Dominant-negative FADD inhibits TNFR60-, Fas/Apo1-  and TRAIL-R/Apo2-mediated cell death but not gene induction

ABSTRACTHPV16 E7 has long been noted to stabilize the TP53 tumor suppressor. However, the molecular mechanism of TP53 stabilization by HPV16 E7 has remained obscure and can occur independent of E2F regulated MDM2 inhibitor, p14ARF. Here, we report that the Damage Induced Noncoding (DINO) lncRNA (DINOL) is the missing link between HPV16 E7 and increased TP53 levels. DINO levels are decreased in cells where TP53 is inactivated, either by HPV16 E6, expression of a dominant negative TP53 minigene or by TP53 depletion. DINO levels are increased in HPV16 E7 expressing cells. HPV16 E7 causes increased DINO expression independent of RB1 degradation and E2F1 activation. Similar to the adjacent CDKN1A locus, DINO expression is regulated by the histone demethylase, KDM6A. DINO stabilizes TP53 in HPV16 E7 expressing cells and as a TP53 transcriptional target, DINO levels further increase. Similar to other oncogenes such as adenovirus E1A or MYC, HPV16 E7 expressing cells are sensitized to cell death under conditions of metabolic stress and in the case of E7, this has been linked to TP53 activation. Consistent with earlier studies, we show that HPV16 E7 expressing keratinocytes are highly sensitive to metabolic stress induced by the antidiabetic drug, metformin. Metformin sensitivity of HPV16 E7 expressing cells is rescued by DINO depletion. This work identifies DINO as a critical mediator TP53 stabilization and activation in HPV16 E7 expressing cells.IMPORTANCEViral oncoproteins, including HPV16 E6 and E7 have been instrumental in elucidating the activities of cellular signaling networks including those governed by the TP53 tumor suppressor. Our study demonstrates that the long noncoding RNA DINO is the long sought missing link between HPV16 E7 and elevated TP53 levels. Importantly, the TP53 stabilizing DINO plays a critical role in the predisposition of HPV16 E7 expressing cells to cell death under metabolic stress conditions from metformin treatment.

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Increased cytochrome P-450 2E1 expression sensitizes hepatocytes to c-Jun-mediated cell death from TNF-α

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00304.2001 ◽

2002 ◽

Vol 282 (2) ◽

pp. G257-G266 ◽

Cited By ~ 60

Author(s):

Hailing Liu ◽

Brett E. Jones ◽

Cynthia Bradham ◽

Mark J. Czaja

Keyword(s):

Cell Death ◽

Oxidant Stress ◽

Nuclear Factor Κb ◽

Dominant Negative ◽

Tnf Α ◽

Cyp2e1 Expression ◽

Factor Α ◽

Jnk Activation ◽

Cytochrome P 450 ◽

Necrosis Factor

The mechanisms underlying hepatocyte sensitization to tumor necrosis factor-α (TNF-α)-mediated cell death remain unclear. Increases in hepatocellular oxidant stress such as those that occur with hepatic overexpression of cytochrome P-450 2E1 (CYP2E1) may promote TNF-α death. TNF-α treatment of hepatocyte cell lines with differential CYP2E1 expression demonstrated that overexpression of CYP2E1 converted the hepatocyte TNF-α response from proliferation to apoptotic and necrotic cell death. Death occurred despite the presence of increased levels of nuclear factor-κB transcriptional activity and was associated with increased lipid peroxidation and GSH depletion. CYP2E1-overexpressing hepatocytes had increased basal and TNF-α-induced levels of c-Jun NH2-terminal kinase (JNK) activity, as well as prolonged JNK activation after TNF-α stimulation. Sensitization to TNF-α-induced cell death by CYP2E1 overexpression was inhibited by antioxidants or adenoviral expression of a dominant-negative c-Jun. Increased CYP2E1 expression sensitized hepatocytes to TNF-α toxicity mediated by c-Jun and overwhelming oxidative stress. The chronic increase in intracellular oxidant stress created by CYP2E1 overexpression may serve as a mechanism by which hepatocytes are sensitized to TNF-α toxicity in liver disease.

