Inhibition of p53-MDM2 Interaction by Small-Molecule Antagonist of MDM2 Effectively Induces Apoptosis in Leukemias.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2536-2536
Author(s):  
Kensuke Kojima ◽  
Marina Konopleva ◽  
Masato Shikami ◽  
Maria Cabreira-Hansen ◽  
C. Ellen Jackson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Annexin V ◽  
Cell Number ◽  
Leukemia Cells ◽  
P53 Mutations ◽  
Wild Type ◽  
Cycle Arrest ◽  
Wild Type P53 ◽  
Induced Apoptosis

Abstract Alteration of the p53 gene is one of the most frequent events in human tumorigenesis and about 50% of all solid tumors have been reported to carry p53 mutations. The inactivation of p53 in cancer has been associated with poor survival, refractory disease and chemoresistance. p53 mutations rarely occur in hematopoietic malignancies. Instead, MDM2 which is a principal cellular antagonist of p53, is overexpressed in the majority of leukemias. Recently, potent and selective small-molecule antagonists of MDM2, Nutlins, have been identified (Science303:844–888, 2004). Nutlins bind MDM2 in the p53-binding pocket and activate the p53 pathway in human cancer cells with wild-type p53, leading to cell cycle arrest, apoptosis, and growth inhibition of human tumor xenografts in nude mice. In this study, we investigated the potential antileukemic activity of the MDM2 antagonist. Treatment of wild-type p53 OCI-AML-3 cells with 5 μM of an active compound (Nutlin-3a) induced cell cycle arrest and apoptosis as evidenced by flow-cytometric analysis (51% reduction of S-phase at 12 h, 27% sub-G1 DNA content and 57% Annexin V positivity at 48 h). Similar proapoptotic effects were observed in MOLM-13 cells which have wild-type p53, but not in p53-null (HL-60 and U937) or mutant p53 (Raji, Jurkat and NB-4) cells. Nutlin-3a induced apoptosis in a dose- and time-dependent manner, and induced maximal effect on cell cycle arrest at 1 μM. Western blot analysis showed that in OCI-AML-3 cells, wild-type p53 accumulated at 1 h after exposure to Nutlin-3a. Increased levels of MDM2, p21 and Noxa proteins were observed at 1 to 3h. This resulted in cleavage of caspase-9 followed by cleavage of caspase-3. A pharmacologic interaction study between MDM2 inhibitor and Ara-C using a fixed-ratio (1:1) experimental design showed highly synergistic cell growth inhibition (CI = 0.44) and induction of apoptosis (CI = 0.83) in OCI-AML-3 cells. Initial studies conducted in primary leukemia cells demonstrated that Nutlin-3a induced apoptosis in 4 of 5 AML samples tested (68–97% Annexin V induction and 65–93% cell number reduction) and 2 CLL samples (>50% Annexin V induction and 37% and 58% cell number reduction). Since MDM2 protein is overexpressed and p53 is not mutated in the majority of primary leukemia cells, this approach may have therapeutic utility in leukemias.

scholarly journals Co-expression of murine double minute 2 siRNA and wild-type p53 induces G1 cell cycle arrest in H1299 cells

2017 ◽  
Vol 16 (6) ◽  
pp. 9137-9142
Author(s):  
Long Liu ◽  
Ping Zhang ◽  
Hua Guo ◽  
Xinyu Tang ◽  
Lianqin Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Wild Type ◽  
Cycle Arrest ◽  
Double Minute ◽  
G1 Cell Cycle Arrest ◽  
Murine Double Minute ◽  
Wild Type P53

scholarly journals The Fanconi anemia complementation group C protein corrects DNA interstrand cross-link-specific apoptosis in HSC536N cells

Blood ◽  
1996 ◽  
Vol 88 (6) ◽  
pp. 2298-2305 ◽  
Author(s):  
UK Marathi ◽  
SR Howell ◽  
RA Ashmun ◽  
TP Brent
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fanconi Anemia ◽  
Complementation Group ◽  
Cross Linking ◽  
Wild Type ◽  
Cross Link ◽  
Cycle Arrest ◽  
Protein Functions ◽  
Induced Apoptosis

