Overexpression of the MDM2 gene by childhood acute lymphoblastic leukemia cells expressing the wild-type p53 gene

The wild-type (wt) p53 tumor suppressor gene is commonly inactivated in human malignancies, either by mutations or by loss of expression. An additional proposed mechanism for inactivation of wt-p53 is amplification of the murine double minute 2 (MDM2) gene and overexpression of the MDM2 protein, which binds to p53 and eliminates its tumor suppressor function. To investigate a potential role for MDM2 in the inactivation of wt-p53 in pediatric acute lymphoblastic leukemia (ALL), we examined the expression of MDM2 and p53, as well as the occurrence of p53 mutations and possible amplification of the MDM2 gene, in 19 pediatric ALL cell lines and one pediatric acute myelogenous leukemia (AML) line. Although we did not find significant amplification of the MDM2 gene in any of the leukemic lines, we detected overexpression of MDM2 in all 10 lines that expressed wt-p53. Of the 10 lines without overexpression of the MDM2 gene, six (including the AML line) did not express p53, and four expressed mutant p53 with single point mutations in exons 7 and 8. To determine whether primary leukemic cells showed a similar correlation, we analyzed the original cryopreserved leukemic bone marrow cells from seven patients from whom cell lines were established. We obtained similar results from both the primary leukemic cells and the corresponding cell lines: overexpression of MDM2 was present in primary cells that expressed wt-p53 but not in cells that lacked expression of wt-p53. These findings suggest an important role for MDM2 in the pathogenesis of pediatric ALL in which leukemic cells express wt-p53.     

Apoptosis of tumoral and nontumoral lymphoid cells is induced by both mdm2 and p53 antisense oligodeoxynucleotides

Following stress signals, the p53 tumor suppressor protein plays a critical role in regulation of cell proliferation, mainly through induction of growth arrest or apoptosis. Therefore, this protein needs to be strictly regulated and numerous studies have shown that the MDM2 protein is an essential element for p53 regulation in normal cells and, most importantly, that overexpression of MDM2 is responsible for p53 inactivation in various types of tumors. A previous study showed that this is the case in some Burkitt lymphoma (BL) cell lines, where enhanced translation of mdm2 messenger RNA results in overexpression of the protein that complexes and inactivates wild-type p53. To further investigate the role of the p53/MDM2 complex in these BL cells, as well as in other lymphoid cells that do not overexpress MDM2, this study used antisense oligodeoxynucleotides directed either against mdm2 or against p53. Results show that the mdm2 antisense oligodeoxynucleotide induces apoptosis of cells that express a high or low level of MDM2 protein, only if they contain wild-type p53. Moreover, apoptosis is independent of the accumulation of p53 following mdm2 antisense treatment. Finally, the p53 antisense oligodeoxynucleotide, which inhibits the expression of wild-type p53, also induces a decrease of the MDM2 level in cells, whether or not they overexpress this protein, and causes apoptosis of these cells. These results indicate that decreasing the MDM2 protein level by directly or indirectly targeting its biosynthesis is a potent tool for the induction of apoptosis.     

Altered expression of p53 and MDM2 proteins in hematological malignancies

In order to define the possible role of the MDM2 gene in the pathogenesis of human leukemia, the expression of MDM2 protein was examined in samples of fixed-permeabilized peripheral blood (PB) or bone marrow (BM) cells of leukemic patients by using flow cytometry. The present study showed, that normal PB and BM cells expressed low levels of MDM2. Overexpression of this protein was more frequently found in leukemic cells, namely in samples of patients with advanced, than those in incipient clinical stage of disease at examination. Of the 34 leukemias tested in our laboratory 24 (70%) showed abnormal expression of the MDM2 protein. This include 8/12 (66%) ALL, 10/13 (76%) B-CLL, and 6/9 (66%) AML. Since MDM2 and p53 are functionally related and overexpression of MDM2 can abrogate wild (wt)-p53 tumor suppressive function, we examined simultaneously with MDM2 protein expression also the expression of both wt-p53 and mutant (mt)-p53 with two MoAbs (Ab5 and Pab240). As measured by flow cytometry only a small part of the observed wt-p53 protein was in true wt-conformation (Ab5+), while most was in mt-conformation (Pab240+), which could mean, that most of the p53 protein in the cells was not functional, as in its usual role as a suppressor of the cell cycle. The MDM2 positive cases were negative for p53 (Pab240-) in hematopoietic cells of patients with B- and T-ALL at diagnosis and in relapsed disease. Samples of patients in remission with immunophenotype of normal cells were p53 and MDM2 negative. The expression of Ki67 antigen a nuclear protein associated with cell proliferation was used to verify the proliferative activity of the leukemic cells. Results of the two-color flow cytometric assay, which allows better definition of pathologic cell populations and nuclear fluorescence data for p53, MDM2 or Ki67 on a population of cells expressing only a given surface blast marker, confirmed their coexpression in the same cell. Our preliminary results supported the view that the expression of p53 is very probably involved in the regulation of leukemic hematopoiesis and that the inhibition of p53 expression could modulate the proliferation of leukemic cells. It appears, that MDM2 overexpression, which may be p53-dependent, or also p53-independent plays an important role in leukemogenesis and/or disease progression.     