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Interaction with Sug1 enables Ipaf ubiquitination leading to caspase 8 activation and cell death

Biochemical Journal ◽

10.1042/bj20091349 ◽

2010 ◽

Vol 427 (1) ◽

pp. 91-104 ◽

Cited By ~ 26

Author(s):

Yatender Kumar ◽

Vegesna Radha ◽

Ghanshyam Swarup

Keyword(s):

Cell Death ◽

Mammalian Cells ◽

Intramolecular Interaction ◽

Dominant Negative ◽

26S Proteasome ◽

Caspase 8 ◽

Interacting Protein ◽

Caspase 1 ◽

Lung Carcinoma Cells ◽

Lrr Domain

Activation of initiator caspases is dependent on interacting proteins, and Ipaf [ICE (interleukin-1β-converting enzyme)-protease activating factor] {NLRC4 [NLR (Nod-like receptor) family CARD (caspase activation and recruitment domain)-containing 4]} an inflammasome component, is involved in caspase 1 activation and apoptosis. Investigating the mechanisms of Ipaf activation, we found that the C-terminal LRR (leucine-rich repeat) domain of Ipaf, through intramolecular interaction, negatively regulates its apoptosis-inducing function. In A549 lung carcinoma cells, expression of Ac-Ipaf (LRR-domain-deleted Ipaf) induced cell death that was dependent on caspase 8, but not on caspase 1. A yeast two-hybrid screen using Ac-Ipaf as bait identified human Sug1 (suppressor of gal 1), a component of the 26S proteasome, as an interacting protein. In mammalian cells Sug1 interacts and co-localizes with Ipaf. Sug1 binds to amino acids 91–253 of Ipaf, which is also the region that the LRR domain binds to. It potentiates cell death induced by Ipaf and Ac-Ipaf, and co-expression of Sug1 and Ipaf induces caspase-8-dependent cell death. Cellular complexes formed by Ipaf and Sug1 contain caspase 8. Expression of Ac-Ipaf or co-expression of Sug1 with Ipaf results in the formation of cytoplasmic aggregates and caspase 8 activation. Sug1 co-expression enabled modification of Ipaf by ubiquitination. Tagging ubiquitin molecules to Ipaf led to aggregate formation, enhanced caspase 8 interaction and activation, resulting in induction of cell death. Using RNAi (RNA interference) and dominant-negative approaches, we have shown that cell death induced by Ac-Ipaf expression or by treatment with TNF-α (tumour necrosis factor α) or doxorubicin is dependent on Sug1. Our results suggest a role for ubiquitination of Ipaf that is enabled by its interaction with Sug1, leading to caspase 8 activation and cell death.

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Dominant-negative ATF5 rapidly depletes survivin in tumor cells

Cell Death and Disease ◽

10.1038/s41419-019-1872-y ◽

2019 ◽

Vol 10 (10) ◽

Author(s):

Xiaotian Sun ◽

James M. Angelastro ◽

David Merino ◽

Qing Zhou ◽

Markus D. Siegelin ◽

...

Keyword(s):

Cell Death ◽

Tumor Cell ◽

Cancer Cells ◽

Dominant Negative ◽

Tumor Cell Death ◽

Cell Penetrating ◽

Selective Death ◽

Tumor Types

Abstract Survivin (BIRC5, product of the BIRC5 gene) is highly expressed in many tumor types and has been widely identified as a potential target for cancer therapy. However, effective anti-survivin drugs remain to be developed. Here we report that both vector-delivered and cell-penetrating dominant-negative (dn) forms of the transcription factor ATF5 that promote selective death of cancer cells in vitro and in vivo cause survivin depletion in tumor cell lines of varying origins. dn-ATF5 decreases levels of both survivin mRNA and protein. The depletion of survivin protein appears to be driven at least in part by enhanced proteasomal turnover and depletion of the deubiquitinase USP9X. Survivin loss is rapid and precedes the onset of cell death triggered by dn-ATF5. Although survivin downregulation is sufficient to drive tumor cell death, survivin over-expression does not rescue cancer cells from dn-ATF5-promoted apoptosis. This indicates that dn-ATF5 kills malignant cells by multiple mechanisms that include, but are not limited to, survivin depletion. Cell-penetrating forms of dn-ATF5 are currently being developed for potential therapeutic use and the present findings suggest that they may pose an advantage over treatments that target only survivin.