Fanconi anemia (FA) cells are hypersensitive to cytotoxicity, cell cycle arrest, and chromosomal aberrations induced by DNA cross-linking agents, such as mitomycin C (MMC) and nitrogen mustard (HN2). Although MMC hypersensitivity is complemented in a subset of FA cells (complementation group C [FA-C]) by wild-type FAC cDNA, the cytoprotective mechanism is unknown. In the current study, we tested the hypothesis that FAC protein functions in the suppression of DNA interstand cross-link (ISC)-induced cell cycle arrest and apoptosis. Comparison of HN2-induced cell cycle arrest and apoptosis with those of its non-cross-linking analogs, diethylaminoethyl chloride and 2- dimethylaminoethyl chloride, delineated the DNA ISC specificity of FAC- mediated cytoprotection. Overexpression of wild-type FAC cDNA in FA-C lymphoblasts (HSC536N cell line) prevented HN2-induced growth inhibition, G2 arrest, and DNA fragmentation that is characteristic of apoptosis. In contrast cytoprotection was not conferred against the effects of the non-cross-linking mustards. Our data show that DNA ISCs induce apoptosis more potently than do DNA monoadducts and suggest that FAC suppresses specifically DNA ISC-induced apoptosis in the G2 phase of the cell cycle.

scholarly journals Enhanced wild-type p53 expression by small activating RNA dsP53-285 induces cell cycle arrest and apoptosis in pheochromocytoma cell line PC12

2017 ◽  
Vol 38 (5) ◽  
pp. 3160-3166 ◽  
Author(s):  
Dengqiang Lin ◽  
Li Meng ◽  
Feifei Xu ◽  
Jianpo Lian ◽  
Yunze Xu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Line ◽  
Wild Type ◽  
P53 Expression ◽  
Cycle Arrest ◽  
Pheochromocytoma Cell ◽  
Wild Type P53

scholarly journals Complementation by wild-type p53 of interleukin-6 effects on M1 cells: induction of cell cycle exit and cooperativity with c-myc suppression.

1993 ◽  
Vol 13 (12) ◽  
pp. 7942-7952 ◽  
Author(s):  
N Levy ◽  
E Yonish-Rouach ◽  
M Oren ◽  
A Kimchi
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cell Cycle Arrest ◽  
Interleukin 6 ◽  
Quiescent State ◽  
Permissive Temperature ◽  
Wild Type ◽  
Antiproliferative Effects ◽  
Cycle Arrest ◽  
Wild Type P53

Stable transfection of M1 myeloid leukemia cells with a temperature-sensitive mutant of p53 results in two phenomena that are manifested exclusively at the permissive temperature. On one hand, activation of wild-type p53 by the temperature shift induced an apoptotic type of cell death which could be inhibited by interleukin-6 (IL-6) (E. Yonish-Rouach, D. Resnitzky, J. Lotem, L. Sachs, A. Kimchi, and M. Oren, Nature 352:345-347, 1991). On the other hand, as reported in this work, activated p53 complemented the antiproliferative effects of IL-6 in M1 cells. A shift to the permissive temperature concomitant with or early after IL-6 treatment imposed a novel pattern of cell cycle arrest in which about 95% of the cells were retained within a G0-like quiescent state. This phase was characterized by 2N DNA content and low RNA and protein content. On the molecular level, activation of wild-type p53 transrepressed the c-myc gene but not the cyclin A, D1, or D2 gene, which are all independently suppressed by IL-6 in M1 cells. To further analyze whether c-myc inhibition mediates or complements p53 effects, the p53-transfected M1 cells were infected with a retroviral vector expressing deregulated c-myc, refractory to p53 or IL-6 action. It was found that the process of cell death was not interrupted at all in these M1 c-myc-p53 double transfectants, suggesting that the transrepression of c-myc is not a major obligatory event mediating p53-induced cell death. In addition, some of the antiproliferative effects of activated p53, manifested in the presence of IL-6, could still be transmitted in the background of constitutive c-myc. Yet the context of deregulated c-myc interfered with the final accumulation of cells within a G0-like phase, suggesting complementary interactions between the outcome of p53 activation and of c-myc suppression in the control of cell cycle arrest.

Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53

Cell ◽  
1992 ◽  
Vol 70 (6) ◽  
pp. 923-935 ◽  
Author(s):  
Laura R. Livingstone ◽  
Alicia White ◽  
Jason Sprouse ◽  
Elizabeth Livanos ◽  
Tyler Jacks ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Gene Amplification ◽  
Wild Type ◽  
Cycle Arrest ◽  
Wild Type P53 ◽  
Altered Cell

The MDM2 Antagonist Nutlin-3 Is Effective to Aggressive NK-Cell Neoplasms with Wild Type p53 in Hypoxia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4999-4999
Author(s):  
Yoko Tabe ◽  
Yasushi Isobe ◽  
Koichi Sugimoto ◽  
Linhua Jin ◽  
Kazuo Oshimi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Nk Cell ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Wild Type ◽  
Cycle Arrest ◽  
G1 Cell Cycle Arrest

Abstract Abstract 4999 Natural killer (NK) cell neoplasms, including extranodal NK/T-cell lymphoma, nasal type (ENKL) and aggressive NK cell leukemia (ANKL), show a highly aggressive clinical course with poor response to chemotherapy, and new treatment approaches are urgently needed to improve cure rates. Patients with NK cell neoplasms cluster in Asia and Latin American countries, and the frequency of p53 mutations has been reported to be various by district. We have demonstrated that MDM2 protein was overexpressed in aggressive subclasses of NK cell neoplasms (Sugimoto et al. Jap J Cancer Res. 2002), which suggests that wild-type p53 expressing malignant NK cells may be a good candidate for biologic therapies that abrogate MDM2-p53 interactions and lead to cell death. Nutlin-3 is a small-molecule antagonist of MDM2 that efficiently blocks the MDM2-p53 interaction. In this study, we investigated the effects of nutlin-3 in 3 cell lines of ENKL and ANKL with known p53 mutation status (wt-p53: NK-YS, HANK-1; mt-p53: KHYG-1). Since aggressive NK-cell neoplasms arise in hypoxic environments and usually show an angiodestructive-infiltration pattern resulting in the tissue necrosis, we tried to assess the anti-proliferative effects and molecular mechanisms of nutlin-3 in the hypoxic condition. For hypoxia experiments, cells were cultured under 1.0% O2 for at least 14 days to assure their continuous proliferation and survival. Under hypoxia, more cells were positive to Annexin V than in normoxia, indicating that hypoxic conditions promote apoptosis in NK cell neoplasms. Nutlin-3 treatment in normoxia resulted in a reduction of cell proliferation with G0/G1 cell cycle arrest in a time and concentration-dependent manner in wt-p53 cells (IC50 at 48 hrs; 3.2 μM for NK-YS and 5.0 μM for HANK-1, MTT test). In hypoxia, nutlin-3 further enhanced cell growth inhibition and G0/G1 cell cycle arrest. An increase in the specific apoptosis (sub G1 and annexin V positivity) by nutlin-3 was observed with similar level between normoxia and hypoxia. The mt-p53 KHYG-1 cells demonstrated neither cell cycle arrest nor increase in the apoptotic cell fraction after nutlin-3 treatment. In the wt-p53 NK-YS and HANK-1 cells, nutlin-3 treatment increased the cellular levels of p53, and p53 dependent proteins including p21, MDM2 itself and the proapoptotic BH3-only proteins Noxa and Puma followed by the activation of caspase-9 and caspase-3 regardless of foxygen level. We observed no significant increase in the p53 targets in the mt-p53 overexpressing KHYG-1 cells. L-asparaginase has been demonstrated to induce apoptosis in aggressive NK cell neopplasms. To determine if inhibition of the TP53-MDM2 interaction by nutlin-3 in NK cell neoplasms might potentiate the effects of L-asparaginase, we assessed the effect of combining the two drugs. However, L-asparaginase induced apoptosis only in NK-YS cells, and no synergistic anti-proliferative effect was observed in any of the cell lines analyzed. These findings demonstrate that nutlin-3 successfully activates wt-p53 in NK cell neoplasms leading to the upregulation of traditional targets such as p21 and proapoptotic proteins including Noxa and Puma, and result in apoptotic cell death regardless of oxygen concentration. The data suggest that p53 activators such as nutlin-3 may be considerable for selected patients with wt-p53 NK cell neoplasms. Disclosures: No relevant conflicts of interest to declare.

