54 results on '"Elia AJ"'
Search Results
2. Identification of KIFC1 as a putative vulnerability in lung cancers with centrosome amplification.
- Author
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Zhang C, Wu BZ, Di Ciano-Oliveira C, Wu YF, Khavkine Binstock SS, Soria-Bretones I, Pham NA, Elia AJ, Chari R, Lam WL, Bray MR, Mak TW, Tsao MS, Cescon DW, and Thu KL
- Abstract
Centrosome amplification (CA), an abnormal increase in the number of centrosomes in the cell, is a recurrent phenomenon in lung and other malignancies. Although CA promotes tumor development and progression by inducing genomic instability (GIN), it also induces mitotic stress that jeopardizes cellular integrity. CA leads to the formation of multipolar mitotic spindles that can cause lethal chromosome segregation errors. To sustain the benefits of CA by mitigating its consequences, malignant cells are dependent on adaptive mechanisms that represent therapeutic vulnerabilities. We aimed to discover genetic dependencies associated with CA in lung cancer. Combining a CRISPR/Cas9 functional genomics screen with tumor genomic analyses, we identified the motor protein KIFC1, also known as HSET, as a putative vulnerability specifically in lung adenocarcinoma (LUAD) with CA. KIFC1 expression was positively correlated with CA in LUAD and associated with worse patient outcomes, smoking history, and indicators of GIN. KIFC1 loss-of-function sensitized LUAD cells with high basal KIFC1 expression to potentiation of CA, which was associated with a diminished ability to cluster extra centrosomes into pseudo-bipolar mitotic spindles. Our work suggests that KIFC1 inhibition represents a novel approach for potentiating GIN to lethal levels in LUAD with CA by forcing cells to divide with multipolar spindles, rationalizing further studies to investigate its therapeutic potential., (© 2024. The Author(s).)
- Published
- 2024
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3. Tumor-specific cholinergic CD4 + T lymphocytes guide immunosurveillance of hepatocellular carcinoma.
- Author
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Zheng C, Snow BE, Elia AJ, Nechanitzky R, Dominguez-Brauer C, Liu S, Tong Y, Cox MA, Focaccia E, Wakeham AC, Haight J, Tobin C, Hodgson K, Gill KT, Ma W, Berger T, Heikenwälder M, Saunders ME, Fortin J, Leung SY, and Mak TW
- Subjects
- Animals, Mice, Programmed Cell Death 1 Receptor genetics, Monitoring, Immunologic, T-Lymphocytes, Regulatory pathology, Carcinoma, Hepatocellular genetics, Liver Neoplasms genetics
- Abstract
Cholinergic nerves are involved in tumor progression and dissemination. In contrast to other visceral tissues, cholinergic innervation in the hepatic parenchyma is poorly detected. It remains unclear whether there is any form of cholinergic regulation of liver cancer. Here, we show that cholinergic T cells curtail the development of liver cancer by supporting antitumor immune responses. In a mouse multihit model of hepatocellular carcinoma (HCC), we observed activation of the adaptive immune response and induction of two populations of CD4
+ T cells expressing choline acetyltransferase (ChAT), including regulatory T cells and dysfunctional PD-1+ T cells. Tumor antigens drove the clonal expansion of these cholinergic T cells in HCC. Genetic ablation of Chat in T cells led to an increased prevalence of preneoplastic cells and exacerbated liver cancer due to compromised antitumor immunity. Mechanistically, the cholinergic activity intrinsic in T cells constrained Ca2+ -NFAT signaling induced by T cell antigen receptor engagement. Without this cholinergic modulation, hyperactivated CD25+ T regulatory cells and dysregulated PD-1+ T cells impaired HCC immunosurveillance. Our results unveil a previously unappreciated role for cholinergic T cells in liver cancer immunobiology., (© 2023. The Author(s).)- Published
- 2023
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4. IDH2 and TET2 mutations synergize to modulate T Follicular Helper cell functional interaction with the AITL microenvironment.
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Leca J, Lemonnier F, Meydan C, Foox J, El Ghamrasni S, Mboumba DL, Duncan GS, Fortin J, Sakamoto T, Tobin C, Hodgson K, Haight J, Smith LK, Elia AJ, Butler D, Berger T, de Leval L, Mason CE, Melnick A, Gaulard P, and Mak TW
- Subjects
- Animals, Humans, Mice, DNA-Binding Proteins genetics, Isocitrate Dehydrogenase genetics, Mutation, T Follicular Helper Cells pathology, T-Lymphocytes, Helper-Inducer, Tumor Microenvironment genetics, Dioxygenases genetics, Immunoblastic Lymphadenopathy genetics, Lymphoma, T-Cell genetics
- Abstract
Angioimmunoblastic T cell lymphoma (AITL) is a peripheral T cell lymphoma that originates from T follicular helper (Tfh) cells and exhibits a prominent tumor microenvironment (TME). IDH2 and TET2 mutations co-occur frequently in AITL, but their contribution to tumorigenesis is poorly understood. We developed an AITL mouse model that is driven by Idh2 and Tet2 mutations. Malignant Tfh cells display aberrant transcriptomic and epigenetic programs that impair TCR signaling. Neoplastic Tfh cells bearing combined Idh2 and Tet2 mutations show altered cross-talk with germinal center B cells that promotes B cell clonal expansion while decreasing Fas-FasL interaction and reducing B cell apoptosis. The plasma cell count and angiogenesis are also increased in the Idh2-mutated tumors, implying a major relationship between Idh2 mutation and the characteristic AITL TME. Our mouse model recapitulates several features of human IDH2-mutated AITL and provides a rationale for exploring therapeutic targeting of Tfh-TME cross-talk for AITL patients., Competing Interests: Declaration of interests A.M. has consulted for Epizyme, Constellation, Daiichi Sankyo, AstraZeneca, BMS, Janssen, and ExoTherapeutics; has research funding from Epizyme, Janssen, Daiichi Sankyo, and AstraZeneca; and is on the advisory board of KDAC. C.E.M. is a cofounder and board member for Biotia and Onegevity Health as well as an advisor or grantee for Abbvie, ArcBio, Daiichi Sankyo, DNA Genotek, Tempus Labs, and Whole Biome. T.W.M. holds equity in Treadwell Therapeutics Inc. and Agios Pharmaceuticals and is a consultant for AstraZeneca and Tessa Therapeutics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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5. Cisplatin toxicity in the developing brain displays an absolute requirement for caspase-3.
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Hui KK, Latif M, Soeandy CD, Huang S, Rodgers CE, Elia AJ, and Henderson JT
- Subjects
- Apoptosis physiology, Brain metabolism, Caspase 3 metabolism, Caspases metabolism, Cisplatin toxicity
- Abstract
Cisplatin is a member of a widely utilized class of chemotherapeutic agent that initiates DNA damage response, cell cycle arrest, and p53-dependent apoptotic cell death in concert with DNA‑platinum adduct formation. While normal programmed cell death (PCD) can occur in the developing neuroepithelium in the absence of caspase-3 within certain genetic backgrounds, we observed an absolute dependency upon this executioner caspase with respect to cisplatin-induced PCD in the developing central nervous system (CNS). We therefore examined the nature of this genotoxic injury in the CNS in vivo, in which cisplatin treatment causes widespread cellular injury consistent with hallmarks of apoptosis which are averted upon caspase-3 inhibition. Examination of cisplatin-mediated injury as a function of time revealed the presence of an alternative, delayed form of necroptosis-like cell death which manifests in Casp3
-/- neuroepithelia for several days following the normal pattern of apoptosis. Together, these findings suggest a coordinated regulation of these disparate PCD pathways in response to genotoxic stress in vivo and highlight the unique and critical role which caspase-3 plays among executioner caspases in coordinating apoptotic versus necroptotic responsiveness of the developing CNS to genotoxic injury., (Copyright © 2022 Elsevier Inc. All rights reserved.)- Published
- 2022
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6. Necroptotic-Apoptotic Regulation in an Endothelin-1 Model of Cerebral Ischemia.
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Dojo Soeandy C, Elia AJ, Cao Y, Rodgers C, Huang S, Elia AC, and Henderson JT
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- Animals, Apoptosis physiology, Brain Ischemia metabolism, Female, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Necrosis chemically induced, Necrosis metabolism, Necrosis pathology, Receptor-Interacting Protein Serine-Threonine Kinases metabolism, Apoptosis drug effects, Brain Ischemia chemically induced, Brain Ischemia pathology, Endothelin-1 toxicity
- Abstract
The primary forms of cell death seen in ischemic stroke are of two major types: a necrotic/necroptotic form, and an apoptotic form that is frequently seen in penumbral regions of injury. Typically apoptotic versus necroptotic programmed cell death is described as competitive in nature, where necroptosis is often described as playing a backup role to apoptosis. In the present study, we examined the relationship between these two forms of cell death in a murine endothelin-1 model of ischemia-reperfusion injury in wildtype and caspase-3 null mice with and without addition of the pharmacologic RIPK1 phosphorylation inhibitor necrostatin-1. Analyses of ischemic brain injury were performed via both cellular and volumetric assessments, electron microscopy, TUNEL staining, activated caspase-3 and caspase-7 staining, as well as CD11b and F4/80 staining. Inhibition of caspase-3 or RIPK1 phosphorylation demonstrates significant neural protective effects which are non-additive and exhibit significant overlap in protected regions. Interestingly, morphologic analysis of the cortex demonstrates reduced apoptosis following RIPK1 inhibition. Consistent with this, RIPK1 inhibition reduces the levels of both caspase-3 and caspase-7 activation. Additionally, this protection appears independent of secondary inflammatory mediators. Together, these observations demonstrate that the necroptotic protein RIPK1 modifies caspase-3/-7 activity, ultimately resulting in decreased neuronal apoptosis. These findings thus modify the traditional exclusionary view of apoptotic/necroptotic signaling, revealing a new form of interaction between these dominant forms of cell death., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2021
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7. Hypoxia-inducible factor 1 alpha limits dendritic cell stimulation of CD8 T cell immunity.
- Author
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Tran CW, Gold MJ, Garcia-Batres C, Tai K, Elford AR, Himmel ME, Elia AJ, and Ohashi PS
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- Animals, Cells, Cultured, Dendritic Cells metabolism, Gene Knockout Techniques, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Interleukin-10 metabolism, Mice, Nitric Oxide Synthase Type II metabolism, Vascular Endothelial Growth Factor A metabolism, Biomarkers metabolism, CD8-Positive T-Lymphocytes immunology, Dendritic Cells cytology, Hypoxia-Inducible Factor 1, alpha Subunit genetics
- Abstract
Dendritic cells are sentinels of the immune system and represent a key cell in the activation of the adaptive immune response. Hypoxia-inducible factor 1 alpha (HIF-1α)-a crucial oxygen sensor stabilized during hypoxic conditions-has been shown to have both activating and inhibitory effects in immune cells in a context- and cell-dependent manner. Previous studies have demonstrated that in some immune cell types, HIF-1α serves a pro-inflammatory role. Genetic deletion of HIF-1α in macrophages has been reported to reduce their pro-inflammatory function. In contrast, loss of HIF-1α enhanced the pro-inflammatory activity of dendritic cells in a bacterial infection model. In this study, we aimed to further clarify the effects of HIF-1α in dendritic cells. Constitutive expression of HIF-1α resulted in diminished immunostimulatory capacity of dendritic cells in vivo, while conditional deletion of HIF-1α in dendritic cells enhanced their ability to induce a cytotoxic T cell response. HIF-1α-expressing dendritic cells demonstrated increased production of inhibitory mediators including IL-10, iNOS and VEGF, which correlated with their reduced capacity to drive effector CD8+ T cell function. Altogether, these data reveal that HIF-1α can promote the anti-inflammatory functions of dendritic cells and provides insight into dysfunctional immune responses in the context of HIF-1α activation., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2020
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8. Endothelin-1-mediated cerebral ischemia in mice: early cellular events and the role of caspase-3.
