Methods in Molecular Biology 1534 Mikhail A Nikiforov Editor OncogeneInduced Senescence Methods and Protocols METHODS IN MOLECULAR BIOLOGY Series Editor John M Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Oncogene-Induced Senescence Methods and Protocols Edited by Mikhail A Nikiforov Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY, USA Editor Mikhail A Nikiforov Department of Cell Stress Biology Roswell Park Cancer Institute Buffalo, NY, USA ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6668-4 ISBN 978-1-4939-6670-7 (eBook) DOI 10.1007/978-1-4939-6670-7 Library of Congress Control Number: 2016955246 © Springer Science+Business Media New York 2017 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A Preface Oncogene-induced senescence is a multistep program triggered in response to aberrant oncoprotein expression and/or activation The eventual function of this fail-safe mechanism is the suppression of the proliferation of cells at the preneoplastic stage ultimately resulting in the prevention of fully malignant progeny On the other hand, senescent cells have been shown to promote cancer initiation and progression in several mouse models Since the discovery of oncogene-induced senescence in 1997 by Serrano et al., many outstanding researchers have been working on this intriguing set of phenotypes In addition to proliferation arrest, cells undergoing oncogene-induced senescence have been initially characterized by changes in the activity of senescence-associated β-galactosidase, cell size, chromatin structure, histone modifications, DNA integrity, etc During the past two decades, new approaches for studying cellular processes underlying senescence-associated phenotypes have emerged leading to the identification of a number of genes that were implicated in the control and/or implementation of oncogene-induced senescence And yet markers of senescence that can be universally applied to all experimental systems have not been identified and might not even exist Conversely, there are virtually no markers that are specific only to the cells undergoing oncogene-induced senescence Therefore, the analysis of phenotypes associated with oncogene-induced senescence requires multiple approaches This book offers in a single volume a unique collection of the state-of-the-art experimental procedures utilized for the induction, detection, and modeling of this complex cellular program The book encompasses protocols for studying oncogene-induced senescence in human specimens and a variety of experimental models including cultured mammalian cells, laboratory mice, and Drosophila melanogaster It also offers a description of high-throughput approaches The book represents a useful asset for the wide audience of medical oncologists and researchers in the fields of oncology, molecular and cellular biology, biochemistry, and animal development The chapters are organized to provide step-by-step guides for experimental procedures including the list of required reagents, equipment, and materials Special attention is paid to the appropriate controls and troubleshooting I would like to thank all the authors whose dedicated work made this book possible, Brittany C Lipchick, Leslie M Paul-Rosner, and my colleagues at Roswell Park Cancer Institute, and the Series Editor, Dr John M Walker, for their invaluable help in editing this book Buffalo, NY, USA Mikhail A Nikiforov v Contents Preface Contributors v ix The Immortal Senescence Anna Bianchi-Smiraglia, Brittany C Lipchick, and Mikhail A Nikiforov Senescence Phenotypes Induced by Ras in Primary Cells Lena Lau and Gregory David Cellular Model of p21-Induced Senescence Michael Shtutman, Bey-Dih Chang, Gary P Schools, and Eugenia V Broude Detecting Markers of Therapy-Induced Senescence in Cancer Cells Dorothy N.Y Fan and Clemens A Schmitt Genome-Wide miRNA Screening for Genes Bypassing Oncogene-Induced Senescence Maria V Guijarro and Amancio Carnero Detection of Dysfunctional Telomeres in Oncogene-Induced Senescence Priyanka L Patel and Utz Herbig RT-qPCR Detection of Senescence-Associated Circular RNAs Amaresh C Panda, Kotb Abdelmohsen, and Myriam Gorospe Autophagy Detection During Oncogene-Induced Senescence Using Fluorescence Microscopy Masako Narita and Masashi Narita Detecting the Senescence-Associated Secretory Phenotype (SASP) by High Content Microscopy Analysis Priya Hari and Juan Carlos Acosta 10 Sudan Black B, The Specific Histochemical Stain for Lipofuscin: A Novel Method to Detect Senescent Cells Konstantinos Evangelou and Vassilis G Gorgoulis 11 Using [U-13C6]-Glucose Tracer to Study Metabolic Changes in Oncogene-Induced Senescence Fibroblasts Katerina I Leonova and David A Scott 12 Detection of the Ubiquitinome in Cells Undergoing Oncogene-Induced Senescence Hengrui Zhu, Linh Le, Hsin-Yao Tang, David W Speicher, and Rugang Zhang 13 Detection of Reactive Oxygen Species in Cells Undergoing Oncogene-Induced Senescence Rabii Ameziane-El-Hassani and Corinne Dupuy vii 17 31 41 53 69 79 89 99 111 121 127 139 viii Contents 14 Detection of Senescent Cells by Extracellular Markers Using a Flow Cytometry-Based Approach Mohammad Althubiti and Salvador Macip 15 Metabolic Changes Investigated by Proton NMR Spectroscopy in Cells Undergoing Oncogene-Induced Senescence Claudia Gey and Karsten Seeger 16 Detection of Nucleotide Disbalance in Cells Undergoing Oncogene-Induced Senescence Mikhail A Nikiforov and Donna S Shewach 17 Senescence-Like Phenotypes in Human Nevi Andrew Joselow, Darren Lynn, Tamara