37 results on '"Zdenek Andrysik"'
Search Results
2. FAM193A is a positive regulator of p53 activity
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Maria M. Szwarc, Anna L. Guarnieri, Molishree Joshi, Huy N. Duc, Madison C. Laird, Ahwan Pandey, Santosh Khanal, Emily Dohm, Aimee K. Bui, Kelly D. Sullivan, Matthew D. Galbraith, Zdenek Andrysik, and Joaquin M. Espinosa
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CP: Molecular biology ,CP: Cancer ,Biology (General) ,QH301-705.5 - Abstract
Summary: Inactivation of the p53 tumor suppressor, either by mutations or through hyperactivation of repressors such as MDM2 and MDM4, is a hallmark of cancer. Although many inhibitors of the p53-MDM2/4 interaction have been developed, such as Nutlin, their therapeutic value is limited by highly heterogeneous cellular responses. We report here a multi-omics investigation of the cellular response to MDM2/4 inhibitors, leading to identification of FAM193A as a widespread regulator of p53 function. CRISPR screening identified FAM193A as necessary for the response to Nutlin. FAM193A expression correlates with Nutlin sensitivity across hundreds of cell lines. Furthermore, genetic codependency data highlight FAM193A as a component of the p53 pathway across diverse tumor types. Mechanistically, FAM193A interacts with MDM4, and FAM193A depletion stabilizes MDM4 and inhibits the p53 transcriptional program. Last, FAM193A expression is associated with better prognosis in multiple malignancies. Altogether, these results identify FAM193A as a positive regulator of p53.
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- 2023
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3. Multi-omics analysis reveals contextual tumor suppressive and oncogenic gene modules within the acute hypoxic response
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Zdenek Andrysik, Heather Bender, Matthew D. Galbraith, and Joaquin M. Espinosa
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Science - Abstract
The response to hypoxia can significantly impact oncogenic processes. Here, the authors define the early transcriptional response to acute hypoxia and identify HIF1A target genes as part of this acute response, providing a resource for investigating context-dependent roles of HIF1A in the biology of cancer.
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- 2021
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4. JAK1 Inhibition Blocks Lethal Immune Hypersensitivity in a Mouse Model of Down Syndrome
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Kathryn D. Tuttle, Katherine A. Waugh, Paula Araya, Ross Minter, David J. Orlicky, Michael Ludwig, Zdenek Andrysik, Matthew A. Burchill, Beth A.J. Tamburini, Colin Sempeck, Keith Smith, Ross Granrath, Dayna Tracy, Jessica Baxter, Joaquin M. Espinosa, and Kelly D. Sullivan
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Down syndrome ,trisomy 21 ,interferon ,cytokine storm ,JAK inhibitors ,autoimmunity ,Biology (General) ,QH301-705.5 - Abstract
Summary: Individuals with Down syndrome (DS; trisomy 21) display hyperactivation of interferon (IFN) signaling and chronic inflammation, which could potentially be explained by the extra copy of four IFN receptor (IFNR) genes encoded on chromosome 21. However, the clinical effects of IFN hyperactivity in DS remain undefined. Here, we report that a commonly used mouse model of DS overexpresses IFNR genes and shows hypersensitivity to IFN ligands in diverse immune cell types. When treated repeatedly with a TLR3 agonist to induce chronic inflammation, these animals overexpress key IFN-stimulated genes, induce cytokine production, exhibit liver pathology, and undergo rapid weight loss. Importantly, the lethal immune hypersensitivity and cytokine production and the ensuing pathology are ameliorated by JAK1 inhibition. These results indicate that individuals with DS may experience harmful hyperinflammation upon IFN-inducing immune stimuli, as observed during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, pointing to JAK1 inhibition as a strategy to restore immune homeostasis in DS.
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- 2020
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5. ΔNp63α Suppresses TGFB2 Expression and RHOA Activity to Drive Cell Proliferation in Squamous Cell Carcinomas
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Christopher G. Abraham, Michael P. Ludwig, Zdenek Andrysik, Ahwan Pandey, Molishree Joshi, Matthew D. Galbraith, Kelly D. Sullivan, and Joaquin M. Espinosa
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Biology (General) ,QH301-705.5 - Abstract
Summary: The transcriptional repressor ΔNp63α is a potent oncogene widely overexpressed in squamous cell carcinomas (SCCs) of diverse tissue origins, where it promotes malignant cell proliferation and survival. We report here the results of a genome-wide CRISPR screen to identify pathways controlling ΔNp63α-dependent cell proliferation, which revealed that the small GTPase RHOA blocks cell division upon ΔNp63α knockdown. After ΔNp63α depletion, RHOA activity is increased, and cells undergo RHOA-dependent proliferation arrest along with transcriptome changes indicative of increased TGF-β signaling. Mechanistically, ΔNp63α represses transcription of TGFB2, which induces a cell cycle arrest that is partially dependent on RHOA. Ectopic TGFB2 activates RHOA and impairs SCC proliferation, and TGFB2 neutralization restores cell proliferation during ΔNp63α depletion. Genomic data from tumors demonstrate inactivation of RHOA and the TGFBR2 receptor and ΔNp63α overexpression in more than 80% of lung SCCs. These results reveal a signaling pathway controlling SCC proliferation that is potentially amenable to pharmacological intervention. : Abraham et al. employ a genome-wide CRISPR screening strategy to characterize the mechanism of action of the ΔNp63α oncogene in SCC. ΔNp63α suppresses TGFB2 expression and RHOA activity to drive SCC proliferation. TGFB2 is sufficient to impair SCC proliferation and necessary to enforce cell cycle arrest upon depletion of ΔNp63α. Keywords: p63, CRISPR screen, p53, lung cancer, head and neck carcinoma
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- 2018
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6. CDK8 Kinase Activity Promotes Glycolysis
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Matthew D. Galbraith, Zdenek Andrysik, Ahwan Pandey, Maria Hoh, Elizabeth A. Bonner, Amanda A. Hill, Kelly D. Sullivan, and Joaquín M. Espinosa
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Biology (General) ,QH301-705.5 - Abstract
Summary: Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancerous tissues. Despite its importance in cancer development, our understanding of mechanisms driving this form of metabolic reprogramming is incomplete. We report here an analysis of colorectal cancer cells engineered to carry a single point mutation in the active site of the Mediator-associated kinase CDK8, creating hypomorphic alleles sensitive to bulky ATP analogs. Transcriptome analysis revealed that CDK8 kinase activity is required for the expression of many components of the glycolytic cascade. CDK8 inhibition impairs glucose transporter expression, glucose uptake, glycolytic capacity and reserve, as well as cell proliferation and anchorage-independent growth, both in normoxia and hypoxia. Importantly, CDK8 impairment sensitizes cells to pharmacological glycolysis inhibition, a result reproduced with Senexin A, a dual inhibitor of CDK8/CDK19. Altogether, these results contribute to our understanding of CDK8 as an oncogene, and they justify investigations to target CDK8 in highly glycolytic tumors. : Galbraith et al. use a chemical genetics approach to examine the role of CDK8 kinase activity in cancer cells. CDK8 activity is required for the transcription of multiple genes encoding enzymes required for glucose metabolism. Impaired CDK8 activity reduces glucose uptake and glycolysis and sensitizes cells to the glucose analog 2-deoxy-D-glucose. Keywords: CDK8, CDK19, Mediator, glycolysis, Warburg effect, chemical genetics, HCT116, SW480, A549, H460
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- 2017
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7. Autophagy Controls the Kinetics and Extent of Mitochondrial Apoptosis by Regulating PUMA Levels
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Jacqueline Thorburn, Zdenek Andrysik, Leah Staskiewicz, Jacob Gump, Paola Maycotte, Andrew Oberst, Douglas R. Green, Joaquín M. Espinosa, and Andrew Thorburn
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Biology (General) ,QH301-705.5 - Abstract
Macroautophagy is thought to protect against apoptosis; however, underlying mechanisms are poorly understood. We examined how autophagy affects canonical death receptor-induced mitochondrial outer membrane permeabilization (MOMP) and apoptosis. MOMP occurs at variable times in a population of cells, and this is delayed by autophagy. Additionally, autophagy leads to inefficient MOMP, after which some cells die through a slower process than typical apoptosis and, surprisingly, can recover and divide afterward. These effects are associated with p62/SQSTM1-dependent selective autophagy causing PUMA levels to be kept low through an indirect mechanism whereby autophagy affects constitutive levels of PUMA mRNA. PUMA depletion is sufficient to prevent the sensitization to apoptosis that occurs when autophagy is blocked. Autophagy can therefore control apoptosis via a key regulator that makes MOMP faster and more efficient, thus ensuring rapid completion of apoptosis. This identifies a molecular mechanism whereby cell-fate decisions can be determined by autophagy.
