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Cell Senescence Methods and Protocols 1st Edition Andrew R. J. Young Cell Senescence Methods and Protocols 1st Edition Andrew R. J. Young Cell Senescence Methods and Protocols 1st Edition Andrew R. J. Young

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ME T H O D S I N MO L E C U L A R BI O L O G Y ™ Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651
Cell Senescence Methods and Protocols 1st Edition Andrew R. J. Young
Cell Senescence Methods and Protocols Edited by Lorenzo Galluzzi, IlioVitale, Oliver Kepp, and Guido Kroemer InstitutGustaveRoussy(IGR),INSERMU848, Villejuif,France
ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-62703-238-4 ISBN 978-1-62703-239-1 (eBook) DOI 10.1007/978-1-62703-239-1 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2012950333 © Springer Science+Business Media, LLC 2013 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. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. 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. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Editors Lorenzo Galluzzi Institut Gustave Roussy Université Paris Descartes Sorbonne Paris Cité Villejuif, France Oliver Kepp Institut Gustave Roussy INSERM U848 Villejuif, France Ilio Vitale Institut Gustave Roussy INSERM U848 Villejuif, France Guido Kroemer Institut Gustave Roussy INSERM U848 Villejuif, France
v v Preface In Vitro and In Vivo Cell Senescence Cell senescence, i.e., the process whereby cells permanently lose the possibility to prolifer- ate without undergoing cell death, can be observed in vitro as well as in vivo, and occurs in a plethora of distinct model organisms. In both cases, cell senescence can be physiological, constituting a safeguard mechanism against cells that have accumulated potentially danger- ous genetic alterations, or can be triggered by exogenous perturbations, such as the admin- istration of DNA-damaging agents at low doses. This book provides a detailed description of the most common techniques for the investigation of cell senescence, in model organ- isms encompassing bacteria (Escherichia coli), fungi (Saccharomyces cerevisiae and Podospora anserina), worms (Caenorhabditis elegans), flies (Drosophila melanogaster), zebrafish (Danio rerio), and mammalian cells. The techniques presented in this book not only cover the study of all the biochemical and functional manifestations of senescence at the cellular level but also include protocols for population analysis and high-throughput approaches in suit- able model organisms, as described by worldwide renowned experts of the field. Chapter Organization The book is composed of three types of chapters. Four review chapters open the book to provide a solid theoretical background on cell senescence, its morphological and biochemi- cal manifestations and its pathophysiological relevance. Twenty-three protocol chapters follow, detailing the methods to investigate the morphological and biochemical features of senescence at the cellular level, in cultured mammalian cells. Finally, seven protocol chap- ters provide techniques for the study of cell senescence in lower model organisms, including methods for population studies. Each of these 30 protocols starts with an Abstract and includes four major sections: Introduction, Materials, Methods, and Notes. The “Abstract” presents an overview of the technique(s) detailed in the chapter. The “Introduction” pro- vides a short theoretical view of the procedure and of its applications. “Materials” recapitu- late the buffers, reagents, solutions, disposables, and equipments necessary to carry out the protocol(s). “Methods” describe step-by-step how the technique(s) must be carried out. Finally, the “Notes” section, which is the hallmark of Methods in Molecular Biology series, indicates not only the sources of problems and how to identify and overcome them, but also safety information, alternative procedures, and hints for the correct interpretation of experimental results.
vi Brief Content of the Chapters Chapter 1 provides an overview on cell senescence and its dynamic links with autophagy, an important cytoprotective mechanism. Chapters 2 and 3 discuss the regulation of cell senes- cence by critical signaling molecules such as the mammalian target of rapamycin (mTOR) and p53. Chapter 4 summarizes the morphological and biochemical markers that have been associated with cell senescence. In Chapters 5–23, protocols for the investigation of senes- cence-associated alterations in cultured cells are provided, including the following: mor- phological features (Chapter 5), cell cycle blockage (Chapter 6), cell cycle-arresting proteins (Chapter 7), senescence-associated β-galactosidase (Chapters 8 and 9), senescence-associ- ated secretory phenotype and chemokyne signaling (Chapters 10 and 11), senescence- associated heterochromatin foci (Chapter 12), DNA damage (Chapter 13), telomerase activity and telomere length (Chapters 14 and 15), alterations of the nuclear envelope (Chapter 16), multiple markers of oxidative stress (Chapters 17–20), BRAF, sirtuin, and p66SHC signaling during senescence (Chapters 21–23). In Chapters 24–27, protocols for the study of cell senescence in global terms are detailed, including a method for the study of metabolomic alterations (Chapter 24), a technique to apply genome-wide RNAi approaches to cell senescence research (Chapter 25), and multiparametric strategies (Chapters 26 and 27). Finally, in Chapters 28–34, protocols applicable to lower model organisms are described, encompassing techniques to assess senescence in Escherichia coli (Chapter 28), Podospora anserina (Chapter 29), Saccharomyces cerevisiae (Chapter 30), Caenorhabditis elegans (Chapters 31 and 32), Drosophila melanogaster (Chapter 33), and Danio rerio (Chapter 34). Potential Audience of This Book In the first instance, this book will be of interest not only for undergraduate and graduate students but also for more experienced scientists who are approaching the study of cell senescence. In addition, the audience of this book encompasses: Libraries of universities and public biological/biomedical research institutions. ● Scientists interested in molecular and cell biology, biochemistry, pharmacology, genet- ● ics, systems biology, medicine, public health, and in life sciences in general. Specialists and experts in model organisms including bacteria, fungi, worms, flies, and ● mammals. Medical oncologists and scientists working in oncology. ● Pharmaceutical companies and developers of new drugs. ● Villejuif, France Lorenzo Galluzzi Ilio Vitale Oliver Kepp Guido Kroemer Preface
vii Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 Cell Senescence as Both a Dynamic and a Static Phenotype. . . . . . . . . . . . . . . . . . 1 Andrew R.J. Young, Masako Narita, and Masashi Narita 2 Senescence Regulation by mTOR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Vjekoslav Dulic 3 Senescence Regulation by the p53 Protein Family. . . . . . . . . . . . . . . . . . . . . . . . . 37 Yingjuan Qian and Xinbin Chen 4 Markers of Cellular Senescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Amancio Carnero 5 Biomarkers of Cell Senescence Assessed by Imaging Cytometry . . . . . . . . . . . . . . 83 Hong Zhao and Zbigniew Darzynkiewicz 6 Cytofluorometric Assessment of Cell Cycle Progression . . . . . . . . . . . . . . . . . . . . 93 Ilio Vitale, Mohamed Jemaà, Lorenzo Galluzzi, Didier Metivier, Maria Castedo, and Guido Kroemer 7 Quantification of Cell Cycle-Arresting Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Oliver Kepp, Isabelle Martins, Laurie Menger, Mickaël Michaud, Sandy Adjemian, Abdul Qader Sukkurwala, Lorenzo Galluzzi, and Guido Kroemer 8 Colorimetric Detection of Senescence-Associated b Galactosidase . . . . . . . . . . . . . 143 Koji Itahana, Yoko Itahana, and Goberdhan P. Dimri 9 Chemiluminescent Detection of Senescence-Associated b Galactosidase . . . . . . . . 157 Vinicius Bassaneze, Ayumi Aurea Miyakawa, and José Eduardo Krieger 10 Detection of the Senescence-Associated Secretory Phenotype (SASP) . . . . . . . . . . 165 Francis Rodier 11 Unbiased Characterization of the Senescence-Associated Secretome Using SILAC-Based Quantitative Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Juan Carlos Acosta, Ambrosius P. Snijders, and Jesús Gil 12 Detection of Senescence-Associated Heterochromatin Foci (SAHF) . . . . . . . . . . . 185 Katherine M. Aird and Rugang Zhang 13 Monitoring DNA Damage During Cell Senescence . . . . . . . . . . . . . . . . . . . . . . . 197 Glyn Nelson and Thomas von Zglinicki 14 Assessment and Quantification of Telomerase Enzyme Activity . . . . . . . . . . . . . . . 215 Michelle F. Maritz, Laura A. Richards, and Karen L. MacKenzie 15 Methods for the Assessment of Telomere Status . . . . . . . . . . . . . . . . . . . . . . . . . . 233 Asako J. Nakamura
viii Contents 16 Detection of Nuclear Envelope Alterations in Senescence . . . . . . . . . . . . . . . . . . . 243 Clea Bárcena, Fernando G. Osorio, and José Maria Pérez Freije 17 Measuring Reactive Oxygen Species in Senescent Cells . . . . . . . . . . . . . . . . . . . . . 253 João F. Passos, Satomi Miwa, and Thomas von Zglinicki 18 Quantification of Protein Carbonylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Nancy B. Wehr and Rodney L. Levine 19 Assays for the Measurement of Lipid Peroxidation . . . . . . . . . . . . . . . . . . . . . . . . 283 Ana Cipak Gasparovic, Morana Jaganjac, Branka Mihaljevic, Suzana Borovic Sunjic, and Neven Zarkovic 20 Raman Spectroscopy for the Detection of AGEs/ALEs . . . . . . . . . . . . . . . . . . . . 297 J. Renwick Beattie, John J. McGarvey, and Alan W. Stitt 21 Monitoring Oncogenic B-RAF-Induced Senescence in Melanocytes . . . . . . . . . . . 313 Sieu L. Tran and Helen Rizos 22 Methods to Investigate the Role of SIRT1 in Endothelial Senescence . . . . . . . . . . 327 Bo Bai and Yu Wang 23 Monitoring Nutrient Signaling Through the Longevity Protein p66SHC1 . . . . . . . . 341 Sofia Chiatamone Ranieri and Giovambattista Pani 24 Profiling the Metabolic Signature of Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . 355 Florian M. Geier, Silke Fuchs, Gabriel Valbuena, Armand M. Leroi, and Jacob G. Bundy 25 Genome-Wide RNAi Screening to Identify Regulators of Oncogene-Induced Cellular Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 Narendra Wajapeyee, Sara K. Deibler, and Michael R. Green 26 An Integrated Approach for Monitoring Cell Senescence . . . . . . . . . . . . . . . . . . . 383 Tatiana V. Pospelova, Zhanna V. Chitikova, and Valery A. Pospelov 27 Robust Multiparametric Assessment of Cellular Senescence. . . . . . . . . . . . . . . . . . 409 Clara Correia-Melo, Diana Jurk, and João F. Passos 28 Assessing Chronological Aging in Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421 Stavros Gonidakis and Valter D. Longo 29 Assessing Organismal Aging in the Filamentous Fungus Podospora anserina . . . . . 439 Heinz D. Osiewacz, Andrea Hamann, and Sandra Zintel 30 Assessing Chronological Aging in Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . 463 Jia Hu, Min Wei, Mario G. Mirisola, and Valter D. Longo 31 Assessing Aging and Senescent Decline in Caenorhabditis elegans: Cohort Survival Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473 Eirini Lionaki and Nektarios Tavernarakis 32 High-Throughput and Longitudinal Analysis of Aging and Senescent Decline in Caenorhabditis elegans . . . . . . . . . . . . . . . . . . . . . . . . . . 485 Eirini Lionaki and Nektarios Tavernarakis 33 Assessing Senescence in Drosophila Using Video Tracking . . . . . . . . . . . . . . . . . . 501 Reza Ardekani, Simon Tavaré, and John Tower 34 Assessing Vascular Senescence in Zebrafish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517 Sandra Donnini, Antonio Giachetti, and Marina Ziche Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
ix Contributors JUAN CARLOS ACOSTA • Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College, London, UK SANDY ADJEMIAN • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France KATHERINE M. AIRD • Women’s Cancer Program, Epigenetics and Progenitor Cells Keystone Program, Fox Chase Cancer Center, Philadelphia, PA, USA REZA ARDEKANI • Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA BO BAI • Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China CLEA BÁRCENA • Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain VINICIUS BASSANEZE • Laboratory of Genetics and Molecular Cardiology/LIM 13, Heart Institute (InCor), University of Sao Paulo Medical School, Sao Paulo, Brazil J. RENWICK BEATTIE • Centre for Vision and Vascular Science, School of Medicine and Dentistry, Queen’s University, Belfast, UK JACOB G. BUNDY • Biomolecular Medicine, Department of Surgery and Cancer, Imperial College, London, UK AMANCIO CARNERO • Instituto de Biomedicina, Hospital Universitario Virgen del Rocio, Sevilla, Spain MARIA CASTEDO • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France XINBIN CHEN • Comparative Oncology Laboratory, University of California, Davis, CA, USA ZHANNA V. CHITIKOVA • Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia; St. Petersburg State University, St. Petersburg, Russia CLARA CORREIA-MELO • Ageing Research Laboratories, Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK ZBIGNIEW DARZYNKIEWICZ • Department of Pathology, Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA SARA K. DEIBLER • Howard Hughes Medical Institute, Chevy Chase, MD, USA; Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA GOBERDHAN P. DIMRI • Department of Biochemistry and Molecular Biology, The George Washington University Medical Center, Washington, DC, USA SANDRA DONNINI • Department of Biotechnology, University of Siena, Siena, Italy VJEKOSLAV DULIC • Institut de Génétique Moléculaire, Montpellier, France; CNRS, UMR5535, Montpellier, France; Université Montpellier 1, Montpellier, France JOSÉ MARIA PÉREZ FREIJE • Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain SILKE FUCHS • Cell and Molecular Biology, Department of Life Sciences, Imperial College, London, UK
