.038). In vitro, MDM2 overexpression significantly enhanced cell invasion, migration and proteolysis compared with control cells, and the converse effects were observed after MDM2-siRNA treatment. MDM2 overexpression induced MMP9 expression in a dose-dependent manner. Taken together, these results suggest that high levels of MDM2 are associated with a poorer prognosis in IDC. This might result from increased tumor invasiveness due to enhanced MMP9 expression causing increased extracellular matrix breakdown.

No evidence for association of the MDM2-309 T/G promoter polymorphism with prostate cancer outcomes

Objectives Mouse double-minute 2 (MDM2) SNP309 polymorphism (T>G) has been correlated with an increased risk of cancer in multiple tumor types. MDM2 overexpression has shown to be weakly associated with distant tumor metastases, and down-regulation of MDM2 via antisense oligonucleotides in vitro has resulted in the radiosensitization of prostate cancer cell lines. Based on these results, we decided to evaluate the role of MDM2 SNP309 in the context of histopathologic parameters and clinical outcomes in prostate cancer tumors. Materials and methods The population consisted of 212 consecutive prostate cancer patients who underwent radical prostatectomy between 1997 and 1999 at Vanderbilt University Medical Center. Two hundred eight of the samples were successfully genotyped for the MDM2 SNP309 polymorphism. Correlations between the polymorphism, recurrence, and survival data were analyzed using univariate and multivariate genetic models. Results The only prognostic factor predictive of overall survival in our study was Gleason score ( P < 0.005). Using χ 2 analysis, we determined that the MDM2 SNP309 polymorphism had no significant association with race ( P = 0.7512), patient's age at diagnosis ( P = 0.6820), pre-prostatectomy PSA level ( P = 0.8606), Gleason's score ( P = 0.4839), surgical margin status ( P = 1.0000), extracapsular extension ( P = 0 .6175), and disease stage ( P = 0.4945). In addition, there was no significant difference in 3-year recurrence-free survival ( P = 0.218), or 8-year overall survival ( P = 0.376). Conclusions Our study finds no evidence for association of the MDM2 SNP309 polymorphism with clinicopathologic variables, recurrence risk, and overall survival outcome in prostate cancer.

The Single Nucleotide Polymorphism IVS1+309 in Mouse Double Minute 2 Does Not Affect Risk of Familial Breast Cancer

The mouse double minute 2 (MDM2) oncoprotein promotes cell survival and cell cycle progression by inhibiting the p53 tumor suppressor protein. Further, MDM2 overexpression can inhibit DNA double-strand break repair in a p53-independent manner. Recently, it was shown that a single nucleotide polymorphism (SNP) in the MDM2 promoter was associated with an accelerated tumor formation in individuals with a p53 mutation. The present case-control study investigated the association of this SNP (IVS1+309) with the risk and the age of onset of familial breast cancer in patients with unknown p53 mutation status. Data from 549 women affected by familial breast cancer and 1,065 healthy controls were analyzed. The cases did not carry BRCA1/2 mutations. Cases and controls showed a similar genotype distribution and the SNP did not seem to modify the age of onset of familial breast cancer. The data were also examined taking into account the presence of any additional cancer after breast cancer and the family history of cases; however, no association was found. These results suggest that the SNP IVS1+309 alone affects neither the risk nor the age of onset of heritable breast cancer.

The Growth Arrest Function of the Human Oncoprotein Mouse Double Minute-2 Is Disabled by Downstream Mutation in Cancer Cells

We have reported earlier that ectopic expression of mouse double minute-2 (MDM2) induces G1 arrest in normal cells. To explain occasional overexpression of MDM2 in cancer cells, we searched for deletion or substitution mutation in the growth suppressor domains of MDM2 in several breast cancer cell lines that overexpress the oncoprotein. Our results suggest the absence of alteration (deletion or substitution) in the open reading frame of MDM2 transcripts in such cells. Because the breast cancer cell line MCF-7 overexpresses MDM2, we isolated the full-length MDM2 transcript from this cell line. The MDM2 cDNA synthesized from transcripts isolated from MCF-7 cells induced inhibition of G1 to S phase transition in normal human diploid cells such as WI38, suggesting that the genetic alterations in breast cancer cells that overexpress MDM2 disable the growth arrest function of the oncoprotein. Consistently, overexpression of full-length MDM2 in MCF-7 cells over its high endogenous level did not inhibit G1-S transition efficiently. Although MDM2 overexpression was accompanied by CDK4 overexpression or absence of cdk4 inhibitor p16 in most breast cancer cells, we found remarkably high levels of cyclin A rather than cyclin E in these cells. Ectopic expression of cyclin A released MDM2-mediated inhibition of G1-S transition in normal human diploid WI38 cells. We propose that cancer cells expressing high levels of cyclin A escape MDM2-mediated G1 arrest, which may account for a selective growth advantage over normal cells.