Terzian, and Neil F Box 18 Detection of Oncogene-Induced Senescence In Vivo Kwan-Hyuck Baek and Sandra Ryeom 19 Detection of Senescence Markers During Mammalian Embryonic Development Mekayla Storer and William M Keyes 20 Induction and Detection of Oncogene-Induced Cellular Senescence in Drosophila Mai Nakamura and Tatsushi Igaki Index 147 155 165 175 185 199 211 219 Contributors KOTB ABDELMOHSEN • Laboratory of Genetics and Genomics, National Institute on AgingIntramural Research Program, National Institutes of Health, Baltimore, MD, USA JUAN CARLOS ACOSTA • Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK MOHAMMAD ALTHUBITI • Mechanisms of Cancer and Aging Laboratory, Department of Molecular and Cell Biology, University of Leicester, Leicester, UK; Cancer Research UK Leicester Centre, Leicester, UK; Department of Biochemistry, Faculty of Medicine, Umm Al-Qura University, Mecca, Saudi Arabia RABBII AMEZIANE-EL-HASSANI • UMR 8200, CNRS, Villejuif, France; Institut Gustave Roussy, Villejuif, France; Unité de Biologie et de Recherche Médicale, Centre National de l’Energie, des Sciences et des Techniques Nucléaires, Rabat, Morocco KWAN-HYUCK BAEK • Department of Molecular and Cellular Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi, Republic of Korea ANNA BIANCHI-SMIRAGLIA • Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY, USA NEIL F BOX • Department of Dermatology, University of Colorado, Aurora, CO, USA; Charles C Gates Center for Regenerative Medicine, University of Colorado, Aurora, USA EUGENIA V BROUDE • Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC, USA AMANCIO CARNERO • Molecular Biology of Cancer Group, Oncohematology and Genetic Department, Instituto de Biomedicina de Sevilla (IBIS/HUVR/CSIC/Universidad de Sevilla), Sevilla, Spain BEY-DIH CHANG • PeptiMed, Inc., Madison, WI, USA GREGORY DAVID • Department of Biochemistry and Molecular Pharmacology, Perlmutter Cancer Institute, New York University School of Medicine, New York, NY, USA CORINNE DUPUY • UMR 8200, CNRS, Villejuif, France; Institut Gustave Roussy, Villejuif, France; University Paris-Saclay, Orsay, France KONSTANTINOS EVANGELOU • Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, Athens, Greece DOROTHY N.Y FAN • Department of Hematology, Oncology and Tumor Immunology, Campus Virchow Clinic, Charité—University Medical Center, Berlin, Germany CLAUDIA GEY • Institute of Chemistry, University of Lübeck, Lübeck, Germany VASSILIS G GORGOULIS • Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, Athens, Greece; Biomedical Research Foundation, Academy of Athens, Athens, Greece; Faculty of Biology, Medicine and Health Manchester Cancer Research Centre, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK ix x Contributors MYRIAM GOROSPE • Laboratory of Genetics, National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, MD, USA MARIA V GUIJARRO • Musculoskeletal and Oncology Lab, Department of Orthopaedics and Rehabilitation, University of Florida, Gainesville, FL, USA PRIYA HARI • Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK UTZ HERBIG • Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School-Cancer Center, Rutgers Biomedical and Health Sciences, Newark, NJ, USA TATSUSHI IGAKI • Laboratory of Genetics, Graduate School of Biostudies, Kyoto University, Kyoto, Japan; PRESTO, Japan Science and Technology Agency (JST), Saitama, Japan ANDREW JOSELOW • Charles C Gates Center for Regenerative Medicine, University of Colorado, Aurora, CO, USA; Department of Dermatology, University of Colorado, Aurora, CO, USA; School of Medicine, Tulane University, New Orleans, LA, USA WILLIAM M KEYES • Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain; Universitat Pompeu Fabra (UPF), Barcelona, Spain LENA LAU • Department of Biochemistry and Molecular Pharmacology, Perlmutter Cancer Institute, New York University School of Medicine, New York, NY, USA LINH LE • Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, PA, USA; Cell and Molecular Biology Graduate Group, Perelman School of Medicine of the University of Pennsylvania, Philadelphia, PA, USA KATERINA I LEONOVA • Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY, USA BRITTANY C LIPCHICK • Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY, USA DARREN LYNN • Charles C Gates Center for Regenerative Medicine, University of Colorado, Aurora, CO, USA; Department of Dermatology, University of Colorado, Aurora, CO, USA SALVADOR MACIP • Mechanisms of Cancer and Aging Laboratory, Department of Molecular and Cell Biology, University of Leicester, Leicester, UK; Cancer Research UK Leicester Centre, Leicester, UK MAI NAKAMURA • Laboratory of Genetics, Graduate School of Biostudies, Kyoto University, Kyoto, Japan MASAKO NARITA • Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK MASASHI NARITA • Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK MIKHAIL A NIKIFOROV • Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY, USA AMARESH C PANDA • Laboratory of Genetics and Genomics, National Institute on AgingIntramural Research Program, National Institutes of Health, Baltimore, MD, USA PRIYANKA L PATEL • Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School-Cancer Center, Rutgers Biomedical and Health Sciences, Newark, NJ, USA SANDRA RYEOM • Department of Cancer Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA CLEMENS A SCHMITT • Department of Hematology, Oncology and Tumor Immunology, Campus Virchow Clinic, Charité—University Medical Center, Berlin, Germany; Contributors xi Molekulares