x Contributors LORENZO GALLUZZI • Institut Gustave Roussy, Villejuif, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France ANA CIPAK GASPAROVIC • Rudjer Boskovic Institute, Zagreb, Croatia FLORIAN M. GEIER • Biomolecular Medicine, Department of Surgery and Cancer, Imperial College, London, UK ANTONIO GIACHETTI • Department of Biotechnology, University of Siena, Siena, Italy JESÚS GIL • Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College, London, UK STAVROS GONIDAKIS • Department of Biological Sciences, Ethel Percy Andrus Gerontology Center, University of Southern California, Los Angeles, CA, USA MICHAEL R. GREEN • Howard Hughes Medical Institute, Chevy Chase, MD, USA; Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA ANDREA HAMANN • Faculty of Biosciences, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany; Frankfurt Cluster of Excellence “Macromolecular Complexes”, Frankfurt, Germany JIA HU • Ethel Percy Andrus Gerontology Center, Davis School of Gerontology, University of Southern California, Los Angeles, CA, USA KOJI ITAHANA • Duke-NUS Graduate Medical School Singapore, Singapore, Singapore YOKO ITAHANA • Duke-NUS Graduate Medical School Singapore, Singapore, Singapore MORANA JAGANJAC • Rudjer Boskovic Institute, Zagreb, Croatia MOHAMED JEMÀA • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France DIANA JURK • Ageing Research Laboratories, Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK OLIVER KEPP • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France JOSÉ EDUARDO KRIEGER • Laboratory of Genetics and Molecular Cardiology/LIM 13, Heart Institute (InCor), University of Sao Paulo Medical School, Sao Paulo, Brazil GUIDO KROEMER • Centre de Recherche des Cordeliers, Paris, France; INSERM, U848, Villejuif, France; Metabolomics Platform, Institut Gustave Roussy, Villejuif, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France ARMAND M. LEROI • Ecology and Evolution, Department of Life Sciences, Imperial College, London, UK RODNEY L. LEVINE • Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA EIRINI LIONAKI • Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Greece VALTER D. LONGO • Davis School of Gerontology, Ethel Percy Andrus Gerontology Center, University of Southern California, Los Angeles, CA, USA CARLOS LÓPEZ-OTÍN • Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain ISABELLE MARTINS • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France KAREN L. MACKENZIE • Cancer Cell Development Group, Children’s Cancer Institute Australia, Randwick, Australia; Lowy Cancer Research Centre, University of New South Wales, Randwick, Australia
xi MICHELLE F. MARITZ • Cancer Cell Development Group, Children’s Cancer Institute Australia, Randwick, Australia; Lowy Cancer Research Centre, University of New South Wales, Randwick, Australia JOHN J. MCGARVEY • School of Chemistry and Chemical Engineering, Queen’s University, Belfast, UK LAURIE MENGER • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France DIDIER METIVIER • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France MICKAËL MICHAUD • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France BRANKA MIHALJEVIC • Rudjer Boskovic Institute, Zagreb, Croatia MARIO G. MIRISOLA • Department of Medical and Forensic Biopathology e Biotechnology, University of Palermo, Palermo, Italy SATOMI MIWA • Ageing Research Laboratories, Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK AYUMI AUREA MIYAKAWA • Laboratory of Genetics and Molecular Cardiology/LIM 13, Heart Institute (InCor), University of Sao Paulo Medical School, Sao Paulo, Brazil ASAKO J. NAKAMURA • Department of Anatomy and Cell Biology, Osaka Medical College, Takatsuki, Japan MASAKO NARITA • Cancer Research UK, Cambridge Research Institute, Cambridge, UK MASASHI NARITA • Cancer Research UK, Cambridge Research Institute, Cambridge, UK GLYN NELSON • Ageing Research Laboratories, Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK HEINZ D. OSIEWACZ • Faculty of Biosciences, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany; Frankfurt Cluster of Excellence “Macromolecular Complexes”, Frankfurt, Germany FERNANDO G. OSORIO • Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain GIOVAMBATTISTA PANI • Institute of General Pathology, Catholic University Medical School, Rome, Italy JOÃO F. PASSOS • Ageing Research Laboratories, Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK VALERY A. POSPELOV • Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia; St. Petersburg State University, St. Petersburg, Russia TATIANA V. POSPELOVA • Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia; St. Petersburg State University, St. Petersburg, Russia YINGJUAN QIAN • Comparative Oncology Laboratory, University of California, Davis, CA, USA SOFIA CHIATAMONE RANIERI • Clinical Chemistry, Laboratory and Endocrinology Unit, Departement of Laboratory Medicine, Azienda Ospedaliera ASMN, Istituto di Ricovero e Cura a Carattere Scientifico, Reggio Emilia, Italy LAURA A. RICHARDS • Cancer Cell Development Group, Children’s Cancer Institute Australia, Randwick, Australia; Lowy Cancer Research Centre, University of New South Wales, Randwick, Australia Contributors
xii HELEN RIZOS • Melanoma Institute Australia, North Sydney, Australia University of Sydney, Sydney, Australia; Westmead Institute for Cancer Research, Westmead Millennium Institute for Medical Research, Westmead, Australia FRANCIS RODIER • Institut du cancer de Montréal, Centre de recherche du CHUM, Montréal, Canada AMBROSIUS P. SNIJDERS • Biomolecular Mass Spectrometry and Proteomics Laboratory, MRC Clinical Sciences Centre, Imperial College, London, UK ALAN W. STITT • Centre for Vision and Vascular Science, School of Medicine and Dentistry, Queen’s University, Belfast, UK ABDUL QADER SUKKURWALA • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France SUZANA BOROVIC SUNJIC • Rudjer Boskovic Institute, Zagreb, Croatia SIMON TAVARÉ • Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA; Department of Oncology, University of Cambridge, Cambridge, UK; Cancer Research UK, Cambridge Research Institute, Cambridge, UK NEKTARIOS TAVERNARAKIS • Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Greece JOHN TOWER • Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA SIEU L. TRAN • Melanoma Institute Australia, North Sydney, Australia; University of Sydney, Sydney, Australia; Westmead Institute for Cancer Research, Westmead Millennium Institute for Medical Research, Westmead, Australia GABRIEL VALBUENA • Biomolecular Medicine, Department of Surgery and Cancer, Imperial College, London, UK ILIO VITALE • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France THOMAS VON ZGLINICKI • Ageing Research Laboratories, Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK NARENDRA WAJAPEYEE • Department of Pathology, Yale University School of Medicine, New Haven, CT, USA YU WANG • Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China NANCY B. WEHR • Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA MIN WEI • Davis School of Gerontology, Ethel Percy Andrus Gerontology Center, University of Southern California, Los Angeles, CA, USA ANDREW R.J. YOUNG • Cancer Research UK, Cambridge Research Institute, Cambridge, UK NEVEN ZARKOVIC • Rudjer Boskovic Institute, Zagreb, Croatia RUGANG ZHANG • Women’s Cancer Program, Epigenetics and Progenitor Cells Keystone Program, Fox Chase Cancer Center, Philadelphia, PA, USA HONG ZHAO • Department of Pathology, Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA MARINA ZICHE • Department of Biotechnology, University of Siena, Siena, Italy SANDRA ZINTEL • Faculty of Biosciences, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany; Frankfurt Cluster of Excellence “Macromolecular Complexes”, Frankfurt, Germany Contributors
1 Lorenzo Galluzzi et al. (eds.), Cell Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 965, DOI 10.1007/978-1-62703-239-1_1, © Springer Science+Business Media, LLC 2013 Chapter 1 Cell Senescence as Both a Dynamic and a Static Phenotype Andrew R.J. Young, Masako Narita, and Masashi Narita Abstract It has been 50 years since cellular senescence was first described in human diploid fibroblasts (HDFs), yet its mechanism as well as its physiological and clinical implications are still not fully appreciated. Recent progress suggests that cellular senescence is a collective phenotype, composed of complex networks of effector programs. The balance and quality within the effector network varies depending on the cell type, the nature of the stress as well as the context. Therefore, understanding each of these effectors in the con- text of the whole network will be necessary in order to fully understand senescence as a whole. Furthermore, searching for new effector programs of senescence will help to define this heterogeneous and complex phenotype according to cellular contexts. Key words: Autophagy, mTOR, Oncogene, SAHFs, SASP, Senescence, SMS, TASCC Cellular senescence is, fundamentally, a stress responsive cell cycle arrest. The senescence phenotype is extremely stable, and thus, senescence is often described as a state of “irreversible” or “permanent” cell cycle arrest, as opposed to the readily revers- ible quiescent state. The senescence phenotype was originally identified in genetically normal, primary, human diploid fibroblasts (HDFs), which “refused” to proliferate indefinitely in culture (1). At that time this behavior was attributed to the cul- ture medium lacking some essential nutrients (2), but it has since been formally attributed to the limits imposed by telomeres (3). When HDFs become senescent, in addition to their stable cell cycle exit, their morphology drastically changes. Typically the cells have enlarged cell and nuclear bodies, prominent nucleoli, and numerous cytoplasmic vacuoles. 1. Introduction
2 A.R.J. Young et al. While it is widely accepted that both the p53 and p16/Rb tumor suppressor pathways are involved in senescence, the precise mechanisms underlying the phenotype are still elusive. Cells respond to cytotoxic stimuli differently depending upon the cell type and the strength of the stress. HDFs are relatively resistant to apoptosis, and are highly prone to senescence. Compared to apop- tosis, senescence is a delayed and much longer process. The con- trast in these two stress responsive phenotypes may be due to the way the signals are conveyed. In apoptosis, a variety of triggers can converge to the executioner caspases through a common mecha- nism (4). In contrast, in senescence a number of effector mecha- nisms, each contributing to the process, have been proposed and it is likely that these effectors collectively define the phenotype. Thus, the senescence process can be viewed as a “matrix” composed of a series of signaling transductions, rather than the single “vector” of signals in apoptosis (Fig. 1). Such effector programs of senescence include senescence associated heterochromatic foci (SAHFs) and epigenetic gene regulation, the DNA damage response, senescence associated secretory phenotype (SASP)/Senescence-messaging secretome (SMS), and macroautophagy (5–16). Due to the highly heterogeneous nature of the senescence phenotype, it has not been possible to identify a universal marker for senescence that applies in all cases. However, the use of a combination of various markers often associated with effector programs of senescence has success- fully contributed to extending the concept of senescence outside the HDF system: a similar phenotype can be induced by various Fig. 1. The juxtaposition of senescence and apoptosis. The contrast between these two stress responsive phenotypes may be due to the way the signals are conveyed. In apop- tosis, a variety of triggers can converge to the executioner caspases through a common mechanism. In contrast, in senescence, a number of effector mechanisms, which can each contribute to the process, have been proposed and it is likely that these effectors collectively define the phenotype, and also that the eventual phenotype maybe different depending on the signals and effectors that induced it. In addition, senescence is typically a delayed and progressive process. Thus, as schematically illustrated here, the senes- cence process can be viewed as a “matrix” composed of a series of signal transductions, rather than the signal “vectors” of apoptosis.
