OBJECTIVE: To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma. METHODS: In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features. RESULTS: The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis. CONCLUSION Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.
Male ; Female ; Humans ; In Situ Hybridization, Fluorescence/methods* ; Cyclin-Dependent Kinase 4/metabolism* ; Liposarcoma/pathology* ; Lipoma/pathology* ; Gene Amplification ; Transcription Factor CHOP/genetics* ; Proto-Oncogene Proteins c-mdm2/metabolism*

OBJECTIVETo study the relationship and clinicopathological significance of Numb,MDM2 and p53 expression in human pancreatic cancer.

METHODSThe expression of Numb,MDM2 and p53 proteins in 65 cases of paired paraffin embedded pancreatic ductal adenocarcinoma (PDAC) specimens and adjacent non-cancerous pancreas was detected by immunohistochemistry (IHC). The relationship among their expression and clinicopathological characters was analyzed.Westem blot was used to examine their expression in 16 paired fresh PDAC specimens and adjacent non-cancerous pancreatic tissues. Meanwhile,Numb expression in Capan-2, PANC-1 and AsPC-1 pancreatic cancer cells with different differentiation were detected by immunofluorescence (IF) , Westem blot and quantitative real-time (qRT) -PCR, respectively. Paired sample t-test, χ(2) test, Kaplan-Meier and Cox regression were used to analyze the results of our experiments, respectively.

RESULTSIHC showed that there was no differential expression of Numb in PDAC and adjacent pancreas (t = 1.746, P = 0.086) , while the expression of MDM2 and p53 was significantly increased in PDAC, compared to that in paired normal pancreas (t = 3.294, P = 0.002; t = 3.152, P = 0.002, respectively) .Numb expression was negatively associated with tumor size (χ² = 5.206, P = 0.023), differentiation (χ² = 7.802, P = 0.005) and UICC stage (χ² = 4.770, P = 0.029), while expression of MDM2 and p53 was positively associated with tumor T and TNM stage, respectively (χ² = 5.182, P = 0.023; χ² = 6.448, P = 0.011) . Correlation analysis showed a negative association between Numb and MDM2 (r = -0.283, P = 0.023) , but there was no relationship of them with p53 (P > 0.05) .Univariate and multivariate analysis revealed that Numb was a protective prognostic indicator for patients with PDAC (χ² = 5.408, P = 0.020). Moreover, patients with Numb positive and MDM2 negative expression had a significantly better overall survival (χ² = 5.868, P = 0.015). Western blot showed that Numb expression was much higher in well differentiated PDAC than that in paired normal pancreas (t = 1.092, P = 0.020) , while the expression of MDM2 and p53 was significantly increased in 16 cases of PDAC (t = 3.263, P = 0.005; t = 3.607, P = 0.003, respectively). Numb expression was gradually increased in pancreatic cancer cells with the increasing degree of cell differentiation detected by IF, Westem blot and qRT-PCR.

CONCLUSIONSNumb acts as a tumor suppressor gene in the development of PDAC. Numb, MDM2 and p53 might coordinately participate in the development of PDAC.

Carcinoma, Pancreatic Ductal ; genetics ; Humans ; Immunohistochemistry ; Kaplan-Meier Estimate ; Membrane Proteins ; metabolism ; Neoplasm Staging ; Nerve Tissue Proteins ; metabolism ; Pancreas ; metabolism ; Pancreatic Neoplasms ; genetics ; Prognosis ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo study the role and possible mechanism of glioma-associated oncogene-1 (Gli1) in regulating the cell invasion and migration of pancreatic cancer cells.

METHODSQuantitative real-time (qRT) -PCR was used to detect the effect of siRNA interference on Gli1, murine double minute 2 (MDM2) and p53 genes. Cell invasion and migration assays were used to observe the effect of Gli1, MDM2 and p53 silence on cell invasion and migration in p53 wild-type Capan-2 pancreatic cancer cells, respectively. Meanwhile, immunoblotting (IB) was used to detect the protein level of matrix metalloproteinase (MMP) -9, phospho-excelluar signal-regulated kinase (pERK) and phosphorylation protein kinase B (pAKT) in Gli1-silencing Capan-2 cells. The data were analyzed by paired t-test.

