Apoptosis ; drug effects ; Hep G2 Cells ; Hepatoblastoma ; metabolism ; Humans ; Oligonucleotides, Antisense ; pharmacology ; Proto-Oncogene Proteins c-mdm2 ; genetics

OBJECTIVETo investigate the relationship between the expression of murine double minute 2 (MDM2) oncogene and non-Hodgkin lymphoma (NHL) in childhood.

METHODSThirty-one cases of NHL were enrolled in this study as patient group and 8 cases of lymphadenitis as control group. (1) Immunohistochemistry ultrasensitive S-P assay was used to detect the expression of MDM2 protein in pathological tissues in all cases. Positive cells were dyed yellow or brown in nuclei. MDM2 positive cell was defined as >/= 10% of the tumor cells were positive, which was overexpression of MDM2 protein. (2) RT-PCR (reverse transcription-polymerase chain reaction) was performed to value the overexpression of MDM2 mRNA in the pathological tissues and mononuclear cells in peripheral blood. While the ratio of MDM2/beta-actin was >16% was defined as overexpression of MDM2 mRNA.

RESULTS(1) Rates of overexpression of MDM2 protein and MDM2 mRNA were 64.5% and 61.3%, respectively, which were significantly different as compared to that of control group (P < 0.05 and P < 0.01, respectively). (2) The relationship analysis among subgroups in the experiment group showed that the overexpression of MDM2 protein did not correlate with classifications of working formulation, cellular origin, sex, clinical stage and involved extranodal sites (P > 0.05), but significantly correlated with classifications of B status and the increased serum LDH level (P < 0.05). It was shown that the overexpression of MDM2 mRNA did not correlate with classifications of working formulation, cellular origin, sex and clinical stage (P > 0.05), significantly correlated with B status (P < 0.05), and was remarkably significantly correlated with the involved extranodal sites and the increased serum LDH level (P < 0.01). (3) It was demonstrated that the overexpression of MDM2 mRNA in the pathological tissues was similar to the overexpression of MDM2 protein in the pathological tissues and MDM2 mRNA in peripheral blood (P > 0.05, kappa = 0.655 and 0.571), and the overexpression of MDM2 protein in the pathological tissues was similar to that of MDM2 mRNA in peripheral blood (P > 0.05, kappa = 0.609).

CONCLUSIONS(1) The rate of MDM2 oncogene overexpression was quite high. (2) The overexpression of MDM2 protein in pathological tissues determined by using immunohistochemistry ultrasensitive S-P assay was similar to that of MDM2 mRNA in pathological tissues detected by using RT-PCR method. Both methods might be used to detect the overexpression of MDM2 oncogene in the cases of childhood NHL. (3) The overexpression of MDM2 oncogene related to the poor status and poor prognosis of patients with childhood NHL.

Biomarkers, Tumor ; analysis ; blood ; Child ; Humans ; Immunohistochemistry ; Lymphoma, Non-Hodgkin ; blood ; genetics ; metabolism ; Neoplasm Proteins ; blood ; genetics ; Oncogenes ; Proto-Oncogene Proteins c-mdm2 ; blood ; genetics ; metabolism ; RNA, Messenger

Animals ; Genes, p53 ; Genetic Therapy ; Humans ; Neoplasms ; genetics ; metabolism ; therapy ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism

Gankyrin is an oncoprotein containing seven ankyrin repeats that is overexpressed in hepatocellular carcinoma (HCC). Gankyrin binds to Mdm2, which results in accelerated ubiquitylation via degradation of p53, and it also plays an important role in cell proliferation. However, little is known about the relationships between p53 levels, cell proliferation, and gankyrin over-expression. In order to investigate the influence of gankyrin protein on p53 and Mdm2 in a zebrafish model, we injected human gankyrin (hgankyrin) containing expression vectors (pCS2-hgankyrin, pCS2-hgankyrin-EGFP) into zebrafish embryos. To measure p53 and Mdm2 expression in hgankyrin-injected embryos, RT-PCR, Northern blot and in-situ hybridization and BrdU immunostaining were used. In addition, to know the effect of hgankyrin on cell proliferation in vitro, cell viability assays such as MTT, trypan blue staining and RT-PCR following transfection of hgankyrin-containing vector into HEK 293 cell line were performed. In vivo results indicated that p53 mRNA levels decreased but those of Mdm2 were not decreased in the presence of hgankyrin. These results suggest that gankyrin downregulates p53 expression and not Mdm2 expression. In the study of cell proliferation, BrdU-positive cells were predominantly increased in the head and tail regions in hgankyrin-injected zebrafish. Additional in vitro studies using trypan blue staining and MTT assay showed that gankyrin-expressing HEK 293 cells proliferated at a faster rate, indicating that gankyrin promotes cell proliferation. Our results demonstrate that hgankyrin overexpression downregulates p53 expression and promotes cell proliferation in zebrafish. Gankyrin may play an important role in tumorigenesis via its effects on p53 and cell proliferation.
Animals ; Cell Line ; Cell Proliferation ; Cell Survival ; Gene Expression ; Humans ; In Situ Hybridization ; Models, Animal ; Proteasome Endopeptidase Complex/*genetics/*metabolism ; Proto-Oncogene Proteins/*genetics/*metabolism ; Proto-Oncogene Proteins c-mdm2/genetics/metabolism ; Tumor Suppressor Protein p53/genetics/*metabolism ; Zebrafish

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

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*

OBJECTIVE: To determine the role of fragile histidine triad (FHIT) and MDM2 in carcinogenesis of oral submucous fibrosis (OSF). METHODS: The expression of FHIT and MDM2 was examined by immunohistochemical S-P method in 44 OSF cases, 15 canceration tissues of OSF, and 10 normal oral mucosa tissues. RESULTS: The expression of FHIT was positive in the normal oral mucosa epithelium. The positive expression of FHIT decreased in the OSF and canceration tissues of the OSF.The rate of FHIT positive expression was significantly lower in canceration tissues of OSF than that of the OSF (P < 0.05). The expression of MDM2 was negative in normal oral mucosa epithelium. The positive expression of MDM2 increased in the OSF and canceration tissues of the OSF, and the rate of MDM2 positive expression was significantly higher in the canceration tissues of OSF than that of the OSF (P < 0.05). CONCLUSION The loss of FHIT and over-expression of MDM2 may play an important role in the carcinogenesis of OSF.
Acid Anhydride Hydrolases ; genetics ; metabolism ; Female ; Humans ; Immunohistochemistry ; Male ; Mouth Mucosa ; metabolism ; Mouth Neoplasms ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; Oral Submucous Fibrosis ; metabolism ; Proto-Oncogene Proteins c-mdm2 ; genetics ; 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

Animals ; Chromosome Deletion ; Chromosomes, Human, Pair 13 ; Genetic Therapy ; Humans ; Liposarcoma ; genetics ; metabolism ; therapy ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Retinoblastoma Protein ; metabolism ; Soft Tissue Neoplasms ; genetics ; metabolism ; therapy