3 1 Dynamic Interaction Between Autophagy and Senescence cellular stresses in different types of somatic cells, as well as different tissues in animals (17–20). Importantly, the effector mechanisms and markers used in these studies were mostly identified in experi- ments using HDFs, and thus, HDFs are still the best-characterized model system for this topic. Therefore, in this review, most descrip- tions are derived from this system unless specifically mentioned otherwise. We also use the term “senescence” to specifically describe the cellular phenotype, distinct from “aging” at the organism level. The complexity of senescence also implies a “time axis,” by which senescence can be understood as a delayed and progressive process. Therefore, it would be sensible to view senescence not only as a static endpoint, but also as a dynamic process of pheno- typic establishment (Fig. 1). This distinction becomes more rele- vant in acute types of senescence, such as oncogene-induced senescence (OIS). Here, we overview the static and dynamic aspects of senescence and discuss our current understanding of the com- plex interactions between the effector programs during senescence, with a particular focus on the regulation of gene expression. It was shown in 1997 that oncogenic Ras could induce premature senescence, in both HDFs and mouse embryonic fibroblasts (MEFs) (21). As the paradoxical name suggests, the Ras-induced senescence (RIS) phenotype is nonlinear. Due to the direct impact of the abnormal mitogenic stimuli, the cells initially proliferate more rapidly (22). This mitotic phenotype is then gradually replaced by the senescence phenotype. Interestingly, the in vivo counterpart of OIS is well represented by some pre-neoplastic or benign tumors, such as melanocytic nevi, lung adenomas, and pan- creatic intraductal neoplasias, and chemically induced skin papillo- mas (23–28) (see ref. 20). These tumors are likely to be a consequence of cell proliferation during this mitotic phase. Thus, OIS/RIS may be a model for the initial steps of tumorigenesis, which include the series of events from the first reaction of the cells to the mitotic stress, following activation of various effector pro- grams, and the maintenance of the phenotype. Importantly, each effector program is typically involved not only in senescence but also in its original, physiological, role outside of senescence. Thus, studying the OIS/RIS system should extend our understanding of each of these effector programs and provide insight into the more general concepts in dynamic alterations of phenotype, such as differentiation and transformation. 2. Oncogenic, Ras-Induced Senescence
4 A.R.J. Young et al. The strong stability of the phenotype, compared to the readily reversible G0 arrest of quiescence, is a critical aspect of senescence, implying its biological relevance in, for instance, tumor suppres- sion and therapy (29, 30), aging (31), and the efficiency of repro- gramming to pluripotent stem cells (32–37). Not surprisingly, one focus of the earlier studies on senescence was on the irreversibility of the cell cycle exit. For example, the Campisi group showed that the refractoriness of cells to mitogens is a hallmark of senescence, and that some growth-related genes are not responsive to growth factors (38, 39). The same group later examined the reversibility of senescence caused by telomere dysfunction using different types of HDFs, with varying levels of the CDK inhibitor p16. They found that the replicative senescence arrest could be reversed by p53 depletion in p16-low cells (such as BJ cells), whereas in p16-high cells (such as WI38 cells) senescence could not be so easily reversed (40). Thus, while p53 contributes to continuous cell cycle arrest signaling, the p16/Rb pathway provides a dominant barrier to the immortalization of human cells. A primary role for the p53 path- way has also been shown in OIS, where it is activated by a persis- tent DNA damage response (41). These data also reinforce the classical view that, in human cells, it is required to abrogate both the p53 and p16/Rb pathways in order to bypass senescence, and that most cancers do indeed have defects in both pathways. We and others have previously shown that HDFs exhibit dra- matic alterations in heterochromatin during senescence (SAHF) in a p16/Rb dependent manner, and have proposed that SAHF for- mation is involved in the stable silencing of at least some cell cycle genes (42, 43). Interestingly, senescent HDFs with high p16 (non- reversible by p53 depletion) show more prominent SAHFs com- pared to p16-low HDFs (reversible by p53 depletion), indicating a correlation between SAHF formation and the stability of senescence arrest (5). Furthermore, we have identified chromatin architectural proteins, HMGA1 and HMGA2, as SAHF components. SAHF dis- ruption by HMGA1 depletion made cellular senescence bypass easier, reinforcing the correlation between phenotype stability and high-order chromatin architecture (44). However, both hetero- chromatin structure and the related epigenetic marks can also be dynamic. Thus, it is conceivable that cells need to have additional mechanisms to achieve the ultimate irreversibility of senescence. The precise relationship between high-order SAHF structure and individual gene regulation is still unclear. To investigate this connection it would be necessary to effectively combine both imag- ing and biochemical data for SAHF markers, which to date has not been managed. It is tempting, however, to speculate that such a clear segregation between euchromatin and heterochromatin in 3. Transcriptional Regulation During Senescence
5 1 Dynamic Interaction Between Autophagy and Senescence the static condition might allow a rather fixed gene expression profile, giving up dynamic and complex gene expression regulation. In this sense, SAHFs may be important not only for gene repres- sion as originally proposed, but may also play a role in the stable maintenance of gene activation in the euchromatic regions. This interpretation would extend our understanding of SAHFs beyond senescence, and rather put the concept of SAHFs in the context of gene plasticity that is associated with distinct cellular phenotypes. Conceivably, senescent cells may exhibit a distinct mRNA expression profile compared to their growing or quiescent coun- terparts (45). A flurry of papers in the late 2000s defined the func- tionality of the plethora of factors secreted by senescent cells. This heterogenous collection of cytokines, chemokines and other fac- tors, termed the SASP or SMS has been shown to have autocrine and paracrine functions (10–13, 45, 46)—the autocrine function being to reinforce the senescence phenotype in the cell of origin and one potential paracrine function being to induce senescence in other cells sharing the same DNA damaging/tumorigenic microen- vironment. Another paracrine function may be the non-serendipi- tous promotion of the transformation of premalignant—but not of normal—cells within the microenvironment (47, 48). Amongtheseriesofpapersdescribingthefunctionalsignificance of SASP/SMS, the Peeper group notably identified SASP/SMS as a “secretome” specifically associated with OIS (10). Microarray experiments showed a set of transcripts highly upregulated in OIS cells, which were upregulated neither in cells that had bypassed OIS nor in quiescent cells (10). Thus, it is tempting to speculate that the “open” areas of chromatin generated by SAHF formation may permit and promote the expression of the secretome as a unit. Interestingly, SASP/SMS can also influence SAHF, in that deple- tion of IL-6 (a central component of SASP/SMS) in OIS corre- lated with reduced formation of SAHF as well as p15, a senescence-related CDK inhibitor (CDKI) (10). It has been shown that over-expression of p16, another CDKI, which is sufficient to induce senescence and SAHFs in HDFs, fails to trigger SASP/ SMS, indicating that SAHF formation per se cannot activate the secretome (49). However, in our time series microarray experi- ments in RIS HDFs, upregulation of SASP/SMS components starts during the transition phase of RIS, when SAHF formation is not yet fully established (15). Thus, the two processes may mutu- ally reinforce each other during the course of OIS establishment: SASP/SMS facilitating SAHF formation (possibly in part through p15 upregulation), while high-order chromatin reorganization might reinforce the stable expression of SASP/SMS components in the late stages of RIS. Thus, it is useful to think of OIS as a whole process, not just a static phenotype, and, along those lines, we recently identified another effector mechanism that affects SASP/SMS during the
6 A.R.J. Young et al. RIS-transition, namely macroautophagy (referred to hereafter as autophagy). As well as transcriptional effects, SASP/SMS is also facilitated at the posttranscription level. To study the process and the dynamic features of the senescence phenotype, 4OHT-inducible RIS is very useful. In the 4OHT RIS system in HDFs, it typically takes around 7 days to establish the senescence phenotype (Fig. 2) (15). In this system, much of the dynamic transcriptional alteration occurs during the RIS transition (15). However, if rapid protein turnover were to happen at the same time it would make achieving gene expression changes far more efficient, and perhaps allow emergent cell remodeling. Consistent with this idea, autophagy is highly active during the RIS transition (15). Autophagy is, essentially, the “trash collector” of the cell, or rather the “recycling collection truck.” Eukaryotic cells have two main known mechanisms for protein degradation: the proteasome and autophagy. The proteasome takes only one protein at a time and targets are signaled for selection, whereas autophagy can col- lect cellular components en masse (although, degrees of specificity are emerging for autophagy too (50)). In autophagy, vesicles form within the cytoplasm and can be seen to have enclosed cytoplasmic content. That cytoplasmic content is then trafficked along micro- tubules to the lysosome. Lysosomes contain the degradative enzymes of the cell: lipases, proteases, DNase, etc. (51). Autophagosomes then fuse with lysosomes, the product of which is termed an autolysosome, and pass their content inside for degra- dation and recycling. That content is then actively released from the lysosome (52), and thus, presumably a gradient of metabolites emanates from the lysosome. 4. Autophagy and SASP/SMS in Senescence Fig. 2. A schematic view of oncogenic Ras-induced senescence (RIS) in human diploid fibroblasts (HDFs). RIS can be acutely induced by adding 4OHT to HDFs stably expressing ER:Ras, mediated by retrovirus-mediated gene transfer. The whole process is typically completed in ~7 days.
7 1 Dynamic Interaction Between Autophagy and Senescence The emerging dogma is thus that autophagy represents the “guardian” of the proteome (53), which could be broadened out to the macromolecule-ome. As macromolecules become damaged, for instance through oxidation, they become misfunctional. Hence constant degradation and resynthesis of macromolecules is required, and is especially required in dire times of cellular stress, such as oxidative or metabolic stress. In addition to that role, however, we have shown that autophagy can have quite unforeseen conse- quences for the cell, impacting on cell fate, in that it can facilitate the establishment of senescence both cell autonomously and non- cell-autonomously through its role in the production of SASP/ SMS in RIS, and beyond. As mentioned in the introduction, the characteristics of senescent cells are that they typically have enlarged cell and nuclear bodies, prominent nucleoli, as well as numerous cytoplasmic vacuoles. Part of this vacuolation may come from enlargement of the ER (54) but a good part comes from the accumulation of autolysosomes (15). Upon the induction of Ras, the HDFs at first proliferate more often, or in other words they go through a “proliferative burst,” and then gradually proliferation declines as they approach the “per- manent” proliferative arrested state of senescence. It is during the transition phase (from this proliferative phase to the senescent, proliferative arrest) that autophagy becomes active. Inhibition of autophagy causes the downregulation of SASP/SMS, such as IL6 and IL8, at the posttranscriptional level. Presumably autophagy plays a role in delivering the “raw material,” the proteins for deg- radation, for their reuse in the synthesis of these SASP/SMS fac- tors, thus supporting the translation of the upregulated SASP/ SMS transcripts. A similar positive role for autophagy in senescence associated secretory protein synthesis has recently been observed in senescent HDFs induced by oxidative stress (55). The authors showed that H2 O2 induced senescence with activated autophagy, and that the inhibition of autophagy not only delayed senescence but also reduced the secretory phenotype. Furthermore, autophagy seems to affect SASP/SMS outside fibroblasts. Sasaki et al. (56) showed that autophagy can mediate cellular senescence in primary biliary cirrhosis (PBC). PBC is a liver-specific autoimmune disease that eventually leads to the extensive loss of small bile ducts. The authors had previously demonstrated cellular senescence in the damaged small bile ducts and suggested that it may be involved in the patho- genesis of progressive bile duct loss in PBC (57). They went on to look for autophagy in their bank of biopsied or surgically resected human liver specimens. Immunohistochemistry staining for autophagy and lysosomal markers showed large vesicles in PBC, chronic viral hepatitis, nonalcoholic steatohepatitis and extrahe- patic biliary obstruction. By far the highest level of these vesicles 4.1. Autophagy in RIS
8 A.R.J. Young et al. was seen in PBC. The autophagy positive small bile ducts also stained for markers of senescence. Moving into cultured biliary epithelial cells (BECs), Sasaki et al. showed that if autophagy was pharmacologically inhibited then the extent of senescence was significantly decreased, as was the BEC SASP/SMS. These studies support the idea that active protein turnover can promote the en masse production of secretory proteins during senes- cence. However, it is also possible that autophagy modulates the senescence phenotype through its ability to maintain the quality of macromolecules. Gamerdinger et al. identified activated autophagy during replicative senescence in HDFs as well as in the neurons of aged rodent brains (14, 58). The authors showed that BAG3, a co- chaperone, together with other chaperons, Hsp70 and HspB8, pro- moted the targeting of misfolded and aggregation prone proteins to autophagosomes. Thus, damaged proteins accumulated during senescence or aging can be eliminated by autophagy. Of note, rep- licative senescence is also associated with activation of a secretome (49), although how autophagy affects SASP/SMS in the context of replicative senescence remains to be tested. Therefore, autophagy would seem to have, at least, two distinct levels of influence on senescence; through its specific role in SASP/SMS and also through its general activity in the quality control of macromolecules. How can we reconcile these roles, or does this suggest another contro- versy, as in the association between autophagy and cell death: do cells become senescent due to autophagy or despite autophagy? The potentially mixed roles for autophagy in senescence might reflect the complex effector networks of senescence mentioned above (Fig. 1). The collective outcome of the combinatorial consequences of those effector programs would shift within the range of the senes- cence phenotype depending on the situation. Therefore, the domi- nant effect of autophagy might be different between RIS (a highly dynamic process) and replicative senescence (a slow and adaptive process). Consistent with this idea, we have recently identified an additional mechanism occurring during RIS by which autophagy can support SASP/SMS, which involves a TOR-autophagy spatial coupling compartment (TASCC) (15). TASCC—more defined mechanistic insight into the relationship between autophagy and senescence. Autophagy and protein synthesis are typically regulated in opposite directions. For example, when cells are starved, autophagy is activated while protein synthesis is suppressed, allowing the degradation products (such as amino acids) to be used as an alternative energy source. This reciprocal regulation of anabolic and catabolic processes is achieved through target of rapamycin (TOR), the master regulator of protein synthe- sis (59). TOR coordinately senses cellular nutrient and energy sta- tus as well as growth factor signals. TOR then integrates those signals and “decides” whether sufficient metabolites and energy are available to synthesize protein (60). TOR is a protein kinase