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- 2014
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8. A Genetic Screen Identifies TCF3/E2A and TRIAP1 as Pathway-Specific Regulators of the Cellular Response to p53 Activation
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Zdenek Andrysik, Jihye Kim, Aik Choon Tan, and Joaquín M. Espinosa
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Biology (General) ,QH301-705.5 - Abstract
The p53 transcription factor participates in diverse cellular responses to stress, including cell-cycle arrest, apoptosis, senescence, and autophagy. The molecular mechanisms defining the ultimate outcome of p53 activation remain poorly characterized. We performed a genome-wide genetic screen in human cells to identify pathway-specific coregulators of the p53 target gene CDKN1A (p21), an inhibitor of cell-cycle progression, versus BBC3 (PUMA), a key mediator of apoptosis. Our screen identified numerous factors whose depletion creates an imbalance in the p21:PUMA ratio upon p53 activation. The transcription factor TCF3, also known as E2A, drives p21 expression while repressing PUMA across cancer cell types of multiple origins. Accordingly, TCF3/E2A depletion impairs the cell-cycle-arrest response and promotes apoptosis upon p53 activation by chemotherapeutic agents. In contrast, TRIAP1 is a specific repressor of p21 whose depletion slows down cell-cycle progression. Our results reveal strategies for driving cells toward specific p53-dependent responses.
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- 2013
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9. Global analysis of p53-regulated transcription identifies its direct targets and unexpected regulatory mechanisms
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Mary Ann Allen, Zdenek Andrysik, Veronica L Dengler, Hestia S Mellert, Anna Guarnieri, Justin A Freeman, Kelly D Sullivan, Matthew D Galbraith, Xin Luo, W Lee Kraus, Robin D Dowell, and Joaquin M Espinosa
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tumor supressor ,genomic ,p21 ,PUMA ,PIG3 ,eRNA ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The p53 transcription factor is a potent suppressor of tumor growth. We report here an analysis of its direct transcriptional program using Global Run-On sequencing (GRO-seq). Shortly after MDM2 inhibition by Nutlin-3, low levels of p53 rapidly activate ∼200 genes, most of them not previously established as direct targets. This immediate response involves all canonical p53 effector pathways, including apoptosis. Comparative global analysis of RNA synthesis vs steady state levels revealed that microarray profiling fails to identify low abundance transcripts directly activated by p53. Interestingly, p53 represses a subset of its activation targets before MDM2 inhibition. GRO-seq uncovered a plethora of gene-specific regulatory features affecting key survival and apoptotic genes within the p53 network. p53 regulates hundreds of enhancer-derived RNAs. Strikingly, direct p53 targets harbor pre-activated enhancers highly transcribed in p53 null cells. Altogether, these results enable the study of many uncharacterized p53 target genes and unexpected regulatory mechanisms.
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- 2014
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10. Cell differentiation modifies the p53 transcriptional program through a combination of gene silencing and constitutive transactivation
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Roubina Tatavosian, Micah G. Donovan, Matthew D. Galbraith, Huy N. Duc, Maria M. Szwarc, Molishree U. Joshi, Amy Frieman, Ganna Bilousova, Yingqiong Cao, Keith P. Smith, Kunhua Song, Angela L. Rachubinski, Zdenek Andrysik, and Joaquin M. Espinosa
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Cell Biology ,Molecular Biology - Abstract
The p53 transcription factor is a master regulator of cellular responses to stress that is commonly inactivated in diverse cancer types. Despite decades of research, the mechanisms by which p53 impedes tumorigenesis across vastly different cellular contexts requires further investigation. The bulk of research has been completed using in vitro studies of cancer cell lines or in vivo studies in mouse models, but much less is known about p53 action in diverse non-transformed human tissues. Here, we investigated how different cellular states modify the p53 transcriptional program in human cells through a combination of computational analyses of publicly available large-scale datasets and in vitro studies using an isogenic system consisting of induced pluripotent stem cells (iPSCs) and two derived lineages. Analysis of publicly available mRNA expression and genetic dependency data demonstrated wide variation in terms of expression and function of a core p53 transcriptional program across various tissues and lineages. To monitor the impact of cell differentiation on the p53 transcriptome within an isogenic cell culture system, we activated p53 by pharmacological inhibition of its negative regulator MDM2. Using cell phenotyping assays and genome wide transcriptome analyses, we demonstrated that cell differentiation confines and modifies the p53 transcriptional network in a lineage-specific fashion. Although hundreds of p53 target genes are transactivated in iPSCs, only a small fraction is transactivated in each of the differentiated lineages. Mechanistic studies using small molecule inhibitors and genetic knockdowns revealed the presence of two major regulatory mechanisms contributing to this massive heterogeneity across cellular states: gene silencing by epigenetic regulatory complexes and constitutive transactivation by lineage-specific transcription factors. Altogether, these results illuminate the impact of cell differentiation on the p53 program, thus advancing our understanding of how this tumor suppressor functions in different contexts.
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- 2023
11. Interferon receptor gene dosage determines diverse hallmarks of Down syndrome
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Katherine A. Waugh, Ross Minter, Jessica Baxter, Congwu Chi, Kathryn D. Tuttle, Neetha P. Eduthan, Matthew D. Galbraith, Kohl T. Kinning, Zdenek Andrysik, Paula Araya, Hannah Dougherty, Lauren N. Dunn, Michael Ludwig, Kyndal A. Schade, Dayna Tracy, Keith P. Smith, Ross E. Granrath, Nicolas Busquet, Santosh Khanal, Ryan D. Anderson, Liza L. Cox, Belinda Enriquez Estrada, Angela L. Rachubinski, Hannah R. Lyford, Eleanor C. Britton, David J. Orlicky, Jennifer L. Matsuda, Kunhua Song, Timothy C. Cox, Kelly D. Sullivan, and Joaquin M. Espinosa
- Abstract
Trisomy 21 causes Down syndrome, a condition characterized by cognitive impairments, immune dysregulation, and atypical morphogenesis. Using whole blood transcriptome analysis, we demonstrate that specific overexpression of four interferon receptors encoded on chromosome 21 associates with chronic interferon hyperactivity and systemic inflammation in Down syndrome. To define the contribution of interferon receptor overexpression to Down syndrome phenotypes, we used genome editing to correct interferon receptor gene dosage in mice carrying triplication of a large genomic region orthologous to human chromosome 21. Normalization of interferon receptor copy number attenuated lethal antiviral responses, prevented heart malformations, decreased developmental delays, improved cognition and normalized craniofacial anomalies. Therefore, interferon receptor gene dosage determines major hallmarks of Down syndrome, indicating that trisomy 21 elicits an interferonopathy amenable to therapeutic intervention.One-Sentence SummaryCorrection of interferon receptor gene dosage rescues multiple key phenotypes in a mouse model of trisomy 21.
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- 2022
12. Global Analyses to Identify Direct Transcriptional Targets of p53
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Matthew D, Galbraith, Zdenek, Andrysik, Kelly D, Sullivan, and Joaquín M, Espinosa
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Transcriptional Activation ,Animals ,Chromatin Immunoprecipitation Sequencing ,Humans ,Genomics ,RNA-Seq ,Tumor Suppressor Protein p53 ,Transcriptome ,Cell Line - Abstract
The transcription factor p53 controls a gene expression program with pleiotropic effects on cell biology including cell cycle arrest and apoptosis. Identifying direct p53 target genes within this network and determining how they influence cell fate decisions downstream of p53 activation is a prerequisite for designing therapeutic approaches that target p53 to effectively kill cancer cells. Here we describe a comprehensive multi-omics approach for identifying genes that are direct transcriptional targets of p53. We provide detailed procedures for measuring global RNA polymerase activity, defining p53 binding sites across the genome, and quantifying changes in steady-state mRNA in response to p53 activation.