- Author
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Dojo Soeandy C, Salmasi F, Latif M, Elia AJ, Suo NJ, and Henderson JT
- Subjects
- Animals, Brain Ischemia pathology, Caspase 3 genetics, Cell Death drug effects, Cerebral Cortex injuries, Cerebral Cortex pathology, Disease Models, Animal, Endothelin-1 administration & dosage, Gene Knockout Techniques, Mice, Mice, Inbred C57BL, Mice, Knockout, Neurons drug effects, Neurons enzymology, Neurons pathology, Brain Ischemia chemically induced, Caspase 3 metabolism, Cerebral Cortex drug effects, Endothelin-1 toxicity
- Abstract
Over the past 30 years a number of animal models of cerebral ischemic injury have been developed. Middle cerebral artery occlusion (MCAO) in particular reproduces both ischemic and reperfusion elements and is widely utilized as a model of ischemic stroke in rodents. However substantial variability exists in this model even in clonal inbred mice due to stochastic elements of the cerebral vasculature. Models such as MCAO thus exhibit significant irreducible variabilities with respect to their zone of injury as well as inducing a sizable volume of injury to the cerebrum with damage to sub-cortical structures, conditions not typically seen for the majority of human clinical strokes. An alternative model utilizes endothelin-1 application focally to cerebral vasculature, resulting in an ischemic reperfusion injury which more closely mimics that seen in human clinical stroke. In order to further define this model we demonstrate that intra-cortical administration of ET-1 results in a highly reproducible pattern of tissue injury which is limited to the cerebral cortex, characterizing the early cellular and molecular events which occur during the first 24 h post-injury. In addition we demonstrate that caspase-3 is both necessary and sufficient to regulate a majority of cortical cell death observed during this period. The enhanced survival effects seen upon genetic deletion of caspase-3 appear to arise as a result of direct modification of cell autonomous PCD signaling as opposed to secondary effectors such as granulocyte infiltration or microglia activation. Taken together these findings detail the early mechanistic features regulating endothelin-1-mediated ischemic injury.
- Published
- 2019
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9. Choline acetyltransferase-expressing T cells are required to control chronic viral infection.
- Author
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Cox MA, Duncan GS, Lin GHY, Steinberg BE, Yu LX, Brenner D, Buckler LN, Elia AJ, Wakeham AC, Nieman B, Dominguez-Brauer C, Elford AR, Gill KT, Kubli SP, Haight J, Berger T, Ohashi PS, Tracey KJ, Olofsson PS, and Mak TW
- Subjects
- Animals, CD4-Positive T-Lymphocytes enzymology, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes enzymology, Cell Movement, Choline O-Acetyltransferase genetics, Female, Lymphocyte Activation, Lymphocytic choriomeningitis virus, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Vasodilation, CD8-Positive T-Lymphocytes immunology, Choline O-Acetyltransferase immunology, Interleukins immunology, Lymphocytic Choriomeningitis immunology
- Abstract
Although widely studied as a neurotransmitter, T cell-derived acetylcholine (ACh) has recently been reported to play an important role in regulating immunity. However, the role of lymphocyte-derived ACh in viral infection is unknown. Here, we show that the enzyme choline acetyltransferase (ChAT), which catalyzes the rate-limiting step of ACh production, is robustly induced in both CD4
+ and CD8+ T cells during lymphocytic choriomeningitis virus (LCMV) infection in an IL-21-dependent manner. Deletion of Chat within the T cell compartment in mice ablated vasodilation in response to infection, impaired the migration of antiviral T cells into infected tissues, and ultimately compromised the control of chronic LCMV clone 13 infection. Our results reveal a genetic proof of function for ChAT in T cells during viral infection and identify a pathway of T cell migration that sustains antiviral immunity., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)- Published
- 2019
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10. E3 ubiquitin ligase Mule targets β-catenin under conditions of hyperactive Wnt signaling.
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Dominguez-Brauer C, Khatun R, Elia AJ, Thu KL, Ramachandran P, Baniasadi SP, Hao Z, Jones LD, Haight J, Sheng Y, and Mak TW
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- Adenomatous Polyposis Coli Protein deficiency, Animals, Axin Protein biosynthesis, Axin Protein genetics, Cell Nucleus metabolism, Cell Nucleus ultrastructure, Colonic Neoplasms metabolism, Cyclin D1 biosynthesis, Cyclin D1 genetics, Down-Regulation, Genes, APC, Genes, Tumor Suppressor, HEK293 Cells, Humans, Mice, Mice, Knockout, Neoplasm Proteins physiology, Organoids metabolism, Organoids ultrastructure, Protein Binding, Protein Processing, Post-Translational, Proteolysis, RNA Interference, RNA, Small Interfering genetics, Recombinant Proteins metabolism, Tumor Suppressor Proteins antagonists & inhibitors, Tumor Suppressor Proteins deficiency, Tumor Suppressor Proteins genetics, Ubiquitin-Protein Ligases antagonists & inhibitors, Ubiquitin-Protein Ligases deficiency, Ubiquitin-Protein Ligases genetics, Ubiquitination, Tumor Suppressor Proteins physiology, Ubiquitin-Protein Ligases physiology, Wnt Signaling Pathway, beta Catenin antagonists & inhibitors
- Abstract
Wnt signaling, named after the secreted proteins that bind to cell surface receptors to activate the pathway, plays critical roles both in embryonic development and the maintenance of homeostasis in many adult tissues. Two particularly important cellular programs orchestrated by Wnt signaling are proliferation and stem cell self-renewal. Constitutive activation of the Wnt pathway resulting from mutation or improper modulation of pathway components contributes to cancer development in various tissues. Colon cancers frequently bear inactivating mutations of the adenomatous polyposis coli ( APC ) gene, whose product is an important component of the destruction complex that regulates β-catenin levels. Stabilization and nuclear localization of β-catenin result in the expression of a panel of Wnt target genes. We previously showed that Mule/Huwe1/Arf-BP1 (Mule) controls murine intestinal stem and progenitor cell proliferation by modulating the Wnt pathway via c-Myc. Here we extend our investigation of Mule's influence on oncogenesis by showing that Mule interacts directly with β-catenin and targets it for degradation under conditions of hyperactive Wnt signaling. Our findings suggest that Mule uses various mechanisms to fine-tune the Wnt pathway and provides multiple safeguards against tumorigenesis.
- Published
- 2017
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11. Metabolic reprogramming during neuronal differentiation.
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Agostini M, Romeo F, Inoue S, Niklison-Chirou MV, Elia AJ, Dinsdale D, Morone N, Knight RA, Mak TW, and Melino G
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- Animals, DNA, Mitochondrial metabolism, Glucose metabolism, Glucose Transporter Type 3 metabolism, Glutamate-Cysteine Ligase deficiency, Glutamate-Cysteine Ligase genetics, Glutamic Acid metabolism, Glutamine metabolism, Mice, Mice, Knockout, Microscopy, Electron, Transmission, Mitochondria genetics, Mitochondria metabolism, Neurons cytology, Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha metabolism, Phosphatidylinositol 3-Kinases metabolism, Proto-Oncogene Proteins c-akt metabolism, Reactive Oxygen Species metabolism, Signal Transduction, TOR Serine-Threonine Kinases metabolism, Cell Differentiation physiology, Metabolic Engineering, Neurons metabolism
- Abstract
Newly generated neurons pass through a series of well-defined developmental stages, which allow them to integrate into existing neuronal circuits. After exit from the cell cycle, postmitotic neurons undergo neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. Lack of a global metabolic analysis during early cortical neuronal development led us to explore the role of cellular metabolism and mitochondrial biology during ex vivo differentiation of primary cortical neurons. Unexpectedly, we observed a huge increase in mitochondrial biogenesis. Changes in mitochondrial mass, morphology and function were correlated with the upregulation of the master regulators of mitochondrial biogenesis, TFAM and PGC-1α. Concomitant with mitochondrial biogenesis, we observed an increase in glucose metabolism during neuronal differentiation, which was linked to an increase in glucose uptake and enhanced GLUT3 mRNA expression and platelet isoform of phosphofructokinase 1 (PFKp) protein expression. In addition, glutamate-glutamine metabolism was also increased during the differentiation of cortical neurons. We identified PI3K-Akt-mTOR signalling as a critical regulator role of energy metabolism in neurons. Selective pharmacological inhibition of these metabolic pathways indicate existence of metabolic checkpoint that need to be satisfied in order to allow neuronal differentiation.
- Published
- 2016
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12. Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2.
- Author
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Inoue S, Li WY, Tseng A, Beerman I, Elia AJ, Bendall SC, Lemonnier F, Kron KJ, Cescon DW, Hao Z, Lind EF, Takayama N, Planello AC, Shen SY, Shih AH, Larsen DM, Li Q, Snow BE, Wakeham A, Haight J, Gorrini C, Bassi C, Thu KL, Murakami K, Elford AR, Ueda T, Straley K, Yen KE, Melino G, Cimmino L, Aifantis I, Levine RL, De Carvalho DD, Lupien M, Rossi DJ, Nolan GP, Cairns RA, and Mak TW
- Subjects
- Animals, Ataxia Telangiectasia Mutated Proteins metabolism, DNA-Binding Proteins metabolism, Dioxygenases, Down-Regulation, Hematopoietic Stem Cells cytology, Humans, Isocitrate Dehydrogenase metabolism, Mice, Mutation, Proto-Oncogene Proteins metabolism, Ataxia Telangiectasia Mutated Proteins genetics, DNA Damage, DNA Repair, DNA-Binding Proteins genetics, Hematopoietic Stem Cells enzymology, Isocitrate Dehydrogenase genetics, Proto-Oncogene Proteins genetics
- Abstract
Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia., Competing Interests: Other authors declare that no conflicts of interest exist., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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13. Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation.
- Author
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Dominguez-Brauer C, Hao Z, Elia AJ, Fortin JM, Nechanitzky R, Brauer PM, Sheng Y, Mana MD, Chio IIC, Haight J, Pollett A, Cairns R, Tworzyanski L, Inoue S, Reardon C, Marques A, Silvester J, Cox MA, Wakeham A, Yilmaz OH, Sabatini DM, van Es JH, Clevers H, Sato T, and Mak TW
- Subjects
- Adenomatous Polyposis Coli pathology, Alleles, Animals, Carcinogenesis metabolism, Carcinogenesis pathology, Cell Proliferation, Colonic Neoplasms genetics, Colonic Neoplasms pathology, Endocytosis, HEK293 Cells, Humans, Mice, Knockout, Models, Biological, Mutation genetics, Paneth Cells pathology, Proto-Oncogene Proteins c-myc metabolism, Tumor Suppressor Proteins, Ubiquitin metabolism, Ubiquitin-Protein Ligases deficiency, Ephrin-B3 metabolism, Intestines cytology, Lysosomes metabolism, Proteasome Endopeptidase Complex metabolism, Proteolysis, Stem Cell Niche, Ubiquitin-Protein Ligases metabolism, Wnt Signaling Pathway
- Abstract
The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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14. Idh1 protects murine hepatocytes from endotoxin-induced oxidative stress by regulating the intracellular NADP(+)/NADPH ratio.
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Itsumi M, Inoue S, Elia AJ, Murakami K, Sasaki M, Lind EF, Brenner D, Harris IS, Chio II, Afzal S, Cairns RA, Cescon DW, Elford AR, Ye J, Lang PA, Li WY, Wakeham A, Duncan GS, Haight J, You-Ten A, Snow B, Yamamoto K, Ohashi PS, and Mak TW
- Subjects
- Animals, Cells, Cultured, Flow Cytometry, Isocitrate Dehydrogenase genetics, Mice, Mice, Knockout, Oxidative Stress drug effects, Oxidative Stress genetics, Reverse Transcriptase Polymerase Chain Reaction, Endotoxins pharmacology, Hepatocytes drug effects, Hepatocytes enzymology, Isocitrate Dehydrogenase metabolism, NADP metabolism
- Abstract
Isocitrate dehydrogenase-1 (Idh1) is an important metabolic enzyme that produces NADPH by converting isocitrate to α-ketoglutarate. Idh1 is known to reduce reactive oxygen species (ROS) induced in cells by treatment with lipopolysaccharide (LPS) in vitro. Here, we used Idh1-deficient knockout (Idh1 KO) mice to investigate the role of Idh1 in antioxidant defense in vivo. Idh1 KO mice showed heightened susceptibility to death induced by LPS and exhibited increased serum levels of inflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The serum of LPS-injected Idh1 KO mice also contained elevated levels of AST, a marker of inflammatory liver damage. Furthermore, after LPS injection, livers of Idh1 KO mice showed histological evidence of elevated oxidative DNA damage compared with livers of wild-type (WT) mice. Idh1 KO livers showed a faster and more pronounced oxidative stress than WT livers. In line with that, Idh1 KO hepatocytes showed higher ROS levels and an increase in the NADP(+)/NADPH ratio when compared with hepatocytes isolated from WT mice. These results suggest that Idh1 has a physiological function in protecting cells from oxidative stress by regulating the intracellular NADP(+)/NADPH ratio. Our findings suggest that stimulation of Idh1 activity may be an effective therapeutic strategy for reducing oxidative stress during inflammatory responses, including the early stages of septic shock.
- Published
- 2015
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15. Pten deletion in RIP-Cre neurons protects against type 2 diabetes by activating the anti-inflammatory reflex.