Krebsforschungszentrum—MKFZ, Berlin, Germany; Max-Delbrück-Center for Molecular Medicine, Berlin, Germany; Max-Delbrück-Center for Molecular Medicine, Berlin, Germany GARY P SCHOOLS • Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolin, Columbia, SC, USA DAVID A SCOTT • Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY, USA KARSTEN SEEGAR • Institute of Chemistry, University of Lübeck, Lübeck, Germany DONNA S SHEWACH • Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA MICHAEL SHTUTMAN • Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolin, Columbia, SC, USA DAVID W SPEICHER • Molecular and Cellular Oncology Program and Proteomics Core, The Wistar Institute, Philadelphia, PA, USA MEKAYLA STORER • Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain; Universitat Pompeu Fabra (UPF), Barcelona, Spain; Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, ON, Canada HSIN-YAO TANG • Molecular and Cellular Oncology Program and Proteomics Core, The Wistar Institute, Philadelphia, PA, USA TAMARA TERZIAN • Charles C Gates Center for Regenerative Medicine, University of Colorado, Aurora, CO, USA; Department of Dermatology, University of Colorado, Aurora, CO, USA RUGANG ZHANG • Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, PA, USA HENGRUI ZHU • Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, PA, USA 206 Mekayla Storer and William M Keyes 11 Permeabilize sections in PBS pH 7.4 with 0.2 % Triton-X for 10 12 Wash slides twice for in PBS pH 7.4 13 Using a PAP pen, outline the tissue sections on the glass slide and add blocking solution (10 % goat serum in PBS, pH 7.4 with 0.1 % Triton-X) to the sections for h (see Note 17) 14 Incubate slides with primary antibody diluted in % goat serum in PBS, pH 7.4 with 0.1 % Triton-X overnight at °C in a humid chamber Antibody concentration may require optimization For p21 staining in embryos, anti-p21 antibody was used at a concentration of 1:200 15 Wash slides twice for in PBS pH 7.4 16 Add biotinylated secondary antibody diluted in % goat serum in PBS, pH 7.4 for h 17 Wash slides twice for in PBS pH 7.4 18 Incubate for 30 with VectaStain ABC Reagent 19 Wash slides twice for in PBS pH 7.4 20 Develop reaction with DAB Kit (see Note 18) 21 Wash slides twice for in PBS pH 7.4 22 Counter-stain samples with haematoxylin and wash slides for in water 23 Dehydrate sections by washing slides for 30 s in the following series of solutions: 96 % ethanol, 96 % ethanol, 96 % ethanol, 100 % ethanol, 100 % ethanol, xylene, and xylene 24 Mount sections in non-water soluble mounting medium Notes Glutaraldehyde can be bought commercially as a 50 % aqueous solution It is both toxic and a strong irritant Aliquots should be made in a fume hood, with the user wearing appropriate personal protective equipment and stored at −20 °C to avoid repeat thawing and freezing of the solution The optimal pH of the PBS supplemented with MgCl2 is a critical step to obtain correct results For staining mouse tissue or chick embryos, pH 5.5 is optimal: however if staining human samples, pH 6.0 is needed This solution must be protected from light, by wrapping with aluminum foil, and can be stored up to month at RT Potassium ferricyanide and potassium hexacyanoferrate II trihydrate both have relatively low toxicity while in powdered form, with the main hazard being mild irritation to the eyes Staining for Cellular Senescence in the Embryo 207 and skin However, under very strong acidic conditions, a highly toxic cyanide gas can be omitted Therefore, appropriate storage of these chemicals away from acids is highly recommended We find that it is best to prepare this fresh each time 5-Bromo4-chloro-3-indolyl β-D-galactopyranosidase (X-gal) is also relatively expensive; therefore, depending on the number of embryos being stained it may be prudent to only make the amount of 40× X-gal required Fresh DMF is also recommended If making % formaldehyde from paraformaldehyde powder, be very careful when manipulating the powder Paraformaldehyde is considered a carcinogen and the powder is very fine and easily inhaled For L, add 800 mL of 1× PBS to a glass beaker on a stir plate in a ventilated hood and heat PBS while stirring to approximately 60 °C Carefully weigh 40 g paraformaldehyde powder using a mask and other personal protective equipment and add to the heated PBS solution Initially, the powder will not dissolve Next, slowly raise the pH by adding M NaOH drop wise until the solution clears Allow the solution to cool and filter using a vacuum filtration unit Finally, adjust the volume of the solution to L with 1× PBS and make aliquots to be stored at −20 °C until needed Chromium Potassium Sulphate is an irritant and may be harmful if absorbed through the skin This chemical should be handled in a fume hood, with the user wearing appropriate personal protective equipment Xylene is both flammable in liquid and vapor forms and is extremely toxic Single exposure can cause respiratory irritation and if swallowed may be fatal This liquid should be handled with extreme caution, in a fume cabinet and the user must wear appropriate personal protective equipment Additionally, xylene is a solvent and should only be poured into glass or appropriate plastic compatible containers Both NaOH and HCl are corrosive reagents and should only be handled wearing appropriate personal protective equipment Hydrogen peroxide is thermodynamically unstable and will decompose to form water and oxygen; therefore, not use the 30 % stock solution if older than months 10 It