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Autophagic Cell Death in Dictyostelium Requires the Receptor Histidine Kinase DhkM

Molecular Biology of the Cell ◽

10.1091/mbc.e09-11-0976 ◽

2010 ◽

Vol 21 (11) ◽

pp. 1825-1835 ◽

Cited By ~ 11

Author(s):

Corinne Giusti ◽

Marie-Françoise Luciani ◽

Sarina Ravens ◽

Alexandre Gillet ◽

Pierre Golstein

Keyword(s):

Cell Death ◽

Late Stage ◽

Histidine Kinase ◽

Insertional Mutagenesis ◽

Dominant Negative ◽

Autophagic Cell Death ◽

Dominant Negative Effect ◽

Cellulose Synthesis ◽

Cell Stage ◽

Negative Effect

Dictyostelium constitutes a genetically tractable model for the analysis of autophagic cell death (ACD). During ACD, Dictyostelium cells first transform into paddle cells and then become round, synthesize cellulose, vacuolize, and die. Through random insertional mutagenesis, we identified the receptor histidine kinase DhkM as being essential for ACD. Surprisingly, different DhkM mutants showed distinct nonvacuolizing ACD phenotypes. One class of mutants arrested ACD at the paddle cell stage, perhaps through a dominant-negative effect. Other mutants, however, progressed further in the ACD program. They underwent rounding and cellulose synthesis but stopped before vacuolization. Moreover, they underwent clonogenic but not morphological cell death. Exogenous 8-bromo-cAMP restored vacuolization and death. A role for a membrane receptor at a late stage of the ACD pathway is puzzling, raising questions as to which ligand it is a receptor for and which moieties it phosphorylates. Together, DhkM is the most downstream-known molecule required for this model ACD, and its distinct mutants genetically separate previously undissociated late cell death events.

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Constitutively Activated Akt-1 Is Vital for the Survival of Human Monocyte-Differentiated Macrophages

Journal of Experimental Medicine ◽

10.1084/jem.194.2.113 ◽

2001 ◽

Vol 194 (2) ◽

pp. 113-126 ◽

Cited By ~ 148

Author(s):

Hongtao Liu ◽

Harris Perlman ◽

Lisa J. Pagliari ◽

Richard M. Pope

Keyword(s):

Cell Death ◽

Ectopic Expression ◽

Human Monocyte ◽

Pi3k Pathway ◽

Dominant Negative ◽

Human Macrophages ◽

Pi3k Inhibitor Ly294002 ◽

Inositol Phosphatase ◽

Pi3k Inhibition ◽

Src Homology 2 Domain

Recent data from mice deficient for phosphatase and tensin homologue deleted from chromosome 10 or src homology 2 domain–containing 5′ inositol phosphatase, phosphatases that negatively regulate the phosphatidylinositol 3-kinase (PI3K) pathway, revealed an increased number of macrophages in these animals, suggesting an essential role for the PI3K pathway for macro-phage survival. Here, we focused on the role of the PI3K-regulated serine/threonine kinase Akt-1 in modulating macrophage survival. Akt-1 was constitutively activated in human macrophages and addition of the PI3K inhibitor, LY294002, suppressed the activation of Akt-1 and induced cell death. Furthermore, suppression of Akt-1 by inhibition of PI3K or a dominant negative (DN) Akt-1 resulted in loss of mitochondrial transmembrane potential, activation of caspases-9 and -3, and DNA fragmentation. The effects of PI3K inhibition were reversed by the ectopic expression of constitutively activated Akt-1 or Bcl-xL. Inhibition of PI3K/Akt-1 pathway either by LY294002 or DN Akt-1 had no effect on the constitutive or inducible activation of nuclear factor (NF)-κB in human macrophages. However, after inhibition of the PI3K/Akt-1 pathway, a marked decrease in the expression of the antiapoptotic molecule Mcl-1, but not other Bcl-2 family members was observed, and Mcl-1 rescued macrophages from LY294002-induced cell death. Further, inhibition of Mcl-1 by antisense oligonucleotides, also resulted in macrophage apoptosis. Thus, our findings demonstrate that the constitutive activation of Akt-1 regulates macrophage survival through Mcl-1, which is independent of caspases, NF-κB, or Bad.

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SLC25A23 augments mitochondrial Ca2+ uptake, interacts with MCU, and induces oxidative stress–mediated cell death

Molecular Biology of the Cell ◽

10.1091/mbc.e13-08-0502 ◽

2014 ◽

Vol 25 (6) ◽

pp. 936-947 ◽

Cited By ~ 84

Author(s):

Nicholas E. Hoffman ◽

Harish C. Chandramoorthy ◽

Santhanam Shanmughapriya ◽

Xueqian Q. Zhang ◽

Sandhya Vallem ◽

...