RESULTSqRT-PCR showed that the expression of Gli1, MDM2 and p53 is down-regulated 70.5% and 74.5%, 61.8% and 65.3%, and 73.8% and 78.2% after siRNA interference, compared with the mock and siRNA control groups, respectively. Cell invasion (94 ± 8) and migration (143 ± 8) in p53 wild-type Capan-2 cells transfected with Gli1siRNA were significantly decreased, compared with the siRNA control group (150 ± 7, 190 ± 10) (t = 6.584, P = 0.022; t = 8.266, P = 0.014) , while MDM2 silence inhibited cell invasion (experiment group:85 ± 12, control group: 138 ± 6) and migration (experiment group: 127 ± 9, control group:180 ± 10) in the same cells, respectively (t = 5.097, P = 0.036;t = 4.860, P = 0.040). However, cell invasion (experiment group: 153 ± 11, control group: 106 ± 7) and migration (experiment group: 209 ± 13, control group: 164 ± 8) in p53-silencing Capan-2 cells were significantly enhanced (t = 4.669, P = 0.043; t = 4.990, P = 0.038). IB showed that Gli1 silence down-regulated MMP-9 but not pERK and pAKT protein expression.

CONCLUSIONGli1 might contribute to the cell invasion and migration in pancreatic cancer via the regulation of MDM2, p53 and MMP-9 expression.

Animals ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Neoplasm Invasiveness ; Oncogene Proteins ; genetics ; metabolism ; Pancreas ; metabolism ; Pancreatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; RNA, Small Interfering ; genetics ; Trans-Activators ; genetics ; metabolism ; Transfection ; Tumor Suppressor Protein p53 ; metabolism ; Zinc Finger Protein GLI1

OBJECTIVETo construct a short hairpin (sh)RNA targeting the gene encoding the MDM2 oncoprotein in order to investigate its role in human hepatocellular carcinoma (HCC) and its potential for use as a gene therapy strategy to inhibit HCC growth in vivo.

METHODSSmall interfering (si)RNAs were designed targeting the MDM2 gene (siMDM2-1 and siMDM2-2) and unrelated sequences (negative control) and cloned into the expression plasmid pGCSilencer-U6-neo-GFP. A HCC mouse model was established by subcutaneous inoculation of HepG2 cells (2 x 10(6) in 0.2 ml) into 20 nude mice. The inoculated mice were divided into four equal groups for tumor-localized injections of saline, negative control siRNA plasmid, siMDM2-1 plasmid, and siMDM2-2 plasmid. Tumor growth was observed daily (by caliper measurement) for one month, when mice were sacrificed by cervical dislocation. The tumor mass was resected for analysis of tumor inhibition rate (% = [(average tumor weight of control group - average tumor weight of treatment group) / average tumor weight of control group x 100]) and effects on MDM2 and p53 mRNA and protein expression (by reverse transcription- PCR and western blotting, both normalized to beta-actin). Significance of between-group differences was assessed by one-way ANOVA or LSD test; pairwise comparisons were made by the Chi-squared test.

RESULTSsiMDM2-1 and siMDM2-2 suppressed the xenografted tumor growth remarkably (60.6% and 54.6% inhibition rates, respectively), significantly reduced the expression ofMDM2 gene (62.8% and 61.6%) and protein (60.7% and 59.5%), and significantly increased p53 gene (47.1% and 45.6%) and protein (45.9% and 44.3%) (all, P < 0.05).

CONCLUSIONshRNA-mediated silencing of the MDM2 gene effectively inhibits HCC tumorigenesis of subcutaneously xenografted HepG2 cells in nude mice, and the mechanism may involve p53.