- Author
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Wang L, Opland D, Tsai S, Luk CT, Schroer SA, Allison MB, Elia AJ, Furlonger C, Suzuki A, Paige CJ, Mak TW, Winer DA, Myers MG Jr, and Woo M
- Subjects
- Animals, Anti-Inflammatory Agents administration & dosage, Central Nervous System metabolism, Diabetes Mellitus, Type 2 complications, Diet, High-Fat, Humans, Inflammation complications, Inflammation drug therapy, Insulin metabolism, Insulin Resistance genetics, Macrophages metabolism, Macrophages pathology, Mice, Neurons drug effects, Neurons metabolism, Neurons pathology, PTEN Phosphohydrolase metabolism, Promoter Regions, Genetic, Rats, Receptors, Muscarinic administration & dosage, Sequence Deletion, Signal Transduction, Diabetes Mellitus, Type 2 drug therapy, Diabetes Mellitus, Type 2 genetics, Inflammation metabolism, Insulin genetics, PTEN Phosphohydrolase genetics
- Abstract
Inflammation has a critical role in the development of insulin resistance. Recent evidence points to a contribution by the central nervous system in the modulation of peripheral inflammation through the anti-inflammatory reflex. However, the importance of this phenomenon remains elusive in type 2 diabetes pathogenesis. Here we show that rat insulin-2 promoter (Rip)-mediated deletion of Pten, a gene encoding a negative regulator of PI3K signaling, led to activation of the cholinergic anti-inflammatory pathway that is mediated by M2 activated macrophages in peripheral tissues. As such, Rip-cre(+) Pten(flox/flox) mice showed lower systemic inflammation and greater insulin sensitivity under basal conditions compared to littermate controls, which were abolished when the mice were treated with an acetylcholine receptor antagonist or when macrophages were depleted. After feeding with a high-fat diet, the Pten-deleted mice remained markedly insulin sensitive, which correlated with massive subcutaneous fat expansion. They also exhibited more adipogenesis with M2 macrophage infiltration, both of which were abolished after disruption of the anti-inflammatory efferent pathway by left vagotomy. In summary, we show that Pten expression in Rip(+) neurons has a critical role in diabetes pathogenesis through mediating the anti-inflammatory reflex.
- Published
- 2014
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16. Role of Nek2 on centrosome duplication and aneuploidy in breast cancer cells.
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Cappello P, Blaser H, Gorrini C, Lin DC, Elia AJ, Wakeham A, Haider S, Boutros PC, Mason JM, Miller NA, Youngson B, Done SJ, and Mak TW
- Subjects
- Animals, Cell Line, Tumor, Chromosome Segregation genetics, Female, Gene Knockdown Techniques, Humans, Lung Neoplasms secondary, Mice, NIMA-Related Kinases, Neoplasm Transplantation, Protein Serine-Threonine Kinases genetics, Aneuploidy, Breast Neoplasms genetics, Breast Neoplasms pathology, Centrosome pathology, Protein Serine-Threonine Kinases physiology
- Abstract
Breast cancer is the most common solid tumor and the second most common cause of death in women. Despite a large body of literature and progress in breast cancer research, many molecular aspects of this complex disease are still poorly understood, hindering the design of specific and effective therapeutic strategies. To identify the molecules important in breast cancer progression and metastasis, we tested the in vivo effects of inhibiting the functions of various kinases and genes involved in the regulation/modulation of the cytoskeleton by downregulating them in mouse PyMT mammary tumor cells and human breast cancer cell lines. These kinases and cytoskeletal regulators were selected based on their prognostic values for breast cancer patient survival. PyMT tumor cells, in which a selected gene was stably knocked down were injected into the tail veins of mice, and the formation of tumors in the lungs was monitored. One of the several genes found to be important for tumor growth in the lungs was NIMA-related kinases 2 (Nek2), a cell cycle-related protein kinase. Furthermore, Nek2 was also important for tumor growth in the mammary fat pad. In various human breast cancer cell lines, Nek2 knockdown induced aneuploidy and cell cycle arrest that led to cell death. Significantly, the breast cancer cell line most sensitive to Nek2 depletion was of the triple negative breast cancer subtype. Our data indicate that Nek2 has a pivotal role in breast cancer growth at primary and secondary sites, and thus may be an attractive and novel therapeutic target for this disease.
- Published
- 2014
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17. Largen: a molecular regulator of mammalian cell size control.
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Yamamoto K, Gandin V, Sasaki M, McCracken S, Li W, Silvester JL, Elia AJ, Wang F, Wakutani Y, Alexandrova R, Oo YD, Mullen PJ, Inoue S, Itsumi M, Lapin V, Haight J, Wakeham A, Shahinian A, Ikura M, Topisirovic I, Sonenberg N, and Mak TW
- Subjects
- Animals, Cell Line, Tumor, Escherichia coli genetics, Escherichia coli metabolism, Genetic Vectors, High-Throughput Screening Assays, Humans, Jurkat Cells, Mice, Mice, Transgenic, Mitochondria genetics, Mitochondria metabolism, Proteins metabolism, RNA, Messenger metabolism, Retroviridae genetics, Retroviridae metabolism, Signal Transduction drug effects, Sirolimus pharmacology, Cell Size drug effects, Gene Expression Regulation, Protein Biosynthesis, Proteins genetics, RNA, Messenger genetics
- Abstract
Little is known about how mammalian cells maintain cell size homeostasis. We conducted a novel genetic screen to identify cell-size-controlling genes and isolated Largen, the product of a gene (PRR16) that increased cell size upon overexpression in human cells. In vitro evidence indicated that Largen preferentially stimulates the translation of specific subsets of mRNAs, including those encoding proteins affecting mitochondrial functions. The involvement of Largen in mitochondrial respiration was consistent with the increased mitochondrial mass and greater ATP production in Largen-overexpressing cells. Furthermore, Largen overexpression led to increased cell size in vivo, as revealed by analyses of conditional Largen transgenic mice. Our results establish Largen as an important link between mRNA translation, mitochondrial functions, and the control of mammalian cell size., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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18. PTPN12 promotes resistance to oxidative stress and supports tumorigenesis by regulating FOXO signaling.
- Author
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Harris IS, Blaser H, Moreno J, Treloar AE, Gorrini C, Sasaki M, Mason JM, Knobbe CB, Rufini A, Hallé M, Elia AJ, Wakeham A, Tremblay ML, Melino G, Done S, and Mak TW
- Subjects
- Animals, Breast Neoplasms genetics, Breast Neoplasms pathology, Cell Division, Cells, Cultured, Female, Humans, Mice, Prognosis, Protein Tyrosine Phosphatase, Non-Receptor Type 12 genetics, RNA, Messenger genetics, Reactive Oxygen Species metabolism, Real-Time Polymerase Chain Reaction, Forkhead Transcription Factors metabolism, Oxidative Stress, Protein Tyrosine Phosphatase, Non-Receptor Type 12 physiology, Signal Transduction
- Abstract
It is well known that protein tyrosine phosphatases (PTPs) that become oxidized due to exposure to reactive oxygen species (ROS) undergo a conformational change and are inactivated. However, whether PTPs can actively regulate ROS levels in order to prevent PTP inhibition has yet to be investigated. Here, we demonstrate that PTP non-receptor type 12 (PTPN12) protects cells against aberrant ROS accumulation and death induced by oxidative stress. Murine embryonic fibroblasts (MEFs) deficient in PTPN12 underwent increased ROS-induced apoptosis under conditions of antioxidant depletion. Cells lacking PTPN12 also showed defective activation of FOXO1/3a, transcription factors required for the upregulation of several antioxidant genes. PTPN12-mediated regulation of ROS appeared to be mediated by phosphoinositide-dependent kinase-1 (PDK1), which was hyperstimulated in the absence of PTPN12. As tight regulation of ROS to sustain survival is a key feature of cancer cells, we examined PTPN12 levels in tumors from a cohort of breast cancer patients. Patients whose tumors showed high levels of PTPN12 transcripts had a significantly poorer prognosis. Analysis of tissues from patients with various breast cancer subtypes revealed that more triple-negative breast cancers, the most aggressive breast cancer subtype, showed high PTPN12 expression than any other subtype. Furthermore, both human breast cancer cells and mouse mammary epithelial tumor cells engineered to lack PTPN12 exhibited reduced tumorigenic and metastatic potential in vivo that correlated with their elevated ROS levels. The involvement of PTPN12 in the antioxidant response of breast cancer cells suggests that PTPN12 may represent a novel therapeutic target for this disease.
- Published
- 2014
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- View/download PDF
19. TAp73 is required for spermatogenesis and the maintenance of male fertility.
- Author
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Inoue S, Tomasini R, Rufini A, Elia AJ, Agostini M, Amelio I, Cescon D, Dinsdale D, Zhou L, Harris IS, Lac S, Silvester J, Li WY, Sasaki M, Haight J, Brüstle A, Wakeham A, McKerlie C, Jurisicova A, Melino G, and Mak TW
- Subjects
- ADAM Proteins genetics, ADAM Proteins metabolism, ADAM17 Protein, Aging pathology, Animals, Apoptosis genetics, Cell Count, Cell Proliferation, DNA Damage genetics, DNA-Binding Proteins deficiency, Female, Gene Expression Regulation, Humans, Infertility, Male blood, Infertility, Male genetics, Infertility, Male pathology, Male, Matrix Metalloproteinase 13 genetics, Matrix Metalloproteinase 13 metabolism, Mice, Mice, Knockout, Nuclear Proteins deficiency, Oxidative Stress genetics, Progesterone blood, RNA, Messenger genetics, RNA, Messenger metabolism, Spermatozoa metabolism, Spermatozoa pathology, Testis metabolism, Testis pathology, Tumor Protein p73, Tumor Suppressor Proteins deficiency, DNA-Binding Proteins metabolism, Fertility genetics, Nuclear Proteins metabolism, Spermatogenesis genetics, Tumor Suppressor Proteins metabolism
- Abstract
The generation of viable sperm proceeds through a series of coordinated steps, including germ cell self-renewal, meiotic recombination, and terminal differentiation into functional spermatozoa. The p53 family of transcription factors, including p53, p63, and p73, are critical for many physiological processes, including female fertility, but little is known about their functions in spermatogenesis. Here, we report that deficiency of the TAp73 isoform, but not p53 or ΔNp73, results in male infertility because of severe impairment of spermatogenesis. Mice lacking TAp73 exhibited increased DNA damage and cell death in spermatogonia, disorganized apical ectoplasmic specialization, malformed spermatids, and marked hyperspermia. We demonstrated that TAp73 regulates the mRNA levels of crucial genes involved in germ stem/progenitor cells (CDKN2B), spermatid maturation/spermiogenesis (metalloproteinase and serine proteinase inhibitors), and steroidogenesis (CYP21A2 and progesterone receptor). These alterations of testicular histology and gene expression patterns were specific to TAp73 null mice and not features of mice lacking p53. Our work provides previously unidentified in vivo evidence that TAp73 has a unique role in spermatogenesis that ensures the maintenance of mitotic cells and normal spermiogenesis. These results may have implications for the diagnosis and management of human male infertility.
- Published
- 2014
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20. Retinoblastoma tumor suppressor protein in pancreatic progenitors controls α- and β-cell fate.
- Author
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Cai EP, Wu X, Schroer SA, Elia AJ, Nostro MC, Zacksenhaus E, and Woo M
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- Animals, Animals, Newborn, Base Sequence, Cell Differentiation genetics, Cell Line, Tumor, Cell Survival genetics, Diabetes Mellitus, Experimental genetics, Diabetes Mellitus, Experimental metabolism, Diabetes Mellitus, Experimental pathology, E2F1 Transcription Factor genetics, E2F1 Transcription Factor metabolism, Female, Glucagon-Secreting Cells cytology, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Immunohistochemistry, Insulin-Secreting Cells cytology, Islets of Langerhans cytology, Male, Mice, Mice, Knockout, Mice, Transgenic, RNA Interference, Retinoblastoma Protein genetics, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Nucleic Acid, Stem Cells cytology, Transcription Factors genetics, Transcription Factors metabolism, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Protein p53 metabolism, Glucagon-Secreting Cells metabolism, Insulin-Secreting Cells metabolism, Islets of Langerhans metabolism, Retinoblastoma Protein metabolism, Stem Cells metabolism
- Abstract
Pancreatic endocrine cells expand rapidly during embryogenesis by neogenesis and proliferation, but during adulthood, islet cells have a very slow turnover. Disruption of murine retinoblastoma tumor suppressor protein (Rb) in mature pancreatic β-cells has a limited effect on cell proliferation. Here we show that deletion of Rb during embryogenesis in islet progenitors leads to an increase in the neurogenin 3-expressing precursor cell population, which persists in the postnatal period and is associated with increased β-cell mass in adults. In contrast, Rb-deficient islet precursors, through repression of the cell fate factor aristaless related homeobox, result in decreased α-cell mass. The opposing effect on survival of Rb-deficient α- and β-cells was a result of opposing effects on p53 in these cell types. As a consequence, loss of Rb in islet precursors led to a reduced α- to β-cell ratio, leading to improved glucose homeostasis and protection against diabetes.
