Molecular Mechanism for the Downregularion of KU70 in Apoptotic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1274-1274
Author(s):  
Vivian Gama ◽  
Tomoyuki Yoshida ◽  
Jose Gomez ◽  
Arthur Haas ◽  
David Basile ◽  
...  
Keyword(s):  
Cell Death ◽  
Molecular Mechanism ◽  
Cell Lines ◽  
Hela Cells ◽  
Rat Kidney ◽  
Cell Types ◽  
Apoptotic Cells ◽  
Kidney Cells ◽  
Etoposide Treatment ◽  
Induced Apoptosis

Abstract Bax is a pro-apoptotic protein that plays a key role in apoptosis. Ku70 has been known as a DNA repair protein in the nucleus. Recently, we found that the cytosolic form of Ku70 has an anti-apoptotic activity to suppress the cytotoxic activity of Bax. Previously, we reported that apoptotic stimuli decreased Ku70 levels in human cancer cell lines such as HeLa cells and human epithelial kidney (HEK) 293T cells, and that the Ku70 disappearance is a caspase-independent event in the early phase of apoptosis in these cell lines. Based on these results, we hypothesized that the down regulation of cytosolic Ku70 levels is one of the mechanisms to trigger Bax-mediated cell death. In this study, (1) we examined whether Ku70 level decrease is a common apoptotic event in various cell types including primary normal (non-cancerous) cells, cancer cells and cells from rat kidney, and (2) we also investigated the molecular mechanism of Ku70 degradation in apoptotic cells. The effects of apoptotic stimuli on the cellular Ku70 levels were examined in 32D (EpoR wt) cells (IL-3-deprivation-induced apoptosis), the primary mouse mast cells (IL-3-deprivation-induced apoptosis), Human Umbilical Vein Endothelial Cells (HUVEC) (doxorubicin treatment), a human megakaryocytic cell line (DAMI cells) (etoposide treatment) and rat kidney cells (ischemia/reperfusion treatment). Ku70 levels were monitored by Western blot analysis. In all the systems examined, the decrease of Ku70 levels was confirmed, supporting our hypothesis that the down regulation of Ku70 is required for the induction of Bax-mediated cell death. To examine the possibility that apoptotic stimuli affect the transcription of Ku70 mRNA, real-time RT-PCR analysis was performed. Apoptotic stimuli did not change Ku70 mRNA levels in HeLa cells (staurosporin treatment) and DAMI cells(etoposide treatment), suggesting that apoptotic stimuli control Ku70 levels by post-translational modification. Although, there is no previous report of the ubiquitination of Ku70, we found that a large proportion of cellular Ku70 proteins are constitutively ubiquitinated in HeLa cells, HEK293T cells, DAMI cells, and in rat kidney cells. Higher molecular weight bands corresponding to ubiquitinated Ku70 become detectable only when Ku70 proteins are enriched by immunoprecipitation. MG132, a proteosome inhibitor, increased the accumulation of ubiquitinated Ku70, suggesting that ubiquitinated Ku70 proteins are subjected to proteosome-dependent degradation. Furthermore, in response to apoptotic stimuli, the levels of ubiquitinated Ku70 increased, probably accounting at least in part, for the decrease of non-modified Ku70 seen during apoptosis. In summary, we found that (1) apoptotic stimuli decrease cellular Ku70 levels in various cell types including cancer and normal cells, and that (2) ubiquitination of Ku70 is one of the mechanisms to down regulate Ku70 levels in apoptotic cells. Ku70 has multiple functions to protect cells from apoptosis and DNA damage. Our results indicate that the stability and probably the function of Ku70 can be regulated by ubiquitination. Identification of the ubiquitin ligase and the de-ubiquitinating enzyme will be needed to understand the precise mechanism of how stress response of cells regulates Ku70 function maintaining cell survival and genomic stability.