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- 2021
13. Global Analyses to Identify Direct Transcriptional Targets of p53
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Matthew D. Galbraith, Kelly D. Sullivan, Zdenek Andrysik, and Joaquín M. Espinosa
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0303 health sciences ,Computational biology ,Biology ,Cell fate determination ,Transcriptome ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,Transcription (biology) ,030220 oncology & carcinogenesis ,RNA polymerase ,Gene expression ,Transcription factor ,Gene ,030304 developmental biology ,P53 binding - Abstract
The transcription factor p53 controls a gene expression program with pleiotropic effects on cell biology including cell cycle arrest and apoptosis. Identifying direct p53 target genes within this network and determining how they influence cell fate decisions downstream of p53 activation is a prerequisite for designing therapeutic approaches that target p53 to effectively kill cancer cells. Here we describe a comprehensive multi-omics approach for identifying genes that are direct transcriptional targets of p53. We provide detailed procedures for measuring global RNA polymerase activity, defining p53 binding sites across the genome, and quantifying changes in steady-state mRNA in response to p53 activation.
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- 2021
14. JAK1 Inhibition Blocks Lethal Immune Hypersensitivity in a Mouse Model of Down Syndrome
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Paula Araya, Keith P Smith, Kelly D. Sullivan, Zdenek Andrysik, Beth A. Jirón Tamburini, Dayna Tracy, Katherine A. Waugh, Kathryn D. Tuttle, Ross E Granrath, Joaquín M. Espinosa, Colin Sempeck, Michael Ludwig, Ross Minter, Matthew A. Burchill, Jessica Baxter, and David J. Orlicky
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0301 basic medicine ,Male ,medicine.medical_treatment ,Inflammation ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,Article ,Autoimmunity ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Immune system ,Interferon ,medicine ,Hypersensitivity ,Animals ,Receptor ,lcsh:QH301-705.5 ,Protein Kinase Inhibitors ,Sulfonamides ,JAK inhibitors ,business.industry ,autoimmunity ,Toll-Like Receptors ,Interferon-alpha ,interferon ,Janus Kinase 1 ,Janus Kinase 2 ,medicine.disease ,Immunity, Innate ,trisomy 21 ,Mice, Inbred C57BL ,030104 developmental biology ,Cytokine ,lcsh:Biology (General) ,Liver ,Purines ,cytokine storm ,Immunology ,TLR3 ,Azetidines ,Pyrazoles ,Female ,medicine.symptom ,Down Syndrome ,Cytokine storm ,business ,030217 neurology & neurosurgery ,medicine.drug - Abstract
Summary: Individuals with Down syndrome (DS; trisomy 21) display hyperactivation of interferon (IFN) signaling and chronic inflammation, which could potentially be explained by the extra copy of four IFN receptor (IFNR) genes encoded on chromosome 21. However, the clinical effects of IFN hyperactivity in DS remain undefined. Here, we report that a commonly used mouse model of DS overexpresses IFNR genes and shows hypersensitivity to IFN ligands in diverse immune cell types. When treated repeatedly with a TLR3 agonist to induce chronic inflammation, these animals overexpress key IFN-stimulated genes, induce cytokine production, exhibit liver pathology, and undergo rapid weight loss. Importantly, the lethal immune hypersensitivity and cytokine production and the ensuing pathology are ameliorated by JAK1 inhibition. These results indicate that individuals with DS may experience harmful hyperinflammation upon IFN-inducing immune stimuli, as observed during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, pointing to JAK1 inhibition as a strategy to restore immune homeostasis in DS.
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- 2020
15. JAK1 inhibition blocks lethal sterile immune responses: implications for COVID-19 therapy
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Katherine A. Waugh, Paula Araya, Michael Ludwig, Joaquín M. Espinosa, David J. Orlicky, Kelly D. Sullivan, Kathryn D. Tuttle, Zdenek Andrysik, Ross Minter, Keith P Smith, Beth A. Jirón Tamburini, Colin Sempeck, and Matthew A. Burchill
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0303 health sciences ,Down syndrome ,education.field_of_study ,Innate immune system ,business.industry ,animal diseases ,medicine.medical_treatment ,Population ,Stimulation ,medicine.disease ,3. Good health ,03 medical and health sciences ,0302 clinical medicine ,Cytokine ,Immune system ,Immunology ,medicine ,Chromosome 21 ,education ,business ,Cytokine storm ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Cytokine storms are drivers of pathology and mortality in myriad viral infections affecting the human population. In SARS-CoV-2-infected patients, the strength of the cytokine storm has been associated with increased risk of acute respiratory distress syndrome, myocardial damage, and death. However, the therapeutic value of attenuating the cytokine storm in COVID-19 remains to be defined. Here, we report results obtained using a novel mouse model of lethal sterile anti-viral immune responses. Using a mouse model of Down syndrome (DS) with a segmental duplication of a genomic region encoding four of the six interferon receptor genes (Ifnrs), we demonstrate that these animals overexpress Ifnrs and are hypersensitive to IFN stimulation. When challenged with viral mimetics that activate Toll-like receptor signaling and IFN anti-viral responses, these animals overproduce key cytokines, show exacerbated liver pathology, rapidly lose weight, and die. Importantly, the lethal immune hypersensitivity, accompanying cytokine storm, and liver hyperinflammation are blocked by treatment with a JAK1-specific inhibitor. Therefore, these results point to JAK1 inhibition as a potential strategy for attenuating the cytokine storm and consequent organ failure during overdrive immune responses. Additionally, these results indicate that people with DS, who carry an extra copy of the IFNR gene cluster encoded on chromosome 21, should be considered at high risk during the COVID-19 pandemic.One Sentence SummaryInhibition of the JAK1 kinase prevents pathology and mortality caused by a rampant innate immune response in mice.
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- 2020
16. Nutlin-Induced Apoptosis Is Specified by a Translation Program Regulated by PCBP2 and DHX30
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Kelly D. Sullivan, Zdenek Andrysik, Sara Zaccara, Annalisa Rossi, Ahwan Pandey, Alberto Inga, Matthew D. Galbraith, Dario Rizzotto, Joaquín M. Espinosa, Alessandro Quattrone, and Erik Dassi
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0301 basic medicine ,Untranslated region ,Cell cycle checkpoint ,Apoptosis ,General Biochemistry, Genetics and Molecular Biology ,Article ,Piperazines ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Transcription (biology) ,Gene expression ,Humans ,Gene Silencing ,RNA, Messenger ,Nucleotide Motifs ,3' Untranslated Regions ,Base Sequence ,Three prime untranslated region ,Imidazoles ,RNA-Binding Proteins ,Nutlin ,Cell Cycle Checkpoints ,Small molecule ,Cell biology ,Neoplasm Proteins ,030104 developmental biology ,Phenotype ,chemistry ,Gene Expression Regulation ,Polyribosomes ,Protein Biosynthesis ,030217 neurology & neurosurgery ,RNA Helicases ,Protein Binding - Abstract
Activation of p53 by the small molecule Nutlin can result in a combination of cell cycle arrest and apoptosis. The relative strength of these events is difficult to predict by classical gene expression analysis, leaving uncertainty as to the therapeutic benefits. In this study, we report a translational control mechanism shaping p53-dependent apoptosis. Using polysome profiling, we establish Nutlin-induced apoptosis to associate with the enhanced translation of mRNAs carrying multiple copies of an identified 3' UTR CG-rich motif mediating p53-dependent death (CGPD-motif). We identify PCBP2 and DHX30 as CGPD-motif interactors. We find that in cells undergoing persistent cell cycle arrest in response to Nutlin, CGPD-motif mRNAs are repressed by the PCBP2-dependent binding of DHX30 to the motif. Upon DHX30 depletion in these cells, the translation of CGPD-motif mRNAs increases, and the response to Nutlin shifts toward apoptosis. Instead, DHX30 inducible overexpression in SJSA1 cells leads to decreased translation of CGPD-motif mRNAs.