- Published
- 2013
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21. BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival.
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Gorrini C, Baniasadi PS, Harris IS, Silvester J, Inoue S, Snow B, Joshi PA, Wakeham A, Molyneux SD, Martin B, Bouwman P, Cescon DW, Elia AJ, Winterton-Perks Z, Cruickshank J, Brenner D, Tseng A, Musgrave M, Berman HK, Khokha R, Jonkers J, Mak TW, and Gauthier ML
- Subjects
- Animals, BRCA1 Protein deficiency, BRCA1 Protein genetics, Breast Neoplasms genetics, Breast Neoplasms metabolism, Cell Line, Cell Transformation, Neoplastic genetics, Cell Transformation, Neoplastic metabolism, Epithelial Cells drug effects, Epithelial Cells metabolism, Estrogens pharmacology, Female, Gene Expression Regulation, Neoplastic, Gene Knockout Techniques, Humans, Mammary Glands, Animal metabolism, Mammary Glands, Animal pathology, Mice, Mutation, NAD(P)H Dehydrogenase (Quinone) genetics, NAD(P)H Dehydrogenase (Quinone) metabolism, NF-E2-Related Factor 2 genetics, Oxidative Stress, Protein Binding, Protein Stability, Reactive Oxygen Species metabolism, Ubiquitination, Antioxidants metabolism, BRCA1 Protein metabolism, Cell Survival genetics, NF-E2-Related Factor 2 metabolism, Signal Transduction
- Abstract
Oxidative stress plays an important role in cancer development and treatment. Recent data implicate the tumor suppressor BRCA1 in regulating oxidative stress, but the molecular mechanism and the impact in BRCA1-associated tumorigenesis remain unclear. Here, we show that BRCA1 regulates Nrf2-dependent antioxidant signaling by physically interacting with Nrf2 and promoting its stability and activation. BRCA1-deficient mouse primary mammary epithelial cells show low expression of Nrf2-regulated antioxidant enzymes and accumulate reactive oxygen species (ROS) that impair survival in vivo. Increased Nrf2 activation rescues survival and ROS levels in BRCA1-null cells. Interestingly, 53BP1 inactivation, which has been shown to alleviate several defects associated with BRCA1 loss, rescues survival of BRCA1-null cells without restoring ROS levels. We demonstrate that estrogen treatment partially restores Nrf2 levels in the absence of BRCA1. Our data suggest that Nrf2-regulated antioxidant response plays a crucial role in controlling survival downstream of BRCA1 loss. The ability of estrogen to induce Nrf2 posits an involvement of an estrogen-Nrf2 connection in BRCA1 tumor suppression. Lastly, BRCA1-mutated tumors retain a defective antioxidant response that increases the sensitivity to oxidative stress. In conclusion, the role of BRCA1 in regulating Nrf2 activity suggests important implications for both the etiology and treatment of BRCA1-related cancers.
- Published
- 2013
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22. Mule/Huwe1/Arf-BP1 suppresses Ras-driven tumorigenesis by preventing c-Myc/Miz1-mediated down-regulation of p21 and p15.
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Inoue S, Hao Z, Elia AJ, Cescon D, Zhou L, Silvester J, Snow B, Harris IS, Sasaki M, Li WY, Itsumi M, Yamamoto K, Ueda T, Dominguez-Brauer C, Gorrini C, Chio II, Haight J, You-Ten A, McCracken S, Wakeham A, Ghazarian D, Penn LJ, Melino G, and Mak TW
- Subjects
- 9,10-Dimethyl-1,2-benzanthracene pharmacology, Animals, Cells, Cultured, Cyclin-Dependent Kinase Inhibitor p15 biosynthesis, Cyclin-Dependent Kinase Inhibitor p15 genetics, Cyclin-Dependent Kinase Inhibitor p16, Cyclin-Dependent Kinase Inhibitor p21 biosynthesis, Cyclin-Dependent Kinase Inhibitor p21 genetics, Female, Genes, ras, Keratinocytes drug effects, Keratinocytes metabolism, Keratinocytes pathology, Male, Mice, Mice, Knockout, Nuclear Proteins deficiency, Nuclear Proteins genetics, Nuclear Proteins metabolism, Oncogene Protein p21(ras) antagonists & inhibitors, Oncogene Protein p21(ras) genetics, Protein Inhibitors of Activated STAT deficiency, Protein Inhibitors of Activated STAT genetics, Protein Inhibitors of Activated STAT metabolism, Proto-Oncogene Proteins c-myc deficiency, Proto-Oncogene Proteins c-myc genetics, Proto-Oncogene Proteins c-myc metabolism, Signal Transduction, Skin Neoplasms chemically induced, Skin Neoplasms genetics, Skin Neoplasms metabolism, Skin Neoplasms pathology, Tetradecanoylphorbol Acetate pharmacology, Tumor Suppressor Protein p53, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases deficiency, Ubiquitin-Protein Ligases genetics, Cell Transformation, Neoplastic genetics, Cyclin-Dependent Kinase Inhibitor p15 metabolism, Cyclin-Dependent Kinase Inhibitor p21 metabolism, Down-Regulation, Nuclear Proteins antagonists & inhibitors, Oncogene Protein p21(ras) metabolism, Protein Inhibitors of Activated STAT antagonists & inhibitors, Proto-Oncogene Proteins c-myc antagonists & inhibitors, Ubiquitin-Protein Ligases metabolism
- Abstract
Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule(flox/flox(y)) (Mule kKO) mice and subjected them to DMBA/PMA-induced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Mule-deficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.
- Published
- 2013
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23. Lipocalin 2 performs contrasting, location-dependent roles in APCmin tumor initiation and progression.
- Author
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Reilly PT, Teo WL, Low MJ, Amoyo-Brion AA, Dominguez-Brauer C, Elia AJ, Berger T, Greicius G, Pettersson S, and Mak TW
- Subjects
- Acute-Phase Proteins deficiency, Acute-Phase Proteins genetics, Animals, Apoptosis physiology, Disease Progression, Female, Genes, APC, Intestinal Neoplasms genetics, Lipocalin-2, Lipocalins genetics, Male, Mice, Mice, Inbred C57BL, Oncogene Proteins deficiency, Oncogene Proteins genetics, Acute-Phase Proteins biosynthesis, Intestinal Neoplasms metabolism, Intestinal Neoplasms pathology, Lipocalins biosynthesis, Oncogene Proteins biosynthesis
- Abstract
Evidence that lipocalin 2 (LCN2) is oncogenic has grown in recent years and comes from both animal models and expression analysis from a variety of human cancers. In the intestine, LCN2 is overexpressed in colitis patients and its overexpression is a negative prognostic indicator in colorectal cancer. Functionally, LCN2 has a number of different activities that may contribute to its oncogenic potential, including increasing matrix metalloproteinase activity, control of iron availability and stimulating inflammation. In this report, we examined APCmin intestinal tumorigenesis in an LCN2-deficient background. We found that the loss of LCN2 increased tumor multiplicity specifically in the duodenum, suggesting a potential tumor-suppressive activity. Concurrently, however, LCN2 increased the average small intestinal tumor size particularly in the distal small intestine. We found that this increase was correlated to tumor iron(II) content, suggesting that an iron-scavenging role is important for LCN2 oncogenic activity in the intestine.
- Published
- 2013
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24. 2-Methoxyestradiol inhibits experimental autoimmune encephalomyelitis through suppression of immune cell activation.
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Duncan GS, Brenner D, Tusche MW, Brüstle A, Knobbe CB, Elia AJ, Mock T, Bray MR, Krammer PH, and Mak TW
- Subjects
- 2-Methoxyestradiol, Animals, Autoimmunity, CD4-Positive T-Lymphocytes cytology, Cytokines biosynthesis, Estradiol pharmacology, Humans, Lymphocyte Activation, Lymphocytes cytology, Mice, Mice, Inbred C57BL, Multiple Sclerosis immunology, NF-kappa B metabolism, NFATC Transcription Factors metabolism, Signal Transduction, Transcription Factor AP-1 metabolism, Tubulin Modulators pharmacology, Encephalomyelitis, Autoimmune, Experimental drug therapy, Estradiol analogs & derivatives
- Abstract
The endogenous metabolite of estradiol, 2-Methoxyestradiol (2ME2), is an antimitotic and antiangiogenic cancer drug candidate that also exhibits disease-modifying activity in animal models of rheumatoid arthritis (RA). We found that 2ME2 dramatically suppresses development of mouse experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). 2ME2 inhibits in vitro lymphocyte activation, cytokine production, and proliferation in a dose-dependent fashion. 2ME2 treatment of lymphocytes specifically reduced the nuclear translocation and transcriptional activity of nuclear factor of activated T-cells (NFAT) c1, whereas NF-κB and activator protein 1 (AP-1) activation were not adversely affected. We therefore propose that 2ME2 attenuates EAE through disruption of the NFAT pathway and subsequent lymphocyte activation. By extension, our findings provide a molecular rationale for the use of 2ME2 as a tolerable oral immunomodulatory agent for the treatment of autoimmune disorders such as MS in humans.
- Published
- 2012
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25. D-2-hydroxyglutarate produced by mutant IDH1 perturbs collagen maturation and basement membrane function.
- Author
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Sasaki M, Knobbe CB, Itsumi M, Elia AJ, Harris IS, Chio II, Cairns RA, McCracken S, Wakeham A, Haight J, Ten AY, Snow B, Ueda T, Inoue S, Yamamoto K, Ko M, Rao A, Yen KE, Su SM, and Mak TW
- Subjects
- Animals, Basement Membrane metabolism, Brain cytology, Brain pathology, Gene Knock-In Techniques, Genotype, Glioma pathology, Mice, Mutation, Protein Stability, Reactive Oxygen Species metabolism, Stress, Physiological, Basement Membrane pathology, Collagen metabolism, Glutarates metabolism, Isocitrate Dehydrogenase genetics, Isocitrate Dehydrogenase metabolism
- Abstract
Isocitrate dehydrogenase-1 (IDH1) R132 mutations occur in glioma, but their physiological significance is unknown. Here we describe the generation and characterization of brain-specific Idh1 R132H conditional knock-in (KI) mice. Idh1 mutation results in hemorrhage and perinatal lethality. Surprisingly, intracellular reactive oxygen species (ROS) are attenuated in Idh1-KI brain cells despite an apparent increase in the NADP(+)/NADPH ratio. Idh1-KI cells also show high levels of D-2-hydroxyglutarate (D2HG) that are associated with inhibited prolyl-hydroxylation of hypoxia-inducible transcription factor-1α (Hif1α) and up-regulated Hif1α target gene transcription. Intriguingly, D2HG also blocks prolyl-hydroxylation of collagen, causing a defect in collagen protein maturation. An endoplasmic reticulum (ER) stress response induced by the accumulation of immature collagens may account for the embryonic lethality of these mutants. Importantly, D2HG-mediated impairment of collagen maturation also led to basement membrane (BM) aberrations that could play a part in glioma progression. Our study presents strong in vivo evidence that the D2HG produced by the mutant Idh1 enzyme is responsible for the above effects.
- Published
- 2012
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26. Caspase-3 deficiency reveals a physiologic role for Smac/DIABLO in regulating programmed cell death.