Caspase-2 is activated at the CD95 death-inducing signaling complex in the course of CD95-induced apoptosis

Blood ◽  
2006 ◽  
Vol 108 (2) ◽  
pp. 559-565 ◽  
Author(s):  
Inna N. Lavrik ◽  
Alexander Golks ◽  
Simone Baumann ◽  
Peter H. Krammer
Keyword(s):  
B Cell ◽  
Molecular Mechanism ◽  
Cell Lines ◽  
Caspase 8 ◽  
Signaling Complex ◽  
Deficient Cell ◽  
Apoptotic Pathways ◽  
Caspase 2 ◽  
Induced Apoptosis

Caspase-2 was reported to be involved in a number of apoptotic pathways triggered by various stimuli. However, the molecular mechanism of procaspase-2 activation in the course of apoptosis remains poorly defined. In this report, we demonstrate that procaspase-2 is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex (DISC) in human T- and B-cell lines. We show that procaspase-2 is activated at the DISC on CD95 stimulation. Despite its presence at the DISC, caspase-2 does not initiate apoptosis on CD95 stimulation in caspase-8–deficient cell lines. Taken together, our data reveal that caspase-2 is activated at the DISC but does not play an initiating role in the CD95-induced apoptosis.

scholarly journals Role of mitochondrial glucocorticoid receptor in glucocorticoid-induced apoptosis

2006 ◽  
Vol 203 (1) ◽  
pp. 189-201 ◽  
Author(s):  
Ronit Vogt Sionov ◽  
Orly Cohen ◽  
Shlomit Kfir ◽  
Yael Zilberman ◽  
Eitan Yefenof
Keyword(s):  
T Cell ◽  
Glucocorticoid Receptor ◽  
Cell Lines ◽  
Cell Types ◽  
Thymic Epithelial Cells ◽  
Dependent Manner ◽  
Localization Signal ◽  
Induced Apoptosis ◽  
T Cell Lines

The mechanisms by which glucocorticoid receptor (GR) mediates glucocorticoid (GC)-induced apoptosis are unknown. We studied the role of mitochondrial GR in this process. Dexamethasone induces GR translocation to the mitochondria in GC-sensitive, but not in GC-resistant, T cell lines. In contrast, nuclear GR translocation occurs in all cell types. Thymic epithelial cells, which cause apoptosis of the PD1.6 T cell line in a GR-dependent manner, induce GR translocation to the mitochondria, but not to the nucleus, suggesting a role for mitochondrial GR in eliciting apoptosis. This hypothesis is corroborated by the finding that a GR variant exclusively expressed in the mitochondria elicits apoptosis of several cancer cell lines. A putative mitochondrial localization signal was defined to amino acids 558–580 of human GR, which lies within the NH2-terminal part of the ligand-binding domain. Altogether, our data show that mitochondrial and nuclear translocations of GR are differentially regulated, and that mitochondrial GR translocation correlates with susceptibility to GC-induced apoptosis.

scholarly journals Upstream Regulatory Role for XIAP in Receptor-Mediated Apoptosis

2004 ◽  
Vol 24 (16) ◽  
pp. 7003-7014 ◽  
Author(s):  
John C. Wilkinson ◽  
Enrique Cepero ◽  
Lawrence H. Boise ◽  
Colin S. Duckett
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Death Receptor ◽  
Full Length ◽  
Proliferative Capacity ◽  
Term Survival ◽  
Truncation Mutant ◽  
Short And Long Term ◽  
Induced Apoptosis

ABSTRACT X-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of cell death that functions by suppressing caspases 3, 7, and 9. Here we describe the establishment of Jurkat-derived cell lines stably overexpressing either full-length XIAP or a truncation mutant of XIAP that can only inhibit caspase 9. Characterization of these cell lines revealed that following CD95 activation full-length XIAP supported both short- and long-term survival as well as proliferative capacity, in contrast to the truncation mutant but similar to Bcl-xL. Full-length XIAP was also able to inhibit CD95-mediated caspase 3 processing and activation, the mitochondrial release of cytochrome c and Smac/DIABLO, and the loss of mitochondrial membrane potential, whereas the XIAP truncation mutant failed to prevent any of these cell death events. Finally, suppression of XIAP levels by RNA interference sensitized Bcl-xL-overexpressing cells to death receptor-induced apoptosis. These data demonstrate for the first time that full-length XIAP inhibits caspase activation required for mitochondrial amplification of death receptor signals and that, by acting upstream of mitochondrial activation, XIAP supports the long-term proliferative capacity of cells following CD95 stimulation.

