Murine Double Minute 2 Overexpression Research Articles

Murine double minute-2 expression in B-cell chronic lymphocytic leukemia: correlation with apoptosis and disease outcome

Introduction B-cell chronic lymphocytic leukemia (B-CLL) is characterized by accumulation of quiescent monoclonal CD5+ B cells, which arise from undefined defects in apoptotic cell death. murine double minute-2 (MDM2) oncoprotein exists in an autoregulatory feedback loop with the tumor suppressor protein p53 and block p53-mediated transactivation and apoptosis. Aim of this work The aim of the study was to evaluate the overexpression of MDM2 in B-CLL patients and correlate it with apoptosis using annexin-V expression and disease outcome. Patients and methods This study was conducted on 55 patients including 45 patients with typical B-CLL; their ages ranged from 55 to 72 years, with a mean age of 58.55 ± 3.9 years. Ten age-matched and sex-matched apparently healthy individuals were included as the control group. The diagnosis of B-CLL was based on clinical, hematological, immunophenotypic, and cytogenetic criteria. Annexin-V and MDM2 oncoprotein expression was assessed by flow cytometric analysis. Results Our study revealed that MDM2 was overexpressed in 53% of B-CLL cases and it was significantly elevated in comparison with the control group (P < 0.001). There was an inverse correlation between MDM2 overexpression and apoptosis (P < 0.001). The mean level of MDM2 expression was significantly reduced after therapy compared with the pretreatment level in the responder group (P < 0.001), whereas there was no significant difference (P>0.05) in the nonresponder group. The expression of MDM2 significantly correlated with the percentage of CD38 expression, LDH level, and B2M level (P < 0.001, P < 0.001, and P < 0.001, respectively) and patients who had responded to treatment showed a significant increase in the level of annexin-V (P < 0.001). MDM2 expression, percentage of CD38 expression, and LDH and B2M levels were significantly higher in the high-risk patients compared with low-risk and intermediate-risk patients (P < 0.001, P < 0.01, P < 0.05, and P < 0.001, respectively).Conclusion MDM2 expression was inversely correlated with apoptosis, with decreased MDM2 expression in responder patients.

Correlation of TP53 and MDM2 genotypes with response to therapy in sarcoma

Relatively few sarcomas harbor TP53 (tumor protein p53) mutations, but in many cases, amplification of MDM2 (murine double minute 2) effectively inactivate p53. The p53 pathway activity can also be affected by normal genetic variation. The mutation status of TP53 and expression of MDM2, TP53, and their genetic variants SNP309 and R72P (Arg72Pro) were investigated in 125 sarcoma patient samples and 18 sarcoma cell lines. Association of the different genotypes and gene aberrations with chemotherapy response and survival, as well as response to MDM2 antagonists in vitro was evaluated. Twenty-two percent of the tumors had mutant TP53 and 20% MDM2 gene amplification. Patients with wild-type TP53 (TP53(Wt) ) tumors had improved survival (P < .001) and TP53(Wt) was an independent prognostic factor (hazard ratio = 0.41; 95% confidence interval = 0.23-0.74; P = .03). Interestingly, there was a trend toward longer time to progression after chemotherapy for tumors with the apoptosis-prone p53 variant R72 (P = .07), which was strongest with doxorubicin/ifosfamide-based regimens (P = .01). Liposarcomas had low R72 frequency (33% versus 56%), but increased levels of MDM2 and MDM4 (51% and 11%, P < .001). MDM2 overexpression on a TP53(Wt) background predicted better response to MDM2 antagonist Nutlin-3a, irrespective of R72P or SNP309 status. Improved survival after chemotherapy was found in patients with TP53(Wt) tumors harboring the R72 variant. MDM2 overexpression in TP53(Wt) tumors predicted good response to MDM2 antagonists, irrespective of R72P or SNP309 status. Thus, detailed TP53 and MDM2 genotype analyses prior to systemic therapy are recommended.