is imperative that all membranes are removed from the embryo as residual membranes will not allow the staining solution to properly permeate the embryo and inconsistent results may be obtained 11 It is possible to fix embryos in a combination of 0.2 % glutaraldehyde and % formaldehyde diluted in PBS that may aid in 208 Mekayla Storer and William M Keyes co-immunostaining on sections or with performing SAβ-gal staining on sections after cutting (see ref 17) We found that fixation in 0.5 % glutaraldehyde gives a very robust signal superior to the combined glutaraldehyde/formaldehyde fixation However, glutaraldehyde is a very strong fixative and makes costaining with antibodies very difficult Please note that fixation times should be optimized for different tissues or different stages of embryonic development, and longer than 16 h of fixation in 0.5 % glutaraldehyde may lead to over-fixation of the tissue and may actually quench the enzymatic activity of the X-gal reaction 12 It is possible to embed the stained embryos in paraffin and subsequently section the embryos following whole mount SAβ-gal staining (see Fig 2) This may be improved by keeping the embryos in X-gal staining solution longer, or overnight at 37 °C, before proceeding to fixation The embryos will generally have some light blue background staining (“overstaining”) However during the process of paraffin embedding this background staining will be cleared 13 Embryos should be imaged immersed in PBS and within a maximum of h following staining and with as minimal exposure to white light as possible Leaving embryos overnight in PBS will result in an increase in a nonspecific light bluish background staining 14 Following overnight fixation in % formaldehyde, embryos can be manually dehydrated by immersing the embryo twice for 15 through a series of 50, 70, 96, and 100 % ethanol Embryos are then placed in two washes of xylene for 7.5 each wash and then infiltrated by paraffin as per standard Fig Wholemount SAβ-gal staining on an embryonic day 11.5 mouse embryo identifies senescence staining in the apical ectodermal ridge of the forelimb (left) Subsequent sectioning of the stained tissue reveals the pattern of staining in the apical ectodermal ridge (right) Staining for Cellular Senescence in the Embryo 209 embedding practices We found that using automated paraffin processing procedures removed the X-gal staining that is readily dissolved to some degree in both ethanol and xylene 15 Use a hammer to crush dry ice so that it becomes powder like This will create a flat surface in which to place your Cryomold ensuring that the sample remains positioned well as the O.C.T freezes 16 SAβ-gal staining may become visible after a few hours; therefore, include a control section and check this section for staining after h and every hour thereafter for absence of background staining Incubating the tissues for too long in SAβ-gal may result in nonspecific background staining 17 Senescent cells tend to have high background and therefore make immunostaining difficult The success of immunostaining with classical markers of senescence is dependent on the quality of the primary and secondary antibody Alternative optimization may include blocking in % BSA with 0.1 % Triton-X diluted in PBS 18 DAB is a hazardous chemical and harmful if swallowed, inhaled, or placed in contact with the skin When finished using the DAB solution, place in 10 % bleach to neutralize and dispose of in an appropriate manner Acknowledgments This work was funded by Grants SAF2010-18829 and SAF201349082-P to W.M.K from the Spanish Ministry for Economy and Competitiveness, the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) from the Generalitat de Catalunya, and CRG core funding References Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid cell strains Exp Cell Res 25:585–621 Serrano M et al (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a Cell 88(5):593–602 Kuilman T et al (2010) The essence of senescence Genes Dev 24(22):2463–2479 Schmitt CA et al (2002) A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy Cell 109(3):335–346 Narita M et al (2003) Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence Cell 113(6):703–716 Zhang R et al (2005) Formation of MacroH2Acontaining senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA Dev Cell 8(1):19–30 Acosta JC et al (2008) Chemokine signaling via the CXCR2 receptor reinforces senescence Cell 133(6):1006–1018 Coppe JP et al (2010) The 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Senescence in Drosophila Mai Nakamura and Tatsushi Igaki Abstract Cellular senescence is induced by various cellular stresses, including activation of the Ras oncogene In Drosophila imaginal epithelia, clones of cells expressing oncogenic Ras (RasV12) show several markers of cellular senescence, such as elevation of SA-β-gal activity, upregulation of the Cdk inhibitor Dacapo (Dap), and heterochromatinization However, these cells not undergo cell cycle arrest or exhibit a DNA damage response (DDR), cellular hypertrophy, or a senescence-associated secretory phenotype (SASP), other essential markers of cellular senescence However, we found that inducing mitochondrial dysfunction within RasV12-expressing cells caused all above-mentioned aspects of cellular senescence This provided the first evidence that cellular senescence occurs in invertebrates and is intriguing because mitochondrial dysfunction is frequently observed in human cancers Here, we describe the procedures for the