9 1 Dynamic Interaction Between Autophagy and Senescence and, if conditions are right, it in turn phosphorylates its substrates, such as eIF4E binding protein (4EBP) and S6 kinase (S6K), and facilitates protein synthesis (59). However, it is possible that the simultaneous activation of pro- tein degradation and synthesis may allow cells to “regenerate” themselves very efficiently, and this seems to be occurring during RIS, where both autophagy and protein synthesis are active (15). In addition, it has also been shown that Rapamycin, which inhibits TOR, can divert senescence into quiescence; thus, mTOR is required for senescence maintenance (61). One potential explana- tion for the apparently paradoxical simultaneous activation of pro- tein degradation and protein synthesis may be the spatial separation of these processes within the cell. By immunostaining, we identified a unique cellular compartment, the TASCC, in which both mam- malian TOR (mTOR) and autolysosomes (the end stage of autophagy) are enriched in the vicinity of the rER-Golgi apparatus (GA). Aside from growth factor signaling, mTOR can also be acti- vated by amino acids (the ultimate end product of autophagic pro- tein degradation). The inhibitory point where mTOR acts on autophagy is the initial step of autophagosome formation; thus, it is possible that the physical coupling of these two opposing pro- cesses causes them to reinforce each other. Indeed, we have shown that amino acids derived from autolysosomes are required for mTOR localization to the TASCC, and that early stage autophago- somes are only detected outside of the TASCC. Our data is highly consistent with emerging evidence from the Sabatini group, indi- cating that mTOR is recruited to, and activated at, the lysosomal surface, where Rheb resides, and that this process is dependent on Rag GTPases, which also localize to lysosomes (62, 63). Furthermore, the TASCC is consistently surrounded by the trans-side of the GA. The rER-GA is the place where both secre- tory proteins as well as lysosomal and other membrane compo- nents are synthesized and processed. Thus, the encompassing of the TASCC by the rER-GA raises an interesting possibility: that the TASCC, together with the associated rER-GA, may form a protein “factory” for both the SASP/SMS to be secreted, and lys- osomal and other membrane proteins (which would, in turn, rein- force TASCC formation itself). This could be tested using the technique of fluorescent noncanonical amino acid tagging (FUNCAT) to specifically visualize newly synthesized proteins in situ (FUNCAT was developed by the Schuman laboratory (64– 66), and is now licensed to Invitrogen). This system is essentially a more modern alternative to radioactive, e.g., 35 S, methionine met- abolic labeling. Pulse–chase experiments with the “tagged” amino acid revealed that as early as 3 min the nascent proteins are enriched around the GA, and subsequently move to the GA, and about 2 h later are detected in the TASCC. In addition, by immunolabelling, IL6 and IL8 were specifically detected in the TASCC as well as the
10 A.R.J. Young et al. surrounding GA, suggesting that the same compartment facilitates the synthesis of proteins for both the degradation machinery and for SASP/SMS. Consistently, inhibition of Rag GTPase activity during RIS blocked mTOR enrichment to the TASCC and reduced the synthesis of SASP/SMS components (15). Although direct evidence for the regulation of protein synthesis within the TASCC by mTOR remains to be shown, the TASCC demonstrates addi- tional complexity in terms of its functional associations between SASP/SMS, senescence and autophagy. Importantly, we also found that the TASCC was in close asso- ciation with the GA in other cellular conditions, such as mac- rophage-like differentiation in culture, during which cells acutely produce abundant IL8 (15). In addition, TASCC-like compart- ments were detected specifically in the glomerular podocytes of mouse kidneys (15). Podocytes are highly enriched for constitutive autophagy as well as prominent GA, and produce abundant secre- tory proteins to support the constant turnover of the glomerular basement membrane (67, 68). The TASCC may thus be associated more generally with the acute, abundant production of secretory proteins, rather than senescence per se. Consistent with this inter- pretation, replicative senescent HDFs do not show a prominent TASCC (Masako Narita & Masashi Narita, unpublished data), sug- gesting that the rate or amount of synthesis of SASP/SMS compo- nents in this adaptive condition may not require such self-reinforcing machinery. Although the exact molecular basis for the necessity of the TASCC in facilitating the production of secretory proteins in some cases but not in others remains to be elucidated, the TASCC might provide a mechanism to modulate the downstream effects of activated autophagy. Taken together, the studies outlined in this review reinforce the higher-order network of the different effector programs of senes- cence and its complexity, in which the same effector might even produce different outcomes through the different aspects— dynamic or static—of senescence. Furthermore when it is consid- ered, as mentioned in Subheading 3, that SASP can affect SAHF, then the new role for autophagy in the regulation of SASP/SMS and its spatial association with mTOR implies a functional coop- eration between nuclear (transcription) and cytoplasmic pheno- types (protein turnover) toward the expression of a subset of genes. While we only focus on the limited aspects of gene regulation in RIS in this review, there are other mechanisms that are also involved in the phenotype and we expect, in this model system, additional unexpected interactions between them, as well as new effector mechanisms to be identified in the future. 5. Conclusions
11 1 Dynamic Interaction Between Autophagy and Senescence Acknowledgments Our work is supported by Cancer Research UK. References 1. Hayflick L (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25: 585–621 2. Shay JW, Wright WE (2000) Hayflick, his limit, and cellular ageing. Nat Rev Mol Cell Biol 1:72–76 3. Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345:458–460 4. Strasser A, Cory S, Adams JM (2011) Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. EMBO J 30:3667–3683 5. Narita M, Nũnez S, Heard E, Narita M, Lin AW, Hearn SA, Spector DL, Hannon GJ, Lowe SW (2003) Rb-mediated heterochromatin for- mation and silencing of E2F target genes dur- ing cellular senescence. Cell 113:703–716 6. Zhang R, Poustovoitov MV, Ye X, Santos HA, ChenW,DaganzoSM,ErzbergerJP,Serebriiskii IG, Canutescu AA, Dunbrack RL, Pehrson JR, Berger JM, Kaufman PD, Adams PD (2005) Formation of MacroH2A-containing senes- cence-associated heterochromatin foci and senescence driven by ASF1a and HIRA. Dev Cell 8:19–30 7. Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, Luise C, Schurra C, Garre M, Giovanni Nuciforo P, Bensimon A, Maestro R, Giuseppe Pelicci P, D’adda Di Fagagna F (2006) Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 444:638–642 8. Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, Vassiliou L-VF, Kolettas E, Niforou K, Zoumpourlis VC, Takaoka M, Nakagawa H, Tort F, Fugger K, Johansson F, Sehested M, Andersen CL, Dyrskjot L, Ørntoft T, Lukas J, Kittas C, Helleday T, Halazonetis TD, Bartek J, Gorgoulis VG (2006) Oncogene- induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 444:633–637 9. Mallette FA, Gaumont-Leclerc M-F, Ferbeyre G (2007) The DNA damage signaling pathway is a critical mediator of oncogene-induced senescence. Genes Dev 21:43–48 10. Kuilman T, Michaloglou C, Vredeveld LCW, Douma S, van Doorn R, Desmet CJ, Aarden LA, Mooi WJ, Peeper DS (2008) Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell 133:1019–1031 11. Acosta JC, O’Loghlen A, Banito A, Guijarro MV, Augert A, Raguz S, Fumagalli M, Da Costa M, Brown C, Popov N, Takatsu Y, Melamed J, D’adda Di Fagagna F, Bernard D, Hernando E, Gil J (2008) Chemokine signal- ing via the CXCR2 receptor reinforces senes- cence. Cell 133:1006–1018 12. Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR (2008) Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell 132:363–374 13. Coppé J-P, Patil CK, Rodier F, Sun Y, Muñoz DP, Goldstein J, Nelson PS, Desprez P-Y, Campisi J (2008) Senescence-associated secre- tory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6:2853–2868 14. Gamerdinger M, Hajieva P, Kaya AM, Wolfrum U, Hartl FU, Behl C (2009) Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3. EMBO J 28:889–901 15. Young ARJ, Narita M, Ferreira M, Kirschner K, Sadaie M, Darot JFJ, Tavaré S, Arakawa S, Shimizu S, Watt FM, Narita M (2009) Autophagy mediates the mitotic senescence transition. Genes Dev 23:798–803 16. Narita M, Young ARJ, Arakawa S, Samarajiwa SA, Nakashima T, Yoshida S, Hong S, Berry LS, Reichelt S, Ferreira M, Tavaré S, Inoki K, Shimizu S, Narita M (2011) Spatial coupling of mTOR and autophagy augments secretory phenotypes. Science 332(6032):966–970 17. Romanov SR, Kozakiewicz BK, Holst CR, Stampfer MR, Haupt LM, Tlsty TD (2001) Normal human mammary epithelial cells spon- taneously escape senescence and acquire genomic changes. Nature 409:633–637 18. Lin AW, Lowe SW (2001) Oncogenic ras acti- vates the ARF-p53 pathway to suppress epithe- lial cell transformation. Proc Natl Acad Sci U S A 98:5025–5030 19. Shay JW, Roninson IB (2004) Hallmarks of senescence in carcinogenesis and cancer ther- apy. Oncogene 23:2919–2933 20. Narita M, Lowe SW (2005) Senescence comes of age. Nat Med 11:920–922
12 A.R.J. Young et al. 21. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW (1997) Oncogenic ras provokes pre- mature cell senescence associated with accumu- lation of p53 and p16INK4a. Cell 88:593–602 22. Lin AW, Barradas M, Stone JC, Van Aelst L, Serrano M, Lowe SW (1998) Premature senes- cence involving p53 and p16 is activated in response to constitutive MEK/MAPK mito- genic signaling. Genes Dev 12:3008–3019 23. Lazzerini Denchi E, Attwooll C, Pasini D, Helin K (2005) Deregulated E2F activity induces hyperplasia and senescence-like fea- tures in the mouse pituitary gland. Mol Cell Biol 25:2660–2672 24. Braig M, Lee S, Loddenkemper C, Rudolph C, Peters AH, Schlegelberger B, Stein H, Dörken B, Jenuwein T, Schmitt CA (2005) Oncogene- induced senescence as an initial barrier in lym- phoma development. Nature 436:660–665 25. Chen Z, Trotman LC, Shaffer D, Lin H-K, Dotan ZA, Niki M, Koutcher JA, Scher HI, Ludwig T, Gerald W, Cordon-Cardo C, Paolo Pandolfi P (2005) Crucial role of p53-depen- dent cellular senescence in suppression of Pten- deficient tumorigenesis. Nature 436:725–730 26. Collado M, Gil J, Efeyan A, Guerra C, Schuhmacher AJ, Barradas M, Benguría A, Zaballos A, Flores JM, Barbacid M, Beach D, Serrano M (2005) Tumour biology: senescence in premalignant tumours. Nature 436:642 27. Michaloglou C, Vredeveld LCW, Soengas MS, Denoyelle C, Kuilman T, Van Der Horst CMAM, Majoor DM, Shay JW, Mooi WJ, Peeper DS (2005) BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436:720–724 28. DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K, Mangal D, Yu KH, Yeo CJ, Calhoun ES, Scrimieri F, Winter JM, Hruban RH, Iacobuzio-Donahue C, Kern SE, Blair IA, Tuveson DA (2011) Oncogene- induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 475: 106–109 29. Schmitt CA, Fridman JS, Yang M, Lee S, Baranov E, Hoffman RM, Lowe SW (2002) A senescence program controlled by p53 and p16INK4a contributes to the outcome of can- cer therapy. Cell 109:335–346 30. te Poele RH, Okorokov AL, Jardine L, Cummings J, Joel SP (2002) DNA damage is able to induce senescence in tumor cells in vitro and in vivo. Cancer Res 62:1876–1883 31. Sharpless NE, Depinho RA (2007) How stem cells age and why this makes us grow old. Nat Rev Mol Cell Biol 8:703–713 32. Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Nakagawa M, Okita K, Yamanaka S (2009) Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460:1132–1135 33. Kawamura T, Suzuki J, Wang YV, Menendez S, Morera LB, Raya A, Wahl GM, Belmonte JCI (2009) Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460:1140–1144 34. Marión RM, Strati K, Li H, Murga M, Blanco R, Ortega S, Fernandez-Capetillo O, Serrano M, Blasco MA (2009) A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 460: 1149–1153 35. Li H, Collado M, Villasante A, Strati K, Ortega S, Cañamero M, Blasco MA, Serrano M (2009) The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 460:1136–1139 36. Krizhanovsky V, Lowe SW (2009) Stem cells: the promises and perils of p53. Nature 460:1085–1086 37. Banito A, Rashid ST, Acosta JC, Li S, Pereira CF, Geti I, Pinho S, Silva JC, Azuara V, Walsh M, Vallier L, Gil J (2009) Senescence impairs successful reprogramming to pluripotent stem cells. Genes Dev 23:2134–2139 38. Seshadri T, Campisi J (1989) Growth-factor- inducible gene expression in senescent human fibroblasts. Exp Gerontol 24:515–522 39. Seshadri T, Campisi J (1990) Repression of c-fos transcription and an altered genetic pro- gram in senescent human fibroblasts. Science 247:205–209 40. Beauséjour CM, Krtolica A, Galimi F, Narita M, Lowe SW, Yaswen P, Campisi J (2003) Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J 22: 4212–4222 41. D’adda Di Fagagna F (2008) Living on a break: cellular senescence as a DNA-damage response. Nat Rev Cancer 8:512–522 42. Narita M (2007) Cellular senescence and chro- matin organisation. Br J Cancer 96:686–691 43. Adams PD (2007) Remodeling of chromatin structure in senescent cells and its potential impact on tumor suppression and aging. Gene 397:84–93 44. Narita M, Narita M, Krizhanovsky V, Nuñez S, Chicas A, Hearn SA, Myers MP, Lowe SW (2006) A novel role for high-mobility group a proteins in cellular senescence and heterochro- matin formation. Cell 126:503–514 45. Shelton DN, Chang E, Whittier PS, Choi D, Funk WD (1999) Microarray analysis of repli- cative senescence. Curr Biol 9:939–945 46. Young ARJ, Narita M (2009) SASP reflects senescence. EMBO Rep 10:228–230