The MDM2 oncoprotein promotes apoptosis in p53-deficient human medullary thyroid carcinoma cells

The MDM2 oncoprotein has been shown to inhibit p53-mediated growth arrest and apoptosis. It also confers growth advantage to different cell lines in the absence of p53. Recently, the ability of MDM2 to arrest the cell cycle of normal human fibroblasts has also been described. We report a novel function for this protein, showing that overexpression of MDM2 promotes apoptosis in p53-deficient, human medullary thyroid carcinoma cells. These cells, devoid of endogenous MDM2 protein, exhibited a significant growth retardation after stable transfection with mdm2. Cell cycle distribution of MDM2 transfectants [medullary thyroid tumor (MTT)-mdm2] revealed a fraction of the cell population in a hypodiploid status, suggesting that MDM2 is sufficient to promote apoptosis. This circumstance is further demonstrated by annexin V labeling. MDM2-induced apoptosis is partially reverted by transient transfection with p53 and p19ARF. Both MTT and MTT-mdm2 cells were tumorigenic when injected into nude mice. However, the percentage ofapoptotic nuclei in tumor sections derived from MDM2-expressing cells was significantly higher relative to that in the parental cell line. MDM2-mediated programmed cell death is at least mediated by a down-regulation of the antiapoptotic protein Bcl-2. Protein levels of caspase-2, which are undetectable in the parental cell line, appear clearly elevated in MTT-mdm2 cells. Caspase-3 activation does not participate in MDM2-induced apoptosis, as determined by protein levels or poly(ADP-ribose) polymerase fragmentation. The results observed in this medullary carcinoma cell line show for the first time that the product of the mdm2 oncogene mediates cell death by apoptosis in p53-deficient tumor cells.     

MDM2 overexpression does not account for stabilization of wild-type p53 protein in non-Hodgkin's lymphomas

p53 protein overexpression is a frequent finding in non-Hodgkin's lymphomas (NHL), being detected in over 25% of the cases. Moreover, some high-grade lymphomas and a large fraction of low-grade tumors show a pattern of scattered p53 accumulation in a limited percentage of neoplastic cells. In contrast, NHLs show a low frequency of p53 gene mutations. To investigate the molecular bases of p53 protein overexpression, a large series of NHLs was analyzed for p53 gene status. The analysis of the entire coding region of the gene (exons 2- 11) and corresponding donor and acceptor splicing sites indicated that a significant proportion of p53-positive tumors overexpresses a wild- type form of p53 protein (wt-p53). To assess whether wt-p53 accumulation was related to the formation of inactive complexes with endogenous proteins, MDM2 oncogene expression and amplification were analyzed. MDM2 overexpression was detected only in one third of the wt- p53-positive cases, thus excluding that MDM2 accounts tout court for the accumulation of a normal p53 protein. However, the fact that MDM2 overexpression was detected in only the p53-positive cases and the observation that MDM2-positive cells were a subpopulation of p53- positive cells suggest a link between the two phenomena. In particular, our results indicate that the accumulation of a wt form of p53 protein could promote the overexpression of the MDM2 gene product. In addition, the prevalence of MDM2 positivity in intermediate/high-grade tumors together with the concordant expression of wt-p53 and MDM2 only in the high-grade component of a 'composite' lymphoma suggests that perturbation in the MDM2/p53 critical ratio could play a role in lymphoma progression.     