Distinct Apoptotic Responses Imparted by c-myc and max

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 1003-1010 ◽  
Author(s):  
Chadd E. Nesbit ◽  
Saijun Fan ◽  
Hong Zhang ◽  
Edward V. Prochownik
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Ectopic Expression ◽  
Drug Induced ◽  
Enhanced Sensitivity ◽  
Contrast Enhanced ◽  
Apoptotic Pathways ◽  
American Society ◽  
Induced Apoptosis ◽  
Cytokine Deprivation

Abstract The c-myc oncoprotein accelerates programmed cell death (apoptosis) after growth factor deprivation or pharmacological insult in many cell lines. We have shown that max, the obligate c-myc heterodimeric partner protein, also promotes apoptosis after serum withdrawal in NIH3T3 fibroblasts or cytokine deprivation in interleukin-3 (IL-3)-dependent 32D murine myeloid cells. We now show that c-myc– and max-overexpressing 32D cells differ in the nature of their apoptotic responses after IL-3 removal or treatment with chemotherapeutic compounds. In the presence of IL-3, c-myc overexpression enhances the sensitivity of 32D cells to Etoposide (Sigma, St Louis, MO), Adriamycin (Pharmacia, Columbus, OH), and Camptothecin (Sigma), whereas max overexpression increases sensitivity only to Camptothecin. Drug treatment of c-myc–overexpressing cells in the absence of IL-3 did not alter the spectrum of drug sensitivity other than to additively accelerate cell death. In contrast, enhanced sensitivity to Adriamycin, Etoposide, and Taxol (Bristol-Meyers Squibb, Princeton, NJ) was revealed in max-overexpressing cells concurrently deprived of IL-3. Differential rates of apoptosis were not strictly correlated with the ability of the drugs to promote G1 or G2/M arrest. Ectopic expression of Bcl-2 or Bcl-XL blocked drug-induced apoptosis in both cell lines. In contrast, whereas Bcl-2 blocked apoptosis in both cell lines in response to IL-3 withdrawal, Bcl-XL blocked apoptosis in max-overexpressing cells but not in c-myc–overexpressing cells. These results provide mechanistic underpinnings for the idea that c-myc and max modulate distinct apoptotic pathways. © 1998 by The American Society of Hematology.

scholarly journals Side-by-side comparison of BH3-mimetics identifies MCL-1 as a key therapeutic target in AML

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Larissa Ewald ◽  
Jessica Dittmann ◽  
Meike Vogler ◽  
Simone Fulda
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Treatment Options ◽  
B Cell Lymphoma ◽  
Bh3 Mimetics ◽  
Domain 3 ◽  
Functional Relevance ◽  
Induced Apoptosis ◽  
Apoptosis Inducing Agents

AbstractDespite advances in the treatment of acute myeloid leukemia (AML), prognosis of AML patients is still dismal and better treatment options are required. B-cell Lymphoma 2 (BCL-2) homology domain 3 (BH3)-mimetics are emerging as a novel class of apoptosis-inducing agents that are currently being tested for the treatment of different hematological malignancies including AML. Particularly, the selective BCL-2 inhibitor ABT-199/Venetoclax is demonstrating clinical responses and has recently been approved in combination for the treatment of AML. Compounds targeting the related protein MCL-1 have recently entered clinical trials, highlighting the urgency to compare the different BH3-mimetics and identify the most promising antiapoptotic target in AML. We performed a side-by-side comparison of different highly selective and potent BH3-mimetics targeting BCL-2 (ABT-199), MCL-1 (S63845) or BCL-xL (A1331852) in a panel of AML cell lines and primary patient cells. Gene knockdown using siRNAs was utilized to investigate the functional relevance of BCL-2 proteins. Western blotting and immunoprecipitations were used to explore the influence of BH3-mimetics on interactions between pro- and antiapoptotic BCL-2 proteins. A1331852 induced apoptosis only in selected cases, indicating that BCL-xL is not a very promising therapeutic target in AML. However, S63845 displayed higher potency than ABT-199, with more cell lines and primary cells responding to S63845 than to ABT-199. MCL-1 dependency in AML cells was confirmed by siRNA-mediated knockdown of MCL-1, which was sufficient to induce apoptosis. S63845-induced cell death was accompanied by a displacement of the BH3-only protein BIM as well as BAK, resulting in BAK-dependent apoptosis. In contrast, ABT-199-induced cell death was mediated by BAX rather than BAK, indicating distinct non-redundant molecular functions of BCL-2 and MCL-1 in AML. Our study reveals that MCL-1 may be a more prevalent therapeutic target than BCL-2 in AML and identifies BIM and BAK as important mediators of S63845-induced apoptosis in AML.