Activation of the EfnB1/EphB1 Forward Signaling Promotes Cell Cycle Arrest in Acute Myeloid Leukemia Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4719-4719
Author(s):  
Kim R Kampen ◽  
Arja ter Elst ◽  
Evelina S. De Bont
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Annexin V ◽  
Cycle Arrest ◽  
Apoptosis Assay ◽  
Ligand Stimulation ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Abstract 4719 Ephrin signaling has been shown to contribute to the pathogenesis of many solid tumors with respect to tumor growth, tumor cell survival, angiogenesis, and metastasizing capacity (Cytokine Growth Factor Rev. Dec;15(6):419-33, et al. Neuro Oncol. 2012). Recently, an aberrant DNA methylation status of ephrin receptors and ligands was described to be associated with outcome in acute lymphoblastic leukemia (Blood.2010 Mar 25;115(12):2412-9). In acute myeloid leukemia (AML), we found an intriguing heterogeneity in membrane receptor expression levels of EphB1. Therefore, we challenged to evaluate the role of EphB1 receptor forward signaling in AML. We investigated the influence of the EphB1 receptor forward signaling in THP-1 (EphB1high), HL60 (EphB1int), and MOLM13 (EphB1low) AML cell lines through exogenous stimulation with the EphB1 ligand; EfnB1. EfnB1 stimulation of the AML cell lines demonstrated to reduce AML growth solely in the EphB1high and EphB1int cell lines (Fig. 1A,P = 0.001 and P =.056). In addition, EfnB1 stimulation induced apoptosis most pronounced in the EphB1high cells (Fig. 1B). Interestingly, MGG stained cytospins of EfnB1 treated THP-1 showed multinucleation of AML cells (Fig. 1C). We hypothesized that these phenotypic effects could be assigned to cell cycle arrest in THP-1 cells. Additionally, cell cycle regulatory proteins CDC2 and CyclinB1 were evaluated by immunoblotting of EfnB1 stimulated THP-1 cells. Phosphorylation of the inactivating CDC2 Tyr15-site demonstrated to be up-regulated in EfnB1 stimulated THP-1 cells, which might be initiated by the increased total CDC2 protein levels that we found (Fig. 1D). CyclinB1 displays enhanced protein expression in EfnB1 treated THP-1 cells. Moreover, quantitative RT-PCR analysis showed that the expression of cell cycle inhibitor p21 is significantly induced by 3-fold in EfnB1 stimulated THP-1 cells, via increasing levels of p53 (Fig. 1E, both P = <0.001). To verify whether the EfnB1 induced cell cycle arrest is EphB1 specific, we enforced EphB1 expression in HL60 EphB1int and MOLM13 EphB1low AML cells by introducing a GFP fused EphB1 overexpression construct. EphB1 overexpression increased EphB1 protein expression levels sufficiently in both AML cell lines, as confirmed by flowcytometric analysis and immunoblots. Exogenous EfnB1 stimulation further increased the apoptosis in EphB1 overexpressing cells in both AML cell lines (Fig. 1F). Again, we found increasing levels of phospho-CDC2Tyr15 and CyclinB1 by immunoblots. From this study, we conclude that AML cells with high EphB1 expression can be forced into a cell cycle arrest upon ligand binding in vitro, while AML cells lacking EphB1 expression have a proliferative and anti-apoptotic survival advantage. The clinical significance and exploitation of EphB1 induced cell cycle arrest in AML will be analyzed in the near future. Figure 1. EfnB1 induced activation of the EphB1 in AML cell lines promotes cell cycle arrest and apoptosis Figure 1. EfnB1 induced activation of the EphB1 in AML cell lines promotes cell cycle arrest and apoptosis (A) Absolute cell counts represent the growth inhibitory effects of EfnB1 ligand stimulation in AML cell lines THP-1 and HL60. (B) Flowcytometric Annexin V/PI apoptosis assay displayed the induction of apoptosis as a result of EfnB1 ligand stimulation in THP-1 and MOLM-13 AML cells. (C) MGG stained cytospins of EfnB1 treated THP-1 cells promotes the induction of multinucleated cells due to cell cycle arrest. (D) Immunoblots showed an enhanced apoptotic BAX/BCL2 ratio, in synergy with an upregulation of cell cycle inactivating checkpoint kinase CDC2Tyr15 upon EfnB1 stimulation in THP-1. (E) qRT-PCR confirms cell cycle inhibition by a 3-fold upregulation of p21 and a 1.5-fold induced expression of p53 in THP-1 EfnB1 stimulated cells. (F) The flowcytometric Annexin V/PI apoptosis assay showed that EfnB1 ligand induced apoptosis is even further induced in EphB1 overexpressing HL60 and MOLM-13 cells. Disclosures: No relevant conflicts of interest to declare.

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