A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control

The p53 tumor suppressor protein and the MDM2 oncoprotein form a feedback-control loop that up-regulates cellular MDM2 production, blocks p53 activity, and promotes p53 decay. tsg101 was discovered as a gene whose deficiency results in neoplastic transformation of NIH 3T3 cells and the ability to generate metastatic tumors in nude mice. Its protein product contains a domain, Ubc, characteristic of the catalytic domain of ubiquitin conjugase (E2) enzymes but lacking an active-site cysteine crucial for ubiquitin conjugase activity. Here we report that TSG101 participates with MDM2 in an autoregulatory loop that modulates the cellular levels of both proteins, and also of p53, by affecting protein decay. We show that the Ubc domain of TSG101 interferes with ubiquitination of MDM2, that TSG101 inhibits MDM2 decay and elevates its steady-state level, and that these events are associated with down-regulation of p53 protein. Conversely, pulse-chase and Western blot experiments in wild-type and mutant fibroblasts indicate that elevation of MDM2 by overexpression of wild-type p53, by amplification of the endogenous MDM2 gene, or by transfection of MDM2-expressing constructs promotes TSG101 loss, which we show occurs by 26S proteasome-dependent decay. Our results identify TSG101 as both a regulator of, and target of, MDM2/p53 circuitry.     

MDM2 induces NF-kappaB/p65 expression transcriptionally through Sp1-binding sites: a novel, p53-independent role of MDM2 in doxorubicin resistance in acute lymphoblastic leukemia

MDM2 protein is thought to exhibit tumorigenic activity by binding to the p53 tumor-suppressor protein and inhibiting its function. Alternatively, MDM2 may have oncogenic roles other than those resulting from p53 interactions. Here we report that MDM2 can induce expression of the p65 subunit of NF-kappaB, which is an anti-apoptotic factor expressed in certain neoplastic cells in response to chemotherapy. Initially, we noted that the overexpression of MDM2 protein in leukemic bone marrow cells of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and an ALL cell line (EU-4) transfected with the MDM2 gene was associated with elevated expression of p65 and in vitro resistance to doxorubicin (Adriamycin). By cotransfection of the MDM2 gene and p65-promoter-reporter constructs into EU-4 cells, we found that transient and high-level MDM2 expression induced p65 promoter activity. In the presence of wild-type (wt) p53, MDM2 increased p65 promoter activity by reversing p53-mediated suppression of p65. In the absence of p53, MDM2 directly increased p65 promoter activity. Deletion and mutation analysis of the p65 promoter indicated that the region between nt -575 and -178, which contains the first and second Sp1-binding sites, was required for activation by MDM2. Further studies using chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assay (EMSA) showed that MDM2 was able to directly bind to the Sp1 site of the p65 promoter. Our findings suggest that by inducing p65 expression, MDM2 has a p53-independent role in tumorigenesis, which may further elucidate the association between MDM2 overexpression and resistant disease in childhood ALL.     

Association of gankyrin protein expression with early clinical stages and insulin-like growth factor-binding protein 5 expression in human hepatocellular carcinoma

Gankyrin (also known as PSMD10) is a liver oncoprotein that interacts with multiple proteins including MDM2 and accelerates degradation of the tumor suppressors p53 and Rb. We produced a monoclonal anti-gankyrin antibody and immunohistochemically assessed the clinicopathological significance of gankyrin overexpression in 43 specimens of human hepatocellular carcinoma (HCC). Specific cytoplasmic staining for gankyrin was observed in 62.8% (27/43) of HCCs, which was significantly associated with low TNM stage (P = 0.004), no capsular invasion (P = 0.018), no portal venous invasion (P = 0.008), and no intrahepatic metastasis (P = 0.012). The cumulative survival rate of patients with gankyrin-positive HCC was significantly higher than that with gankyrin-negative HCC (P = 0.037). p53 and MDM2 were positively stained by antibodies in 30.2% and 23.3%, respectively, of HCCs, but neither was inversely associated with gankyrin expression. In the Huh-7 human HCC cell line, overexpression of gankyrin up-regulated expression of insulin-like growth factor binding protein 5 (IGFBP-5), whereas suppression of gankyrin expression by siRNA down-regulated it. Supression of IGFBP-5 expression inhibited proliferation of Huh-7 cells as well as U-2 OS osteosarcoma cells. In HCC specimens, positive staining for IGFBP-5 was observed by immunohistochemistry in 41.9% (18/43), and the level of expression was significantly correlated with that of gankyrin (rho = 0.629, P < 0.001). CONCLUSION: These results suggest that gankyrin plays an oncogenic role(s) mainly at the early stages of human hepatocarcinogenesis, and that IGFBP-5 inducible by gankyrin overexpression may be involved in it.     