induction and detection of cellular senescence in Drosophila epithelia Key words Cellular senescence, Drosophila, SASP, Ras, Mitochondrial dysfunction Introduction Cellular senescence is an irreversible cell cycle arrest that can be induced by various oncogenic stresses such as activation of the oncogene Ras Therefore, cellular senescence has been considered to act as a tumor suppression mechanism In contrast to this view, recent studies have indicated that cellular senescence can act as a tumor promoter, as senescent cells highly express various oncogenic secreted proteins such as growth factors and inflammatory cytokines, a phenomenon called the senescence-associated secretory phenotype (SASP) or the senescence-messaging secretome (SMS) [1–7] We have found in Drosophila imaginal epithelia that mutations in mitochondrial respiratory complexes, alterations common to human cancers, combined with Ras activation (RasV12/mito−/−) triggers secretion of the inflammatory cytokine Unpaired (Upd, an IL-6 homologue) which induces tumor growth and progression in neighboring tissue [8–12] This nonautonomous tumor Mikhail A Nikiforov (ed.), Oncogene-Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol 1534, DOI 10.1007/978-1-4939-6670-7_20, © Springer Science+Business Media New York 2017 211 212 Mai Nakamura and Tatsushi Igaki progression driven by Upd is reminiscent of the SASP Indeed, we found that RasV12/mito−/− cells exhibit all hallmarks of cellular senescence: elevation of senescence-associated β-galactosidase (SA-β-gal) activity, upregulation of the Cdk inhibitor Dacapo (Dap, a p21/p27 homologue), cell cycle arrest, heterochromatinization, DNA damage response (DDR), cellular hypertrophy, and the SASP [13] Intriguingly, although Ras activation alone is sufficient to cause cellular senescence in mammalian cultured cells, it only causes elevation of SA-β-gal activity, Cdk inhibitor expression, and heterochromatinization in Drosophila epithelia This suggests that mitochondrial dysfunction acts as an essential factor or enhancer of the oncogene-induced cellular senescence in vivo Here, we describe detailed procedures for the induction and detection of cellular senescence in Drosophila imaginal epithelia 2.1 Materials Flies and Larvae 2.2 Detection of SA-β-gal Activity Drosophila melanogaster: Culture flies at 25 °C in vials containing a standard fly food Drosophila larvae: Collect all larvae at third instar and dissect them in 1× PBS To induce cellular senescence in the eye imaginal disc, prepare flies with the following genotypes In these flies, GFP-labeled RasV12/mito−/− clones are generated in the eye-antennal disc Pdswk10101 and CoVatend are loss-of-function mutations for the genes encoding components of the mitochondrial respiratory complexes I and IV, respectively Fluorescently labeled mitotic clones are induced in the imaginal disc by the MARCM system [14–16] Genotypes: eyFLP5, Act>y+>Gal4, UAS-GFP/UAS-RasV12; FRT82B, Tub-Gal80/FRT82B, CoVatend (see Fig 1A, E) Tub-Gal80, FRT40A, UAS-RasV12/P{w+mc=lacW} Pdswk10101, FRT40A; eyFLP6, Act>y+>Gal4, UAS-GFP/+ (see Fig 1B–D, F) The Senescence Cells Histochemical Staining Kit (Sigma-Aldrich) Filtered ultrapure water: Filter ultrapure water using 0.2 μm filter 1× Fixation Buffer: Dilute 10× Fixation buffer provided in The Senescence Cells Histochemical Staining Kit with filtered ultrapure water 1× Staining Solution: Dilute 10× Staining Solution provided in The Senescence Cells Histochemical Staining Kit with filtered ultrapure water 10× Phosphate-buffered saline (PBS): Dissolve 160 g of NaCl, g of KCl, 58 g of Na2HPO4·12H2O, and g of KH2PO4 in Fig Detection of oncogene-induced cellular senescence in Drosophila imaginal epithelia (A–D) Eye-antennal disc bearing GFP-labeled RasV12/Pdsw−/− clones was subjected to the SA-β-gal assay, or stained with anti-Dap antibody (B), anti-Histone-H3-trimethyl-K9 antibody (C), or Phalloidin (D) (E) Eye-antennal disc bearing GFPlabeled RasV12/CoVa−/− clones was stained with anti-y-H2Av antibody (F) Eye-antennal disc bearing GFPlabeled RasV12/Pdsw−/− clones was stained with anti-Upd antibody 214 Mai Nakamura and Tatsushi Igaki L of ultrapure water and autoclave for 15 at 121 °C To make 1× PBS, dilute 10× PBS with filtered ultrapure water Warm X-gal Solution provided in The Senescence Cells Histochemical Staining Kit at 37 °C for h before performing the assay 2.3 Detection of Dap, TriMe-H3K9, γ-H2Av, and Upd 1× PBS: Dilute 10× PBS with ultrapure water % Paraformaldehyde (PFA): Dissolve g of paraformaldehyde, 5.8 g of Na2HPO4·12H2O, and 0.592 g of NaH2PO4·2H2O in 200 mL of ultrapure water and incubate for 1–2 h at 60 °C (shake every 30 min) Store at −20 °C PBT: 1× PBS containing 0.1 % Triton X-100 PBTn (blocking buffer, use 150 μL/sample): 1× PBT plus % Normal Donkey serum (Jackson ImmunoResearch) Store at °C (see Note 1) Antibody dilution buffer: PBTn or Can Get Signal Immunoreaction Enhancer Solution (TOYOBO) (see Note 2) Primary antibodies: mouse anti-Dap monoclonal antibody NP1 (Developmental Studies Hybridoma Bank), rabbit antiHistone H3 tri-methyl K9 (TriMe-H3K9) polyclonal antibody (Abcam), mouse anti-y-H2Av (phosphorylated histone H2Av) monoclonal antibody UNC93-5.2.1, (Developmental Studies Hybridoma Bank), and rabbit anti-Upd polyclonal antibody (a gift from D Harrison) (see Table 1) Secondary antibodies: Use fluorescently labeled anti-mouse/ rabbit antibodies at 1:250 dilution with PBTn or Can Get Signal Immunoreaction Enhancer Solution A (see Table 1) DAPI-containing