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- 2020
17. Mechanisms of transcriptional regulation by p53
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Matthew D. Galbraith, Kelly D. Sullivan, Joaquín M. Espinosa, and Zdenek Andrysik
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Transcriptional Activation ,0301 basic medicine ,biology ,Repressor ,Review ,Cell Biology ,Chromatin ,Cell biology ,Repressor Proteins ,Mice ,03 medical and health sciences ,Transactivation ,030104 developmental biology ,Protein Domains ,biology.protein ,Transcriptional regulation ,Animals ,Humans ,Mdm2 ,Tumor Suppressor Protein p53 ,Enhancer ,Molecular Biology ,Gene ,Transcription factor - Abstract
p53 is a transcription factor that suppresses tumor growth through regulation of dozens of target genes with diverse biological functions. The activity of this master transcription factor is inactivated in nearly all tumors, either by mutations in the TP53 locus or by oncogenic events that decrease the activity of the wild-type protein, such as overexpression of the p53 repressor MDM2. However, despite decades of intensive research, our collective understanding of the p53 signaling cascade remains incomplete. In this review, we focus on recent advances in our understanding of mechanisms of p53-dependent transcriptional control as they relate to five key areas: (1) the functionally distinct N-terminal transactivation domains, (2) the diverse regulatory roles of its C-terminal domain, (3) evidence that p53 is solely a direct transcriptional activator, not a direct repressor, (4) the ability of p53 to recognize many of its enhancers across diverse chromatin environments, and (5) mechanisms that modify the p53-dependent transcriptional program in a context-dependent manner.
- Published
- 2017
18. CDK8 Kinase Activity Promotes Glycolysis
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Zdenek Andrysik, Amanda A. Hill, Maria Hoh, Kelly D. Sullivan, Joaquín M. Espinosa, Elizabeth A. Bonner, Ahwan Pandey, and Matthew D. Galbraith
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0301 basic medicine ,Kinase ,Cell growth ,Glucose transporter ,Biology ,Warburg effect ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Glycolysis Inhibition ,030104 developmental biology ,lcsh:Biology (General) ,Anaerobic glycolysis ,Cancer research ,Glycolysis ,Kinase activity ,lcsh:QH301-705.5 - Abstract
Summary: Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancerous tissues. Despite its importance in cancer development, our understanding of mechanisms driving this form of metabolic reprogramming is incomplete. We report here an analysis of colorectal cancer cells engineered to carry a single point mutation in the active site of the Mediator-associated kinase CDK8, creating hypomorphic alleles sensitive to bulky ATP analogs. Transcriptome analysis revealed that CDK8 kinase activity is required for the expression of many components of the glycolytic cascade. CDK8 inhibition impairs glucose transporter expression, glucose uptake, glycolytic capacity and reserve, as well as cell proliferation and anchorage-independent growth, both in normoxia and hypoxia. Importantly, CDK8 impairment sensitizes cells to pharmacological glycolysis inhibition, a result reproduced with Senexin A, a dual inhibitor of CDK8/CDK19. Altogether, these results contribute to our understanding of CDK8 as an oncogene, and they justify investigations to target CDK8 in highly glycolytic tumors. : Galbraith et al. use a chemical genetics approach to examine the role of CDK8 kinase activity in cancer cells. CDK8 activity is required for the transcription of multiple genes encoding enzymes required for glucose metabolism. Impaired CDK8 activity reduces glucose uptake and glycolysis and sensitizes cells to the glucose analog 2-deoxy-D-glucose. Keywords: CDK8, CDK19, Mediator, glycolysis, Warburg effect, chemical genetics, HCT116, SW480, A549, H460
- Published
- 2017
19. p53-induced apoptosis is specified by a translation program regulated by PCBP2 and DHX30
- Author
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Dario Rizzotto, Annalisa Rossi, Joaquín M. Espinosa, Erik Dassi, Sara Zaccara, Zdenek Andrysik, Kelly D. Sullivan, Ahwan Pandey, Alessandro Quattrone, Matthew D. Galbraith, and Alberto Inga
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0303 health sciences ,050208 finance ,Cell cycle checkpoint ,Three prime untranslated region ,05 social sciences ,Translation (biology) ,Nutlin ,Small molecule ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,Transcription (biology) ,Apoptosis ,030220 oncology & carcinogenesis ,0502 economics and business ,Gene expression ,050207 economics ,Polysome profiling ,030304 developmental biology - Abstract
Activation of p53 by the small molecule Nutlin can result in a combination of cell cycle arrest and apoptosis. The relative strength of these events is difficult to predict by classical gene expression analysis, leaving uncertainty as to the therapeutic benefits of Nutlin. Here, we report a new translational control mechanism shaping p53-dependent apoptosis. Using polysome profiling, we establish Nutlin-induced apoptosis to be associated with the enhanced translation of mRNAs carrying multiple copies of a newly identified 3’UTR CG-rich motif mediating p53-dependent death (CGPD-motif). We identified PCBP2 and DHX30 as CGPD-motif interactors. We found that in cells undergoing persistent cell cycle arrest in response to Nutlin, CGPD-motif mRNAs are repressed by the PCBP2-dependent binding of DHX30 to the motif. Thus, upon DHX30 depletion in these cells, the translation of CGPD-motif mRNAs is increased, and the response to Nutlin shifts towards apoptosis. Instead, DHX30 inducible overexpression in SJSA1 cells, that undergo Nutlin-induced apoptosis, leads to decreased translation of CGPD-motif mRNAs. Overall, this work establishes the role of PCBP2-DHX30 in controlling the translation of CGPD-motif mRNAs and thus provide a new mechanism to modulate the induction of p53-dependent apoptosis.
- Published
- 2019
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20. Acetylation and deacetylation of Cdc25A constitutes a novel mechanism for modulating Cdc25A functions with implications for cancer
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Nicholas Redilla, Peter J. Stambrook, Zdenek Andrysik, Moying Yin, Kathryn Rice, and Enerlyn M. Lozada
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0301 basic medicine ,CDC25A ,Cell cycle checkpoint ,DNA damage ,Apoptosis ,Biology ,Histone Deacetylases ,03 medical and health sciences ,0302 clinical medicine ,Neoplasms ,HDAC11 ,Dual-specificity phosphatase ,ARD1 ,cancer ,Humans ,cdc25 Phosphatases ,N-Terminal Acetyltransferase E ,Phosphorylation ,Cells, Cultured ,N-Terminal Acetyltransferase A ,Cell Proliferation ,Cell Cycle ,Ubiquitination ,Acetylation ,Cell cycle ,3. Good health ,HEK293 Cells ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,Cancer research ,biology.protein ,Cdc25A acetylation ,Histone deacetylase ,Protein Processing, Post-Translational ,Research Paper - Abstract
// Enerlyn M. Lozada 1 , Zdenek Andrysik 1, 2 , Moying Yin 1 , Nicholas Redilla 1 , Kathryn Rice 1 , Peter J. Stambrook 1 1 Department of Molecular Genetics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA 2 Current affiliation: Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, USA Correspondence to: Enerlyn M. Lozada, e-mail: lozadaen@ucmail.uc.edu and enerlyn@gmail.com Keywords: Cdc25A acetylation, ARD1, HDAC11, DNA damage, cancer Received: December 22, 2015 Accepted: February 16, 2016 Published: March 07, 2016 ABSTRACT The dual specificity phosphatase Cdc25A is a key regulator of the cell cycle that promotes cell cycle progression by dephosphorylating and activating cyclin-dependent kinases. In response to genotoxicants, Cdc25A undergoes posttranslational modifications which contribute to its proteasome-mediated degradation and consequent cell cycle checkpoint arrest. The most thoroughly studied Cdc25A modification is phosphorylation. We now provide the first evidence that Cdc25A can be acetylated and that it directly interacts with the ARD1 acetyltransferase which acetylates Cdc25A both biochemically and in cultured cells. When acetylated, Cdc25A has an extended half-life. We have also identified the class IV histone deacetylase, HDAC11, as a Cdc25A deacetylase. We further show that DNA damage, such as exposure to methyl methanesulfonate (MMS), etoposide or arsenic, increases Cdc25A acetylation. Importantly, this acetylation modulates Cdc25A phosphatase activity and its function as a cell cycle regulator, and may reflect a cellular response to DNA damage. Since Cdc25A, ARD1, and HDAC11 are frequently dysregulated in multiple types of cancer, our findings may provide insight into a novel mechanism in carcinogenesis.
- Published
- 2016
21. Transcriptional Responses to IFN-γ Require Mediator Kinase-Dependent Pause Release and Mechanistically Distinct CDK8 and CDK19 Functions
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Terezia Vcelkova, Heather Bender, Iris Steinparzer, Giulio Superti-Furga, Matthew D. Galbraith, Vitaly Sedlyarov, Lucy Sneezum, Renata Kleinova, Dylan J. Taatjes, Robin D. Dowell, Kevin Eislmayr, Zdenek Andrysik, Jonathan D. Rubin, Joaquín M. Espinosa, Fabian Amman, Florian Wascher, Cecilia B. Levandowski, and Pavel Kovarik
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0303 health sciences ,biology ,Kinase ,RNA polymerase II ,Cell Biology ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Mediator ,Transcription (biology) ,biology.protein ,Cyclin-dependent kinase 8 ,STAT1 ,Kinase activity ,Molecular Biology ,Transcription factor ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Summary Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to “reprogram” cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.