A Splicing Variant of MDM4 Overexpression Plays an Indicator of p53 Aberrations and Marks a Potential Therapeutic Target in Chronic Lymphocytic Leukemia

Abstract 4564 ObjectiveHuman homolog of murine double minute 4 (MDM4) belongs to murine double minute (MDM) family. Splicing variant of MDM4 (S-MDM4) is obtained from the deletion of exon 6, which results in an internal deletion of 68 bp. Murine double minute 2 (MDM2) is an analogy of MDM4 and shares highly similar structure with each other. The MDM4 gene plays a crucial role in regulating p53 activity and has been found to overexpress in chronic lymphocytic leukemia (CLL). The purpose of this study was to investigate the prognostic significance of MDM4, and characterize the role of MDM4 in p53 pathway. MethodsFull-length MDM4 (FL-MDM4), S-MDM4 and MDM2 mRNA expressions were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in 140 Chinese patients with CLL. The correlation between those MDM expressions and CLL prognostic marker such as clinical stage, immunoglobulin heavy-chain variable region (IGHV) mutational status, ZAP-70, CD38, and chromosomal abnormalities were analyzed. Furthermore, primary CLL cells were treated in vitro with either fludarabine or Nutlin-3, to explore the interaction between p53 status and those MDMs. ResultsFL-MDM4 and S-MDM4 expressions were significantly increased with the del(17p13) (P=0.037 and P=0.006), p53 mutations (P=0.023 and P<0.001) and p53 aberrations (P=0.024 and P<0.001). The correlation between the level of MDM2 expression and p53 status was not observed (P=0.196, P=0.095 and P=0.092, respectively). High level of S-MDM4 mRNA expression was associated with short treatment free survival (TFS) (P=0.004). FL-MDM4 expression was significantly decreased after 24 h treatment with fludarabine (P=0.001), but increased after Nutlin-3-treated (P=0.008) in primary CLL cells without p53 aberrations. Both S-MDM4 and MDM2 expressions were significantly increased after fludarabine-treated in CLL cells without p53 aberrations (P=0.013 and P=0.030). Besides, MDM2 overexpression also occurred in CLL cells with p53 wild type after Nutlin-3-treated (P=0.018). In the primary CLL cells with p53 aberrations or p53 dysfunction, the levels of FL-MDM4, S-MDM4 and MDM2 expressions were not significantly increased or decreased after fludarabine or Nutlin-3 treatment. ConclusionS-MDM4 overexpression is an indicator of p53 aberrations in CLL patients, suggesting those patients have a poor prognosis. FL-MDM4 inhibitory effect on p53 can be removed by MDM2-p53 and saved by Nutlin-3, and S-MDM4 overexpression marks a potential therapeutic target in CLL. Disclosures:No relevant conflicts of interest to declare.

S-MDM4 mRNA overexpression indicates a poor prognosis and marks a potential therapeutic target in chronic lymphocytic leukemia

The purpose of the present study was to investigate the prognostic significance of murine double minute 4 (MDM4) in chronic lymphocytic leukemia (CLL) and to characterize the role of MDM4 in the p53 pathway. Full-length MDM4 (FL-MDM4), a splicing variant of MDM4 (S-MDM4) and murine double minute 2 (MDM2) mRNA expressions were detected by quantitative PCR in 140 Chinese patients with CLL, and primary CLL cells were treated in vitro with either fludarabine or Nutlin-3 to explore the interaction between p53 status and MDM4 or MDM2 expression. A marked increase of FL-MDM4 and S-MDM4 expressions were observed in the CLL patients with p53 aberrations (deletion and/or mutation) (P = 0.024, P < 0.001). A high level of S-MDM4 mRNA expression was associated with short treatment free survival (TFS) (P = 0.004). FL-MDM4 expression was significantly decreased after fludarabine treatment (P = 0.001) but increased after Nutlin-3 treatment (P = 0.008) of primary CLL cells without p53 aberrations. Both S-MDM4 and MDM2 expressions were significantly increased after fludarabine treatment of CLL cells without p53 aberrations (P = 0.013 and P = 0.030). MDM2 overexpression also occurred in CLL cells with p53 wild type after Nutlin-3 treatment (P = 0.018). FL-MDM4 and S-MDM4 overexpression are indicators of p53 aberrations in CLL patients, suggesting that those patients have a poor prognosis. FL-MDM4 inhibitory effects on p53 can be removed by MDM2-p53 and saved by Nutlin-3.