Animals ; Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Proliferation ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; pathology ; Male ; Mice ; Mice, Nude ; Plasmids ; Proto-Oncogene Proteins c-mdm2 ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Transfection ; Tumor Suppressor Protein p53 ; metabolism ; Xenograft Model Antitumor Assays

Mdm2 and Mdm4 are two key negative regulators of the tumor suppressor p53. Deletion of either Mdm2 or Mdm4 induces p53-dependent early embryonic lethality in knockout mouse models. The tissue-specific deletion of Mdm2 induces p53-dependent apoptosis, whereas the deletion of Mdm4 induces both p53-dependent apoptosis and cell cycle arrest. Compared to Mdm4 deletion, Mdm2 deletion causes more severe phenotypic defects. Disrupting the Mdm2 and Mdm4 interaction using knockin mice models causes embryonic lethality that can be completely rescued by the concomitant loss of p53, suggesting that Mdm2 and Mdm4 heterodimerization is critical to inhibit p53 activity during embryogenesis. Overexpression of Mdm2 and Mdm4 in mice induces spontaneous tumorigenesis, which clearly indicates that Mdm2 and Mdm4 are bona fide oncogenes. Studies from these mouse models strongly suggest that blocking Mdm2- and Mdm4-mediated p53 inhibition is an appealing therapeutic strategy for cancer patients with wild-type p53 alleles.
Animals ; Apoptosis ; Cell Cycle Checkpoints ; Mice ; Mice, Knockout ; Models, Animal ; Proto-Oncogene Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-mdm2 ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; antagonists & inhibitors ; genetics ; metabolism ; Ubiquitin-Protein Ligases ; genetics ; metabolism

OBJECTIVETo investigate the relationship between the expressions of p53 pathway genes and EZH2 in nasopharyngeal carcinoma (NPC).

METHODSThe expression levels of p53, mdm2, p63 and EZH2 proteins were detected by immunohistochemistry in 47 cases of undifferentiated NPC and 12 cases of chronic nasopharyngitis, and their correlation to the clinical parameters and prognosis were analyzed.

RESULTSThe protein expressions of p53, mdm2, p63 and EZH2 in NPC were 31.9%, 85.1%, and 95.7%, respectively. mdm2 and EZH2 was not correlated to p53 protein expression (P>0.05); positive correlations was found between EZH2 and p63 expressions and between p53 and p63. The high expression of EZH2 and p63 proteins was correlated to advanced T stage and clinical stage of NPC (P<0.05). The five-year disease-free survival rate in patients with high EZH2 protein expression was significantly lower than that in patients with low EZH2 expression.

CONCLUSIONmdm2 does not show an obvious correlation to p53 protein inactivation in NPC. p63 protein overexpression may be associated with p53 protein inactivation. The overexpression of EZH2 is correlated to NPC progression and poor prognosis.

Adult ; Aged ; Carcinoma ; Enhancer of Zeste Homolog 2 Protein ; Female ; Humans ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Polycomb Repressive Complex 2 ; genetics ; metabolism ; Prognosis ; Proto-Oncogene Proteins c-mdm2 ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism

p53 (encoded by TP53) is undoubtedly one of the most extensively studied genes and proteins. It is a highly potent transcription factor which, under normal circumstances, is maintained at low level. Both genotoxic and non-genotoxic stresses can induce p53 stabilized leading to changes in the expression of p53-responsive genes. The biological outcome inducing this pathway can be either growth arrest and apoptosis or senescence to maintain the integrity of the genome or to delete the damaged cells. The biochemical activity of p53 itself and the cellular environment govern the choice between these outcomes in a cell type- and stress-specific manner. So, p53 is a pivotal tumour suppressor and a mainstay of our body's natural anticancer defence. This review could provide some useful information for further study on the mechanisms of tumorigenesis and its progression, and also could contribute to the discovery of antitumor agents.
Animals ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; DNA Damage ; DNA Repair ; Genes, p53 ; Humans ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Signal Transduction ; Tumor Suppressor Protein p53 ; genetics ; physiology

OBJECTIVETo investigate the transcriptional activity of P53 in gastric cancer.