- Author
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Hui KK, Kanungo AK, Elia AJ, and Henderson JT
- Subjects
- Animals, Apoptosis Regulatory Proteins, Carrier Proteins genetics, Caspase 3 genetics, Caspase 6 genetics, Caspase 6 metabolism, Caspase 7 genetics, Caspase 7 metabolism, Caspase Inhibitors, Cell Line, Cell Lineage, Fibroblasts metabolism, Inhibitor of Apoptosis Proteins antagonists & inhibitors, Inhibitor of Apoptosis Proteins genetics, Inhibitor of Apoptosis Proteins metabolism, Mice, Mice, Knockout, Mitochondrial Proteins genetics, Poly(ADP-ribose) Polymerases metabolism, RNA Interference, RNA, Small Interfering metabolism, X-Linked Inhibitor of Apoptosis Protein antagonists & inhibitors, X-Linked Inhibitor of Apoptosis Protein genetics, X-Linked Inhibitor of Apoptosis Protein metabolism, Apoptosis, Carrier Proteins metabolism, Caspase 3 deficiency, Caspase 3 metabolism, Mitochondrial Proteins metabolism
- Abstract
Inhibitor of apoptosis protein (IAP)-binding proteins such as Grim, Reaper and HID have been shown to exert a critical role in regulating caspase activity in species such as D. Melanogaster. However, a comparable role for the mammalian homologue of second mitochondrial-derived activator of caspase/direct IAP-binding protein with low pI (Smac/DIABLO) has yet to be clearly established in vivo. Despite tremendous interest in recent years in the use of so-called Smac mimetics to enhance chemotherapeutic potency, our understanding of the true physiologic nature of Smac/DIABLO in regulating programmed cell death (PCD) remains elusive. In order to critically evaluate the role of Smac/DIABLO in regulating mammalian PCD, deficiency of caspase-3 was used as a sensitizing mutation in order to reduce aggregate levels of executioner caspase activity. We observe that combinatorial deletion of Diablo and Casp3, but neither alone, results in perinatal lethality in mice. Consistent with this, examination of both intrinsic and extrinsic forms of PCD in lines of murine embryonic fibroblasts demonstrate that loss of Smac/DIABLO alters both caspase-dependent and caspase-independent intrinsic PCD. Comparative small interfering RNA inhibition studies of X-linked inhibitor of apoptosis, cellular inhibitor of apoptosis (cIAP)-1, cIAP-2, caspase-6 and -7 in both wild-type and Casp3/Diablo DKO mouse embryonic fibroblast lineages, supports a model in which Smac/DIABLO acts to enhance the early phase executioner caspase activity through the modulation of inhibitory interactions between specific IAP family members and executioner caspases-3 and -7.
- Published
- 2011
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27. Acidic nuclear phosphoprotein 32kDa (ANP32)B-deficient mouse reveals a hierarchy of ANP32 importance in mammalian development.
- Author
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Reilly PT, Afzal S, Gorrini C, Lui K, Bukhman YV, Wakeham A, Haight J, Ling TW, Cheung CC, Elia AJ, Turner PV, and Mak TW
- Subjects
- Animals, Apoptosis, Biomarkers, Tumor metabolism, Breast Neoplasms metabolism, Breast Neoplasms pathology, Cell Proliferation, Cells, Cultured, Embryo, Mammalian anatomy & histology, Female, Fibroblasts cytology, Fibroblasts physiology, Gene Targeting, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mutation, RNA, Messenger genetics, RNA, Messenger metabolism, Survival Rate, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Embryo, Mammalian physiology, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Nuclear Proteins genetics, Nuclear Proteins metabolism
- Abstract
The highly conserved ANP32 proteins are proposed to function in a broad array of physiological activities through molecular mechanisms as diverse as phosphatase inhibition, chromatin regulation, caspase activation, and intracellular transport. On the basis of previous analyses of mice bearing targeted mutations of Anp32a or Anp32e, there has been speculation that all ANP32 proteins play redundant roles and are dispensable for normal development. However, more recent work has suggested that ANP32B may in fact have functions that are not shared by other ANP32 family members. Here we report that ANP32B expression is associated with a poor prognosis in human breast cancer, consistent with the increased levels of Anp32b mRNA present in proliferating wild-type (WT) murine embryonic fibroblasts and stimulated WT B and T lymphocytes. Moreover, we show that, contrary to previous assumptions, Anp32b is very important for murine embryogenesis. In a mixed genetic background, ANP32B-deficient mice displayed a partially penetrant perinatal lethality that became fully penetrant in a pure C57BL/6 background. Surviving ANP32B-deficient mice showed reduced viability due to variable defects in various organ systems. Study of compound mutants lacking ANP32A, ANP32B, and/or ANP32E revealed previously hidden roles for ANP32A in mouse development that became apparent only in the complete absence of ANP32B. Our data demonstrate a hierarchy of importance for the mammalian Anp32 genes, with Anp32b being the most critical for normal development.
- Published
- 2011
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28. Combined deficiency for MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) delays tumor development.
- Author
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Ueda T, Sasaki M, Elia AJ, Chio II, Hamada K, Fukunaga R, and Mak TW
- Subjects
- Animals, Cell Line, Tumor, Eukaryotic Initiation Factor-4E metabolism, Glioma pathology, Lymphoma etiology, Mice, Mice, Knockout, Mice, Nude, Neoplasm Transplantation, PTEN Phosphohydrolase deficiency, Phosphorylation, Protein Serine-Threonine Kinases physiology, Transplantation, Heterologous, Neoplasms etiology, Protein Serine-Threonine Kinases deficiency
- Abstract
MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) are protein-serine/threonine kinases that are activated by ERK or p38 and phosphorylate eIF4E, which is involved in cap-dependent translation initiation. However, Mnk1/2 double knockout (Mnk-DKO) mice show normal cell growth and development despite an absence of eIF4E phosphorylation. Here we show that the tumorigenesis occurring in the Lck-Pten mouse model (referred to here as tPten(-/-) mice) can be suppressed by the loss of Mnk1/2. Phosphorylation of eIF4E was greatly enhanced in lymphomas of parental tPten(-/-) mice compared with lymphoid tissues of wild-type mice, but was totally absent in lymphomas of tPten(-/-); Mnk-DKO mice. Notably, stable knockdown of Mnk1 in the human glioma cell line U87MG resulted in dramatically decreased tumor formation when these cells were injected into athymic nude mice. Our data demonstrate an oncogenic role for Mnk1/2 in tumor development, and highlight these molecules as potential anticancer drug targets that could be inactivated with minimal side effects.
- Published
- 2010
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29. Smg1 is required for embryogenesis and regulates diverse genes via alternative splicing coupled to nonsense-mediated mRNA decay.
- Author
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McIlwain DR, Pan Q, Reilly PT, Elia AJ, McCracken S, Wakeham AC, Itie-Youten A, Blencowe BJ, and Mak TW
- Subjects
- Animals, Cells, Cultured, Embryo, Mammalian cytology, Embryo, Mammalian embryology, Embryonic Stem Cells cytology, Embryonic Stem Cells metabolism, Female, Gene Expression Profiling, Genes, Lethal, Immunoblotting, Male, Mice, Mice, Inbred C57BL, Mice, Inbred Strains, Mice, Knockout, Phosphatidylinositol 3-Kinases genetics, Phosphorylation, Protein Serine-Threonine Kinases genetics, Reverse Transcriptase Polymerase Chain Reaction, Alternative Splicing, Embryo, Mammalian metabolism, Phosphatidylinositol 3-Kinases metabolism, Protein Serine-Threonine Kinases metabolism, RNA Stability
- Abstract
Smg1 is a PI3K-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbor premature termination codons (PTCs) as a result of events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. By using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5. High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from approximately 9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signaling, membrane dynamics, cell death, and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.
- Published
- 2010
- Full Text
- View/download PDF
30. Identification of BERP (brain-expressed RING finger protein) as a p53 target gene that modulates seizure susceptibility through interacting with GABA(A) receptors.
- Author
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Cheung CC, Yang C, Berger T, Zaugg K, Reilly P, Elia AJ, Wakeham A, You-Ten A, Chang N, Li L, Wan Q, and Mak TW
- Subjects
- Animals, Base Sequence, Cells, Cultured, Convulsants toxicity, DNA Primers genetics, Genes, p53, Humans, Male, Mice, Mice, Knockout, Nerve Tissue Proteins deficiency, Neurons metabolism, Pentylenetetrazole toxicity, RNA, Messenger genetics, RNA, Messenger metabolism, Receptors, GABA-A genetics, Seizures chemically induced, Up-Regulation, Carrier Proteins genetics, Nerve Tissue Proteins genetics, Receptors, GABA-A metabolism, Seizures genetics, Seizures metabolism, Tumor Suppressor Protein p53 metabolism
- Abstract
p53 is a central player in responses to cellular stresses and a major tumor suppressor. The identification of unique molecules within the p53 signaling network can reveal functions of this important transcription factor. Here, we show that brain-expressed RING finger protein (BERP) is a gene whose expression is up-regulated in a p53-dependent manner in human cells and in mice. We generated BERP-deficient mice by gene targeting and demonstrated that they exhibit increased resistance to pentylenetetrazol-induced seizures. Electrophysiological and biochemical studies of cultured cortical neurons of BERP-deficient mice showed a decrease in the amplitude of GABA(A) receptor (GABA(A)R)-mediated miniature inhibitory postsynaptic currents as well as reduced surface protein expression of GABA(A)Rs containing the gamma2-subunit. However, BERP deficiency did not decrease GABA(A)Rgamma2 mRNA levels, raising the possibility that BERP may act at a posttranscriptional level to regulate the intracellular trafficking of GABA(A)Rs. Our results indicate that BERP is a unique p53-regulated gene and suggest a role for p53 within the central nervous system.
- Published
- 2010
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31. Disruption of the Lcn2 gene in mice suppresses primary mammary tumor formation but does not decrease lung metastasis.
- Author
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Berger T, Cheung CC, Elia AJ, and Mak TW
- Subjects
- Animals, Blotting, Western, Crosses, Genetic, Electrophoresis, Polyacrylamide Gel, Female, Histological Techniques, Lipocalin-2, Lung Neoplasms secondary, Matrix Metalloproteinase 9 metabolism, Mice, Mice, Transgenic, Acute-Phase Proteins genetics, Lipocalins genetics, Lung Neoplasms genetics, Mammary Neoplasms, Animal genetics, Neoplasm Metastasis genetics, Oncogene Proteins genetics
- Abstract
Based largely on studies in xenograft models, lipocalin-2 (Lcn2) has been implicated in the progression of multiple types of human tumors, including breast cancer. Here we examine the role of Lcn2 in mammary tumorigenesis and lung metastasis using an in vivo molecular genetics approach. We crossed a well-characterized transgenic mouse model of breast cancer, the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) mouse, with two independent gene-targeted Lcn2(-/-) mouse strains of the 129/Ola or C57BL/6 genetic background. The onset and progression of mammary tumor development and lung metastasis in the female progeny of these crosses were monitored over a 20-week period. Female Lcn2(-/-)MMTV-PyMT mice of the 129/Ola background (Lcn2(-/-)PyMT(129)) showed delayed onset of mammary tumors, and both Lcn2(-/-)PyMT(129) mice and Lcn2(-/-)MMTV-PyMT mice of the C57BL/6 background (Lcn2(-/-)PyMT(B6)) exhibited significant decreases in multiplicity and tumor burden (approximately 2- to 3-fold), as measured by total tumor weight and volume. At the molecular level, mammary tumors derived from Lcn2(-/-)PyMT(B6) females showed reduced matrix metalloproteinase-9 (MMP-9) activity and a lack of high molecular weight MMP activity. However, although increased MMP-9 activity has been linked to tumor progression, neither Lcn2(-/-)PyMT(B6) nor Lcn2(-/-)PyMT(129) female mice showed a reduction in lung metastases compared to Lcn2(+/+)PyMT controls. Our results demonstrate, using an in vivo animal model approach, that Lcn2 is a potent inducer of mammary tumor growth but not a significant promoter of lung metastasis.
- Published
- 2010
- Full Text
- View/download PDF
32. Inhibition of apoptosome activation protects injured motor neurons from cell death.
- Author
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Kanungo AK, Hao Z, Elia AJ, Mak TW, and Henderson JT
- Subjects
- Animals, Axotomy, Caspase 3 genetics, Cell Death, Cytochromes c genetics, Gene Expression Regulation, Developmental, Mice, Mice, Inbred C57BL, Receptor, Nerve Growth Factor genetics, Apoptosomes metabolism, Motor Neurons cytology, Motor Neurons metabolism
- Abstract
Within the mammalian central nervous system many forms of neurodegenerative injury are regulated via programmed cell death, a highly conserved program of cellular suicide. Programmed cell death is regulated by multiple signaling pathways, which have been identified within mammalian cells, although several lines of evidence suggest that the intrinsic pathway predominantly regulates the death of motor neurons following acute injury in vivo. We have tested this hypothesis by performing facial axotomies on cytochrome c knock-in mice containing a point mutation in the genomic locus of cytochrome c resulting in a lysine to alanine conversion at position 72 of the protein. The introduced mutation inhibits the ability of cytochrome c to induce the formation of the apoptosome, a protein complex that is principally required for the activation of the intrinsic pathway, but does not alter its function in oxidative phosphorylation. Homozygous cytochrome c knock-in mutants displayed a significant enhancement in motor neuron survival following injury when compared with littermate controls, thus establishing the apoptosome as a viable target for protecting motor neurons from neural injury. However, protection of facial motor neurons differs from that previously reported in mice either overexpressing anti-apoptotic or lacking pro-apoptotic members of the Bcl-2 family, which are thought to regulate several aspects of mitochondrial dysfunction including the release of cytochrome c from the mitochondria to the cytoplasm. Therefore, these results directly demonstrate for the first time the influence of the apoptosome on injury-induced neuronal programmed cell death in vivo isolated from upstream Bcl-2 family-mediated effects.