13 1 Dynamic Interaction Between Autophagy and Senescence 47. Krtolica A, Parrinello S, Lockett S, Desprez PY, Campisi J (2001) Senescent fibroblasts pro- mote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci U S A 98:12072–12077 48. Campisi J, D’adda Di Fagagna F (2007) Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 8:729–740 49. Rodier F, Coppé J-P, Patil CK, Hoeijmakers WAM, Muñoz DP, Raza SR, Freund A, Campeau E, Davalos AR, Campisi J (2009) Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion. Nature 11:973–979 50. Kraft C, Peter M, Hofmann K (2010) Selective autophagy: ubiquitin-mediated recognition and beyond. Nature 12:836–841 51. Holtzman E (1989) Lysosomes. Springer, New York, p 439 52. Mancini GM, Havelaar AC, Verheijen FW (2000) Lysosomal transport disorders. J Inherit Metab Dis 23:278–292 53. Ryan KM (2011) p53 and autophagy in cancer: guardian of the genome meets guardian of the proteome. Eur J Cancer 47:44–50 54. Denoyelle C, Abou-Rjaily G, Bezrookove V, Verhaegen M, Johnson TM, Fullen DR, Pointer JN, Gruber SB, Su LD, Nikiforov MA, Kaufman RJ, Bastian BC, Soengas MS (2006) Anti- oncogenic role of the endoplasmic reticulum differentially activated by mutations in the MAPK pathway. Nat Cell Biol 8:1053–1063 55. Luo Y, Zou P, Zou J, Wang J, Zhou D, Liu L (2011) Autophagy regulates ROS-induced cel- lular senescence via p21 in a p38 MAPKα dependent manner. Exp Gerontol 46:860–867 56. Sasaki M, Miyakoshi M, Sato Y, Nakanuma Y (2010) Autophagy mediates the process of cel- lular senescence characterizing bile duct dam- ages in primary biliary cirrhosis. Lab Invest 90:835–843 57. Sasaki M, Ikeda H, Haga H, Manabe T, Nakanuma Y (2005) Frequent cellular senes- cence in small bile ducts in primary biliary cir- rhosis: a possible role in bile duct loss. J Pathol 205:451–459 58. Gamerdinger M, Kaya AM, Wolfrum U, Clement AM, Behl C (2011) BAG3 mediates chaperone-based aggresome-targeting and selective autophagy of misfolded proteins. EMBO Rep 12:149–156 59. Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism. Cell 124:471–484 60. Sengupta S, Peterson TR, Sabatini DM (2010) Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell 40:310–322 61. Korotchkina LG, Leontieva OV, Bukreeva EI, Demidenko ZN, Gudkov AV, Blagosklonny MV (2010) The choice between p53-induced senescence and quiescence is determined in part by the mTOR pathway. Aging (Albany NY) 2:344–352 62. Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, Sabatini DM (2008) The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320:1496–1501 63. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM (2010) Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141:290–303 64. Dieterich DC, Link AJ, Graumann J, Tirrell DA, Schuman EM (2006) Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc Natl Acad Sci U S A 103:9482–9487 65. Dieterich DC, Lee JJ, Link AJ, Graumann J, Tirrell DA, Schuman EM (2007) Labeling, detection and identification of newly synthe- sized proteomes with bioorthogonal non- canonical amino-acid tagging. Nat Protoc 2: 532–540 66. Dieterich DC, Hodas JJL, Gouzer G, Shadrin IY, Ngo JT, Triller A, Tirrell DA, Schuman EM (2010) In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat Neurosci 13:897–905 67. Hartleben B, Gödel M, Meyer-Schwesinger C, Liu S, Ulrich T, Köbler S, Wiech T, Grahammer F, Arnold SJ, Lindenmeyer MT, Cohen CD, Pavenstädt H, Kerjaschki D, Mizushima N, Shaw AS, Walz G, Huber TB (2010) Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. J Clin Invest 120:1084–1096 68. Pavenstädt H, Kriz W, Kretzler M (2003) Cell biology of the glomerular podocyte. Physiol Rev 83:253–307
15 Lorenzo Galluzzi et al. (eds.), Cell Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 965, DOI 10.1007/978-1-62703-239-1_2, © Springer Science+Business Media, LLC 2013 Chapter 2 Senescence Regulation by mTOR Vjekoslav Dulic Abstract The senescence program is activated in response to diverse stress stimuli potentially compromising genetic stability and leads to an irreversible cell cycle arrest. The mTOR pathway plays a crucial role in the regula- tion of cell metabolism and cellular growth. The goal of this chapter is to present evidence linking these two processes, which have one common regulator—the tumor suppressor p53. While the role of mTOR in senescence is still controversial, recent papers have shed new light onto this issue. This review, far from being exhaustive given the complexity of the field, will hopefully stimulate further research in this domain, whose relevance for ageing is becoming increasingly documented. Key words: AKT, CDK, CKI, Cell cycle exit, D-type cyclins, mTOR, pRB, Quiescence, Senescence Abbreviations ARF Alternative reading frame ATM Ataxia telangiectasia mutated ATR ATM and Rad3-related kinase Bmi1 B lymphoma Mo-MLV insertion region 1 Cdc25 Cell division cycle 25 (CDK-activating phosphatase) CDK Cyclin-dependent kinase Chk1/2 Checkpoint kinase 1/2 CKI CDK inhibitor ERK1/2 Extracellular regulated kinase (also called MAPK) FOXO Forkhead box protein O FKBP12 Peptidyl-prolyl cis/trans isomerase that forms a complex with rapamycin HES1 Hairy enhancer of Split1 IGF Insulin-like growth factors IRS Insulin receptor substrate INK4A Inhibitor of Cdk4 A MAPK Mitogen-activated protein kinase mTOR Mechanistic (mammalian) target of rapamycin TORC1/2 TOR complex 1 and 2 PcG Polycomb group
16 V. Dulic PDK1 3-Phosphoinositide-dependent kinase 1 PI3K Phosphoinositide 3-kinase PIKK Phosphoinositide 3-kinase (PI3K)-related protein serine/threonine kinase family PML Promyelocytic leukemia protein PTEN Phosphatase and tensin homologue pRb Retinoblastoma protein RHEB Ras homologue enriched in brain SAHF Senescence-associated heterochromatin foci SASP Senescence-associated secretory phenotype SGK Serum- and glucocorticoid-regulated kinase TSC1/2 Tuberous sclerosis 1,2 GTPase TGFb Transforming growth factor b Cellular senescence was originally described by Leonard Hayflick, who showed that, after a finite number of population doublings (mitotic clock), primary human cells irreversibly cease to prolifer- ate in vitro (1). This so-called Hayflick limit was later shown to be provoked, at least in part, by telomere erosion (2), the gradual loss of DNA at the ends of chromosomes, a consequence of the “replication problem” predicted by Olovnikov in the early 1970— hence “replicative senescence” (3). It has been therefore proposed that telomere erosion could activate a DNA damage response (DDR) and cause G1 cell cycle arrest similar to the one elicited by ionizing radiation (4). Moreover, following an excellent intuition, Hayflick associated cellular senescence with both cancer and ageing— with cancer, because cells must acquire certain characteristics of tumor cells to escape senescence, and with ageing, because accu- mulation of senescent cells could contribute to the global deterio- ration of an organism. We now know that these original hypotheses were fundamentally right, and since then, senescence, from a “tis- sue culture artifact,” became an ever-expanding field of intense research largely due to its tumor suppression potential and contri- bution to age-related pathologies. In addition to telomere dys- function, senescence can be induced by a large variety of stress stimuli, including strong mitogenic signals generated by hyper- activation of certain oncogenes (Ras, Raf, Myc), irreparable DNA damage, or oxidative stress. This invariably inhibits cyclin- dependent kinases (CDK), key cell division regulators, leading to the cell cycle arrest that requires activities of two major tumor suppressors, p53 and pRb (Fig. 1). As a number of excellent recent reviews extensively covered this topic (5–7), we shall here briefly outline the aspects of cellular senescence and the pathways implicated in its implementation that are relevant for this chapter. 1. Introduction
17 2 mTOR, Metabolism and Cell Senescence Senescence is a viable and metabolically active state, which is char- acterized by a virtually permanent (irreversible) cessation of cell division. It is associated with dramatic changes in cell morphology (large flat cells), metabolism, gene expression and secretion patterns (senescence-associated secretory phenotype or SASP), originally described by Campisi and colleagues (8). Senescence has recently been shown to play a crucial role in age-related pathologies associ- ated with accumulation of senescent cells (9, 10). Both in vitro and in vivo, senescent cells can be detected by virtue of β-galactosidase activity (β-gal) (11), a lysosomal enzyme, which probably does not play a direct role in senescence (12), but serves as a reliable biomarker 2. How is Permanent Cell Cycle Arrest Achieved? Fig. 1. Molecular pathways leading to senescence-associated irreversible cell cycle arrest. Pro-senescence stimuli activate DNA damage response pathway (DDR) leading to perma- nent inactivation of cyclin-dependent kinases (CDK) that control DNA replication and mito- sis (K1/2). In addition, together with Cdk4/6 (K4/6), they keep inactive tumor suppressor pRb (and related pocket proteins p107 and p130). Genotoxic stress and activated onco- genes induce expression of CDK inhibitors p21Waf1/Cip1 (p21) and p16Ink4A (p16) that block cell cycle progression and pRb inactivation. Active pRb blocks expression of genes controlling cell cycle (in part by sequestering E2F family of transcription factors) and contributes to chromatin reorganization in the form of SAHF or PML nuclear bodies.The pathways leading to induction of p16 are not entirely elucidated but, in the case of OIS (oncogene-induced senescence), it has been shown that demethylase JMJD3 blocks repression of Ink4A locus by PcG protein Bmi1.Arf (p19ARF ), another product of this locus, plays a major role in senes- cence in mice, while p16 is more important in humans. Figure adapted from ref. 119.
18 V. Dulic (see Chapter 4 and Fig. 2). In addition, senescent cells often exhibit dramatic rearrangement of chromatin structure, in the forms of PML (promyelocytic leukemia protein) nuclear bodies (13, 14) and senescence-associated heterochromatin foci (SAHF) (15). These heterochromatin structures, whose formation requires active pRb, are thought to play an important role in conferring irreversibility of the cell cycle arrest by repressing the genes controlling cell prolif- eration (Figs. 1 and 2). However, SAHFs are not observed in all cell types and in all forms of senescence (15), and recent work associates SAHF formation with oncogene induced senescence and repression of DNA damage signaling (16). The pro-senescence stimuli all converge on one major pathway to permanently arrest the cell cycle (Fig. 1). The senescence pro- gram is initiated by inactivation of CDK (17), the key cell cycle regulators. CDKs play two distinct roles. First, they control the onset and progression of DNA replication and mitosis, thus enabling and orchestrating the cell cycle. Second, CDKs, particu- larly Cdk4/6 associated with D-type cyclins, phosphorylate, thus keeping inactive, the retinoblastoma (pRb) tumor suppressor and related “pocket-proteins” (p107, p130). This is essential for cell cycle progression, since active (hypo-phosphorylated) pocket pro- teins drive cell cycle exit by sequestering E2F family transcription factors and by repressing the genes required for cell division (18). Fig. 2. Senescence versus quiescence. Quiescence (or G0) is reversible and hypotrophic cell cycle exit that occurs in G1 prior to restriction (R) point. It is mediated preferentially by p27-dependent CDK inactivation and cyclin D1 downregulation. Some results suggest that genes expressed in quiescent cells can block senescence (HES1). By contrast, senescence is irreversible and hypertrophic cell cycle exit that takes place either in G1 or G2, depending on the activated checkpoint. Cell cycle arrest is mediated by p16/p21-dependent CDK inactivation (Fig. 1) and senescent cells contain high levels of G1 cyclins (cyclin D and cyclin E) associated with inactive CDK.While the cytoplasm could be frequently stained for β-galacto- sidase (see Chapters 4, 8 and 9), senescent nuclei are often large and sometimes binuclear (due to endoreplication or defective cytokinesis) and could contain heterochromatin in the forms of SAHF (see Chapter 12) or PML bodies. Both events require active pRb family pocket proteins but in senescence only pRb is essential (ref. 19). GF growth factors.
19 2 mTOR, Metabolism and Cell Senescence Despite often-overlapping functions between different members of the pocket protein family, pRb was shown to play a unique and nonredundant role in senescence by repressing E2F target genes involved in DNA replication (19) (Fig. 1). Recent data suggest that this process requires a recruitment of promoter-bound pRb and E2Fs to PML nuclear bodies (14). More recently, p130/E2F4 complex was also implicated in senescence by repressing, via PML, T-box protein 2 (TBX2) frequently overexpressed in cancer (20). The presence of diverse genotoxic stresses that induce senescence activates a DDR network, controlled by ATM/ATR kinases, which blocks CDK activation via CDC25 family phosphatases, thereby temporarily halting cell cycle progression (Fig. 1). While not required for this transient arrest, p53 and its transcriptional target p21Waf1/Cip1/Sdi1 (p21), a CDK inhibitor (CKI), are essential parts of irreversible cell cycle arrest by the senescence program (21, 22). Permanent CDK inactivation by p21 stably blocks DNA synthesis and mitotic entry and, via activation of pRb, drives exit from the cell cycle (19, 23) (Fig. 1). Another CKI, p16Ink4A (p16), which targets specifically pRb kinases Cdk4/6, also plays an important role in senescence and, along with β-gal, it is often used as a bio- marker both in vitro and in vivo (9, 24). However, unlike p21, p16 does not seem to be involved in the cell cycle arrest associated with replicative senescence (21) and its late induction, which occurs after p21-mediated cell cycle arrest, might promote a stabilization of senescent state (25, 26). Nevertheless, p16 is a key regulator of oncogene-induced senescence (OIS) (27) and recent work in a mouse model strongly implicated this inhibitor in acquisition of age-related pathologies (9). Although p16 is widely considered as an essential part of the senescence program, the pathways regulat- ing its induction in response to senescence-promoting stimuli are still not entirely elucidated. It has been suggested that, in the case of OIS, activation of histone demethylase JMJD3 suppresses the repression of the INK4A-ARF locus by Polycomb group proteins (PcG), Bmi1; (5). Another protein encoded by this locus is tumor suppressor p19ARF (ARF) that plays a prominent role in senescence in mice but not in humans (27). ARF stabilizes p53 by inhibiting MDM2 and, via its effector p21 blocks cell cycle. Thus, ultimate targets of both products of the INK4A-ARF locus are CDKs that inactivate pRb, and their dysregulation invariably compromises genome integrity (Fig. 1). What distinguishes senescence from another non-proliferating state, quiescence, also referred to as G0 or G0/G1 phase (28)? In contrast to senescence, quiescence is a reversible cell cycle arrest, 3. Quiescence Versus Senescence