SlowFade Gold Antifade Reagent (Molecular Probes) 2.4 Measurement of the Size of Senescent Cells Prepare the materials listed in Subheading 2.3, items 1–4 and Alexa546-conjugated Phalloidin (Molecular Probes) Table Antibodies for the detection of cellular senescence in Drosophila Antibody Animal Dilution ratio Dilute solution Staining time Source Dap Mouse 1:6 PBTn days DSHB TriMe-H3K9 Rabbit 1:50 Can Get Signal Overnight Abcam γH2Av Mouse 1:200 PBTn Overnight DSHB Upd Rabbit 1:500 PBTn Overnight D Harrison DSHB: Developmental Studies Hybridoma Bank Induction and Detection of Oncogene-Induced Cellular Senescence in Drosophila 215 Methods 3.1 Detection of SA-β-gal Activity Dissect third instar larvae in 1× PBS by inverting the head section and removing the fat body and other tissues, leaving the eye-antennal discs and brain attached to the larval body Place dissected larvae into mL of 1× PBS in the 1.5 mL microtube on ice during the dissection process Remove PBS from the microtube and add 150 μL of 1× Fixation Buffer, then incubate for 15 at room temperature Remove 1× Fixation Buffer from the microtube and wash the fixated larval tissues with mL of 1× PBS for three times 10 on a rotator at room temperature During the washing process, prepare the following Staining Mixture using the reagents provided in The Senescence Cells Histochemical Staining Kit Components of the Staining Mixture (150 μL/sample) (see Note 3) Staining Solution 10× μL Reagent B 1.125 μL Reagent C 1.125 μL X-gal Solution 2.25 μL Staining Solution 1× 60 μL Filtered ultrapure water 76.5 μL Remove 1× PBS from the microtube and add the Staining Mixture, then incubate for 90 h at 37 °C with protection from light Remove the Staining Mixture from the microtube and wash with mL of 1× PBS for three times 10 on a rotator at room temperature Mount eye-antennal discs on a glass slide with 1× PBS (as mounting media) and seal the coverslip with nail polish (store at °C if needed) Take images with a light microscope as quickly as possible (see Fig 1A) 3.2 Detection of Dap, TriMe-H3, γ-H2Av, and Upd Dissect third instar larvae in 1× PBS by inverting the head section and removing the fat body and other tissues, leaving the eye-antennal discs and brain attached to the larval body Place dissected larvae into mL of 1× PBS in the 1.5 mL microtube on ice during the dissection process Remove 1× PBS from the microtube and add 150 μL of % PFA, then incubate for on ice and for 20 at room temperature 216 Mai Nakamura and Tatsushi Igaki Remove % PFA from the microtube and wash with mL of 1× PBT for three times 20 on a rotator at room temperature Remove PBT from the microtube and add 150 μL of PBTn, then incubate for 30 on a rotator at room temperature Remove PBTn from the microtube and add 50 μL of the primary antibody solution (mouse anti-Dap (1:6), rabbit antiHistone H3 trimethyl K9 (TriMe-H3K9) (1:50), mouse anti-y-H2Av (1:200), or rabbit anti-Upd (1:500)) diluted with PBTn or Can Get Signal Immunoreaction Enhancer Solution A (see Table 1), then incubate at °C for 16 h to days with protection from light (see Table 1) Remove the primary antibody solution from the microtube and wash with mL of 1× PBT for three times 20 on a rotator at room temperature Add 50 μL of the secondary antibody solution diluted with PBTn or Can Get Signal Immunoreaction Enhancer Solution A, then incubate at room temperature for h with protection from light (see Table 1) Remove the secondary antibody solution from the microtube and wash with mL of 1× PBT for three times 20 on a rotator at room temperature Remove PBT from the microtube and add 50 μL of DAPIcontaining SlowFade Gold Antifade Reagent (as mounting media) 10 Mount eye-antennal discs on a glass slide in the mounting media and seal the coverslip with nail polish (store at °C if needed) 11 Take images with a confocal laser microscope as quickly as possible (see Fig 1B, C, E, F) 3.3 Measurement of the Size of Senescent Cells Perform the same steps 1–4 as in Subheading 3.2 Remove PBTn from the microtube and add 50 μL of Phalloidin solution (diluted with PBTn), then incubate for h at room temperature on a rotator Remove Phalloidin solution from the microtube and wash with mL of 1× PBT for three times 20 on a rotator at room temperature Remove PBT from the microtube and add 50 μL of DAPIcontaining SlowFade Gold Antifade Reagent (as mounting media) Mount eye-antennal discs on a glass slide with the mounting media and seal the coverslip with nail polish Take images with a confocal laser microscope Measure the area of each cell manually in the image of a confocal section using ImageJ (see Note 4) (see Fig 1D) Induction and Detection of Oncogene-Induced Cellular Senescence in Drosophila 217 Notes PBTn should be prepared freshly for each experiment Can Get Signal Immunoreaction Enhancer Solution is a solution containing an accelerator for antigen-antibody reactions, which improves sensitivity and specificity For cells that not cause the SASP (e.g., clones of cells expressing RasV12), SA-β-gal can be detected using the following Staining Mixture: 15 μL of 10× Staining Solution, 1.875 μL of Reagent B, 1.875 μL of Reagent C, 3.75 μL of X-gal Solution, 127.5 μL of ultrapure water (150 μL in total) Protect from light for 24 h and perform the same steps 1–7 as in Subheading 3.1 ImageJ is a free software for image processing Acknowledgment This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology-Japan (MEXT) to T.I., the Japan Society for the Promotion of Science to