- Published
- 2008
- Full Text
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33. Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury.
- Author
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Berger T, Togawa A, Duncan GS, Elia AJ, You-Ten A, Wakeham A, Fong HE, Cheung CC, and Mak TW
- Subjects
- Acute-Phase Proteins genetics, Animals, Apoptosis, Cells, Cultured, Escherichia coli Infections genetics, Escherichia coli Infections immunology, Immunity, Innate immunology, Kidney metabolism, Kidney pathology, Lipocalin-2, Lipocalins, Mice, Mice, Knockout, Oncogene Proteins genetics, Reperfusion Injury immunology, Acute-Phase Proteins deficiency, Acute-Phase Proteins metabolism, Escherichia coli physiology, Escherichia coli Infections metabolism, Escherichia coli Infections microbiology, Oncogene Proteins deficiency, Oncogene Proteins metabolism, Reperfusion Injury metabolism
- Abstract
Diverse functions have been reported for lipocalin 2. To investigate these functions in vivo, we generated gene-targeted lipocalin 2-deficient mice (Lcn2-/- mice). In vitro studies have suggested that lipocalin 2 is important for cellular apoptosis induced by IL-3 withdrawal, and for the induction of kidney differentiation during embryogenesis. Analysis of Lcn2-/- mice showed normal cell death upon IL-3 withdrawal and normal kidney development. However, we found that Lcn2-/- mice exhibited an increased susceptibility to bacterial infections, in keeping with the proposed function of lipocalin 2 in iron sequestration. Neutrophils isolated from Lcn2-/- mice showed significantly less bacteriostatic activity compared with WT controls. The bacteriostatic property of the WT neutrophils was abolished by the addition of exogenous iron, indicating that the main function of lipocalin 2 in the antibacterial innate immune response is to limit this essential element. Another important function ascribed to lipocalin 2 has been its protective role against kidney ischemia-reperfusion injury. We analyzed Lcn2-/- mice using a mouse model for severe renal failure and could not detect any significant differences compared with their WT littermates.
- Published
- 2006
- Full Text
- View/download PDF
34. Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner.
- Author
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Hirao A, Cheung A, Duncan G, Girard PM, Elia AJ, Wakeham A, Okada H, Sarkissian T, Wong JA, Sakai T, De Stanchina E, Bristow RG, Suda T, Lowe SW, Jeggo PA, Elledge SJ, and Mak TW
- Subjects
- 9,10-Dimethyl-1,2-benzanthracene, Alanine chemistry, Animals, Ataxia Telangiectasia Mutated Proteins, CD4-Positive T-Lymphocytes metabolism, CD8-Positive T-Lymphocytes metabolism, Carcinogens, Cell Cycle, Cell Cycle Proteins, Checkpoint Kinase 2, DNA-Binding Proteins, Dose-Response Relationship, Radiation, Female, Genes, p53, Male, Mice, Mice, Knockout, Models, Biological, Models, Genetic, Mutation, Neoplasms, Experimental prevention & control, Phosphorylation, Protein Binding, Protein Kinases metabolism, Thymus Gland cytology, Time Factors, Tissue Distribution, Tumor Suppressor Protein p53 metabolism, Apoptosis, Protein Kinases physiology, Protein Serine-Threonine Kinases metabolism, Tumor Suppressor Proteins physiology
- Abstract
In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.
- Published
- 2002
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35. Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells.
- Author
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Hitoshi S, Alexson T, Tropepe V, Donoviel D, Elia AJ, Nye JS, Conlon RA, Mak TW, Bernstein A, and van der Kooy D
- Subjects
- Animals, Brain metabolism, Bromodeoxyuridine pharmacology, Cell Cycle, Cell Differentiation, Central Nervous System embryology, Central Nervous System metabolism, Immunohistochemistry, Mice, Microscopy, Fluorescence, Mutation, Neurons metabolism, Presenilin-1, Prosencephalon metabolism, Protein Binding, Receptor, Notch1, Receptors, Notch, Retroviridae genetics, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, Time Factors, Membrane Proteins metabolism, Receptors, Cell Surface, Transcription Factors
- Abstract
Neural stem cells, which exhibit self-renewal and multipotentiality, are generated in early embryonic brains and maintained throughout the lifespan. The mechanisms of their generation and maintenance are largely unknown. Here, we show that neural stem cells are generated independent of RBP-Jkappa, a key molecule in Notch signaling, by using RBP-Jkappa(-/-) embryonic stem cells in an embryonic stem cell-derived neurosphere assay. However, Notch pathway molecules are essential for the maintenance of neural stem cells; they are depleted in the early embryonic brains of RBP-Jkappa(-/-) or Notch1(-/-) mice. Neural stem cells also are depleted in embryonic brains deficient for the presenilin1 (PS1) gene, a key regulator in Notch signaling, and are reduced in PS1(+/-) adult brains. Both neuronal and glial differentiation in vitro were enhanced by attenuation of Notch signaling and suppressed by expressing an active form of Notch1. These data are consistent with a role for Notch signaling in the maintenance of the neural stem cell, and inconsistent with a role in a neuronal/glial fate switch.
- Published
- 2002
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36. Signal transducer and activator of transcription 6 is frequently activated in Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma.
- Author
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Skinnider BF, Elia AJ, Gascoyne RD, Patterson B, Trumper L, Kapp U, and Mak TW
- Subjects
- Adaptor Proteins, Signal Transducing, Adolescent, Adult, Aged, Aged, 80 and over, Apoptosis, Autocrine Communication, Carrier Proteins biosynthesis, Carrier Proteins genetics, Cell Division, Cytokines biosynthesis, Cytokines genetics, Cytoskeletal Proteins, DNA-Binding Proteins metabolism, Epstein-Barr Virus Infections genetics, Epstein-Barr Virus Infections metabolism, Female, Herpesvirus 4, Human genetics, Hodgkin Disease genetics, Hodgkin Disease pathology, Hodgkin Disease virology, Humans, Inflammation, Interleukin-13 Receptor alpha1 Subunit, Intracellular Signaling Peptides and Proteins, LIM Domain Proteins, Lymphoma, Non-Hodgkin genetics, Lymphoma, Non-Hodgkin metabolism, Lymphoma, Non-Hodgkin pathology, Male, Middle Aged, Paracrine Communication, Phosphorylation, Receptors, Interleukin-13, Reed-Sternberg Cells pathology, STAT3 Transcription Factor, STAT5 Transcription Factor, STAT6 Transcription Factor, Signal Transduction, Tumor Cells, Cultured metabolism, Gene Expression Regulation, Neoplastic, Hodgkin Disease metabolism, Interleukin-13 physiology, Milk Proteins, Neoplasm Proteins metabolism, Protein Processing, Post-Translational, Receptors, Interleukin physiology, Reed-Sternberg Cells metabolism, Trans-Activators metabolism
- Abstract
The unique clinicopathologic features of Hodgkin lymphoma (HL) are due to the multiple cytokines produced by its neoplastic cells, the Hodgkin and Reed-Sternberg (HRS) cells. Cytokine signaling is mediated through the signal transducer and activator of transcription (STAT) family of transcription factors. Immunoblotting and immunohistochemistry were used to examine cell lines and tissue sections derived from patients with HL and non-Hodgkin lymphoma (NHL) for expression of activated STAT proteins. Constitutive phosphorylation of STAT6 and STAT3 was common in HL. STAT6 was constitutively phosphorylated in 5 of 5 HL cell lines and in HRS cells from 25 of 32 (78%) classical HL cases. STAT3 was constitutively phosphorylated in 4 of 5 HL cell lines and in HRS cells from 27 of 31 (87%) classical HL cases. Only 4 of 24 NHL cases demonstrated constitutive STAT6 activation, whereas STAT3 activation was observed in 6 of 13 (46%) cases of B-cell NHL and 8 of 11 (73%) cases of T-cell NHL. Constitutive STAT5 phosphorylation was not a common feature of HL or NHL. STAT6 mediates signaling by interleukin 13 (IL-13), a cytokine frequently expressed by HRS cells. Antibody-mediated neutralization of IL-13 resulted in significant decreases in both cellular proliferation and levels of phosphorylated STAT6 of HL cell lines. In conclusion, constitutive STAT6 phosphorylation is a common and distinctive feature of HRS cells in classical HL, whereas STAT3 activation was regularly present in both HL and NHL. These results suggest that IL-13 signaling is largely responsible for the constitutive STAT6 activation observed in HRS cells and further implicate IL-13 as an important growth factor in classical HL.
- Published
- 2002
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37. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death.
- Author
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Joza N, Susin SA, Daugas E, Stanford WL, Cho SK, Li CY, Sasaki T, Elia AJ, Cheng HY, Ravagnan L, Ferri KF, Zamzami N, Wakeham A, Hakem R, Yoshida H, Kong YY, Mak TW, Zúñiga-Pflücker JC, Kroemer G, and Penninger JM
- Subjects
- Animals, Apoptosis Inducing Factor, Apoptotic Protease-Activating Factor 1, Caspase 9, Caspases metabolism, Cell Differentiation, Chimera, Embryo, Mammalian cytology, Embryonic and Fetal Development physiology, Female, Flavoproteins genetics, Gene Targeting, Male, Membrane Proteins genetics, Mice, Mice, Inbred C57BL, Morphogenesis, Proteins physiology, Recombination, Genetic, Stem Cells, Apoptosis physiology, Flavoproteins physiology, Membrane Proteins physiology, Mitochondria physiology
- Abstract
Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome-a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies-the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.
- Published
- 2001
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38. Interleukin 13 and interleukin 13 receptor are frequently expressed by Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma.
- Author
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Skinnider BF, Elia AJ, Gascoyne RD, Trümper LH, von Bonin F, Kapp U, Patterson B, Snow BE, and Mak TW
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, DNA metabolism, Female, Gene Expression, Gene Library, Hodgkin Disease etiology, Hodgkin Disease pathology, Humans, In Situ Hybridization, Interleukin-13 Receptor alpha1 Subunit, Lymphoma, Non-Hodgkin genetics, Male, Middle Aged, Polymerase Chain Reaction, RNA, Messenger metabolism, Receptors, Interleukin metabolism, Receptors, Interleukin-13, Reed-Sternberg Cells metabolism, Tumor Cells, Cultured, Hodgkin Disease genetics, Interleukin-13 genetics, Receptors, Interleukin genetics, Reed-Sternberg Cells chemistry
- Abstract
Hodgkin lymphoma (HL) is characterized by the abnormal expression of multiple cytokines, accounting for its unique clinicopathologic features. We have previously shown that interleukin-13 (IL-13) is secreted by HL cell lines and may serve as an autocrine growth factor. To determine the frequency of IL-13 expression in lymphoma patients, tissue sections from 36 patients with classical HL, 5 patients with nodular lymphocyte predominance HL (NLPHL), and 23 patients with non-Hodgkin lymphoma (NHL) were subjected to in situ hybridization. In 31 of 36 cases (86%) of classical HL patients of all histologic subtypes, between 25% to almost 100% of Hodgkin and Reed Sternberg (HRS) cells were positive for IL-13 expression. In contrast, in no case of NLPHL and in only 4 of 23 NHL cases (1 of 5 T-cell-rich B-cell lymphomas, 2 of 5 anaplastic large cell lymphomas, and 1 of 5 peripheral T-cell lymphomas) did the neoplastic cells express IL-13. The expression of the IL-13 receptor chain alpha1 (IL-13Ralpha1) was also analyzed by in situ hybridization. In 24 of 27 (89%) cases of classical HL, between 25% to 75% of HRS cells, as well as a high frequency of lymphocytes and histiocytes, were positive for IL-13Ralpha1 expression. These results were confirmed by the construction of complementary DNA libraries from single HRS cells, followed by polymerase chain reaction analysis, in which IL-13Ralpha1 transcripts were found to be present in all 6 cases of HL. These data indicate that expression of IL-13 and IL-13Ralpha1 is a common feature of HRS cells in HL, consistent with the hypothesis that IL-13 may play a role in autocrine growth in classical HL.
- Published
- 2001
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39. The mammalian ShcB and ShcC phosphotyrosine docking proteins function in the maturation of sensory and sympathetic neurons.