- Published
- 2019
22. The miR-106b-25 cluster mediates breast tumor initiation through activation of NOTCH1 via direct repression of NEDD4L
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Joaquín M. Espinosa, Zdenek Andrysik, E. S. Linklater, Ahwan Pandey, Heide L. Ford, David J. Drasin, Anna L. Guarnieri, Michael U.J. Oliphant, Rebecca L. Vartuli, Taylor J. Hotz, Santosh Khanal, and Christina G. Towers
- Subjects
0301 basic medicine ,Cancer Research ,Nedd4 Ubiquitin Protein Ligases ,Population ,Notch signaling pathway ,Estrogen receptor ,Triple Negative Breast Neoplasms ,Biology ,Article ,tumor-initiating cell ,03 medical and health sciences ,Breast cancer ,breast cancer ,Downregulation and upregulation ,miR-106b-25 ,NOTCH1 ,Cell Line, Tumor ,microRNA ,Genetics ,medicine ,Humans ,RNA, Messenger ,Receptor, Notch1 ,education ,Molecular Biology ,Triple-negative breast cancer ,miRNA ,education.field_of_study ,Cancer ,medicine.disease ,3. Good health ,Up-Regulation ,Gene Expression Regulation, Neoplastic ,MicroRNAs ,030104 developmental biology ,Receptors, Estrogen ,Cancer research ,MCF-7 Cells ,Female ,Neoplasm Recurrence, Local ,Signal Transduction - Abstract
Tumor initiating cells (TIC) represent a subset of tumor cells with increased self-renewal capability. TICs display resistance to frontline cancer treatment and retain the ability to repopulate a tumor after therapy, leading to cancer relapse. NOTCH signaling has been identified as an important driver of the TIC population, yet mechanisms governing regulation of this pathway in cancer remain to be fully elucidated. Here, we identify a novel mechanism of NOTCH regulation and TIC induction in breast cancer, via the miR-106b-25 miRNA cluster. We show that the miR-106b-25 cluster upregulates NOTCH1 in multiple breast cancer cell lines, representing both estrogen receptor (ER+) and triple negative breast cancer (TNBC), through direct repression of the E3 ubiquitin ligase, NEDD4L. We further show that upregulation of NOTCH1 is necessary for TIC induction downstream of miR-106b-25 in both ER+ and TNBC breast cancer cells, and that re-expression of NEDD4L is sufficient to reverse miR106b-25-mediated NOTCH1 upregulation and TIC induction. Importantly, we demonstrate a significant positive correlation between miR-106b-25 and NOTCH1 protein, yet a significant inverse correlation between miR-106b-25 and NEDD4L mRNA in human breast cancer, suggesting a critical role for the miR106b-25/NEDD4L/NOTCH1 axis in the disease. Further, we show for the first time that NEDD4L expression alone is significantly associated with a better relapse free prognosis for breast cancer patients. These data expand our knowledge of the mechanisms underlying NOTCH activation and TIC induction in breast cancer, and may provide new avenues for the development of therapies targeting this resistant subset of tumor cells.
- Published
- 2017
23. Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity
- Author
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Sara Zaccara, Kelly D. Sullivan, Zdenek Andrysik, Anna L. Guarnieri, Joaquín M. Espinosa, Alberto Inga, Morgan MacBeth, Ahwan Pandey, and Matthew D. Galbraith
- Subjects
0301 basic medicine ,Transcription, Genetic ,endocrine system diseases ,Cell Cycle Proteins ,Gene product ,Small hairpin RNA ,03 medical and health sciences ,Transcription (biology) ,Neoplasms ,Proto-Oncogene Proteins ,Genetics ,Humans ,Enhancer ,neoplasms ,Gene ,Transcription factor ,Genetics (clinical) ,biology ,Research ,Nuclear Proteins ,Proto-Oncogene Proteins c-mdm2 ,Chromatin ,Cell biology ,Enhancer Elements, Genetic ,030104 developmental biology ,MCF-7 Cells ,biology.protein ,Mdm2 ,Tumor Suppressor Protein p53 - Abstract
The tumor suppressor TP53 is the most frequently mutated gene product in human cancer. Close to half of all solid tumors carry inactivating mutations in the TP53 gene, while in the remaining cases, TP53 activity is abrogated by other oncogenic events, such as hyperactivation of its endogenous repressors MDM2 or MDM4. Despite identification of hundreds of genes regulated by this transcription factor, it remains unclear which direct target genes and downstream pathways are essential for the tumor suppressive function of TP53. We set out to address this problem by generating multiple genomic data sets for three different cancer cell lines, allowing the identification of distinct sets of TP53-regulated genes, from early transcriptional targets through to late targets controlled at the translational level. We found that although TP53 elicits vastly divergent signaling cascades across cell lines, it directly activates a core transcriptional program of ∼100 genes with diverse biological functions, regardless of cell type or cellular response to TP53 activation. This core program is associated with high-occupancy TP53 enhancers, high levels of paused RNA polymerases, and accessible chromatin. Interestingly, two different shRNA screens failed to identify a single TP53 target gene required for the anti-proliferative effects of TP53 during pharmacological activation in vitro. Furthermore, bioinformatics analysis of thousands of cancer genomes revealed that none of these core target genes are frequently inactivated in tumors expressing wild-type TP53. These results support the hypothesis that TP53 activates a genetically robust transcriptional program with highly distributed tumor suppressive functions acting in diverse cellular contexts.
- Published
- 2017
24. PO-077 Identification of DHX30 as an inhibitor of the translation of pro-apoptotic mRNAS after P53 activation by nutlin
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Joaquín M. Espinosa, Erik Dassi, Sara Zaccara, Bartolomeo Bosco, Zdenek Andrysik, Annalisa Rossi, Matthew D. Galbraith, Alessandro Quattrone, Alberto Inga, and Dario Rizzotto
- Subjects
Cloning ,Cancer Research ,chemistry.chemical_compound ,Oncology ,chemistry ,Consensus sequence ,RNA ,Gene silencing ,Luciferase ,Nutlin ,Gene ,Transcription factor ,Cell biology - Abstract
Introduction The transcription factor p53 can be efficiently activated by the small molecule Nutlin-3 without inducing genotoxic stress. Treatment of different cell lines with this small molecule can result in different phenotypes, ranging from cell cycle arrest to apoptosis. HCT116 (colon cancer-derived cells) and SJSA1 (osteosarcoma-derived cells) were used to model the opposite behaviour respectively, by analysing the transcriptional and translational responses after Nutlin-3 treatment. Material and methods Total and polysomal-bound mRNAs were collected and sequenced after 12 hour of 10 uM Nutlin-3 treatment. A bioinformatics analysis of the polysome-enriched mRNAs using Weeder allowed the identification of a 3’UTR motif (‘CG-rich’) which is enriched in the translationally upregulated genes of SJSA1. The effect of the motif on translation was evaluated after cloning its consensus sequence in the 3’UTR of the b-globin gene, which was put downstream the luciferase reporter. The activity of the construct was evaluated after 12 or 24 hours of Nutlin-3. The same consensus was used for a pull-down experiment followed by mass spectrometry to identify proteins interacting with it. Results and discussions RNA-seq data indicate that HCT116 and SJSA1, although sharing almost completely the transcriptional program lead by p53, show almost no overlap at a translation level. SJSA1 present different pro-apoptotic translationally-upregulated genes after Nutlin-3, which have one or more instances of a CG-rich motif in the 3’UTR. The impact of the motif is to enhance the activity of the luciferase reported when cloned in two copies flanking the 3’UTR of the b-globin gene, but only in SJSA1. A pull-down experiment using an RNA bait with the consensus motif was used to identify interactors, among which DHX30 was deeply studied. DHX30 silencing in HCT116 causes: 1) enhanced the activity of the reporter construct after Nutlin; 2) polysomal association of selected mRNAs containing the motif; 3) induction of apoptosis as assessed by Annexin-V staining. In addition, silencing of DHX30 in U2OS cells decreased their survival after Nutlin-3 treatment. Conclusion We show how a p53-dependent transcriptional program can be shaped at a translational level thanks to the action of a CG-rich motif which is enriched in the 3’UTR of some pro-apoptotic mRNAs and that can be bound by DHX30. This protein acts as a translational repressor of mRNAs containing the motif. The exact mechanism and the generalisation of the model are currently being investigated.