Ceramide-induced G2 arrest in rhabdomyosarcoma (RMS) cells requires p21Cip1/Waf1 induction and is prevented by MDM2 overexpression

The sphingoplipid ceramide is responsible for a diverse range of biochemical and cellular responses including a putative role in modulating cell cycle progression. Herein, we describe that an accumulation of ceramide, achieved through the exogenous application of C(6)-ceramide or exposure to sphingomyelinase, induces a G(2) arrest in Rhabdomyosarcoma (RMS) cell lines. Utilizing the RMS cell line RD, we show that this G(2) arrest required the rapid induction of p21(Cip1/Waf1) independent of DNA damage. This was followed at later time points (48 h) by the commitment to apoptosis. Apoptosis was prevented by Bcl-2 overexpression, but permitted the maintenance of elevated p21(Cip1/Waf1) protein expression and the stabilization of the G(2) arrest response. Inhibition of p21(Cip1/Waf1) protein synthesis with cyclohexamide (CHX) or silencing of p21(Cip1/Waf1) with siRNA, prevented ceramide-mediated G(2) arrest and the late induction of apoptosis. Further, adopting the recent discovery that murine double minute 2 (MDM2) controls p21(Cip1/Waf1) expression by presenting this CDK inhibitor to the proteasome for degradation, RD cells overexpressing MDM2 abrogated ceramide-mediated p21(Cip1/Waf1) induction, G(2) arrest and the late ensuing apoptosis. Collectively, these data further support the notion that ceramide accumulation can modulate cell cycle progression. Additionally, these observations highlight MDM2 expression and proteasomal activity as key determinants of the cellular response to ceramide accumulation.

Alterations of G1‐S checkpoint in chordoma

To the authors' knowledge, little is known regarding the alterations of G(1)-S checkpoint and their significance in chordoma, a rare bone tumor. The authors investigated the clinicopathologic relevance of cell cycle abnormalities in chordoma. The expression levels of p53, murine double minute 2 (MDM2), retinoblastoma protein (pRb), cyclin D1, p16(INK4a), and p27(Kip1) were investigated using immunohistochemical techniques; p53 mutations were studied by polymerase chain reaction (PCR)-single-strand conformation polymorphism, and mdm2 amplification was analyzed using real-time quantitative PCR. The results were compared with clinicopathologic parameters in 101 lesions. Approximately 10-45% of primary tumors presented alterations of p53, MDM2, cyclin D1, and pRb proteins; most tumors lacked expression of p16(INK4a) and p27(Kip1). Alterations of p53, MDM2, cyclin D1, and pRb proteins were found to have cooperative effects on both higher proliferative ability (MIB-1 labeling index [LI]) and increased nuclear pleomorphism, a previously described prognostic indicator for patients with chordoma. Multivariate analyses revealed that, among these alterations, p53 overexpression was the only independent factor for higher MIB-1 LI. At the genetic level, mdm2 gene amplification was detected in 15.4% of the lesions but did not correlate with MDM2 overexpression or other clinicopathologic parameters. No p53 mutations were detected in the current series. Survival analysis revealed that p53 overexpression, but no other cell cycle alterations, was associated with a reduced overall survival. Accumulation of cell cycle alterations led to an increased MIB-1 LI and nuclear pleomorphism, a previously described prognostic indicator in chordoma. The authors believe that p53 overexpression in particular is associated with an unfavorable prognosis in patients with chordoma.

Overexpression of MDM2 oncoprotein correlates with possession of estrogen receptor alpha and lack of MDM2 mRNA splice variants in human breast cancer.

To evaluate the significance of murine double minute 2 (MDM2) oncoprotein in human breast cancer as a nuclear-cytoplasmic shuttling protein, an estrogen receptor (ER) alpha regulator, and a prognostic marker and to study how MDM2 is overexpressed, we investigated its status in tissue samples and examined the correlation between overexpression and MDM2 gene abnormalities, status, and clinicopathological parameters. We detected MDM2 oncoprotein in both nucleus and cytoplasm by frozen-section immunohistochemistry. There was a significant correlation between MDM2 overexpression and low-grade nuclear atypia, absence of lymph node involvement, and increased levels of ER alpha protein. Our molecular assays found no point mutations in Ser17, but there was a correlation between MDM2 overexpression and the lack of splice variant mRNAs. These results suggest that the distribution of MDM2 reflects its nuclear-cytoplasmic shuttling ability; that interaction between p53 and MDM2 for tumor progression is not enhanced by point mutations at codon 17; and that the expression of MDM2 splice variants is a reason for the lack of its overexpression. MDM2 overexpression correlates with favorable prognostic parameters. A decreased level of MDM2 will lead to a deviation from the ER alpha signaling pathway.