Keyword(s):

Cell Death ◽

Ectopic Expression ◽

Dominant Negative ◽

Inner Mitochondrial Membrane ◽

Solute Carriers ◽

Metabolic Functions ◽

Evolutionarily Conserved ◽

Ef Hand ◽

Solute Carrier ◽

Negative Phenotype

Emerging findings suggest that two lineages of mitochondrial Ca2+ uptake participate during active and resting states: 1) the major eukaryotic membrane potential–dependent mitochondrial Ca2+ uniporter and 2) the evolutionarily conserved exchangers and solute carriers, which are also involved in ion transport. Although the influx of Ca2+ across the inner mitochondrial membrane maintains metabolic functions and cell death signal transduction, the mechanisms that regulate mitochondrial Ca2+ accumulation are unclear. Solute carriers—solute carrier 25A23 (SLC25A23), SLC25A24, and SLC25A25—represent a family of EF-hand–containing mitochondrial proteins that transport Mg-ATP/Pi across the inner membrane. RNA interference–mediated knockdown of SLC25A23 but not SLC25A24 and SLC25A25 decreases mitochondrial Ca2+ uptake and reduces cytosolic Ca2+ clearance after histamine stimulation. Ectopic expression of SLC25A23 EF-hand–domain mutants exhibits a dominant-negative phenotype of reduced mitochondrial Ca2+ uptake. In addition, SLC25A23 interacts with mitochondrial Ca2+ uniporter (MCU; CCDC109A) and MICU1 (CBARA1) while also increasing IMCU. In addition, SLC25A23 knockdown lowers basal mROS accumulation, attenuates oxidant-induced ATP decline, and reduces cell death. Further, reconstitution with short hairpin RNA–insensitive SLC25A23 cDNA restores mitochondrial Ca2+ uptake and superoxide production. These findings indicate that SLC25A23 plays an important role in mitochondrial matrix Ca2+ influx.

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α-Synuclein binds and sequesters PIKE-L into Lewy bodies, triggering dopaminergic cell death via AMPK hyperactivation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618627114 ◽

2017 ◽

Vol 114 (5) ◽

pp. 1183-1188 ◽

Cited By ~ 13

Author(s):

Seong Su Kang ◽

Zhentao Zhang ◽

Xia Liu ◽

Fredric P. Manfredsson ◽

Li He ◽

...

Keyword(s):

Cell Death ◽

Molecular Mechanisms ◽

Critical Role ◽

Lewy Bodies ◽

Cell Loss ◽

Dominant Negative ◽

Dependent Manner ◽

Ph Domain ◽

Dopaminergic Cell ◽

Neuroprotective Actions

The abnormal aggregation of fibrillar α-synuclein in Lewy bodies plays a critical role in the pathogenesis of Parkinson’s disease. However, the molecular mechanisms regulating α-synuclein pathological effects are incompletely understood. Here we show that α-synuclein binds phosphoinositide-3 kinase enhancer L (PIKE-L) in a phosphorylation-dependent manner and sequesters it in Lewy bodies, leading to dopaminergic cell death via AMP-activated protein kinase (AMPK) hyperactivation. α-Synuclein interacts with PIKE-L, an AMPK inhibitory binding partner, and this action is increased by S129 phosphorylation through AMPK and is decreased by Y125 phosphorylation via Src family kinase Fyn. A pleckstrin homology (PH) domain in PIKE-L directly binds α-synuclein and antagonizes its aggregation. Accordingly, PIKE-L overexpression decreases dopaminergic cell death elicited by 1-methyl-4-phenylpyridinium (MPP+), whereas PIKE-L knockdown elevates α-synuclein oligomerization and cell death. The overexpression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or α-synuclein induces greater dopaminergic cell loss and more severe motor defects in PIKE-KO and Fyn-KO mice than in wild-type mice, and these effects are attenuated by the expression of dominant-negative AMPK. Hence, our findings demonstrate that α-synuclein neutralizes PIKE-L’s neuroprotective actions in synucleinopathies, triggering dopaminergic neuronal death by hyperactivating AMPK.