- Published
- 2018
25. A DR4:tBID axis drives the p53 apoptotic response by promoting oligomerization of poised BAX
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Zdenek Andrysik, Joaquín M. Espinosa, Ryan E. Henry, Matthew D. Galbraith, and Ramiro París
- Subjects
0303 health sciences ,Programmed cell death ,General Immunology and Microbiology ,biology ,Cell growth ,General Neuroscience ,Cytochrome c ,Cell ,Cell fate determination ,biology.organism_classification ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,Apoptosis ,030220 oncology & carcinogenesis ,Puma ,medicine ,biology.protein ,Molecular Biology ,Caspase ,030304 developmental biology - Abstract
The cellular response to p53 activation varies greatly in a stimulus- and cell type-specific manner. Dissecting the molecular mechanisms defining these cell fate choices will assist the development of effective p53-based cancer therapies and also illuminate fundamental processes by which gene networks control cellular behaviour. Using an experimental system wherein stimulus-specific p53 responses are elicited by non-genotoxic versus genotoxic agents, we discovered a novel mechanism that determines whether cells undergo proliferation arrest or cell death. Strikingly, we observe that key mediators of cell-cycle arrest (p21, 14-3-3σ) and apoptosis (PUMA, BAX) are equally activated regardless of outcome. In fact, arresting cells display strong translocation of PUMA and BAX to the mitochondria, yet fail to release cytochrome C or activate caspases. Surprisingly, the key differential events in apoptotic cells are p53-dependent activation of the DR4 death receptor pathway, caspase 8-mediated cleavage of BID, and BID-dependent activation of poised BAX at the mitochondria. These results reveal a previously unappreciated role for DR4 and the extrinsic apoptotic pathway in cell fate choice following p53 activation.
- Published
- 2012
26. Autophagy Inhibition Mediates Apoptosis Sensitization in Cancer Therapy by Relieving FOXO3a Turnover
- Author
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Jim O'Prey, Zdenek Andrysik, Michael J. Morgan, Brent E. Fitzwalter, Michael Ludwig, Kelly D. Sullivan, Christina G. Towers, Maria Hoh, Joaquín M. Espinosa, Andrew Thorburn, and Kevin M. Ryan
- Subjects
0301 basic medicine ,Programmed cell death ,Apoptosis ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Puma ,Autophagy ,Humans ,Molecular Biology ,Transcription factor ,biology ,Forkhead Box Protein O3 ,Forkhead Transcription Factors ,Cell Biology ,biology.organism_classification ,030104 developmental biology ,Gene Expression Regulation ,chemistry ,030220 oncology & carcinogenesis ,Cancer cell ,Cancer research ,biology.protein ,Mdm2 ,Growth inhibition ,Apoptosis Regulatory Proteins ,Developmental Biology - Abstract
Macroautophagy (autophagy) is intimately linked with cell death and allows cells to evade apoptosis. This has prompted clinical trials to combine autophagy inhibitors with other drugs with the aim of increasing the likelihood of cancer cells dying. However, the molecular basis for such effects is unknown. Here, we describe a transcriptional mechanism that connects autophagy to apoptosis. The autophagy-regulating transcription factor, FOXO3a, is itself turned over by basal autophagy creating a potential feedback loop. Increased FOXO3a upon autophagy inhibition stimulates transcription of the pro-apoptotic BBC3/PUMA gene to cause apoptosis sensitization. This mechanism explains how autophagy inhibition can sensitize tumor cells to chemotherapy drugs and allows an autophagy inhibitor to change the action of an MDM2-targeted drug from growth inhibition to apoptosis, reducing tumor burden in vivo. Thus, a link between two processes mediated via a single transcription factor binding site in the genome can be leveraged to improve anti-cancer therapies.
- Published
- 2018
27. The novel mouse Polo-like kinase 5 responds to DNA damage and localizes in the nucleolus
- Author
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Peter J. Stambrook, El Mustapha Bahassi, Ya-Qin Li, Zdenek Andrysik, William Z. Bernstein, Jay A. Tischfield, Li Deng, and David L. Myer
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DNA damage ,Nucleolus ,Apoptosis ,Polo-like kinase ,Biology ,Genome Integrity, Repair and Replication ,Protein Serine-Threonine Kinases ,PLK3 ,Cell Line ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Genetics ,Animals ,Humans ,Cloning, Molecular ,Gene ,030304 developmental biology ,0303 health sciences ,DNA synthesis ,G1 Phase ,Cell cycle ,Molecular biology ,030220 oncology & carcinogenesis ,Ectopic expression ,Tumor Suppressor Protein p53 ,Sequence Alignment ,Sequence Analysis ,Cell Nucleolus ,DNA Damage - Abstract
Polo-like kinases (Plk1-4) are emerging as an important class of proteins involved in many aspects of cell cycle regulation and response to DNA damage. Here, we report the cloning of a fifth member of the polo-like kinase family named Plk5. DNA and protein sequence analyses show that Plk5 shares more similarities with Plk2 and Plk3 than with Plk1 and Plk4. Consistent with this observation, we show that mouse Plk5 is a DNA damage inducible gene. Mouse Plk5 protein localizes predominantly to the nucleolus, and deletion of a putative nucleolus localization signal (NoLS) within its N-terminal moiety disrupts its nucleolar localization. Ectopic expression of Plk5 leads to cell cycle arrest in G1, decreased DNA synthesis, and to apoptosis, a characteristic it shares with Plk3. Interestingly, in contrast to mouse Plk5 gene, the sequence of human Plk5 contains a stop codon that produces a truncated protein lacking part of the kinase domain.
- Published
- 2010
28. The aryl hydrocarbon receptor-dependent deregulation of cell cycle control induced by polycyclic aromatic hydrocarbons in rat liver epithelial cells
- Author
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Lenka Svihálková-Sindlerová, Cornelia Dietrich, Carsten Weiss, Dagmar Faust, Anne Kranz, Jan Vondráček, Zdenek Andrysik, Alois Kozubík, Miroslav Machala, and Pavel Krcmar
- Subjects
Health, Toxicology and Mutagenesis ,Cyclin A ,Gene Expression ,Apoptosis ,Cell Cycle Proteins ,Cell Line ,Benz(a)Anthracenes ,Benzo(a)pyrene ,Cytochrome P-450 CYP1A1 ,polycyclic compounds ,Genetics ,Animals ,Rat liver ‘stem-like’ cells ,RNA, Messenger ,Polycyclic Aromatic Hydrocarbons ,RNA, Small Interfering ,Molecular Biology ,Aryl hydrocarbon receptor ,Cell proliferation ,Carcinogen ,Cell Proliferation ,Fluorenes ,Base Sequence ,biology ,Chemistry ,Cell growth ,Cell Cycle ,Cyclin-Dependent Kinase 2 ,Contact inhibition ,Epithelial Cells ,Transfection ,Molecular biology ,Polycyclic aromatic hydrocarbons ,Polycyclic Hydrocarbons, Aromatic ,Rats ,Receptors, Aryl Hydrocarbon ,Biochemistry ,Multiprotein Complexes ,Mutation ,Hepatocytes ,biology.protein ,CDK inhibitor ,Mutagens - Abstract
Disruption of cell proliferation control by polycyclic aromatic hydrocarbons (PAHs) may contribute to their carcinogenicity. We investigated role of the aryl hydrocarbon receptor (AhR) in disruption of contact inhibition in rat liver epithelial WB-F344 'stem-like' cells, induced by the weakly mutagenic benz[a]anthracene (BaA), benzo[b]fluoranthene (BbF) and by the strongly mutagenic benzo[a]pyrene (BaP). There were significant differences between the effects of BaA and BbF, and those of the strongly genotoxic BaP. Both BaA and BbF increased percentage of cells entering S-phase and cell numbers, associated with an increased expression of Cyclin A and Cyclin A/cdk2 complex activity. Their effects were significantly reduced in cells expressing a dominant-negative AhR mutant (dnAhR). Roscovitine, a chemical inhibitor of cdk2, abolished the induction of cell proliferation by BbF. However, neither BaA nor BbF modulated expression of the principal cdk inhibitor involved in maintenance of contact inhibition, p27(Kip1), or pRb phosphorylation. The strongly mutagenic BaP induced apoptosis, a decrease in total cell numbers and significantly higher percentage of cells entering S-phase than either BaA or BbF. Given that BaP induced high levels of Cyclin A/cdk2 activity, downregulation of p27(Kip1) and hyperphosphorylation of pRb, the accumulation of cells in S-phase was probably due to cell proliferation, although S-phase arrest due to blocked replication forks can not be excluded. Both types of effects of BaP were significantly attenuated in dnAhR cells. Transfection of WB-F344 cells with siRNA targeted against AhR decreased induction of Cyclin A induced by BbF or BaP, further supporting the role of AhR in proliferative effects of PAHs. This suggest that activation of AhR plays a significant role both in disruption of contact inhibition by weakly mutagenic PAHs and in genotoxic effects of BaP possibly leading to enhanced cell proliferation. Thus, PAHs may increase proliferative rate and the likelihood of fixation of mutations. This work was partly supported by ECNIS, European Union 6th Framework Program, Priority 5: “Food Quality and Safety” (Contract No. 513943).