- Author
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Sakai R, Henderson JT, O'Bryan JP, Elia AJ, Saxton TM, and Pawson T
- Subjects
- Animals, COS Cells, Cell Differentiation genetics, Cells, Cultured, Cloning, Molecular, Gene Targeting, Membrane Proteins genetics, Membrane Proteins metabolism, Mice, Mice, Knockout, Nerve Tissue Proteins genetics, Neurons, Afferent cytology, Organ Specificity, Phosphotyrosine metabolism, Proteins genetics, Sequence Homology, Amino Acid, Shc Signaling Adaptor Proteins, Signal Transduction, Src Homology 2 Domain-Containing, Transforming Protein 1, Src Homology 2 Domain-Containing, Transforming Protein 2, Src Homology 2 Domain-Containing, Transforming Protein 3, Sympathetic Nervous System cytology, Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport, Nerve Tissue Proteins metabolism, Neurons, Afferent metabolism, Neuropeptides, Sympathetic Nervous System metabolism, src Homology Domains genetics
- Abstract
Shc proteins possess SH2 and PTB domains and serve a scaffolding function in signaling by a variety of receptor tyrosine kinases. There are three known mammalian Shc genes, of which ShcB and ShcC are primarily expressed in the nervous system. We have generated null mutations in ShcB and ShcC and have obtained mice lacking either ShcB or ShcC or both gene products. ShcB-deficient animals exhibit a loss of peptidergic and nonpeptidergic nociceptive sensory neurons, which is not enhanced by additional loss of ShcC. Mice lacking both ShcB and ShcC exhibit a significant loss of neurons within the superior cervical ganglia, which is not observed in either mutant alone. The results indicate that these Shc family members possess both unique and overlapping functions in regulating neural development and suggest physiological roles for ShcB/ShcC in TrkA signaling.
- Published
- 2000
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40. Tumor necrosis factor receptor-associated factor 6 (TRAF6) deficiency results in exencephaly and is required for apoptosis within the developing CNS.
- Author
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Lomaga MA, Henderson JT, Elia AJ, Robertson J, Noyce RS, Yeh WC, and Mak TW
- Subjects
- Animals, Antigens, Differentiation biosynthesis, Apoptosis genetics, Bromodeoxyuridine, Cell Division genetics, Cell Movement genetics, Central Nervous System metabolism, Central Nervous System pathology, Genotype, Heterozygote, Homozygote, Immunohistochemistry, In Situ Nick-End Labeling, Inbreeding, Mice, Mice, Inbred ICR, Mice, Knockout, Neural Tube Defects metabolism, Neural Tube Defects pathology, Organ Specificity genetics, Penetrance, Phenotype, Proteins metabolism, Receptor, Nerve Growth Factor metabolism, TNF Receptor-Associated Factor 6, Neural Tube Defects genetics, Proteins genetics
- Abstract
Tumor necrosis factor receptor-associated factors (TRAFs) are adaptor proteins important in mediating intracellular signaling. We report here that targeted deletion of traf6 greatly increases the frequency of failure of neural tube closure and exencephaly in traf6 (-/-) mice. The penetrance of this defect is influenced by genetic background. Neural tube fusion requires the coordination of several biological processes, including cell migration invoked by contact-dependent signaling, cell proliferation, and programmed cell death (PCD). To gain greater insight into the role of TRAF6 in these processes, neural development and migration within the CNS of traf6 (-/-) mice and controls were assessed through temporal examination of a number of immunohistochemical markers. In addition, relative levels of cellular proliferation and PCD were examined throughout embryonic development using bromodeoxyuridine (BrdU) and in situ terminal deoxynucleotidyl transferase-mediated dUTP biotinylated nick end labeling (TUNEL), respectively. The data suggest that loss of TRAF6 does not significantly alter the level of cellular proliferation or the pattern of neural differentiation per se, but rather regulates the level of PCD within specific regions of the developing CNS. Substantial reductions in TUNEL were observed within the ventral diencephalon and mesencephalon in exencephalic traf6 (-/-) embryos. Our results demonstrate a novel and prominent role for TRAF6 in the regional control of PCD within the developing CNS.
- Published
- 2000
41. Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription.
- Author
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Bonnard M, Mirtsos C, Suzuki S, Graham K, Huang J, Ng M, Itié A, Wakeham A, Shahinian A, Henzel WJ, Elia AJ, Shillinglaw W, Mak TW, Cao Z, and Yeh WC
- Subjects
- Amino Acid Sequence, Animals, Blotting, Southern, Cycloheximide pharmacology, Dose-Response Relationship, Drug, Female, Fibroblasts metabolism, Flow Cytometry, Gene Targeting, Genes, Reporter, Genotype, Heterozygote, I-kappa B Kinase, In Situ Nick-End Labeling, Interleukin-1 pharmacology, Ligases metabolism, Liver pathology, Lymphocytes metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Models, Genetic, Molecular Sequence Data, Phosphorylation, Precipitin Tests, Protein Binding, Protein Serine-Threonine Kinases metabolism, Protein Serine-Threonine Kinases physiology, Protein Synthesis Inhibitors pharmacology, Proteins metabolism, Recombinant Proteins metabolism, TNF Receptor-Associated Factor 2, Thymus Gland cytology, Time Factors, Transcription, Genetic, Tumor Necrosis Factor-alpha metabolism, Apoptosis, Liver metabolism, NF-kappa B biosynthesis, NF-kappa B genetics
- Abstract
Induction of NF-kappaB-dependent transcription requires phosphorylation and subsequent degradation of I-kappaB, an inhibitor of NF-kappaB, followed by nuclear translocation and DNA binding of NF-kappaB. Tumor necrosis factor receptor-associated factor 2 (TRAF2) plays a role in NF-kappaB activation in response to cytokines such as tumor necrosis factor alpha (TNFalpha). In this study, we purified and characterized a novel kinase (T2K, also known as TBK1 or NAK), which associates with TRAF2 and exhibits kinase activity towards I-kappaBalpha in vitro. The physiological function of T2K was investigated using T2K-deficient mice. Heterozygotes appear normal, but t2k(-/-) animals die at approximately E14.5 of massive liver degeneration and apoptosis. Never theless, hematopoietic progenitors from T2K-deficient fetal liver support normal lymphocyte development. Furthermore, t2k(-/-) embryonic fibroblasts and thymocytes do not display increased sensitivity to TNFalpha-induced apoptosis. In response to either TNFalpha or IL-1 induction, t2k(-/-) embryonic fibroblasts exhibit normal degradation of I-kappaB and kappaB-binding activity. However, NF-kappaB-directed transcription is dramatically reduced. These results demonstrate that, like I-kappaB kinase beta and the RelA subunit of NF-kappaB, T2K is critical in protecting embryonic liver from apoptosis. However, T2K has a unique role in the activation of NF-kappaB-directed transcription, apparently independent of I-kappaB degradation and NF-kappaB DNA binding.
- Published
- 2000
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42. Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development.
- Author
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Yeh WC, Itie A, Elia AJ, Ng M, Shu HB, Wakeham A, Mirtsos C, Suzuki N, Bonnard M, Goeddel DV, and Mak TW
- Subjects
- Animals, Apoptosis genetics, CASP8 and FADD-Like Apoptosis Regulating Protein, Carrier Proteins genetics, Caspase 3, Caspase 8, Caspase 9, Caspases metabolism, Cell Line, Embryonic and Fetal Development genetics, Enzyme Activation immunology, Female, Heart embryology, JNK Mitogen-Activated Protein Kinases, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mitogen-Activated Protein Kinases metabolism, Myocardium enzymology, Myocardium immunology, Myocardium metabolism, Myocardium pathology, NF-kappa B metabolism, Stem Cells enzymology, Stem Cells immunology, Stem Cells metabolism, Stem Cells pathology, Tumor Necrosis Factor-alpha physiology, Apoptosis immunology, Carrier Proteins physiology, Embryonic and Fetal Development immunology, Intracellular Signaling Peptides and Proteins, Receptors, Tumor Necrosis Factor physiology
- Abstract
Casper (c-FLIP) associates with FADD and caspase-8 in signaling complexes downstream of death receptors like Fas. We generated Casper-deficient mice and cells and noted a duality in the physiological functions of this molecule. casper-/- embryos do not survive past day 10.5 of embryogenesis and exhibit impaired heart development. This phenotype is reminiscent of that reported for FADD-/- and caspase-8-/- embryos. However, unlike FADD-/- and caspase-8-/- cells, casper-/- embryonic fibroblasts are highly sensitive to FasL- or TNF-induced apoptosis and show rapid induction of caspase activities. NF-kappaB and JNK/SAPK activation is intact in TNF-stimulated casper-/- cells. These results suggest that Casper has two distinct roles: to cooperate with FADD and caspase-8 during embryonic development and to mediate cytoprotection against death factor-induced apoptosis.
- Published
- 2000
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43. Severe liver degeneration and lack of NF-kappaB activation in NEMO/IKKgamma-deficient mice.
- Author
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Rudolph D, Yeh WC, Wakeham A, Rudolph B, Nallainathan D, Potter J, Elia AJ, and Mak TW
- Subjects
- Animals, Apoptosis, Cell Survival drug effects, Cells, Cultured, Embryo, Mammalian, Fetal Death, Fibroblasts cytology, Fibroblasts metabolism, I-kappa B Kinase, Liver embryology, Mice, Mice, Knockout, Protein Serine-Threonine Kinases deficiency, Protein Serine-Threonine Kinases genetics, Tumor Necrosis Factor-alpha toxicity, Liver pathology, NF-kappa B metabolism, Protein Serine-Threonine Kinases metabolism
- Abstract
Phosphorylation of IkappaB, an inhibitor of NF-kappaB, is an important step in the activation of the transcription factor NF-kappaB. Phosphorylation is mediated by the IkappaB kinase (IKK) complex, known to contain two catalytic subunits: IKKalpha and IKKbeta. A novel, noncatalytic component of this kinase complex called NEMO (NF-kappaB essential modulator)/IKKgamma was identified recently. We have generated NEMO/IKKgamma-deficient mice by gene targeting. Mutant embryos die at E12.5-E13.0 from severe liver damage due to apoptosis. NEMO/IKKgamma-deficient primary murine embryonic fibroblasts (MEFs) lack detectable NF-kappaB DNA-binding activity in response to TNFalpha, IL-1, LPS, and Poly(IC) and do not show stimulus-dependent IkappaB kinase activity, which correlates with a lack of phosphorylation and degradation of IkappaBalpha. Consistent with these data, mutant MEFs show increased sensitivity to TNFalpha-induced apoptosis. Our data provide in vivo evidence that NEMO/IKKgamma is the first essential, noncatalytic component of the IKK complex.
- Published
- 2000
44. Retinal stem cells in the adult mammalian eye.
- Author
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Tropepe V, Coles BL, Chiasson BJ, Horsford DJ, Elia AJ, McInnes RR, and van der Kooy D
- Subjects
- Animals, Cell Count, Cell Differentiation, Cell Division, Cell Lineage, Cell Size, Cell Survival, Cells, Cultured, Clone Cells, Colony-Forming Units Assay, Fibroblast Growth Factor 2 pharmacology, Homeodomain Proteins biosynthesis, Intermediate Filament Proteins biosynthesis, Mice, Nestin, Neuroglia cytology, Neuroglia metabolism, Neurons cytology, Neurons metabolism, Pigment Epithelium of Eye cytology, Pigment Epithelium of Eye embryology, Retina embryology, Retina metabolism, Retinal Rod Photoreceptor Cells cytology, Retinal Rod Photoreceptor Cells metabolism, Stem Cells metabolism, Transcription Factors biosynthesis, Nerve Tissue Proteins, Retina cytology, Stem Cells cytology
- Abstract
The mature mammalian retina is thought to lack regenerative capacity. Here, we report the identification of a stem cell in the adult mouse eye, which represents a possible substrate for retinal regeneration. Single pigmented ciliary margin cells clonally proliferate in vitro to form sphere colonies of cells that can differentiate into retinal-specific cell types, including rod photoreceptors, bipolar neurons, and Müller glia. Adult retinal stem cells are localized to the pigmented ciliary margin and not to the central and peripheral retinal pigmented epithelium, indicating that these cells may be homologous to those found in the eye germinal zone of other nonmammalian vertebrates.
- Published
- 2000
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45. In vivo evidence that caspase-3 is required for Fas-mediated apoptosis of hepatocytes.