Caspase-dependent and -independent pathways for cadmium-induced apoptosis in cultured kidney proximal tubule cells

2006 ◽  
Vol 291 (4) ◽  
pp. F823-F832 ◽  
Author(s):  
Wing-Kee Lee ◽  
Marouan Abouhamed ◽  
Frank Thévenod
Keyword(s):  
Cell Death ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Cadmium Concentration ◽  
Nuclear Staining ◽  
Kidney Proximal Tubule ◽  
Cell Death Pathways ◽  
Cyt C ◽  
Induced Apoptosis ◽  
Apoptosis Inducing Factor

The nephrotoxic metal cadmium at micromolar concentrations induces apoptosis of rat kidney proximal tubule (PT) cells within 3–6 h of exposure. The underlying cell death pathways remain poorly defined. Using Hoechst 33342/ethidium bromide nuclear staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell death assays, 10–50 μM cadmium induced apoptosis of immortalized rat kidney cells derived from the S1-segment of PT at 6 and 24 h, but necrosis at 24 h only. Cadmium (10–50 μM) also caused mitochondrial cytochrome c (cyt. c)- and apoptosis-inducing factor release at 24 h, but not at 6 h, as measured by immunofluorescence imaging and immunoblotting. Caspases-9 and -3 were activated only by 10 μM cadmium for 24 h, and accordingly apoptosis was significantly reduced by the respective inhibitors (z-LEHD-fmk, z-DEVD-fmk; 10 μg/ml) at 24 h, but not at 6 h, without affecting necrosis. At 6 h, 10 μM cadmium increased the activity of the calcium-activated protease calpain, but not at 24 h, and calpain inhibitors (ALLN, PD 150606; 10–30 μM) blocked apoptosis by 10 μM cadmium at 3–6 h. However, PD-150606 also attenuated caspase-3 activity and apoptosis at 24 h, suggesting calpain-dependent caspase activation. Thus cadmium-induced apoptosis of PT cells involves a complex and sensitive interplay of signaling cascades involving mitochondrial proapoptotic factors, calpains and caspases, whose activation is also determined by cadmium concentration and the duration of cadmium exposure.

Uncovering Cardioprotective Strategies For Anthracycline Therapy By Analysis Of Redox and Apoptotic Effects

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1293-1293
Author(s):  
Daniela E. Egas Bejar ◽  
Joy M. Fulbright ◽  
Fernando F. Corrales-Medina ◽  
Mary E. Irwin ◽  
Blake Johnson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Vitamin E ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Cell Types ◽  
Leukemia Cells ◽  
Soluble Form ◽  
Leukemia Cell Lines