- Published
- 2007
29. Global analysis of p53-regulated transcription identifies its direct targets and unexpected regulatory mechanisms
- Author
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Hestia Mellert, Justin A. Freeman, Robin D. Dowell, Joaquín M. Espinosa, Matthew D. Galbraith, Veronica L. Dengler, Xin Luo, Mary A. Allen, Zdenek Andrysik, W. Lee Kraus, Kelly D. Sullivan, and Anna L. Guarnieri
- Subjects
Programmed cell death ,Transcription, Genetic ,Cell division ,QH301-705.5 ,Science ,Cell ,eRNA ,Apoptosis ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,genomic ,chemistry.chemical_compound ,Transcription (biology) ,tumor supressor ,PUMA ,genomics ,medicine ,Humans ,human ,Biology (General) ,Human Biology and Medicine ,Gene ,Genetics ,Binding Sites ,General Immunology and Microbiology ,p21 ,General Neuroscience ,Proto-Oncogene Proteins c-mdm2 ,General Medicine ,Suicide gene ,3. Good health ,medicine.anatomical_structure ,chemistry ,Genes and Chromosomes ,Cancer cell ,RNA ,Medicine ,PIG3 ,Tumor Suppressor Protein p53 ,DNA ,Research Article - Abstract
The p53 transcription factor is a potent suppressor of tumor growth. We report here an analysis of its direct transcriptional program using Global Run-On sequencing (GRO-seq). Shortly after MDM2 inhibition by Nutlin-3, low levels of p53 rapidly activate ∼200 genes, most of them not previously established as direct targets. This immediate response involves all canonical p53 effector pathways, including apoptosis. Comparative global analysis of RNA synthesis vs steady state levels revealed that microarray profiling fails to identify low abundance transcripts directly activated by p53. Interestingly, p53 represses a subset of its activation targets before MDM2 inhibition. GRO-seq uncovered a plethora of gene-specific regulatory features affecting key survival and apoptotic genes within the p53 network. p53 regulates hundreds of enhancer-derived RNAs. Strikingly, direct p53 targets harbor pre-activated enhancers highly transcribed in p53 null cells. Altogether, these results enable the study of many uncharacterized p53 target genes and unexpected regulatory mechanisms. DOI: http://dx.doi.org/10.7554/eLife.02200.001, eLife digest The growth, division and eventual death of the cells in the body are processes that are tightly controlled by hundreds of genes working together. If any of these genes are switched on (or off) in the wrong cell or at the wrong time, it can lead to cancer. It has been known for many years that the protein encoded by one gene in particular—called p53—is nearly always switched off in cancer cells. The p53 protein normally acts like a ‘brake’ to slow the uncontrolled division of cells, and some researchers are working to find ways to switch on this protein in cancer cells. However, this approach appears to only work in specific cases of this disease. For better results, we need to understand how p53 is normally switched on, and what other genes this protein controls once it is activated. Allen et al. have now identified the genes that are directly switched on when cancer cells are treated with a drug that artificially activates the p53 protein. Nearly 200 genes were switched on, and almost three quarters of these genes had not previously been identified as direct targets of p53. Although p53 tends to act as a brake to slow cell division, it is not clear how it distinguishes between its target genes—some of which promote cell survival, while others promote cell death. Allen et al. found that survival genes are switched on more strongly than cell death genes via a range of different mechanisms; this may explain why most cancers can survive drug treatments that reactivate p53. Also, Allen et al. revealed that some p53 target genes are primed to be switched on, even before the p53 protein is activated, by proteins (and other molecules) acting in regions of the DNA outside of the genes. By uncovering many new gene targets for the p53 protein, the findings of Allen et al. could help researchers developing new drugs or treatments for cancer. DOI: http://dx.doi.org/10.7554/eLife.02200.002
- Published
- 2014
30. Author response: Global analysis of p53-regulated transcription identifies its direct targets and unexpected regulatory mechanisms
- Author
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Matthew D. Galbraith, Xin Luo, Mary A. Allen, Joaquín M. Espinosa, Justin A. Freeman, Hestia Mellert, Veronica L. Dengler, W. Lee Kraus, Kelly D. Sullivan, Anna L. Guarnieri, Zdenek Andrysik, and Robin D. Dowell
- Subjects
Transcription (biology) ,Biology ,Cell biology - Published
- 2014
31. Autophagy controls the kinetics and extent of mitochondrial apoptosis by regulating PUMA levels
- Author
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Andrew Oberst, Jacqueline Thorburn, Jacob M. Gump, Zdenek Andrysik, Joaquín M. Espinosa, Andrew Thorburn, Leah Staskiewicz, Paola Maycotte, and Douglas R. Green
- Subjects
Programmed cell death ,Cell Membrane Permeability ,Population ,Regulator ,Apoptosis ,Mitochondrion ,Biology ,Transfection ,General Biochemistry, Genetics and Molecular Biology ,Article ,Puma ,Cell Line, Tumor ,Proto-Oncogene Proteins ,Autophagy ,Humans ,education ,lcsh:QH301-705.5 ,education.field_of_study ,biology.organism_classification ,Cell biology ,Mitochondria ,Kinetics ,lcsh:Biology (General) ,Mitochondrial Membranes ,Apoptosis Regulatory Proteins ,HeLa Cells - Abstract
SummaryMacroautophagy is thought to protect against apoptosis; however, underlying mechanisms are poorly understood. We examined how autophagy affects canonical death receptor-induced mitochondrial outer membrane permeabilization (MOMP) and apoptosis. MOMP occurs at variable times in a population of cells, and this is delayed by autophagy. Additionally, autophagy leads to inefficient MOMP, after which some cells die through a slower process than typical apoptosis and, surprisingly, can recover and divide afterward. These effects are associated with p62/SQSTM1-dependent selective autophagy causing PUMA levels to be kept low through an indirect mechanism whereby autophagy affects constitutive levels of PUMA mRNA. PUMA depletion is sufficient to prevent the sensitization to apoptosis that occurs when autophagy is blocked. Autophagy can therefore control apoptosis via a key regulator that makes MOMP faster and more efficient, thus ensuring rapid completion of apoptosis. This identifies a molecular mechanism whereby cell-fate decisions can be determined by autophagy.