Analysis of the p53 and MDM-2 gene in acute myeloid leukemia.

The MDM-2 (murine double minute 2) gene codes for a cellular protein that can bind to the p53 tumor suppressor gene product, thereby functioning as a negative regulator of p53. In order to define the role of the MDM-2 gene in the pathogenesis of human acute myeloid leukemia, the expression and the sequence of the MDM-2 gene were examined in samples of bone marrow and/or peripheral mononuclear cells of 38 patients by using immunostaining, polymerase chain reaction (PCR), single strand conformation polymorphism, and sequencing. Immunohistochemical staining detected a weak accumulation of the MDM-2 protein in AML patients of FAB classification M4 and M5. RT-PCR analysis revealed a heterogeneous expression pattern of MDM-2 mRNA in AML samples of different FAB classification. An increased level of MDM-2 mRNA expression was observed in 17 of 38 AML patients when compared to normal controls. No structural changes in a 488 bp region extending from nucleotide 890 to 1378 of the MDM-2 cDNA were detected using RT-SSCP and sequence analysis. In addition, heterogeneous expression of p53 transcripts was found with the highest p53 mRNA levels in AML M4 and M5. Interestingly, there seems to be a correlation between the relative ratios of p53 and MDM-2 mRNA levels in AML M4 and M5: in 15 of 23 cases high p53 mRNA expression was directly associated with high levels of MDM-2 transcripts. An exclusively intranuclear p53 immunostaining pattern was found in 10 of 16 (58%) AML FAB M4 and M5, whereas the remaining AML samples tested were negative for p53 (0/10). Using RT-SSCP analysis and direct sequencing of the RT-PCR amplification products of p53 exon 5-8, we observed that only 1 of 38 AML patients showed a point mutation in the p53 gene. This missense mutation occurred in the evolutionary highly conserved region of p53 at codon 255 (Ile to Phe). These data indicated that structural alterations of the p53 gene do not play an important role in the initiation and progression of AML. However, abrogation of p53 tumor suppressor function due to MDM-2 overexpression may be an alternative molecular mechanism by which a subset of AMLs may escape from p53-regulated growth control.

MDM2 overexpression is rare in ovarian carcinoma irrespective of TP53 mutation status

Somatic mutations in TP53 are seen in many human cancers. In addition, the protein product of the wild-type TP53 can be sequestered by the protein MDM2 (murine double minute 2). This protein is commonly overexpressed in human sarcomas and gliomas, usually as a result of gene amplification. In this study, 43 ovarian carcinomas (OCs) were analysed for aberrations in the TP53 gene by immunohistochemistry (IHC), loss of heterozygosity (LOH) or mutation analysis. The MDM2 gene and its product was studied by Southern blotting and IHC. Over 50% of the OCs studied showed mutations in TP53 by either direct sequencing (19/36, 53%), positive IHC (23,43, 53%) or both, whereas 0/32 had amplification of MDM2 and only 1/37 tumours had positive IHC using the anti-MDM2 antibody IF-2. The solitary example of positive IHC in this series was seen in a mixed müllerian tumour with sarcomatous differentiation and was not accompanied by MDM2 DNA amplification. These results support previous data showing that around 50% of OCs have mutations in TP53 and in addition, suggest that MDM2 is not amplified in OC, but the presence of sarcomatous features in mixed müllerian tumours may result in positive immunohistochemistry with IF-2.

The MDM2 Oncogene Overexpression in Chronic Lymphocytic Leukemia and Low-Grade Lymphoma of B-Cell Origin

The expression of the murine double minute-2 (MDM2) gene, the product of which binds to and inactivates p53, was studied in 60 patients with B-cell chronic lymphocytic leukemia (B-CLL) or non-Hodgkin's lymphoma (B-NHL). Northern blot analysis showed that the level of MDM2 gene expression was low in normal human B-cells, whereas 17 of the patients (28.3%) with B-CLL or NHL had more than 10-fold higher levels of MDM2 gene expression than that observed in normal B cells. Immunohistochemical analysis confirmed MDM2 overexpression at the cellular protein level. MDM2 gene overexpression was found more frequently in patients with the low-grade type of lymphoma (56.5%) than in those with intermediate-/high-grade types (10.8%) (P = .001). Moreover, MDM2 overexpression was found significantly more frequently in patients at advanced clinical stages. Simultaneous analysis of p53 gene mutation showed that three patients had both MDM2 gene overexpression and p53 gene mutation. The results of the present study suggest that MDM2 gene overexpression may play an important role in the tumorigenicity and/or disease progression of CLL and low-grade lymphomas of B-cell origin.