Abstract Anthracyclines are among the most powerful drugs used for the treatment of leukemia, however their use has been associated with cardiotoxicity. Reactive oxygen species (ROS) are generated in both cancer and normal cells after anthracycline exposure and have been implicated in both early and late onset cardiotoxicity. Counteracting this ROS generation are intracellular antioxidants such as the ubiquitous antioxidant glutathione (GSH), levels of which are depleted upon anthracycline exposure. Basal expression of GSH pathway components and other antioxidants vary greatly between different cell types. Due to this differential expression of cellular antioxidants in cardiomyocytes versus leukemia cells, we posit that anthracyclines exert distinct effects on oxidative stress and consequent apoptosis induction in leukemia cells and nontransformed hematopoietic cells (PBMC) relative to cardiomyocytes. As a result, we expect potentially varied mechanisms of cell death induction in these cell lines after anthracycline treatment. To test this hypothesis, the acute leukemia cell lines Jurkat and ML-1 and the cardiomyocyte line H9C2 were used. Dose responses with the anthracyclines, doxorubicin and daunorubicin, were carried out and trypan blue exclusion and propidium iodide staining followed by flow cytometry were used to assess viability and DNA fragmentation respectively. Cardiomyocytes had a 25-150 fold higher IC50 value than the acute leukemia cell lines, indicating selectivity. To assess whether apoptosis was induced by anthracyclines, caspase 3 activity was measured and found to be increased at 24 hours in Jurkat cells which preceded decreases in viability, supporting an apoptotic mechanism of cell death. GSH levels also decreased markedly after 24 hours of treatment with anthracyclines in this cell line, however, a pan-caspase inhibitor did not block GSH depletion, indicating that these events occur independent of each other. To evaluate whether antioxidants conferred protection against loss of viability in all cell types, cells were pretreated for at least 30 minutes with antioxidants and then treated with doxorubicin and daunorubicin for 24 hours. Antioxidants used were N-acetylcysteine (NAC, a GSH precursor and amino acid source), GSH ethyl ester (cell permeable form of GSH), tiron (free radical scavenger) and trolox (a water soluble form of vitamin E). GSH ethylester did not prevent cytotoxicity of anthracyclines in acute leukemia lines or cardiomyocytes. Therefore boosting GSH levels in leukemia cells does not reverse cytotoxicity. Trolox, however, did block anthracycline induced cell death in ML-1 cells, suggesting that vitamin E supplementation would counteract leukemia cell specific effects of anthracyclines on AML cells. Tiron protected PBMC from doxorubicin cytotoxicity but did not protect leukemia cells or cardiomyocytes, hinting at a protective strategy for normal non-leukemia blood cells. Interestingly, NAC did not interfere with the cytotoxic effects of anthracyclines on acute leukemia cells or PBMC, but protected H9C2 cells from daunorubicin cytotoxicity. Taken together, these data reveal differential protective effects of antioxidants in cardiomyocytes and PBMCs relative to ALL and AML cells. Our work indicates that NAC can protect cardiomyocytes without interfering with anthracycline cytotoxicity in acute leukemia cells. In humans, one randomized control trial tested the addition of NAC to doxorubicin therapy, detecting no evidence of cardioprotective activity by chronic administration of NAC. However, the schedule used for administration of NAC in that study may not have been optimal, and biomarkers for oxidative stress reduction by NAC were not incorporated into the trial. Previously, other antioxidants have been used with very limited clinical success and possible contributing factors include inadequate sample size, choice of agent, dose used, duration of intervention and the lack of biomarker endpoints. Designing a cardioprotective and antioxidant strategy with attention to these factors may prove to be efficacious in protecting cardiac cells without interfering with the antitumoral effect of anthracyclines. To this end, our data suggests that trolox and vitamin E analogues should not be used in acute leukemia as they may interfere with the cytotoxic action of anthracyclines but NAC or cysteine may be used as cardioprotectants. Disclosures: No relevant conflicts of interest to declare.

The mTOR Inhibitor Everolimus Overcomes CXCR4-Mediated Resistance to HDAC Inhibitor Panobinostat through Inhibition of p21 and Mitosis Regulators, Sensitizing MM Cells to DNA-Damaged Induced Apoptosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 891-891
Author(s):  
Katia Beider ◽  
Valeria Voevoda ◽  
Hanna Bitner ◽  
Evgenia Rosenberg ◽  
Hila Magen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Cell Lines ◽  
Hdac Inhibitor ◽  
Mtor Inhibitor ◽  
Mtor Inhibition ◽  
Induced Apoptosis