20 V. Dulic which is induced by the absence of mitogens or growth factors, nutrient starvation, or increasing cell density (confluence) (Fig. 2). Typical examples of quiescent cells are lymphocytes, whose activation is part of the immune response, adult stem cells, or dermal fibroblasts, which actively participate in wound healing (29). In general, quiescence is characterized by low metabolism and protein synthesis, lack of cellular growth, and, the absence of global het- erochromatin structures such as PML bodies (30) or SAHF (15). The latter feature is probably responsible for the reversible nature of quiescence. However, much against the prevailing view, recent results showed that fibroblasts rendered quiescent by contact inhi- bition exhibit comparable metabolic activity to actively proliferat- ing cells. In addition to promote recycling of damaged macromolecules via autophagy, high metabolic activity might serve for biosynthesis and secretion of extracellular matrix proteins (31). Importantly, while downregulating expression of genes involved in cell division, quiescent cells upregulate genes, such as HES1, that inhibit senescence, differentiation and apoptosis (32, 33). Unlike senescence, which can occur either in the G1 or G2 phase of the cell cycle (34), depending on when the damage is detected and the efficiency of the checkpoints (23, 35), quiescence essentially takes place in G1, prior to the restriction (R) checkpoint (36). A teleo- logical explanation for this observation could be given by the uni- directionality of the cell cycle—G0 state always precedes DNA replication (S phase). Therefore, if cells became quiescent in the G2 phase, cell cycle entry upon stimulation would result in genome reduplication giving rise to undesirable tetraploidy. Like senescence, quiescence is characterized by CDK inactivation, the absence of S-phase- and Mitosis-promoting cyclins and the pres- ence of hypo-phosphorylated pocket proteins. However, unlike senescence, where CDKs are inhibited by p21 or/and p16, the major CKI involved in quiescent arrest is p27Kip1 (p27) (37–39). Unlike p21, p27 induction is independent of the p53 pathway (39) and its levels/activity is primarily regulated by translation, phos- phorylation, and Skp2-mediated degradation (40). Although p53 and p21 were also implicated in the cell cycle arrest following growth factor removal (41, 42), p53 integrity does not seem to be essential for quiescence (43) (Fig. 2). However, a redundancy of p27 with p21 might explain why contact inhibition is not impaired in p27−/− cells (44). Interestingly, the expression of D-type cyclins, regulatory subunits of Cdk4/6 kinases and key signal inte- grators, strikingly differs between quiescence and senescence (Fig. 2). Downregulation of cyclin Ds is a hallmark of quiescence and their rapid mitogen-dependent induction is invariably associated with cell cycle entry (42, 45, 46). In sharp contrast, D cyclins are stabi- lized (cyclin D1) and even overexpressed (cyclin D2) in senescent cells (17, 35, 43, 47). In conjunction with the large-cell phenotype that is observed in senescent cells, these results suggest that the
21 2 mTOR, Metabolism and Cell Senescence mitogenic pathways involved in cell growth are active in senescent cells (see below) and might even have a positive role in senescence regulation. Indeed, several recent publications have connected the “hypertrophic” phenotype of senescent cells with activity of mTOR, a master cell growth regulator (48), although this topic is still controversial (49). Originally identified in budding yeast mutants conferring resis- tance to a potent antifungal metabolite (50), the target of rapamy- cin (TOR) is at the core of a vast signaling pathway regulating cell growth and metabolism in virtually all eukaryotes. The complexity of this pathway, which couples energy and nutrient abundance to the execution of cellular growth and division, relies on the fact that TOR simultaneously senses energy (ATP state), nutrients, stress, as well as growth factors (51, 52). In addition to its growth-related functions, the TOR pathway is also wired to the regulators of cell cycle machinery such as G1 cyclins (D-type and E-type) control- ling G1/S-phase progression (see below and Fig. 3). It is therefore not surprising that TOR is essential for many developmental and physiological processes (53) while deregulation in its signaling has been implicated in a wide variety of diseases (52). mTOR (“m” stands for mammalian or, more recently, mechanistic), which belongs to the phosphoinositide 3-kinase (PI3K)-related protein serine/threonine kinase family (PIKK), forms two distinct multi- protein complexes, having distinctive physical structures and functions: mTOR complex 1 (mTORC1), which is sensitive to rapamycin owing to association with the peptidyl-prolyl cis/trans isomerase FKBP12 (50), and mTORC2, which is not sensitive to rapamycin in most cases. Their assembly, substrate specificity, and regulation are defined by specific accessory proteins: Raptor (regu- latory associated protein of mTOR) being the most prominent for mTORC1, and Rictor (rapamycin-insensitive companion of mTOR), which is specific for mTORC2. Since the regulatory inputs and the cellular actions of mTOR are far too complex for the scope of this chapter (and are reviewed in ref. 53, 54), I outline only some “essentials” focusing mainly on mTORC1, the better characterized of the two. mTORC1, which is activated by nutrients, growth factors, and cellular energy status, regulates temporal aspects of cellular growth including protein synthesis, ribosome biogenesis, lipid synthesis, nutrient import, and autophagy. Best-known substrates of mTORC1 are p70 ribosomal protein S6 kinases (S6K1 and S6K2) and the eIF4E binding proteins (4E-BP1 and 4E-BP2) by which it controls protein synthesis. The positive and negative control of 4. The mTOR Pathway Essentials
22 V. Dulic mTORC1 in response to intracellular and extracellular stimuli is mediated by the phosphorylation and subsequent inhibition of Tuberous sclerosis 1 (TSC1, hamartin)/TSC2 (tuberin) complex (Fig. 3). TSC2 subunit, a hub for a variety of cues impinging on mTORC1, serves as GTPase activating protein (GAP), which itself inhibits the small GTPase Rheb, a direct mTORC1 activator (51, 53, 55). TSC2 activity is regulated by several kinases: negatively, by Akt and ERK1/2 (activate mTORC1) and positively, by AMPK and GSK3β (inhibit mTORC1). Importantly, the Wnt pathway also regulates TSC1/TSC2, via inhibition of GSK3β (53) (see Fig. 4). mTORC2 is thought to regulate mainly cytoskeleton orga- nization and cell survival by phosphorylating of AGC kinase family members, including Akt, SGK1, and PKC (52, 53) (Fig. 3). Although little is known about upstream regulators of mTORC2 (56), recent reports showing that its activation requires association with ribosomes suggest that it is active only in growing cells (57). Fig. 3. Interactions between mTOR signaling, p53 and cell cycle regulators.While mTORC1 controls cell growth/division and metabolism,TORC2 mainly controls cell shape and activates Akt.TSC1/TSC2 complex plays a pivotal role as a hub for both positive and negative cues that control mTORC1 activity via signaling kinases (see ref. 53). mTOR controls cell division by regulating synthesis of cyclin D and activity/localization of CDK inhibitor p27. While in the nucleus, p27 inhibits CDKs (cD- K4/K6 and cE-K2) thereby blocking cell cycle progression (like in quiescence). Growth factors stimulate mTOR and ERK1/2 that, via Akt, RSK and SGK, mediate p27 phosphorylation leading to its cytoplasmic sequestration. Cytoplasmic p27 pro- motes proliferation by stabilizing cyclin D-Cdk4/6 assembly and cell motility by inhibiting RhoA/ROCK (see ref. 71). In response to DNA damage or other stresses, p53 negatively regulates mTORC1 by activating AMPK and PTEN and inducing TSC2. Constitutive activation of mTOR (asterisk) could induce p53 translation (see ref. 86). Figure adapted from ref. 119. GF growth factors (insulin, IGF1); Sesn, sestrin.
23 2 mTOR, Metabolism and Cell Senescence As eluded above, mTOR pathway is also implicated in cell division, in part by controlling the synthesis of D-type cyclins (58, 59), major regulators of G1-S phase progression, whose induction in response to growth factors is regulated by the Ras-ERK1/2 path- way (60, 61) (Fig. 3). Recent data show that mTORC1 controls cell proliferation, but not growth, via its targets 4E-BPs, by stimu- lating the translation of proteins involved in cell cycle progression, including cyclin D3 (62). In addition, in the fission yeast model, TOR was also shown to control mitotic entry in response to nutrients. In this system, both rapamycin (by inhibiting TOR) and poor nitrogen sources stimulate the stress MAPK pathway (Spc1/Sty1), which, by activating Polo kinase and Cdc2 (Cdk1 ortholog, the main mitotic regulator) accelerated mitosis with reduced cell size as consequence (63). While no equivalent path- way was yet reported in other models, these observations are con- sistent with earlier results that linked mTOR signaling with cell-size control (64). Nevertheless, despite general downregulation of 5. mTOR and Cell Cycle Fig. 4. Possible roles of p53 and mTOR in senescence. Genotoxic stress (via p53–p21) and activated oncogenes (via p16) induce senescence by inactivating CDK and activating pRb (see Fig. 1). Classically, p53 and activated oncogenes induce autophagy by inhibiting mTOR pathway (grey box).Autophagy is implicated in senescence by stimulating specific secretory phenotype (SASP) that also promotes senescence. However, recent data suggest that mTOR might positively regulate senescence (grey dotted lines) as in the case of persistent Wnt1 signaling or PTEN knockdown. Pro-senescence mTOR activity could be also activated via oncogenic stress—by inducing autophagy, or via low p53 levels (grey dotted cross)—by promoting cell growth and D-type cyclin synthesis. Hypertrophy of senescent cells could be explained by deregulated cell signaling (not responding to extracellular cues) resulting in permanent mTOR activation. This is in agreement with results showing that in senescent cells cell growth and cell division are uncoupled events. For more details regarding mTOR regu- lation, see Fig. 3 and ref. 53. Figure adapted from ref. 119. GF growth factors.
24 V. Dulic protein translation in mitosis (65), several recent papers suggest that mTORC1 might also control protein synthesis in mitosis by regulating expression of human Cdk1, which phosphorylates eukaryotic elongation factor 2 (eEF2); (66). Moreover, it seems that, at G2/M transition, Cdk1 might also regulate mTORC1 activity by phosphorylating Raptor (67, 68). The mTOR pathway also positively controls cell cycle by regulating the activity and subcellular localization of the CKI p27, negative regulator of G1/S progression (Fig. 3). In the presence of growth factors, Akt (activated by mTORC2), RSK (activated by ERK1/2), and SKG (activated by mTORC1, mTORC2 and Pdk1) phosphorylate p27, resulting in its cyto- plasmic retention (69–71). This not only prevents p27 to exert its nuclear role as CDK inhibitor, by inactivating CDKs control- ling DNA replication (as cyclin E-Cdk2), but also promote cell proliferation by stabilizing assembly of cyclin D-CDK4/6 com- plexes and increasing cell motility, via inhibition of RhoA signal- ing (71) (Fig. 3). In addition, mTORC2 regulates cell proliferation via AKT-mediated inhibition of forkhead box pro- tein O (FOXO) transcription factors playing a key role in pro- moting apoptosis (72). Importantly, the FOXO family members were also shown to block cell cycle by inducing p27 (73) or repressing D-type cyclins (74). In the light of increasing evidence that altered metabolism is not only a common feature of many cancer cells but that it can also greatly contribute to malignant transformation (75), the discov- ery that p53 also regulates metabolic pathways and interacts with mTOR/AKT pathways is not that surprising (reviewed in refs. 76, 77) (Fig. 3 and 4). Indeed, it has been shown that, in response to diverse stress signals, activation of p53 directly or indirectly inhibits mTOR activity, thus regulating its downstream targets, including those involved in the activation of autophagy, a recently described tumor suppression mechanism that is involved in senes- cence (54, 78) (see also Chapter 3). The role of autophagy in senescence is discussed in more detail in the chapter by M. Narita (see Chapter 1). In addition to its housekeeping role in the main- tenance of energy homeostasis, autophagy, which is negatively regulated by mTOR, is also induced by a variety of stress stimuli, such as nutrient depletion (when mTOR is inhibited), playing a vital role in preserving cellular viability through the degradation of cellular proteins and organelles (53, 79). 6. TOR, p53, and Genotoxic Stress
25 2 mTOR, Metabolism and Cell Senescence The mechanism by which p53 negatively regulates mTOR involves, in part, the activation of AMPK and requires its target TSC1/TSC2 complex (Fig. 3), both of which respond to energy deprivation in cells (53, 80). In response to genotoxic stress caused by DNA damage, p53 inhibits mTORC1 pathway, by phosphory- lating Sestrin 1 and 2 that activate AMPK (81), as well as the IGF-1/Akt pathway, by inducing transcription of PTEN (82) (Fig. 3). In addition, p53 can repress mTORC1 by inducing directly TSC2 (83) or AMPK, which suppresses translation by acti- vating TSC2 (80) or by directly inhibiting mTORC1 (54, 84). However, it has been reported that AMPK might also control the p53 activity, like in the case of glucose starvation, which induces the transient AMPK–mediated phosphorylation and activation of p53 leading to reversible cell cycle arrest (85, 86). There are sev- eral examples showing that conversely, mTOR can also control p53. Upon glucose removal, mTOR negatively regulates p53 by activating PP2A (87). However, some results suggest that deregu- lated (constitutive) mTOR activity might, in response to nutrient or genomic stress, induce p53 translation, which in conjunction with activation by AMPK, induces apoptosis (88). This implies that, at least in certain biological contexts, mTOR downregula- tion/inactivation is required to prevent cell death caused by stress stimuli. Thus, p53 and mTOR signaling networks can cross-talk and coordinately regulate cell growth, proliferation and death. The mTOR pathway has been shown to have opposing roles on cellular senescence. On the one hand, by inhibiting autophagy (78, 86, 89), mTOR is considered as a negative regulator of senescence (Fig. 4), which is in agreement with its widely accepted negative role in organismal ageing (90). Moreover, as mentioned above, mTOR is inhibited by p53, a bona fide pro-senescence positive reg- ulator (Figs. 3 and 4). On the other hand, the hypertrophic pheno- type of senescent cells (Fig. 2) and increased production of secretory proteins (SASP; Fig. 4), which requires high metabolic activity, are in apparent contradiction with cellular shrinkage that is usually associated with autophagy and mTOR inhibition (as in quiescence). Indeed, in the case of oncogene or DNA damage-induced senes- cence, the onset of both senescence and autophagy correlates with inhibition of mTORC1 and mTORC2 activity (91). This might suggest a negative feedback between mTOR and senescence. These findings are also in agreement with earlier observations that acti- vation of p53 by genotoxic stress inhibits mTOR pathway by acti- vating both AMPK and PTEN (Fig. 3) (81, 86). Likewise, persistent 7. mTOR’ s Role in Senescence: Repressor or Inducer?