- Author
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Woo M, Hakem A, Elia AJ, Hakem R, Duncan GS, Patterson BJ, and Mak TW
- Subjects
- Animals, Antibodies, Monoclonal administration & dosage, Caspase 3, Caspases genetics, Cytochrome c Group metabolism, In Situ Nick-End Labeling, Injections, Intraperitoneal, Liver metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Protein Processing, Post-Translational immunology, Proto-Oncogene Proteins c-bcl-2 antagonists & inhibitors, Proto-Oncogene Proteins c-bcl-2 biosynthesis, Staining and Labeling, Survival Analysis, fas Receptor immunology, Apoptosis immunology, Caspases physiology, Liver enzymology, Liver immunology, fas Receptor physiology
- Abstract
Caspase-3 is essential for Fas-mediated apoptosis in vitro. We investigated the role of caspase-3 in Fas-mediated cell death in vivo by injecting caspase-3-deficient mice with agonistic anti-Fas Ab. Wild-type controls died rapidly of fulminant hepatitis, whereas the survival of caspase-3-/- mice was increased due to a delay in hepatocyte cell death. Bcl-2 expression in the liver was dramatically decreased in wild-type mice following anti-Fas injection, but was unchanged in caspase-3-/- mice. Hepatocytes from anti-Fas-injected wild-type, but not caspase-3-/-, mice released cytochrome c into the cytoplasm. Western blotting confirmed the lack of caspase-3-mediated cleavage of Bcl-2. Presumably the presence of intact Bcl-2 in caspase-3-/- hepatocytes prevents the release of cytochrome c from the mitochondria, a required step for the mitochondrial death pathway. We also show by Western blot that Bcl-xL, caspase-9, caspase-8, and Bid are processed by caspase-3 in injected wild-type mice but that this processing does not occur in caspase-3-/- mice. This study thus provides novel in vivo evidence that caspase-3, conventionally known for its downstream effector function in apoptosis, also modifies Bcl-2 and other upstream proteins involved in the regulation of Fas-mediated apoptosis.
- Published
- 1999
46. Interleukin 13 is secreted by and stimulates the growth of Hodgkin and Reed-Sternberg cells.
- Author
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Kapp U, Yeh WC, Patterson B, Elia AJ, Kägi D, Ho A, Hessel A, Tipsword M, Williams A, Mirtsos C, Itie A, Moyle M, and Mak TW
- Subjects
- Cell Division drug effects, Gene Expression Regulation, Neoplastic, Genes, Neoplasm, Herpesvirus 4, Human genetics, Hodgkin Disease genetics, Hodgkin Disease pathology, Humans, Immunohistochemistry, In Situ Hybridization, Interleukin-13 genetics, Interleukin-13 pharmacology, Interleukin-15 metabolism, Lymph Nodes pathology, Lymphoma, Non-Hodgkin genetics, RNA, Messenger analysis, Reed-Sternberg Cells pathology, Tumor Cells, Cultured, Hodgkin Disease immunology, Interleukin-13 metabolism, Reed-Sternberg Cells immunology
- Abstract
Gene expression patterns can provide vital clues to the pathogenesis of neoplastic diseases. We investigated the expression of 950 genes in Hodgkin's disease (HD) by analyzing differential mRNA expression using microarrays. In two independent microarray experiments, the HD-derived cell lines L428 and KMH2 were compared with an Epstein-Barr virus (EBV)-immortalized lymphoblastoid B cell line, LCL-GK. Interleukin (IL)-13 and IL-5 were found to be highly expressed in the HD-derived cell lines. Examination of IL-13 and IL-5 expression by Northern blot analysis and enzyme-linked immunosorbent assay confirmed these results and revealed the expression of IL-13 in a third HD-derived cell line, HDLM2. Control LCL and EBV-negative non-Hodgkin lymphoma-derived cell lines did not express IL-13. In situ hybridization of lymph node tissue from HD patients showed that elevated levels of IL-13 were specifically expressed by Hodgkin/Reed-Sternberg (H/RS) tumor cells. Treatment of a HD-derived cell line with a neutralizing antibody to IL-13 resulted in a dose-dependent inhibition of H/RS cell proliferation. These data suggest that H/RS cells produce IL-13 and that IL-13 plays an important role in the stimulation of H/RS cell growth, possibly by an autocrine mechanism. Modulation of the IL-13 signaling pathway may be a logical objective for future therapeutic strategies.
- Published
- 1999
- Full Text
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47. Interaction between Notch signalling and Lunatic fringe during somite boundary formation in the mouse.
- Author
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Barrantes IB, Elia AJ, Wünsch K, Hrabe de Angelis MH, Mak TW, Rossant J, Conlon RA, Gossler A, and de la Pompa JL
- Subjects
- Animals, Body Patterning, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Embryonic and Fetal Development, Female, Immunoglobulin J Recombination Signal Sequence-Binding Protein, Intracellular Signaling Peptides and Proteins, Male, Membrane Proteins genetics, Mesoderm, Mice, Mutagenesis, Receptors, Notch, Somites, Glycosyltransferases, Membrane Proteins metabolism, Nuclear Proteins, Proteins metabolism, Signal Transduction
- Abstract
Background: The process of somitogenesis can be divided into three major events: the prepatterning of the mesoderm; the formation of boundaries between the prospective somites; and the cellular differentiation of the somites. Expression and functional studies have demonstrated the involvement of the murine Notch pathway in somitogenesis, although its precise role in this process is not yet well understood. We examined the effect of mutations in the Notch pathway elements Delta like 1 (Dll1), Notch1 and RBPJkappa on genes expressed in the presomitic mesoderm (PSM) and have defined the spatial relationships of Notch pathway gene expression in this region., Results: We have shown that expression of Notch pathway genes in the PSM overlaps in the region where the boundary between the posterior and anterior halves of two consecutive somites will form. The Dll1, Notch1 and RBPJkappa mutations disrupt the expression of Lunatic fringe (L-fng), Jagged1, Mesp1, Mesp2 and Hes5 in the PSM. Furthermore, expression of EphA4, mCer 1 and uncx4.1, markers for the anterior-posterior subdivisions of the somites, is down-regulated to different extents in Notch pathway mutants, indicating a global alteration of pattern in the PSM., Conclusions: We propose a model for the mechanism of somite border formation in which the activity of Notch in the PSM is restricted by L-fng to a boundary-forming territory in the posterior half of the prospective somite. In this region, Notch function activates a set of genes that are involved in boundary formation and anterior-posterior somite identity.
- Published
- 1999
- Full Text
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48. Expression of human FALS SOD in motorneurons of Drosophila.
- Author
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Elia AJ, Parkes TL, Kirby K, St George-Hyslop P, Boulianne GL, Phillips JP, and Hilliker AJ
- Subjects
- Animals, Animals, Genetically Modified, Drosophila growth & development, Gene Expression, Gene Targeting, Humans, Longevity genetics, Longevity physiology, Mice, Oxidative Stress, Phenotype, Amyotrophic Lateral Sclerosis enzymology, Amyotrophic Lateral Sclerosis genetics, Drosophila enzymology, Drosophila genetics, Motor Neurons enzymology, Mutation, Superoxide Dismutase genetics
- Abstract
Mutations in human CuZn superoxide dismutase (SOD) have been associated with familial amyotrophic lateral sclerosis (FALS). Although leading to many experimental advances, this finding has not yet led to a clear understanding of the biochemical mechanism by which mutations in SOD promote the degeneration of motorneurons that causes this incurable paralytic disease. To explore the biochemical mechanism of FALS SOD-mediated neuropathogenesis, we used transgenic methodology to target the expression of a human FALS SOD to motorneurons of Drosophila, an organism known for its phenotypic sensitivity to genetic manipulation of SOD. Earlier, we showed that targeted expression of human SOD in motorneurons of Drosophila causes a dramatic extension of adult lifespan (>40%) and rescues most of the phenotypes of SOD-null mutants. Using the same genetic system, we now ask if targeted expression of a mutant allele of human SOD that is associated with FALS causes paralysis and premature death, or is otherwise injurious in Drosophila as it is in humans and transgenic mice. Here we report that high-level expression of a human FALS SOD in motorneurons is not detrimental and does not promote paralysis and premature death when expressed in motorneurons of Drosophila. In sharp contrast, the expression of FALS SOD in Drosophila actually extends lifespan, augments resistance to oxidative stress and partially rescues SOD-null mutants in a manner predicted by our earlier studies on the expression of wildtype human SOD in Drosophila motorneurons.
- Published
- 1999
- Full Text
- View/download PDF
49. High cancer susceptibility and embryonic lethality associated with mutation of the PTEN tumor suppressor gene in mice.
- Author
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Suzuki A, de la Pompa JL, Stambolic V, Elia AJ, Sasaki T, del Barco Barrantes I, Ho A, Wakeham A, Itie A, Khoo W, Fukumoto M, and Mak TW
- Subjects
- Animals, Embryonic and Fetal Development genetics, Exons, Female, Fetal Death genetics, Gamma Rays, Genotype, Mice, Mice, Mutant Strains, PTEN Phosphohydrolase, Phenotype, Polymerase Chain Reaction, Pregnancy, Recombination, Genetic, Genes, Tumor Suppressor, Genetic Predisposition to Disease genetics, Lymphoma, T-Cell genetics, Neoplasms genetics, Phosphoric Monoester Hydrolases genetics, Proto-Oncogenes, Sequence Deletion, Tumor Suppressor Proteins
- Abstract
Background: Germ-line and sporadic mutations in the tumor suppressor gene PTEN (also known as MMAC or TEP1), which encodes a dual-specificity phosphatase, cause a variety of cancers such as Cowden disease, glioblastoma, endometrial carcinoma and prostatic cancer. PTEN is widely expressed, and Cowden disease consistently affects various organ systems, suggesting that the PTEN protein must have an important, although as yet poorly understood, function in cellular physiology., Results: Homozygous mutant mice lacking exons 3-5 of the PTEN gene (mPTEN3-5) had severely expanded and abnormally patterned cephalic and caudal regions at day 8.5 of gestation. Embryonic death occurred by day 9.5 and was associated with defective chorio-allantoic development. Heterozygous mPTEN3-5 mice had an increased incidence of tumors, especially T-cell lymphomas; gamma-irradiation reduced the time lapse of tumor formation. DNA analysis of these tumors revealed the deletion of the mPTEN gene due to loss of heterozygosity of the wild-type allele. Tumors associated with loss of heterozygosity in mPTEN showed elevated phosphorylation of protein kinase B (PKB, also known as Akt kinase), thus providing a functional connection between mPTEN and a murine proto-oncogene (c-Akt) involved in the development of lymphomas., Conclusions: The mPTEN gene is fundamental for embryonic development in mice, as mPTEN3-5 mutant embryos died by day 9.5 of gestation, with patterning defects in cephalic and caudal regions and defective placentation. Heterozygous mice developed lymphomas associated with loss of heterozygosity of the wild-type mPTEN allele, and tumor appearance was accelerated by gamma-irradiation. These lymphomas had high levels of activated Akt/PKB, the protein product of a murine proto-oncogene with anti-apoptotic function, associated with thymic lymphomas. This suggests that tumors associated with mPTEN loss of heterozygosity may arise as a consequence of an acquired survival advantage. We provide direct evidence of the role of mPTEN as a tumor suppressor gene in mice, and establish the mPTEN mutant mouse as an experimental model for investigating the role of PTEN in cancer progression.
- Published
- 1998
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50. Apaf1 is required for mitochondrial pathways of apoptosis and brain development.
- Author
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Yoshida H, Kong YY, Yoshida R, Elia AJ, Hakem A, Hakem R, Penninger JM, and Mak TW
- Subjects
- Animals, Apoptotic Protease-Activating Factor 1, Brain Chemistry physiology, Caspase 2, Caspase 3, Caspase 8, Caspase 9, Cells, Cultured, Cysteine Endopeptidases metabolism, Cytochrome c Group metabolism, Embryo, Mammalian abnormalities, Enzyme Precursors metabolism, Fibroblasts cytology, Fibroblasts enzymology, Gene Expression, Head abnormalities, Membrane Potentials physiology, Mice, Mice, Knockout, Phenotype, Proteins immunology, Proteins metabolism, Skin Abnormalities, Stem Cells cytology, Stem Cells metabolism, T-Lymphocytes chemistry, T-Lymphocytes cytology, T-Lymphocytes radiation effects, Thymus Gland chemistry, Thymus Gland cytology, Thymus Gland radiation effects, Ultraviolet Rays, fas Receptor physiology, Apoptosis physiology, Brain cytology, Brain embryology, Caspases, Mitochondria enzymology, Proteins genetics
- Abstract
Apoptosis is essential for the precise regulation of cellular homeostasis and development. The role in vivo of Apaf1, a mammalian homolog of C. elegans CED-4, was investigated in gene-targeted Apaf1-/- mice. Apaf1-deficient mice exhibited reduced apoptosis in the brain and striking craniofacial abnormalities with hyperproliferation of neuronal cells. Apaf1-deficient cells were resistant to a variety of apoptotic stimuli, and the processing of Caspases 2, 3, and 8 was impaired. However, both Apaf1-/- thymocytes and activated T lymphocytes were sensitive to Fas-induced killing, showing that Fas-mediated apoptosis in these cells is independent of Apaf1. These data indicate that Apaf1 plays a central role in the common events of mitochondria-dependent apoptosis in most death pathways and that this role is critical for normal development.
- Published
- 1998
- Full Text
- View/download PDF
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