M.N and T.I., the Japan Science and Technology Agency to T.I., and the Takeda Science Foundation to T.I References Rodier F, Campisi J (2011) Four faces of cellular senescence J Cell Biol 192:547–556 Ohtani N, Hara E (2013) Roles and mechanisms of cellular senescence in regulation of tissue homeostasis Cancer Sci 104:525–530 Coppe JP, Desprez PY, Krtolica A, Campisi J (2010) The senescence-associated secretory phenotype: the dark side of tumor suppression Annu Rev Pathol 5:99–118 Young AR, Narita M (2009) SASP reflects senescence EMBO Rep 10:228–230 Davalos AR, Coppe JP, Campisi J, Desprez PY (2010) Senescent cells as a source of inflammatory factors for tumor progression Cancer Metastasis Rev 29:273–283 Kuilman T, Michaloglou C, Mooi WJ, Peeper DS (2010) The essence of senescence Genes Dev 24:2463–2479 Kuilman T, Peeper DS (2009) Senescencemessaging secretome: SMS-ing cellular stress Nat Rev Cancer 9:81–94 Brandon M, Baldi P, Wallace DC (2006) Mitochondrial mutations in cancer Oncogene 25:4647–4662 Carew JS, Huang P (2002) Mitochondrial defects in cancer Mol Cancer 1:9 10 Modica-Napolitano JS, Kulawiec M, Singh KK (2007) Mitochondria and human cancer Curr Mol Med 7:121–131 11 Pedersen PL (1978) Tumor mitochondria and the bioenergetics of cancer cells Prog Exp Tumor Res 22:190–274 12 Ohsawa S, Sato Y, Enomoto M, Nakamura M, Betsumiya A, Igaki T (2012) Mitochondrial defect drives non-autonomous tumor progression through Hippo signaling in Drosophila Nature 490:547–551 13 Nakamura M, Ohsawa S, Igaki T (2014) Mitochondrial defects trigger proliferation of neighbouring cells via a senescence-associated secretory phenotype in Drosophila Nat Commun 5:5264 218 Mai Nakamura and Tatsushi Igaki 14 Owusu-Ansah E, Yavari A, Mandal S, Banerjee U (2008) Distinct mitochondrial retrograde signals control the G1-S cell cycle checkpoint Nat Genet 40:356–361 15 Mandal S, Guptan P, Owusu-Ansah E, Banerjee U (2005) Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila Dev Cell 9:843–854 16 Lee T, Luo L (1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis Neuron 22:451–461 Printed on acid-free paperBiomedicine INDEX A F Adenomas 186–188, 190–194 Amplex Red 140–143 Antibodies 7, 19, 22, 25, 27, 29, 33, 36, 37, 46, 48, 49, 60, 69, 71, 75, 78, 95, 96, 100–103, 108, 133–134, 148, 149, 151, 176, 179–182, 187, 191–193, 196, 197, 202, 206, 208, 209, 213, 214, 216, 217 Apoptosis 8, 43, 175 Autophagosome 6, 90, 91 Autophagy 6, 8, 42, 89–97 Flow cytometry 147–151 Fluorescence in situ hybridization (FISH) 7, 70–72, 75, 76 B Beta-galactosidase 187, 190–191 BRAF 7, 55, 176 BrdU (5-Bromo-2-Deoxyuridine) 7, 19, 21–22, 24–26, 29, 44, 51, 59, 60, 62, 131, 186, 188, 193–194, 197, 200 C Cancer v, 2, 3, 8, 41–43, 45–51, 55–58, 70, 111, 121, 122, 175, 186–190, 211 CDK inhibitor 19, 20, 28, 32, 42, 56, 212 Cell cycle 1, 3, 7, 19, 22–23, 27, 32, 53, 56, 57, 89, 175, 177 Cell cycle arrest 7, 17, 32, 34, 41, 43, 56, 99, 147, 175, 176, 186, 199, 211, 212 Chemotherapy 42, 44, 49 Choline metabolism 155 Circular RNAs (circRNAs) 3, 79–84, 86, 87 C-MYC 2, 3, 5, 57, 59, 166, 167, 177 Cyclin E 55, 166 Cytokines 4, 6, 19, 53, 99, 211 D DNA damage 2, 4, 7, 17, 19, 53, 54, 56, 70, 75–76, 99, 127, 147, 166, 177, 178, 199 DNA damage response (DDR) 5, 7, 8, 42, 44, 54, 55, 69, 70, 72–76, 100, 165, 166, 212 Drosophila v, 7, 211–216 H H3K9me3 8, 42, 44–46, 48–51, 197, 214, 216 Heterochromatin 19, 50, 56 Heterochromatinization 212 High content microscopy 4, 99–107, 109 HPV-16 166 HRasG12V 2, 7, 70, 91, 93, 165–167, 186 Human Nevi 175–182 Hydrogen peroxide 188, 202, 205, 207 I IL-1alpha 20, 28, 29, 100, 101 IL-1beta 20, 29, 100 Immunofluorescence 19, 28, 60, 62–63, 69, 71, 72, 91, 93, 95–96, 100–103, 192, 193 Inflammasome 100 Inosine monophosphate dehydrogenase (IMPDH2) 166 Interleukin-6 (IL-6) 2, 20, 28, 29, 100–102, 105, 212 Interleukin-8 (IL-8) 20, 28, 29, 100–102, 105 Isopropyl-β-thiogalactopyranoside(IPTG) 32–37 K Ki67 19, 20, 28, 29, 42, 44–51, 177, 179, 200 L Lac-repressor 32, 34 LC3 91–93, 97 Lipofuscin 4, 111–115, 117 Liquid chromatography tandem mass spectrometry (LC-MS/MS) 128, 129, 131–134 Long noncoding RNAs (lncRNAs) 79 Lysosome 4, 6, 27, 54, 90, 93, 112 M E Embryo 200–204, 206–209 Enzyme-linked immunosorbent assays (ELISA) 4, 19, 34 Extracellular markers 147–151 Mammalian target of rapamycin (mTOR) 6, 32, 56, 90, 91, 97 Mass spectrometry (MS) 121–124, 134, 155 Melanoma 2, 5, 7, 8, 167, 176–179, 186 Mikhail A Nikiforov (ed.), Oncogene-Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol 1534, DOI 10.1007/978-1-4939-6670-7, © Springer Science+Business Media New York 2017 219 ONCOGENE-INDUCED SENESCENCE: METHODS AND PROTOCOLS 220 Index Metabolic flux analysis (MFA) 121 Metabolomics 155–162 Micro RNAs (miRNAs) 3, 53–63, 79 Mitochondrial dysfunction 2, 7, 212 mRNA .28, 56, 79, 82, 200 N Neural tube .199 NMR Spectroscopy 155–162 Noncoding RNAs (ncRNAs) 3, 56 Nucleotide pools 5, 7, 166 O Oncogenic activation 53, 99 P p16Ink4A 2, 3, 7, 8, 19, 29, 32, 42, 51, 56, 177, 179, 185, 186 p19ARF 56, 58, 185–188, 191–192 p21Waf1/Cip1/Sdi1 32 p53 2, 3, 8, 31, 44, 56, 57, 177, 187–188, 191–192, 200 Phosphoribosyl pyrophosphate synthetase (PRPS2) 166 Post-translational modification (PTM) 6, 127 Proliferative capacity 99 Q Quantitative-real-time PCR (qRT-PCR) 4, 19, 20, 22–23, 27 Quiescence 53, 166, 175 R Ras 3, 5, 7, 17, 19–27, 29, 55, 100, 102, 127–131, 152, 176, 186, 211 Rb-E2F pathway 166 Reactive Oxygen Species (ROS) 4, 7, 32, 42, 54, 57, 140–144, 165, 176 Ribonucleotide reductase (RR) 5, 166 