26 V. Dulic AMPK activation leads to senescence in mouse embryo fibroblasts (MEF) in the presence of functional p53 (85), which is consistent with earlier observation that AMP levels strongly increase in senes- cent cells (92). However, it is also possible that mTOR activation via different stimuli could be involved in the induction of senescence. This pro- senescence role of mTOR was first uncovered by manipulation of its upstream regulators or downstream targets. Pandolfi and col- leagues found that overexpression of an mTOR target eIF-4E induced senescence both in vitro and in vivo (93). More recently, activation of the PI3K/Akt and mTOR pathways by PTEN knock- down has been shown to induce senescence by promoting accu- mulation of p53 and p21 (94). Similarly, in an elegant in vivo skin model, Gutkind and colleagues showed that continuous mTOR activation induced by persistent Wnt1 signaling (via inhibiting GSK3; (95)) promoted epithelial stem cell exhaustion by provok- ing senescence (96). Importantly, senescence and hair loss were abolished by the presence of the mTOR inhibitor rapamycin sug- gesting that mTOR stimulation is a direct consequence of Wnt1 expression (96) (Fig. 4). These results connect persistent mitogen stimulation and mTOR activation with senescence. Moreover, they are in agreement with accumulated evidence connecting accumulation of senescent cells with ageing (7, 9). Indeed, increased mTOR activity was also observed in hematopoietic stem cells from old mice (97), whereas mTOR inhibition by rapamycin could extend lifespan both in invertebrates (98, 99) and in mice (97, 100, 101). In the context of OIS, mTOR promotes lysosome biogenesis, which is required for autophagy and senescence-specific secretory phenotype, in a newly identified cytoplasmic compartment (termed TOR-autophagy spatial coupling compartment or TASCC) (102). While these results clearly indicate that mTOR has pro-senescence functions (Fig. 4), they are surprising in light of earlier report, con- necting the induction of senescence by oncogenic ras with mTOR downregulation (91). However, in this previous report it was clearly shown that increased phosphorylation of both mTORC1 and mTORC2 substrates (S6K and FoxO3a, respectively) coin- cided more with the onset of cycle arrest (and senescence) than with ras-induced cell cycle entry, as proposed by the authors. Indeed, transient activation of mTOR was also observed in cells in which senescence was triggered by the genotoxic agent etoposide. Therefore, while the results showing that appearance of late senes- cence and autophagy markers coincided with downregulation of mTOR activity, overall this work supports a positive role of mTOR pathway in the induction of autophagy at the onset of senescence. Although mainly focused on autophagy-related roles of mTOR, the above results are consistent with the model proposed by Blagosklonny and coworkers, whose recent experiments suggest
27 2 mTOR, Metabolism and Cell Senescence that cellular growth and persistent mTOR activity might be implicated in the onset of the senescence program. Consistent with their finding that serum stimulation is required for both the senes- cent phenotype (large flat cells) and irreversibility of the cell cycle arrest induced by overexpression of the CDK inhibitor p21 or DNA damage, they observed that a key mTOR target, S6K, is strongly phosphorylated in senescent cells (103). Moreover, in agreement with our earlier observation (17), senescent cells also accumulated another mTOR target, cyclin D1, but the biological significance of its presence is not clear (see below). Interestingly, while rapamycin, like serum starvation, prevented S6K phosphory- lation and diminished the senescent marker β-gal staining, it failed to completely abolish large flat cell morphology and to inhibit cyclin D1 induction. While these results imply that mTORC1 is not essential for these events, it would be interesting to test whether they are controlled by mTORC2, which is less sensitive to rapamy- cin and which is implicated in both cell shape and, via its target Akt, in cyclin D1 stability (Figs. 3 and 4). In addition, rapamycin prevents the onset of senescence if added simultaneously with senescence inducers (p21 induction, genotoxic agents), but fails to revert the already established senescence (104). Taken together, the above results suggest that (transient?) mTOR activity might positively control senescence but it is still unclear how DDR network controls activity of this pathway. How one can reconcile the data suggesting a positive role of mTOR in senescence with those showing that p53 inhibits the mTOR pathway (76, 77)? In the work of Narita’s team the possible inter- actions between p53 and the mTOR pathway were not examined, because in OIS cell cycle arrest is mainly mediated by the CDK inhibitor p16, which is not regulated by p53 (91) (Fig. 1). In the quest to decipher the respective roles of mTOR and p53 in the senescence program, Blagosklonny and coworkers took advantage of nutlin-3A, a specific small-molecule MDM2 antagonist, which strongly induces p53 expression thereby triggering its transcrip- tional activity in a non-genotoxic manner (105). Surprisingly, while previous work has shown that nutlin-3A induces senescence in mouse fibroblasts (106), in human fibrosarcoma cells HT1080 and WI-38 human fibroblasts, nutlin-3A induces reversible cell cycle exit resembling quiescence (107). By exploiting a HT1080- derived cell line in which p21 is expressed from an inducible promoter, Demidenko et al. showed that nutlin-3A or p53 overex- pression could “convert” p21-induced senescence into a quies- cence-like state (108). Nutlin-3A was, however, much less efficient 8. Connections Between mTOR and p53 in Senescence— Lessons from Nutlin-3A
28 V. Dulic in preventing senescence in the presence of DNA damage by H2 O2 , which also stimulates the phosphorylation of mTOR (108). In addition, both nutlin-3A and rapamycin suppress senescence with- out interfering with cell cycle arrest, which led the authors to sug- gest that high p53 levels preclude the onset of senescence by inhibitingmTORandinducingquiescence(108,109).Accordingly, knockdown of TSC2, a negative mTORC1 regulator (Fig. 3), par- tially compromises nutlin-3-induced quiescence, resulting in accu- mulation of senescent cells (110). In agreement with the positive role of mTOR in senescence, in cell lines in which nutlin-3A did induce senescence (termed “senescence-prone”), mTOR was not inhibited, whereas the quiescence was “restored” in the presence of rapamycin (110). Unfortunately, these studies did not provide insight into molecular mechanisms explaining differences between “senescence-prone” and “quiescence-prone” cells. In agreement with the above experiments, when exposed to etoposide, serum- deprived or rapamycin-treated fibroblasts (or epithelial cells) failed to become senescent despite p21 induction, as drug removal, con- comitant with serum addition, enabled proliferation (111). In con- trast, serum addition in the presence of etoposide induced senescence, presumably by activating mTOR. These results sup- port the hypothesis that quiescence (or mTOR inhibition) com- promises senescence. However, it is not clear why the checkpoints were not activated once cells exposed to etoposide reentered the cell cycle (assuming that the DNA damage had not been repaired) and how the cells got rid of high p21 levels. This work, however, did not address the role of the CKI p27 in p53-induced quiescence. This point is relevant for understand- ing the mechanism that induces quiescence, since mTOR inhibi- tion by rapamycin or p53 overexpression might also block SGK/ Akt-mediated p27 phosphorylation and cytoplasmic localization leading to its nuclear accumulation and activation (70, 71) (Fig. 3). One could imagine that, in the presence of rapamycin or absence of serum, p27-dependent CDK inactivation together with cyclin D1 down-regulation could also contribute to quiescence. Reactivation of mTOR (by serum addition) would revert this pro- cess, enabling cell cycle entry and progression, probably by degrad- ing p27 and p21 and inducing G1 cyclins (D- and E-type). Overall, the results of Blagosklonny’s team are consistent with the idea that mTOR positively regulates senescence, which might be because p53 levels are not sufficient to inhibit mTOR-dependent cell growth. According to their hypothesis, the “true role” of p53 (when highly expressed) would be to induce quiescence by inhibit- ing mTOR, which in turn would block the onset of senescence. Consequently, senescence occurs in situations where “the condi- tions for quiescence are not met” (low p53 levels) and where p53 “fails” to suppress the mTOR pathway (108, 112). This hypothesis is consistent with the data showing that quiescence prevents
29 2 mTOR, Metabolism and Cell Senescence p21-induced senescence or inappropriate differentiation due to expression of the transcriptional repressor HES1, playing a key role in the reversibility of this non-proliferative state (33) (Fig. 2). There are, however, several caveats regarding the use of nutlin- 3A as a tool to study the role of p53 and mTOR in senescence. For example, strong p53 induction by nutlin-3A is not usually observed in either quiescence or senescence and it is not clear which biologi- cal event produces equivalent amounts of p53. As a matter of fact, primary fibroblasts expressing the HPV16-E6 (hereafter E6) onco- gene that degrades p53 (113) can become quiescent in the absence of serum or confluence, whereas strong p21 induction in senescent cells occurs in the absence of significant increases in p53 protein levels (43). Therefore, the hypothesis that high p53 levels induce quiescence while low p53 levels lead to senescence (108, 109) should be verified in other experimental models. For example, our recent results showed that irreversible cell cycle arrest by genotoxic drugs in non-transformed human cells is independent on the degree of p53 activation (or levels) or even p21 levels but rather on the efficiency of p21 to inhibit different CDKs (Lossaint, 2011). Finally, as mentioned above, this model is in apparent contradic- tion with the results showing that in other cell lines nutlin-3A does not induce quiescence but instead elicits endoreduplication, giving rise to tetraploid cells (114), senescence—due to persistent p21 expression (106, 115), or even apoptosis (116). One explanation could be that nutlin-3A might have other targets than p53 (pRb?) or that its effects might be dependent on the experimental proto- col and cell type. The above-mentioned work mostly addressed the role of mTOR in premature (induced) senescence but less is known regarding its implication in replicative senescence. Several earlier observations, however, are consistent with the notion that the mTOR pathway might be active in senescent cells and that it is not controlled by p53 or affected by its status. In fact, in human fibroblasts aged in vitro a suppression of the p53/p21 pathway compromises cell cycle arrest, senescence-specific cyclin D2 induction (see below), as well as formation of SAHF, but it does not prevent large flat cell morphology or cyclin D1 accumulation (43). This implies that these two sets of key events associated with senescence, i.e. irre- versible cell cycle arrest and cell growth, might be uncoupled, which can also explain the senescent phenotype (Fig. 4). In addi- tion, some data suggest that mitogenic pathways in senescent cells might be also deregulated. Firstly, in sharp contrast to early passage fibroblasts, serum withdrawal in senescent fibroblasts does not 9. mTOR and Senescence: Constitutive Activation or Deregulation?
30 V. Dulic affect cyclin D1 mRNA or cyclin E1 expression or protein synthesis. The CDKs associated with these cyclins are, however, inhibited by p21, explaining the absence of DNA replication (17, 26) (Fig. 1). Interestingly, in agreement with other publications (35, 117), late senescence is associated with strong accumulation of cyclin D2, whose levels are not affected by serum withdrawal. The presence of G1 cyclins, and especially of cyclin D2, in senescent cells is intrigu- ing and it is not clear whether they might play a role in senescence or they merely reflect deregulated mTOR activity. One possibility is that cyclin D1 is part of the DNA repair machinery, as suggested recently (118). Secondly, late-passage p53-deficient fibroblasts (E6) failed to become quiescent upon serum withdrawal, which is documented by both the absence of cyclin D1 or cyclin A down- regulation and p27 induction (or activation). Consequently, CDKs controlling DNA replication were highly active even in the absence of serum. However, these cells failed to proliferate due to deleteri- ous effects of DNA damage resulting in aberrant mitoses, endorep- lication, or cell death (43). It is therefore possible that, concomitant with increased population doubling, gradual deregulation of the mTOR pathway contributes to the senescent phenotype. In the absence of the p53/pRb safeguard system, this deregulation pro- vides a fertile ground for tumorigenesis. In conclusion, the role of the mTOR pathway in senescence is still controversial, partly due to the various models and experimen- tal designs employed in different studies. Apart from the studies manipulating upstream mTOR regulators (PTEN, Wnt1) that reveal pro-senescence mTOR-functions, most of the work has focused on the autophagy-related aspects of senescence, which appeared to exclude mTOR as an important regulator of senes- cence. Few researchers, however, considered a hypertrophic phe- notype of senescent cells and elevated levels of D-type cyclins as an evidence of mTOR activity. Future work will show whether or not this activity is actually required for the onset of senescence, as some investigators proposed, or it is merely the result of senescence- associated deregulation of the mTOR pathway. If the former is the case, it will be interesting to learn whether, and if so, how p53 and/or pRb networks contribute to its induction. Acknowledgments I am grateful to Drs Anne Brunet (Stanford University, Palo Alto, USA) and Jacques Piette (CRBM, Montpellier, France) for their insightful comments.