Multiple patterns of MDM-2 deregulation in human leukemias: implications in leukemogenesis and prognosis.

The human analogue of the mouse double minute-2 (MDM-2) protein binds to p53 protein and abrogates its tumor-suppressing activity. MDM-2 overexpression may represent an alternative mechanism to p53 mutation for escaping the p53-mediated growth control. Interestingly, multiple MDM-2 protein isoforms have been described and the possibility of functional differences between various isoforms has been raised. Previously, we demonstrated significant MDM-2 mRNA overexpression in human leukemias and suggested that MDM-2 overexpression may be a marker of aggressiveness of the disease. Polyclonal antibodies (Ab) have been generated to detect various isoforms of the MDM-2 protein. Using these Abs, we confirmed MDM-2 protein overexpression in leukemias. Furthermore, we observed heterogeneity in the isoforms expressed in various types of leukemias. In addition, we demonstrated that analysis by flow cytometry could be used as a diagnostic tool for detecting altered MDM-2 protein expression in leukemias. Here we review and expand our initial observations and confirm MDM-2 mRNA and protein overexpression by reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, and western blot analyses. Understanding the possible role of MDM-2 oncogene expression in leukemias may establish the scientific basis for new therapeutic approaches.

The Human MDM-2 Oncogene Is Overexpressed in Leukemias

AbstractThe human homologue of the mouse double minute 2 (MDM-2) gene codes for a cellular protein that forms a complex with the mutant and wild-type p53 protein and modulates its trans-activation activity. Overexpression of the MDM-2 gene in cells increases their tumorigenic potential and overcomes the growth-suppressive activity of p53. Previous reports have shown that the MDM-2 gene is amplified in approximately one third of human sarcomas. To examine the role of MDM-2 in leukemia, we analyzed MDM-2 gene amplification and mRNA expression in various types of leukemias. We did not detect gene amplification in any of the 48 cases of leukemia that we examined. In contrast, we observed significant MDM-2 mRNA overexpression in 34 of 64 cases (53%). The level of mRNA overexpression in some cases of leukemias was comparable to that observed in some cases of sarcomas, which demonstrate more than 50-fold MDM-2 gene amplification. Furthermore, we divided these cases into different prognostic groups according to their karyotypic abnormalities. MDM-2 overexpression seemed to be associated with unfavorable chromosomal abnormalities. These findings suggest that the expression of the MDM-2 gene is altered in a significant fraction of human leukemias and MDM-2 may play a significant role in leukemogenesis. In addition, these results suggest that mechanisms other than gene amplification may play a significant role in deregulating the MDM-2 expression.

The human MDM-2 oncogene is overexpressed in leukemias

The human homologue of the mouse double minute 2 (MDM-2) gene codes for a cellular protein that forms a complex with the mutant and wild-type p53 protein and modulates its trans-activation activity. Overexpression of the MDM-2 gene in cells increases their tumorigenic potential and overcomes the growth-suppressive activity of p53. Previous reports have shown that the MDM-2 gene is amplified in approximately one third of human sarcomas. To examine the role of MDM-2 in leukemia, we analyzed MDM-2 gene amplification and mRNA expression in various types of leukemias. We did not detect gene amplification in any of the 48 cases of leukemia that we examined. In contrast, we observed significant MDM-2 mRNA overexpression in 34 of 64 cases (53%). The level of mRNA overexpression in some cases of leukemias was comparable to that observed in some cases of sarcomas, which demonstrate more than 50-fold MDM-2 gene amplification. Furthermore, we divided these cases into different prognostic groups according to their karyotypic abnormalities. MDM-2 overexpression seemed to be associated with unfavorable chromosomal abnormalities. These findings suggest that the expression of the MDM-2 gene is altered in a significant fraction of human leukemias and MDM-2 may play a significant role in leukemogenesis. In addition, these results suggest that mechanisms other than gene amplification may play a significant role in deregulating the MDM-2 expression.