RNA-binding proteins (RBPs) 79 S Secretome 211 Secretory proteins 89 Senescence 1–9, 17–29, 31–37, 41–51, 53–64, 69–87, 89–97, 99–109, 111–118, 121–124, 127–136, 139–144, 147–152, 155–163, 165–173, 175–183, 185–209, 211–217 Senescence Associated Secretory Phenotype (SASP) 4, 6, 8, 19, 20, 22–23, 27–29, 42, 44, 55, 89, 90, 99–107, 109, 111, 211, 212, 217 Senescence-associated heterochromatin foci (SAHF) 19, 22, 26, 29, 42, 54, 56, 161, 177, 186, 188, 191–193, 197 Sponge circRNAs 79 Stable isotope labeling with amino acids in cell culture (SILAC) 128, 129, 132 Sudan Black B (SBB) 4, 111–115, 117 T Telomere attrition 99, 111, 185 Telomere dysfunction 55, 77 Telomere dysfunction-induced DNA damage foci (TIF) 5, 70, 73, 76–77 Therapy-induced senescence (TIS) 7–9, 41–51 Thymidylate synthase (TS) 5, 166 U Ubiquitin 6, 128 Ubiquitinome 127–136 Ubiquitin-proteasome pathway 127 [U-13 C6]-glucose 121–124 [...]... combination of HRasV12 and mitochondrial dysfunction was necessary to induce oxidative stress and activate c-Jun amino (N)-terminal kinase (JNK) signaling Ras and JNK together suppressed the Hippo pathway and induced senescence [132] Another form of senescence highly reminiscent of OIS is the therapy -induced senescence (TIS) TIS is often a consequence of anticancer therapy and has been shown to be induced. .. few extrinsic factors have been implicated in the establishment/support of the senescent phenotype; these include the matricellular protein CCN1 (also known as CYR61) [20] and other ECM-related components such as integrin β1 [21] and plasminogen inhibitor-1 (PAI-1) [22] and secreted factors such as insulin-like growth factorbinding proteins (IGFBPs) [23] and interleukin-6 (IL-6) (reviewed in ref 24)... and thus induces Ras activity We use the Mikhail A Nikiforov (ed.), Oncogene- Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol 1534, DOI 10.1007/978-1-4939-6670-7_2, © Springer Science+Business Media New York 2017 17 18 Lena Lau and Gregory David Fig 1 Telomerase reverse transcriptase (TERT)-immortalized primary human lung embryonic fibroblasts (IMR90T) expressing an inducible... can alter a protein function (monoubiquitination) [107, 108] The process of ubiquitination is highly dynamic, being regulated by both ubiquitin ligases (E1, E2, and E3 enzymes), which add ubiquitin moieties to proteins, and deubiquitinating enzymes (DUBs) which instead remove the tag [109] A recent paper profiled the changes in protein ubiquitination patterns occurring during OIS and identified most... cycle and become unresponsive to the action of mitogens Furthermore, senescent cells undergo morphological and metabolic alterations Mikhail A Nikiforov (ed.), Oncogene- Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol 1534, DOI 10.1007/978-1-4939-6670-7_1, © Springer Science+Business Media New York 2017 1 2 Anna Bianchi-Smiraglia et al which lead to enlarged cell and organelles... cells in vivo and in vitro OIS has been shown to play major roles at both the cellular and organismal levels in biological processes ranging from embryonic development to barrier to cancer progression Here we will briefly outline major advances in methodologies that are being utilized for induction, identification, and characterization of molecular processes in cells undergoing oncogene- induced senescence. .. were able to visualize senescence in premalignant tumors using SA-β-Gal staining and BrdU incorporation, as well as with antibodies toward OIS effectors (including p16INK4a and p15INK4b) [128, 129] Lower organisms such as zebrafish (Danio rerio) and Drosophila have been used as well for studying OIS In zebrafish, expression of a heat shock-inducible human HRASV12 was shown to result in robust accumulation... of the key players which are induced by and in turn sustain and propagate the senescence phenotype belong to the family of the interleukins (especially the pro-inflammatory IL-6 and IL-1, as well as IL-8) [8–10, 89, 90] In addition, components of the tumor growth factor (TGF)-β and insulin-like growth factor (IGF)/IGF receptor pathways have shown to play a prominent role in the SASP [8–10, 89, 90] However,... multiubiquitin chain is confined to specific lysine in a targeted short-lived protein Science 243(4898):1576–1583 Mittal R, McMahon HT (2009) Arrestins as adaptors for ubiquitination in endocytosis and sorting EMBO Rep 10(1):41–43 Neutzner M, Neutzner A (2012) Enzymes of ubiquitination and deubiquitination Essays Biochem 52:37–50 Magnuson B, Ekim B, Fingar DC (2012) Regulation and function of ribosomal protein... sustained form of DNA damage and triggers cellular senescence Ras can be introduced into primary cells by retroviral infection Constitutively active and inducible Ras constructs are readily available Inducible Ras chimeric proteins consist of the activated oncogene fused to the ligand-binding domain of estrogen receptor (ER), whereby addition of 4-hydroxytamoxifen (4-OHT) stabilizes the fusion protein and ... (ed.), Oncogene- Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol 1534, DOI 10.1007/978-1-4939-6670-7_2, © Springer Science+Business Media New York 2017 17 18 Lena Lau and. .. such as integrin β1 [21] and plasminogen inhibitor-1 (PAI-1) [22] and secreted factors such as insulin-like growth factorbinding proteins (IGFBPs) [23] and interleukin-6 (IL-6) (reviewed in ref... Changes in Oncogene- Induced Senescence Fibroblasts Katerina I Leonova and David A Scott 12 Detection of the Ubiquitinome in Cells Undergoing Oncogene- Induced Senescence