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Role of MKK3 and p38 MAPK in cytokine-induced death of insulin-producing cells

Biochemical Journal ◽

10.1042/bj20050814 ◽

2005 ◽

Vol 393 (1) ◽

pp. 129-139 ◽

Cited By ~ 15

Author(s):

Natalia Makeeva ◽

Jason W. Myers ◽

Nils Welsh

Keyword(s):

Nitric Oxide ◽

Cell Death ◽

Protein Kinase ◽

P38 Mapk ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Nitric Oxide Donor ◽

Cell Leukaemia ◽

Dominant Negative Mutant

The aim of the present investigation was to elucidate further the importance of p38 MAPK (mitogen-activated protein kinase) in nitric oxide- and cytokine-induced β-cell death. For this purpose, isolated human islets were treated with d-siRNA (diced small interfering RNA) and then exposed to the nitric oxide donor DETA/NONOate [2,2′-(hydroxynitrosohydrazono)bis-ethanamine]. We observed that cells treated with p38α-specific d-siRNA, but not with d-siRNA targeting GL3 (a firefly luciferase siRNA plasmid) or PKCδ (protein kinase Cδ), were protected against nitric oxide-induced death. This was paralleled by an increased level of Bcl-XL (B-cell leukaemia/lymphoma-X long). For an in-depth study of the mechanisms of p38 activation, MKK3 (MAPK kinase 3), MKK6 and their dominant-negative mutants were overexpressed in insulin-producing RIN-5AH cells. In transient transfections, MKK3 overexpression resulted in increased p38 phosphorylation, whereas in stable MKK3-overexpressing RIN-5AH clones, the protein levels of p38 and JNK (c-Jun N-terminal kinase) were decreased, resulting in unaffected phospho-p38 levels. In addition, a long-term MKK3 overexpression did not affect cell death rates in response to the cytokines interleukin-1β and interferon-γ, whereas a short-term MKK3 expression resulted in increased cytokine-induced RIN-5AH cell death. The MKK3-potentiating effect on cytokine-induced cell death was abolished by a nitric oxide synthase inhibitor, and MKK3-stimulated p38 phosphorylation was enhanced by inhibitors of phosphatases. Finally, as the dominant-negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild-type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide. In addition, it is likely that a long-term increase in p38 activity is counteracted by both a decreased expression of the p38, JNK and p42 genes as well as an increased dephosphorylation of p38.

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Abstract 223: Parkin is Critical for the Clearance of Damaged Mitochondria via an Autophagy-independent Pathway

Circulation Research ◽

10.1161/res.119.suppl_1.223 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Babette C Hammerling ◽

Melissa Q Cortez ◽

Rita H Najor ◽

Åsa B Gustafsson

Keyword(s):

Cell Death ◽

Ischemia Reperfusion ◽

Degradation Pathway ◽

Dominant Negative ◽

Upstream Regulator ◽

Late Endosomes ◽

Early Endosomes ◽

Response To Stress ◽

Autophagy Pathway ◽

Endosomal Pathway

Functional mitochondria are essential for highly metabolic organs such as the heart. When mitochondria are damaged they can release pro-death factors and reactive oxygen species which in turn can result in cell death. The E3 ubiquitin ligase Parkin plays an important role in clearing damaged mitochondria via the autophagy pathway to protect cells against unnecessary cell death. Interestingly, we have found that Parkin can mediate clearance of damaged mitochondria via an autophagy-independent pathway. In fact, Parkin promotes clearance of depolarized mitochondria at the same rate in both wild-type (WT) and autophagy deficient Atg5-/- mouse embryonic fibroblasts (MEFs) in response to the mitochondrial uncoupler FCCP. We also found that Parkin-mediated ubiquitination is critical for this process as disease associated mutants of Parkin were incapable of inducing mitochondrial clearance in Atg5-/- MEFs. Upon further investigation, we observed a significant increase in the number of Rab5+ early- and Rab7+ late endosomes in both WT and Atg5-/- MEFs after depolarization of mitochondria with FCCP or valinomycin, indicating activation of the endosomal-lysosomal degradation pathway. We did not observe activation of the endosomal pathway after exposure to actinomycin D, an inhibitor RNA synthesis and activator of apoptosis, confirming that mitochondrial damage specifically activates the endosomal degradation pathway. We also observed activation of the endosomal pathway in neonatal myocytes in response to FCCP treatment or after exposure to simulated ischemia/reperfusion (sI/R). Overexpression of the dominant negative Rab5S34N significantly enhanced sI/R-mediated cell death, suggesting that this is a protective pathway activated by cells in response to stress. Moreover, Beclin1 is well known to regulate activation of autophagy. Here, we found that knockdown of Beclin1 inhibited both the number of Rab5+ early endosomes and their colocalization with mitochondria in response to either FCCP or sI/R in myocytes, suggesting that Beclin1 is a critical upstream regulator of the endosomal degradation pathway. Thus, our data suggest that Parkin mediates clearance of damaged mitochondria via both the autophagy and endosomal pathways in cells.

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