The MDM2 oncogene overexpression in chronic lymphocytic leukemia and low-grade lymphoma of B-cell origin

The expression of the murine double minute-2 (MDM2) gene, the product of which binds to and inactivates p53, was studied in 60 patients with B-cell chronic lymphocytic leukemia (B-CLL) or non-Hodgkin's lymphoma (B-NHL). Northern blot analysis showed that the level of MDM2 gene expression was low in normal human B-cells, whereas 17 of the patients (28.3%) with B-CLL or NHL had more than 10-fold higher levels of MDM2 gene expression than that observed in normal B cells. Immunohistochemical analysis confirmed MDM2 overexpression at the cellular protein level. MDM2 gene overexpression was found more frequently in patients with the low-grade type of lymphoma (56.5%) than in those with intermediate-/high-grade types (10.8%) (P = .001). Moreover, MDM2 overexpression was found significantly more frequently in patients at advanced clinical stages. Simultaneous analysis of p53 gene mutation showed that three patients had both MDM2 gene overexpression and p53 gene mutation. The results of the present study suggest that MDM2 gene overexpression may play an important role in the tumorigenicity and/or disease progression of CLL and low-grade lymphomas of B-cell origin.

MDM2 gene amplification and transcript levels in human sarcomas: relationship to TP53 gene status.

Alterations of the TP53 tumor suppressor gene appear to be implicated in the tumorigenesis and progression of several types of human cancer, including different histologic subtypes of sarcomas. The MDM2 (murine double minute-2) gene encodes a nuclear phosphoprotein that may interact with both mutant and wild-type p53 proteins, thereby inhibiting p53-mediated transactivation in a dose-dependent manner. Recently it has been suggested that mdm2 and p53 proteins are components of an autoregulatory loop in which the MDM2 gene is transactivated by p53. Our purpose was to examine the frequency of MDM2 amplifications in larger panels of sarcomas, determine if the mRNA level could be elevated in tumors without concomitant gene amplification, and relate MDM2 findings to the TP53 status of the tumors. Sarcoma tissue of different histologic subtypes was obtained from 68 patients at the time of surgery and from 26 human xenografts in nude mice. In addition, two human sarcoma cell lines (OSA and U2OS) were studied. Genomic DNA from tumor tissue, in vitro cell lines, and peripheral blood cells were isolated by Southern-blot analysis methods to determine MDM2 gene amplification. Tumor DNA was analyzed for possible TP53 gene mutations in exons 5, 7, and 8 by constant denaturing gel electrophoresis. To determine the MDM2 and TP53 mRNA levels, Northern-blot analysis was performed. Amplification of the MDM2 gene was detected in 10 tumors (10.3%). Whereas MDM2 amplification and/or over-expression were found only in two (U2OS and OSA cell lines) of 18 osteosarcomas, one of 20 malignant fibrous histiocytomas (MFHs), and in none of 14 leiomyosarcomas, such alterations were observed in two of two fibrosarcomas, three of six malignant schwannomas, three of 19 liposarcomas, and in the one hemangiopericytoma examined. MDM2 overexpression was found in all nine examined cases with and in three tumors without amplification. TP53 mutations were detected in 12 cases (five osteosarcomas, four MFHs, and three leiomyosarcomas), of which none showed amplification, but one had increased levels of MDM2 mRNA. None of the fibrosarcomas, malignant schwannomas, and liposarcomas examined had mutated TP53. The six sarcomas that showed high TP53 mRNA expression in the absence of gene mutation also had elevated levels of MDM2 mRNA. The present data provide further indications that increased MDM2 expression level, caused by gene amplification or altered regulation of transcription, is involved in tumor progression of some, but not all, sarcoma subtypes.