- Published
- 2014
32. A genetic screen identifies TCF3/E2A and TRIAP1 as pathway-specific regulators of the cellular response to p53 activation
- Author
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Jihye Kim, Zdenek Andrysik, Joaquín M. Espinosa, and Aik Choon Tan
- Subjects
Cyclin-Dependent Kinase Inhibitor p21 ,Repressor ,Apoptosis ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Mediator ,Puma ,Cell Line, Tumor ,Proto-Oncogene Proteins ,Basic Helix-Loop-Helix Transcription Factors ,Humans ,RNA, Small Interfering ,lcsh:QH301-705.5 ,Transcription factor ,030304 developmental biology ,0303 health sciences ,biology ,Autophagy ,Intracellular Signaling Peptides and Proteins ,Cell Cycle Checkpoints ,biology.organism_classification ,HCT116 Cells ,lcsh:Biology (General) ,030220 oncology & carcinogenesis ,TCF3 ,Cancer research ,RNA Interference ,RNA Polymerase II ,Signal transduction ,Tumor Suppressor Protein p53 ,Apoptosis Regulatory Proteins ,Genetic screen ,DNA Damage ,Signal Transduction - Abstract
SummaryThe p53 transcription factor participates in diverse cellular responses to stress, including cell-cycle arrest, apoptosis, senescence, and autophagy. The molecular mechanisms defining the ultimate outcome of p53 activation remain poorly characterized. We performed a genome-wide genetic screen in human cells to identify pathway-specific coregulators of the p53 target gene CDKN1A (p21), an inhibitor of cell-cycle progression, versus BBC3 (PUMA), a key mediator of apoptosis. Our screen identified numerous factors whose depletion creates an imbalance in the p21:PUMA ratio upon p53 activation. The transcription factor TCF3, also known as E2A, drives p21 expression while repressing PUMA across cancer cell types of multiple origins. Accordingly, TCF3/E2A depletion impairs the cell-cycle-arrest response and promotes apoptosis upon p53 activation by chemotherapeutic agents. In contrast, TRIAP1 is a specific repressor of p21 whose depletion slows down cell-cycle progression. Our results reveal strategies for driving cells toward specific p53-dependent responses.
- Published
- 2013
33. A DR4:tBID axis drives the p53 apoptotic response by promoting oligomerization of poised BAX
- Author
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Ryan E, Henry, Zdenek, Andrysik, Ramiro, París, Matthew D, Galbraith, and Joaquín M, Espinosa
- Subjects
p53 ,Caspase 8 ,apoptosis ,cell fate choice ,Article ,Mitochondria ,Protein Transport ,Receptors, TNF-Related Apoptosis-Inducing Ligand ,BAX ,Proto-Oncogene Proteins ,Humans ,DR4 ,Tumor Suppressor Protein p53 ,Apoptosis Regulatory Proteins ,BH3 Interacting Domain Death Agonist Protein ,Cell Proliferation ,bcl-2-Associated X Protein - Abstract
A DR4:tBID axis drives the p53 apoptotic response by promoting oligomerization of poised BAX Depending on the context, cells either arrest or undergo apoptosis in response to p53 activation. Key mediators of both pathways are activated irrespective of the fate choice, but apoptotic cells selectively induce the DR4 death receptor pathway, caspase 8-mediated BID cleavage and activation of BAX., The cellular response to p53 activation varies greatly in a stimulus- and cell type-specific manner. Dissecting the molecular mechanisms defining these cell fate choices will assist the development of effective p53-based cancer therapies and also illuminate fundamental processes by which gene networks control cellular behaviour. Using an experimental system wherein stimulus-specific p53 responses are elicited by non-genotoxic versus genotoxic agents, we discovered a novel mechanism that determines whether cells undergo proliferation arrest or cell death. Strikingly, we observe that key mediators of cell-cycle arrest (p21, 14-3-3σ) and apoptosis (PUMA, BAX) are equally activated regardless of outcome. In fact, arresting cells display strong translocation of PUMA and BAX to the mitochondria, yet fail to release cytochrome C or activate caspases. Surprisingly, the key differential events in apoptotic cells are p53-dependent activation of the DR4 death receptor pathway, caspase 8-mediated cleavage of BID, and BID-dependent activation of poised BAX at the mitochondria. These results reveal a previously unappreciated role for DR4 and the extrinsic apoptotic pathway in cell fate choice following p53 activation.
- Published
- 2011
34. Apoptosis and inhibition of gap-junctional intercellular communication induced by LA-12, a novel hydrophobic platinum(IV) complex
- Author
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Radek Tesarik, Jaroslav Turánek, Petr Sova, Alois Kozubík, Jiri Neuzil, Pavlina Polaskova, Frantisek Zak, Zdenek Andrysik, Pavlína Turánek Knotigová, Miroslav Machala, and Lubomir Prochazka
- Subjects
MAPK/ERK pathway ,Organoplatinum Compounds ,Biophysics ,Hyperphosphorylation ,Antineoplastic Agents ,Apoptosis ,Platinum Compounds ,Biology ,Biochemistry ,Connexon ,Cell Line ,Cell Line, Tumor ,Extracellular ,medicine ,Amantadine ,Animals ,Phosphorylation ,Molecular Biology ,Cisplatin ,Dose-Response Relationship, Drug ,Kinase ,Gap Junctions ,Epithelial Cells ,Cell biology ,Rats ,Connexin 43 ,Hydrophobic and Hydrophilic Interactions ,Intracellular ,medicine.drug - Abstract
A new hydrophobic platinum(IV) complex, LA-12, a very efficient anticancer drug lacking cross-resistance with cisplatin (CDDP), is now being tested in clinical trials. Here we investigated the apoptogenic activity of LA-12 and its effect on gap-junctional intercellular communication (GJIC) in the rat liver epithelial cell line WB-F344. LA-12 induced apoptosis much more efficiently than did CDDP due to a combination of rapid penetration into the cell and attack on DNA, leading to fast activation of p53 and caspase-3. Exposure of WB-F344 cells to LA-12 led to rapid induction of the time- and dose-dependent decrease in GJIC. On the molecular level, loss of GJIC induced by LA-12 was mediated by activation of extracellular signal-regulated kinase (ERK)-1 and ERK-2, as demonstrated by the use of inhibitors of ERK activation. Inhibition of GJIC was linked to rapid hyperphosphorylation of connexin-43 and disappearance of connexon clusters from membranes, which was not observed in the case of CDDP.
- Published
- 2007
35. 447 Acute exposure to polycyclic aromatic hydrocarbons affects multiple cell signaling components in rat liver epithelial cell line
- Author
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Miroslav Machala, Brad L. Upham, Jiří Neča, Zdenek Andrysik, Katerina Pencikova, Luděk Bláha, Jan Vondráček, and Petra Kapplová
- Subjects
Cell signaling ,medicine.anatomical_structure ,Biochemistry ,Chemistry ,Rat liver ,Acute exposure ,medicine ,General Medicine ,Line (text file) ,Toxicology ,Epithelium ,Cell biology - Published
- 2003
36. 449 AHR-activating polycyclic aromatic hydrocarbons induce a release from contact inhibition or apoptosis in rat liver epithelial cell line
- Author
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Katerina Chramostova, Zdenek Andrysik, Miroslav Machala, Alois Kozubík, Jan Vondráček, Karel Souček, and B. Vojtesek
- Subjects
medicine.anatomical_structure ,Apoptosis ,Chemistry ,Rat liver ,medicine ,Contact inhibition ,Organic chemistry ,General Medicine ,Line (text file) ,Toxicology ,Epithelium ,Cell biology - Published
- 2003
37. Antimigraine Drug Avitriptan Is a Ligand and Agonist of Human Aryl Hydrocarbon Receptor that Induces CYP1A1 in Hepatic and Intestinal Cells
- Author
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Barbora Vyhlídalová, Kristýna Krasulová, Petra Pečinková, Karolína Poulíková, Radim Vrzal, Zdeněk Andrysík, Aneesh Chandran, Sridhar Mani, and Zdenek Dvorak
- Subjects
Aryl Hydrocarbon Receptor ,Antimigraine drugs ,Triptans ,repurposing ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The efforts for therapeutic targeting of the aryl hydrocarbon receptor (AhR) have emerged in recent years. We investigated the effects of available antimigraine triptan drugs, having an indole core in their structure, on AhR signaling in human hepatic and intestinal cells. Activation of AhR in reporter gene assays was observed for Avitriptan and to a lesser extent for Donitriptan, while other triptans were very weak or no activators of AhR. Using competitive binding assay and by homology docking, we identified Avitriptan as a low-affinity ligand of AhR. Avitriptan triggered nuclear translocation of AhR and increased binding of AhR in CYP1A1 promotor DNA, as revealed by immune-fluorescence microscopy and chromatin immune-precipitation assay, respectively. Strong induction of CYP1A1 mRNA was achieved by Avitriptan in wild type but not in AhR-knockout, immortalized human hepatocytes, implying that induction of CYP1A1 is AhR-dependent. Increased levels of CYP1A1 mRNA by Avitriptan were observed in human colon carcinoma cells LS180 but not in primary cultures of human hepatocytes. Collectively, we show that Avitriptan is a weak ligand and activator of human AhR, which induces the expression of CYP1A1 in a cell-type specific manner. Our data warrant the potential off-label therapeutic application of Avitriptan as an AhR-agonist drug.
- Published
- 2020
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