Kinase requirements in human cells: V. Synthetic lethal interactions between p53 and the protein kinases SGK2 and PAK3

Cervical carcinomas are initiated through a series of well-defined stages that rely on the expression of human papillomavirus (HPV) oncogenes. A panel of 100 small hairpin RNAs that target essential kinases in many tumor types was used to study the stepwise appearance of kinase requirements during cervical tumor development. Twenty-six kinases were commonly required in three cell lines derived from frank carcinomas, and each kinase requirement was traced to the specific stage in which the requirement emerged. Six kinases became required following HPV-induced immortalization, and the requirement for two kinases, SGK2 and PAK3, was mapped to the inactivation of p53 in primary human epithelial cells. Loss of the p53 tumor suppressor in other primary epithelial cells also induced dependence on SGK2 and PAK3. Hence, SGK2 and PAK3 provide important cellular functions following p53 inactivation, fulfilling the classical definition of synthetic lethality; loss of p53, SGK2, or PAK3 alone has little effect on cell viability, whereas loss of p53 together with either SGK2 or PAK3 loss leads to cell death. Whereas tumor suppressor gene mutations are not directly druggable, other proteins or pathways that become obligatory to cell viability following tumor suppressor loss provide theoretical targets for tumor suppressor-specific drug discovery efforts. The kinases SGK2 and PAK3 may thus represent such targets for p53-specific drug development.     

Nanosensing protein allostery using a bivalent mouse double minute two (MDM2) assay

The tumor suppressor protein, p53, is either mutated or absent in >50% of cancers and is negatively regulated by the mouse double minute (MDM2) protein. Understanding and inhibition of the MDM2-p53 interaction are, therefore, critical for developing novel chemotherapeutics, which are currently limited because of a lack of appropriate study tools. We present a nanosensing approach to investigate full-length MDM2 interactions with p53, thus providing an allosteric assay for identifying binding ligands. Surface-enhanced Raman scattering (SERS)-active nanoparticles, functionalized with a p53 peptide mimic (peptide 12.1), display biologically specific aggregation following addition of MDM2. Nanoparticle assembly is competitively inhibited by the N-terminal MDM2-binding ligands peptide 12.1 and Nutlin-3. This study reports nanoparticle assembly through specific protein-peptide interactions that can be followed by SERS. We demonstrate solution-based MDM2 allosteric interaction studies that use the full-length protein.     

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.     

Reduced murine double minute 2 and 4 protein,but not messenger RNA,expression is associated with more severe disease in myelodysplastic syndromes and acute myeloblastic leukaemia

The human homologues of murine double minute 2 (MDM2) and 4 (MDM4) negatively regulate p53 tumour suppressor activity and are reported to be frequently overexpressed in human malignancies, prompting clinical trials with drugs that prevent interactions between MDM2/MDM4 and p53. Bone marrow samples from 111 patients with acute myeloblastic leukaemia, myelodysplastic syndrome or chronic myelomonocytic leukaemia were examined for protein (fluorescence-activated cell sorting) and messenger RNA (mRNA) expression (quantitative polymerase chain reaction) of MDM2, MDM4 and tumour protein p53 (TP53). Low protein expression of MDM2 and MDM4 was observed in immature cells from patients with excess of marrow blasts (>5%) compared with CD34⁺/CD45^low cells from healthy donors and patients without excess of marrow blasts (<5%). The mRNA levels were indistinguishable in all samples examined regardless of disease status or blast levels. Low MDM2 and MDM4 protein expression were correlated with poor survival. These data show a poor correlation between mRNA and protein expression levels, suggesting that quantitative flow cytometry analysis of protein expression levels should be used to predict and validate the efficacy of MDM2 and MDM4 inhibitors. These findings show that advanced disease is associated with reduced MDM2 and MDM4 protein expression and indicate that the utility of MDM2 and MDM4 inhibitors may have to be reconsidered in the treatment of advanced myeloid malignancies.     

A role of thymidine phosphorylase and P53 tissue protein expression in biology of endometrial cancer

Tumor suppressor gene p53, the most commonly mutated gene in human cancers has been shown to play an important role in the biology of gynaecological carcinomas. Thymidine phosphorylase is one of the most important angiogenic factors, which is connected with tumor expansion and invasiveness. The aim of the study was an evaluation of thymidine phosphorylase and p-53 tissue protein expression in human endometrial cancer cells by immunohistochemistry and comparison obtained data with clinicopathological factors as FIGO stage of disease and histopathologic grade. Endometrial cancer specimens were obtained from 55 postmenopausal patients (aged 52 to 74 years) operated by total abdominal hysterectomy with bilateral salpingo-oophorectomy. None of patients received preoperative pelvic irradiation. Histopathological typing and grading of the endometrial tumors (G-1, G-2, G-3) as well as myometrial invasion (<1/2, >1/2) were assessed using standard criteria, on hematoxylin-eosin sections. FIGO clinical stage of disease was determined. at the surgery. Thymidine phosphorylase overexpression was observed in 23 (41,8 %) cases. Although we found no statistically significant differences in TP expression between histopathologic grades, particular FIGO stages showed a significant trend of increase TP tumor overexpression. P53 protein overexpression was observed in tumor tissue in 21 cases (35,2%). It tended to be more frequent in cases of advanced disease as well as in low differentiated tumors. Although we found no statistically significant differences in p53 gene expression between groups of FIGO stage and histopathologic grade, we obtained a significant trend of increasing the P53 positive rate with both FIGO and tumour differentiation grading Joint assessment of thymidine phosphorylase and tumor suppressor genes expression may be of additional value in determining the biology of endometrial carcinoma. Key words: endometrial cancer, thymidine phosphorylase, p-53.     

