MDMX is essential for the regulation of p53 protein levels in the absence of a functional MDM2 C-terminal tail

Background MDM2 is an E3 ubiquitin ligase that is able to ubiquitinate p53, targeting it for proteasomal degradation. Its homologue MDMX does not have innate E3 activity, but is able to dimerize with MDM2. Although mouse models have demonstrated both MDM2 and MDMX are individually essential for p53 regulation, the significance of MDM2-MDMX heterodimerization is only partially understood and sometimes controversial. MDM2C462A mice, where the C462A mutation abolishes MDM2 E3 ligase activity as well as its ability to dimerize with MDMX, die during embryogenesis. In contrast, the MDM2Y487A mice, where the Y487A mutation at MDM2 C-terminus significantly reduces its E3 ligase activity without disrupting MDM2-MDMX binding, survive normally even though p53 is expressed to high levels. This indicates that the MDM2-MDMX heterodimerization plays a critical role in the regulation of p53. However, it remains unclear whether MDMX is essential for the regulation of p53 protein levels in the context of an endogenous MDM2 C-terminal tail mutation. Results Here, we studied the significance of MDM2-MDMX binding in an MDM2 E3 ligase deficient context using the MDM2Y487A mouse embryonic fibroblast (MEF) cells. Surprisingly, down-regulation of MDMX in MDM2Y487A MEFs resulted in a significant increase of p53 protein levels. Conversely, ectopic overexpression of MDMX reduced p53 protein levels in MDM2Y487A MEFs. Mutations of the RING domain of MDMX prevented MDMX-MDM2 binding, and ablated MDMX-mediated suppression of p53 protein expression. Additionally, DNA damage treatment and nuclear sequestration of MDMX inhibited MDMX activity to suppress p53 protein expression. Conclusions These results suggest that MDMX plays a key role in suppressing p53 protein expression in the absence of normal MDM2 E3 ligase activity. We found that the ability of MDMX to suppress p53 levels requires MDM2 binding and its cytoplasmic localization, and this ability is abrogated by DNA damage. Hence, MDMX is essential for the regulation of p53 protein levels in the context of an MDM2 C-terminal mutation that disrupts its E3 ligase activity but not MDMX binding. Our study is the first to examine the role of MDMX in the regulation of p53 in the context of endogenous MDM2 C-terminal mutant MEF cells.

P53 Protein Expression in Epithelial Lining of Odontogenic Cysts: An Immunohistochemical Descriptive Case Study

OBJECTIVES: The current study was conducted to analyze immunohistochemical appearance of P53 protein in odontogenic cysts. METHODOLOGY: Thirty paraffin blocks of confirmed case were prepared to investigate the immunohistochemical appearance of P53 protein. RESULTS: Sixteen out of thirty odontogenic cysts (53.3%) showed P53, four out of ten dentigerous cyst (40%) had P53, twelve out of fifteen odontogenic keratocysts (80%) expressed P53 while none of the five radicular cysts (0%) showed P53 protein. CONCLUSION: Reclassification of OKC as keratocystic odontogenic tumor was supported by the present study and its findings.

HPV, p16 and p53 As Indicators of Response to Survival in Larynx Squamous Cell Carcinoma

Purpose: To analyze the relationship between the prognosis of patients with larynx squamous cell carcinoma (LSCC) and human papillomavirus (HPV) infection, p16 and p53 protein expression. Methods: All patients were treated at the department of radiation oncology, Anhui provincial hospital, between May 2005 and May 2012. The 41 consecutive patients with LSCC were treated surgically and received postoperative radiotherapy. Analyses of pathology specimen were surgically removed and performed on formalin-fixed, paraffin-embedded tissue samples. HPV DNA sequences in tumor tissues were screened by a commercial Luminex technique for HPVs and HPV-specific PCR assays. P16 and p53 protein expression were detected by immunohistochemical staining. Overall survival(OS)and progression-free survival(PFS)for HPV-positive and HPV-negative patients, p16-positive and p16-negative patients, p53-positive and p53-negative patients were estimated by Kaplan-Meier analysis. Cox regression model was used for multivariate analysis. Results: HPV-DNA was detected in 4(9.7%)of all specimens. Among them, 3 were positive for HPV-56,1 for HPV-16. With the follow-up of 3-78 months(a median of 34 months),patients with HPV-positive tumors had better overall survival than patients with HPV-positive tumors(75% vs 61%, P>0.05). Multivariate analysis by Cox regression model showed that nodal status was independent prognostic factors for patients with LSCC(P<0.05). Conclusions: HPV status is not an independent prognostic factor. Nodal status was independent prognostic factors for patients with LSCC.

Cytotoxic potentials of silibinin assisted silver nanoparticles on human colorectal HT-29 cancer cells

It is of interest to study the cytotoxicity of silibinin assisted silver nanoparticles in human colorectal (HT-29) cancer cells. Silver nanoparticles were synthesized using silibinin as a reducing agent. The synthesized silibinin assisted silver nanoparticles (SSNPs) were characterized and analyzed using a transmission electron microscope and spectrophotometer. The SSNPs synthesized in this study are spherical and their size ranges from 10 to 80 nm. HT-29 cells were treated with different concentrations (2, 4, 6, 8 and 10 ng/mL) of SSNPs and cytotoxicity was evaluated. The apoptosis was using flow cytometry. p53 protein expression using western blot. SSNPs are induced a decrease in viability and increased concentration-dependent cytotoxicity in HT-29 cells. SSNPs treatment also caused apoptosis-related morphological changes. SSNPs treatments at 8 and 16 ng/ml showed a prominent apoptotic change i.e., 70.3% and 83.6% respectively, and decreased viability of HT-29 cells 20% and 11.2% respectively as compared to control cells. SSNPs treatments induced p53 expression in HT-29 cells. Data shows that SSNPs have the potential to induce apoptosis in colorectal cancer cells. This provides insights for the further evaluation of SSNPs in fighting colon cancer.