Abstract Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Despite the initial efficacious treatment, MM patients often become refractory to common anti-MM drugs, therefore novel therapies are in need. Pan-histone deacetylase (HDAC) inhibitor panobinostat exerts multiple cytotoxic actions in MM cells in vitro, and was approved for the treatment of relapsed/refractory MM in combination with bortezomib and dexamethasone. Although having promising anti-MM properties, panobinostat lacks therapeutic activity as monotherapy. The aim of the current study was to elucidate the mechanisms underlying MM resistance to panobinostat and to define strategies to overcome it. Results: Panobinostat at the low concentrations (IC50 5-30 nM) suppressed the viability in MM cell lines (n=7) and primary CD138+ cells from MM patients (n=8) in vitro. Sensitivity to panobinostat correlated with reduced expression of chemokine receptor CXCR4, while overexpression of CXCR4 or its ligand CXCL12 in RPMI8226 and CAG MM cell lines significantly (p<0.001) increased their resistance to panobinostat, pointing to the role of the CXCR4 axis in HDACi response. Notably, similar expression levels of class I HDACs (HDAC1-3) were detected in MM cells with either low or high CXCR4. Interaction with BM stromal cells that represent the source of CXCL12 also protected MM cells from panobinostat-induced apoptosis, further strengthening a role for CXCR4 downstream pathway. Decreased sensitivity to cytotoxic effect was concomitant with reduced histone (H3K9 and H4K8) acetylation in response to panobinostat treatment. In addition, resistance to HDACi was associated with the reversible G0/G1 cell growth arrest, whereas sensitivity was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators involved in CXCR4-mediated HDACi resistance revealed the pro-survival AKT/mTOR pathway to be regulated by both CXCR4 over-expression and interaction with BMSCs. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance and induced synergistic cell death of MM cell lines and primary MM cells, but not of normal mononuclear cells (CI<0.4). This effect was concurrent with the increase in DNA double strand breaks, histone H2AX phosphorylation, loss of Dψm, cytochrome c release, caspase 3 activation and PARP cleavage. The increase in DNA damage upon combinational treatment was not secondary to the apoptotic DNA fragmentation, as it occurred similarly when apoptosis onset was blocked by caspase inhibitor z-VAD-fmk. Kinetics studies also confirmed that panobinostat-induced DNA damage preceded apoptosis induction. Strikingly, combined panobinostat/everolimus treatment resulted in sustained DNA damage and irreversible suppression of MM cell proliferation accompanied by robust apoptosis, in contrast to the modest effects induced by single agent. Gene expression analysis revealed distinct genetic profiles of single versus combined exposures. Whereas panobinostat increased the expression of cell cycle inhibitors GADD45G and p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated DNA repair genes, including RAD21, Ku70, Ku80 and DNA-PKcs. Furthermore, combined treatment markedly decreased both mRNA and protein expression of anti-apoptotic factors survivin and BCL-XL, checkpoint regulator CHK1, and G2/M-specific factors PLK1, CDK1 and cyclin B1, therefore suppressing the DNA damage repair and inhibiting mitotic progression. Given the anti-apoptotic role of p21, the synergistic lethal effect of everolimus could be attributed to its ability to suppress p21 induction by panobinostat ensuing the shift in the DNA damage response toward apoptosis. Conclusions: Collectively, our findings indicate that CXCR4/CXCL12 activity promotes the resistance of MM cells to HDACi with panobinostat through mTOR activation. Inhibition of mTOR by everolimus synergizes with panobinostat by simultaneous suppression of p21, G2/M mitotic factors and DNA repair machinery, rendering MM cells incapable of repairing accumulated DNA damage and promoting their apoptosis. Our results unravel the mechanism responsible for strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel therapeutic strategy to eradicate MM. Disclosures No relevant conflicts of interest to declare.

Targeting mutant p53 with PK11007: A new approach for the treatment of patients with triple-negative breast cancer?

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e14099-e14099 ◽  
Author(s):  
Naoise C Synnott ◽  
Matthias R Bauer ◽  
Stephen F. Madden ◽  
Alyson M. Murray ◽  
Rut Klinger ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Death ◽  
Cell Lines ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
P53 Protein ◽  
Mutant P53 ◽  
Inhibition Of Proliferation ◽  
Tnbc Cell ◽  
Induced Apoptosis