Effects of MDM2 inhibitors on vascular endothelial growth factor-mediated tumor angiogenesis in human breast cancer

Background

Mouse double minute 2 (MDM2) is overexpressed in many malignant tumors, and MDM2 levels are associated with poor prognosis of several human cancers, including breast cancer. In the present study, we investigated the function of MDM2 in vascular endothelial growth factor (VEGF)-mediated tumor angiogenesis of breast cancer and the potential value of MDM2 as an anti-angiogenic therapy target for cancer therapy by inhibiting MDM2 with antisense oligonucleotides (ASO) or other antagonist nutlin-3.

Methods

Anti-MDM2 ASO and nutlin-3 were evaluated for their in vitro and in vivo anti-angiogenesis activities in different human breast cancer models with a different p53 status: MCF-7 cell line containing wild-type p53 and MDA-MB-468 cell line containing mutant p53. MCF-7 and MDA-MB-468 cells were incubated with different concentrations of ASO or nutlin-3 for various periods of time. VEGF gene and protein expression in tumor cells was measured by qPCR and Western blot. The level of VEGF protein secreted in the culture supernatant of treated cells was quantified by enzyme-linked immunosorbent assay (ELISA). Nude mouse xenograft models were further established to determine their effects on tumor growth and angiogenesis. Serum levels of VEGF were measured by ELISA. VEGF expression and microvessel density in tumor tissues were studied by immunohistochemistry. Both angiogenesis and tumor growth were digitally quantified.

Results

In both MCF-7 and MDA-MB-468 cells, VEGF expression and secretion were reduced, resulting from specific inhibition of MDM2 expression by ASO. In vivo assay, after administration of ASO, VEGF production reduced and anti-angiogenesis activity occurred in nude mice bearing MCF-7 or MDA-MB-468 xenograft. However, in both models treated with nutlin-3, VEGF production was not changed and anti-angiogenesis activity was not observed.

Conclusion

In summary, the ASO construct targeting MDM2 specifically suppresses VEGF expression in vitro and VEGF-mediated tumor angiogenesis in vivo in breast cancer. Furthermore, the suppression of VEGF expression subsequent to inhibition of MDM2 in p53 mutant cells suggests that MDM2 has a regulatory role on VEGF expression through a p53-independent mechanism.     

D-peptide inhibitors of the p53–MDM2 interaction for targeted molecular therapy of malignant neoplasms

The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53, conferring tumor development and survival. Antagonists targeting the p53-binding domains of MDM2 and MDMX kill tumor cells both in vitro and in vivo by reactivating the p53 pathway, promising a class of antitumor agents for cancer therapy. Aided by native chemical ligation and mirror image phage display, we recently identified a D-peptide inhibitor of the p53-MDM2 interaction termed ^DPMI-α (TNWYANLEKLLR) that competes with p53 for MDM2 binding at an affinity of 219 nM. Increased selection stringency resulted in a distinct D-peptide inhibitor termed ^DPMI-γ (DWWPLAFEALLR) that binds MDM2 at an affinity of 53 nM. Structural studies coupled with mutational analysis verified the mode of action of these D-peptides as MDM2-dependent p53 activators. Despite being resistant to proteolysis, both ^DPMI-α and ^DPMI-γ failed to actively traverse the cell membrane and, when conjugated to a cationic cell-penetrating peptide, were indiscriminately cytotoxic independently of p53 status. When encapsulated in liposomes decorated with an integrin-targeting cyclic-RGD peptide, however, ^DPMI-α exerted potent p53-dependent growth inhibitory activity against human glioblastoma in cell cultures and nude mouse xenograft models. Our findings validate D-peptide antagonists of MDM2 as a class of p53 activators for targeted molecular therapy of malignant neoplasms harboring WT p53 and elevated levels of MDM2.     

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.