METHODSThe activity of p53 in gastric cancer was investigated by dual-luciferase reporter assay. The coding sequence of the p53 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. The expression of P21WAF1/Cip1, Gadd45alpha and Mdm2 was detected by immunohistochemistry in 76 samples of gastric carcinoma, and their adjacent tissues were analyzed as control.

RESULTSThe p53 activity was higher in human normal cell lines than that of gastric cancer. Furthermore, the transcriptional activity of P53 was lowest in MKN28 cells in which p53 was mutated. The expression level of P21WAF1/Cip1, Gadd45alpha and Mdm2 in the adjacent tissues was higher than that of cancer tissues (P<0.05), and there was a tendency of decline in the positive ratio with the poor differentiation of the carcinoma (P<0.05). In addition, a strong linear correlation was observed between P21WAF1/Cip1, Gadd45alpha and Mdm2 (P<0.05).

CONCLUSIONChanges of P53 transcriptional activity play an important role in the development of gastric cancer. p53 mutation could affect its transcriptional activity. P21WAF1/Cip1, Gadd45alpha and Mdm2 are a group of effecters that could reflect P53 transcriptional activity when detected together in cancer tissues.

Adult ; Aged ; Carcinoma ; genetics ; metabolism ; pathology ; Cell Cycle Proteins ; Cell Line ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Nuclear Proteins ; Proto-Oncogene Proteins c-mdm2 ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Transcriptional Activation ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Young Adult

Adult ; Aged ; Animals ; Apoptosis ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Female ; Gene Expression ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; metabolism ; pathology ; Liver Neoplasms ; metabolism ; pathology ; Male ; Matrix Metalloproteinase 7 ; genetics ; metabolism ; Mice ; Middle Aged ; Prognosis ; Proto-Oncogene Proteins c-mdm2 ; genetics ; metabolism ; RNA, Messenger ; metabolism

Mouse double minute 2 (Mdm2) gene was isolated from a cDNA library derived from transformed mouse 3T3 cells, and was classified as an oncogene as it confers 3T3 and Rat2 cells tumorigenicity when overexpressed. It encodes a nucleocytoplasmic shuttling ubiquitin E3 ligase, with its main target being tumor suppressor p53, which is mutated in more than 50% of human primary tumors. Mdm2's oncogenic activity is mainly mediated by p53, which is activated by various stresses, especially genotoxic stress, via Atm (ataxia telangiectasia mutated) and Atr (Atm and Rad3-related). Activated p53 inhibits cell proliferation, induces apoptosis or senescence, and maintains genome integrity. Mdm2 is also a target gene of p53 transcription factor. Thus, Mdm2 and p53 form a feedback regulatory loop. External and internal cues, through multiple signaling pathways, can act on Mdm2 to regulate p53 levels and cell proliferation, death, and senescence. This review will focus on how Mdm2 is regulated under genotoxic stress, and by the Akt1-mTOR-S6K1 pathway that is activated by insulin, growth factors, amino acids, or energy status.
3T3 Cells ; Animals ; Apoptosis ; Cell Proliferation ; Cellular Senescence ; DNA Damage ; Energy Metabolism ; Feedback, Physiological ; Gene Library ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Mice ; Molecular Targeted Therapy ; Mutation ; Neoplasms ; genetics ; metabolism ; Proto-Oncogene Proteins c-mdm2 ; genetics ; metabolism ; Ribosomal Protein S6 Kinases, 90-kDa ; genetics ; metabolism ; Signal Transduction ; genetics ; TOR Serine-Threonine Kinases ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Ubiquitin-Protein Ligases ; genetics ; metabolism