e14099 Background:The identification of a targeted therapy for patients with triple-negative breast cancer (TNBC) is one of the most urgent needs in breast cancer therapeutics. Since the p53 gene is mutated in approximately 80% of TNBC patients, it is a potential therapeutic target for this form of breast cancer. PK11007 is a 2-sulfonypyrimidine that stabilizes and reactivates mutant p53 (Bauer et al, PNAS 2016). The compound recently was reported to preferentially decrease viability in p53-compromised cancer cells. The aim of this investigation was to evaluate PK11007 as a potential new treatment for TNBC. Methods: Cell viability was determined using the MTT assay. Apoptosis was detected using Annexin V Apoptosis Detection Kit. Migration was determined by Transwell migration assay. Knockdowns of p53 protein were carried out using predesigned Flexitube sequences (Qiagen). Results: IC50 values for inhibition of proliferation by PK11007 in the panel of 17 breast cell lines ranged from 2.3 to 42.2 μM. There were significantly lower IC50values for TNBC than for non-TNBC cell lines (p = 0.03) and for p53-mutated cell lines compared with p53 WT cells (p = 0.003). Response to PK11007 however, was independent of ER or HER2 status of the cells. In addition, PK11007 induced apoptosis and inhibited migration in p53 mutant cell lines. Using RNAseq and gene ontogeny analysis, we found that PK11007 altered the expression of genes enriched in pathways involved in regulated cell death, regulation of apoptosis, signal transduction, protein refolding and locomotion. To establish if PK11007 acts by targeting mutant p53, we used siRNA to knockdown p53 in 3 p53-mutated TNBC cell lines. Reduction in p53 protein levels resulted in a significant decrease in the growth inhibitory effects of PK11007, in all 3 cell lines investigated, suggesting that PK11007 mediates growth inhibition via p53. The observations that PK11007 inhibited cell growth, induced apoptosis, blocked cell migration and altered genes involved in cell death, are all consistent with the ability of PK11007 to activate mutant p53. Conclusions: Based on our data, we conclude that targeting mutant p53 with PK11007 is a potential approach for treating p53-mutated TNBC.

scholarly journals Flow cytometric analysis of spontaneous and dexamethasone-induced apoptosis in thymocytes from severely malnourished rats

2001 ◽  
Vol 86 (5) ◽  
pp. 545-548 ◽  
Author(s):  
Rocío Ortiz ◽  
Leticia Corté ◽  
Humberto Gonz´lez–M´rquez ◽  
José Luis Gómez ◽  
Cristina Gonz´lez ◽  
...  
Keyword(s):  
Cell Death ◽  
Flow Cytometric Analysis ◽  
Experimental Models ◽  
Severe Malnutrition ◽  
Apoptotic Cells ◽  
Principal Mechanism ◽  
High Prevalence ◽  
Thymocyte Apoptosis ◽  
Induced Apoptosis

Severe malnutrition is widely distributed throughout the world, showing a high prevalence in developing countries. Experimental animal models have been useful to study the effects of malnutrition at different levels and ages. Apoptosis is a well recognised process of cell death occurring under several physiological and pathological conditions. It represents the principal mechanism involved in cell selection in the thymus. Thymocyte apoptosis induction by dexamethasone is one of the best characterised experimental models of programmed cell death. The aim of the present study was to determine whether severe malnutrition increased spontaneous and/or dexamethasone-induced apoptosisin vivoin thymocytes of experimentally malnourished rats during lactation. Thymocytes were obtained from malnourished rats at weaning (21d of age). Apoptosis frequency was estimated by the terminal transferase-mediated dUTP nick end labelling assay. Spontaneous apoptosis was 1·9 (SD 1·0) % IN WELL NOURISHED RATS IN CONTRAST TO 13·3 (sd 3·8) % in malnourished animals; this is seven times greater (P<0·001). Interestingly, the frequency of dexamethasone-induced apoptosis was similar in both groups of animals (47·9 (sd 10·1) % in well nourished rats and 53·8 (sd 8·0) % in malnourished rats). The results obtained in the present study indicate that malnutrition is associated with a significant increase of spontaneously apoptotic cells. In addition, the data showed that the fraction of thymocytes susceptible to dexamethasone-induced apoptosis was similar in well nourished and malnourished animals. The greater levels of spontaneously apoptotic cells associated with malnutrition could be related to alterations of the microenvironment of the thymus and/or to an obstruction of early thymocyte maturation.

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