31 2 mTOR, Metabolism and Cell Senescence References 1. Hayflick L (1965) The limited in vitro life- time of human diploid cell strains. Exp Cell Res 37:614–636 2. Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345:458–460 3. Olovnikov AM (1973) A theory of margino- tomy. The incomplete copying of template margin in enzymic synthesis of polynucle- otides and biological significance of the phe- nomenon. J Theor Biol 41:181–190 4. Stein GH, Dulic V (1995) Origins of G1 arrest in senescent human fibroblasts. Bioessays 17:537–543 5. Kuilman T, Michaloglou C, Mooi WJ, Peeper DS (2010) The essence of senescence. Genes Dev 24:2463–2479 6. Adams PD (2009) Healing and hurting: molecular mechanisms, functions, and pathol- ogies of cellular senescence. Mol Cell 36: 2–14 7. Rodier F, Campisi J (2011) Four faces of cel- lular senescence. J Cell Biol 192:547–556 8. Rodier F, Coppe JP, Patil CK, Hoeijmakers WA, Munoz DP, Raza SR, Freund A, Campeau E, Davalos AR, Campisi J (2009) Persistent DNA damage signalling triggers senescence- associated inflammatory cytokine secretion. Nat Cell Biol 11:973–979 9. Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B, Kirkland JL, van Deursen JM (2011) Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479:232–236 10. Kang TW, Yevsa T, Woller N, Hoenicke L, Wuestefeld T, Dauch D, Hohmeyer A, Gereke M, Rudalska R, Potapova A, Iken M, Vucur M, Weiss S, Heikenwalder M, Khan S, Gil J, Bruder D, Manns M, Schirmacher P, Tacke F, Ott M, Luedde T, Longerich T, Kubicka S, Zender L (2011) Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 479:547–551 11. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O et al (1995) A bio- marker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92:9363–9367 12. Lee BY, Han JA, Im JS, Morrone A, Johung K, Goodwin EC, Kleijer WJ, DiMaio D, Hwang ES (2006) Senescence-associated beta- galactosidase is lysosomal beta-galactosidase. Aging Cell 5:187–195 13. Bernardi R, Pandolfi PP (2007) Structure, dynamics and functions of promyelocytic leu- kaemia nuclear bodies. Nat Rev Mol Cell Biol 8:1006–1016 14. Vernier M, Bourdeau V, Gaumont-Leclerc MF, Moiseeva O, Begin V, Saad F, Mes-Masson AM, Ferbeyre G (2011) Regulation of E2Fs and senescence by PML nuclear bodies. Genes Dev 25:41–50 15. Narita M, Nunez S, Heard E, Narita M, Lin AW, Hearn SA, Spector DL, Hannon GJ, Lowe SW (2003) Rb-mediated heterochromatin forma- tion and silencing of E2F target genes during cellular senescence. Cell 113:703–716 16. Di Micco R, Sulli G, Dobreva M, Liontos M, Botrugno OA, Gargiulo G, Dal Zuffo R, Matti V, d’Ario G, Montani E, Mercurio C, Hahn WC, Gorgoulis V, Minucci S, d’Adda di Fagagna F (2011) Interplay between onco- gene-induced DNA damage response and heterochromatin in senescence and cancer. Nat Cell Biol 13:292–302 17. Dulic V, Drullinger LF, Lees E, Reed SI, Stein GH (1993) Altered regulation of G1 cyclins in senescent human diploid fibroblasts: accumulation of inactive cyclin E-Cdk2 and cyclin D1-Cdk2 complexes. Proc Natl Acad Sci U S A 90:11034–11038 18. Burkhart DL, Sage J (2008) Cellular mecha- nisms of tumour suppression by the retino- blastoma gene. Nat Rev Cancer 8:671–682 19. Chicas A, Wang X, Zhang C, McCurrach M, Zhao Z, Mert O, Dickins RA, Narita M, Zhang M, Lowe SW (2010) Dissecting the unique role of the retinoblastoma tumor sup- pressor during cellular senescence. Cancer Cell 17:376–387 20. Martin N, Benhamed M, Nacerddine K, Demarque MD, van Lohuizen M, Dejean A, Bischof O (2012) Physical and functional interaction between PML and TBX2 in the establishment of cellular senescence. EMBO J 31:95–109 21. Herbig U, Jobling WA, Chen BP, Chen DJ, Sedivy JM (2004) Telomere shortening trig- gers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a). Mol Cell 14:501–513 22. Vousden KH, Prives C (2009) Blinded by the light: the growing complexity of p53. Cell 137:413–431 23. Baus F, Gire V, Fisher D, Piette J, Dulic V (2003) Permanent cell cycle exit in G2 phase after DNA damage in normal human fibroblasts. EMBO J 22:3992–4002
32 V. Dulic 24. Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L, Sharpless NE (2004) Ink4a/Arf expression is a biomarker of aging. J Clin Invest 114:1299–1307 25. Alcorta DA, Xiong Y, Phelps D, Hannon G, Beach D, Barrett JC (1996) Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc Natl Acad Sci U S A 93:13742–13747 26. Stein GH, Drullinger LF, Soulard A, Dulic V (1999) Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mecha- nisms of senescence and differentiation in human fibroblasts. Mol Cell Biol 19: 2109–2117 27. Gil J, Peters G (2006) Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol 7:667–677 28. Coller HA (2011) Cell biology. The essence of quiescence. Science 334:1074–1075 29. Coller HA (2007) What’s taking so long? S-phase entry from quiescence versus prolif- eration. Nat Rev Mol Cell Biol 8:667–670 30. Ferbeyre G, de Stanchina E, Querido E, Baptiste N, Prives C, Lowe SW (2000) PML is induced by oncogenic ras and promotes premature senescence. Genes Dev 14: 2015–2027 31. Lemons JM, Feng XJ, Bennett BD, Legesse- Miller A, Johnson EL, Raitman I, Pollina EA, Rabitz HA, Rabinowitz JD, Coller HA (2010) Quiescent fibroblasts exhibit high metabolic activity. PLoS Biol 8:e1000514 32. Coller HA, Sang L, Roberts JM (2006) A new description of cellular quiescence. PLoS Biol 4:e83 33. Sang L, Coller HA, Roberts JM (2008) Control of the reversibility of cellular quies- cence by the transcriptional repressor HES1. Science 321:1095–1100 34. Blagosklonny MV (2006) Cell senescence: hypertrophic arrest beyond the restriction point. J Cell Physiol 209:592–597 35. Shelton DN, Chang E, Whittier PS, Choi D, Funk WD (1999) Microarray analysis of repli- cative senescence. Curr Biol 9:939–945 36. Pardee AB (1989) G1 events and regulation of cell proliferation. Science 246:603–608 37. Polyak K, Kato JY, Solomon MJ, Sherr CJ, Massague J, Roberts JM, Koff A (1994) p27Kip1, a cyclin-Cdk inhibitor, links trans- forming growth factor-beta and contact inhi- bition to cell cycle arrest. Genes Dev 8:9–22 38. Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM (1996) A syndrome of multior- gan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 85:733–744 39. Sherr CJ, Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13:1501–1512 40. Chu IM, Hengst L, Slingerland JM (2008) The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to antican- cer therapy. Nat Rev Cancer 8:253–267 41. Itahana K, Dimri GP, Hara E, Itahana Y, Zou Y, Desprez PY, Campisi J (2002) A role for p53 in maintaining and establishing the qui- escence growth arrest in human cells. J Biol Chem 277:18206–18214 42. Pajalunga D, Mazzola A, Salzano AM, Biferi MG, De Luca G, Crescenzi M (2007) Critical requirement for cell cycle inhibitors in sus- taining nonproliferative states. J Cell Biol 176:807–818 43. Dulic V, Beney GE, Frebourg G, Drullinger LF, Stein GH (2000) Uncoupling between phe- notypic senescence and cell cycle arrest in aging p21-deficient fibroblasts. Mol Cell Biol 20:6741–6754 44. Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, Horii I, Loh DY (1996) Mice lacking p27(Kip1) display increased body size, multiple organ hyperpla- sia, retinal dysplasia, and pituitary tumors. Cell 85:707–720 45. Matsushime H, Roussel MF, Ashmun RA, Sherr CJ (1991) Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell 65:701–713 46. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G (1993) Cyclin D1 is a nuclear pro- tein required for cell cycle progression in G1. Genes Dev 7:812–821 47. Atadja P, Wong H, Veillete C, Riabowol K (1995) Overexpression of cyclin D1 blocks proliferation of normal diploid fibroblasts. Exp Cell Res 217:205–216 48. Pani G (2011) From growing to secreting: new roles for mTOR in aging cells. Cell Cycle 10:2450–2453 49. Blagosklonny MV (2011) Cell cycle arrest is not senescence. Aging (Albany NY) 3:94–101 50. Heitman J, Movva NR, Hall MN (1991) Targets for cell cycle arrest by the immuno- suppressant rapamycin in yeast. Science 253:905–909 51. Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism. Cell 124:471–484
33 2 mTOR, Metabolism and Cell Senescence 52. Dazert E, Hall MN (2011) mTOR signaling in disease. Curr Opin Cell Biol 23:744–755 53. Zoncu R, Efeyan A, Sabatini DM (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 12:21–35 54. Sengupta S, Peterson TR, Sabatini DM (2010) Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell 40:310–322 55. Conn CS, Qian SB (2011) mTOR signaling in protein homeostasis: less is more? Cell Cycle 10:1940–1947 56. Oh WJ, Jacinto E (2011) mTOR complex 2 signaling and functions. Cell Cycle 10: 2305–2316 57. Zinzalla V, Stracka D, Oppliger W, Hall MN (2011) Activation of mTORC2 by association with the ribosome. Cell 144:757–768 58. Averous J, Fonseca BD, Proud CG (2008) Regulation of cyclin D1 expression by mTORC1 signaling requires eukaryotic initi- ation factor 4E-binding protein 1. Oncogene 27:1106–1113 59. Balcazar N, Sathyamurthy A, Elghazi L, Gould A, Weiss A, Shiojima I, Walsh K, Bernal-Mizrachi E (2009) mTORC1 activa- tion regulates beta-cell mass and proliferation by modulation of cyclin D2 synthesis and sta- bility. J Biol Chem 284:7832–7842 60. Lavoie JN, L’Allemain G, Brunet A, Muller R, Pouyssegur J (1996) Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK path- way. J Biol Chem 271:20608–20616 61. Hitomi M, Stacey DW (1999) Cyclin D1 pro- duction in cycling cells depends on ras in a cell- cycle- specific manner. Curr Biol 9:1075–1084 62. Dowling RJ, Topisirovic I, Alain T, Bidinosti M, Fonseca BD, Petroulakis E, Wang X, Larsson O, Selvaraj A, Liu Y, Kozma SC, Thomas G, Sonenberg N (2010) mTORC1- mediated cell proliferation, but not cell growth, controlled by the 4E-BPs. Science 328:1172–1176 63. Petersen J, Nurse P (2007) TOR signalling regulates mitotic commitment through the stress MAP kinase pathway and the Polo and Cdc2 kinases. Nat Cell Biol 9:1263–1272 64. Fingar DC, Salama S, Tsou C, Harlow E, Blenis J (2002) Mammalian cell size is con- trolled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev 16: 1472–1487 65. Pyronnet S, Dostie J, Sonenberg N (2001) Suppression of cap-dependent translation in mitosis. Genes Dev 15:2083–2093 66. Smith EM, Proud CG (2008) cdc2-cyclin B regulates eEF2 kinase activity in a cell cycle- and amino acid-dependent manner. EMBO J 27:1005–1016 67. Ramirez-Valle F, Badura ML, Braunstein S, Narasimhan M, Schneider RJ (2010) Mitotic raptor promotes mTORC1 activity, G(2)/M cell cycle progression, and internal ribosome entry site-mediated mRNA translation. Mol Cell Biol 30:3151–3164 68. Gwinn DM, Asara JM, Shaw RJ (2010) Raptor is phosphorylated by cdc2 during mitosis. PLoS One 5:e9197 69. Hong F, Larrea MD, Doughty C, Kwiatkowski DJ, Squillace R, Slingerland JM (2008) mTOR-raptor binds and activates SGK1 to regulate p27 phosphorylation. Mol Cell 30: 701–711 70. Toker A (2008) mTOR and Akt signaling in cancer: SGK cycles in. Mol Cell 31:6–8 71. Wander SA, Zhao D, Slingerland JM (2011) p27: a barometer of signaling deregulation and potential predictor of response to tar- geted therapies. Clin Cancer Res 17:12–18 72. Guertin DA, Stevens DM, Thoreen CC, Burds AA, Kalaany NY, Moffat J, Brown M, Fitzgerald KJ, Sabatini DM (2006) Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. Dev Cell 11: 859–871 73. Medema RH, Kops GJ, Bos JL, Burgering BM (2000) AFX-like Forkhead transcription fac- tors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404:782–787 74. Schmidt M, Fernandez de Mattos S, van der Horst A, Klompmaker R, Kops GJ, Lam EW, Burgering BM, Medema RH (2002) Cell cycle inhibition by FoxO forkhead transcrip- tion factors involves downregulation of cyclin D. Mol Cell Biol 22:7842–7852 75. Kroemer G, Pouyssegur J (2008) Tumor cell metabolism: cancer’s Achilles’ heel. Cancer Cell 13:472–482 76. Vousden KH, Ryan KM (2009) p53 and metabolism. Nat Rev Cancer 9:691–700 77. Feng Z (2010) p53 regulation of the IGF-1/ AKT/mTOR pathways and the endosomal compartment. Cold Spring Harb Perspect Biol 2:a001057 78. Young AR, Narita M (2010) Connecting autophagy to senescence in pathophysiology. Curr Opin Cell Biol 22:234–240 79. Kroemer G, Marino G, Levine B (2010) Autophagy and the integrated stress response. Mol Cell 40:280–293
34 V. Dulic 80. Inoki K, Zhu T, Guan KL (2003) TSC2 mediates cellular energy response to control cell growth and survival. Cell 115:577–590 81. Budanov AV, Karin M (2008) p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling. Cell 134: 451–460 82. Feng Z, Hu W, de Stanchina E, Teresky AK, Jin S, Lowe S, Levine AJ (2007) The regula- tion of AMPK beta1, TSC2, and PTEN expression by p53: stress, cell and tissue specificity, and the role of these gene products in modulating the IGF-1-AKT-mTOR path- ways. Cancer Res 67:3043–3053 83. Feng Z, Zhang H, Levine AJ, Jin S (2005) The coordinate regulation of the p53 and mTOR pathways in cells. Proc Natl Acad Sci U S A 102:8204–8209 84. Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk BE, Shaw RJ (2008) AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 30:214–226 85. Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, Birnbaum MJ, Thompson CB (2005) AMP-activated protein kinase induces a p53- dependent metabolic checkpoint. Mol Cell 18:283–293 86. Feng Z, Levine AJ (2010) The regulation of energy metabolism and the IGF-1/mTOR pathways by the p53 protein. Trends Cell Biol 20:427–434 87. Kong M, Fox CJ, Mu J, Solt L, Xu A, Cinalli RM, Birnbaum MJ, Lindsten T, Thompson CB (2004) The PP2A-associated protein alpha4 is an essential inhibitor of apoptosis. Science 306:695–698 88. Lee CH, Inoki K, Karbowniczek M, Petroulakis E, Sonenberg N, Henske EP, Guan KL (2007) Constitutive mTOR activa- tion in TSC mutants sensitizes cells to energy starvation and genomic damage via p53. EMBO J 26:4812–4823 89. Kim J, Kundu M, Viollet B, Guan KL (2011) AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 13:132–141 90. Selman C, Tullet JM, Wieser D, Irvine E, Lingard SJ, Choudhury AI, Claret M, Al-Qassab H, Carmignac D, Ramadani F, Woods A, Robinson IC, Schuster E, Batterham RL, Kozma SC, Thomas G, Carling D, Okkenhaug K, Thornton JM, Partridge L, Gems D, Withers DJ (2009) Ribosomal pro- tein S6 kinase 1 signaling regulates mamma- lian life span. Science 326:140–144 91. Young AR, Narita M, Ferreira M, Kirschner K, Sadaie M, Darot JF, Tavare S, Arakawa S, Shimizu S, Watt FM, Narita M (2009) Autophagy mediates the mitotic senescence transition. Genes Dev 23:798–803 92. Zwerschke W, Mazurek S, Stockl P, Hutter E, Eigenbrodt E, Jansen-Durr P (2003) Metabolic analysis of senescent human fibroblasts reveals a role for AMP in cellular senescence. Biochem J 376:403–411 93. Ruggero D, Montanaro L, Ma L, Xu W, Londei P, Cordon-Cardo C, Pandolfi PP (2004) The translation factor eIF-4E pro- motes tumor formation and cooperates with c-Myc in lymphomagenesis. Nat Med 10: 484–486 94. Astle MV, Hannan KM, Ng PY, Lee RS, George AJ, Hsu AK, Haupt Y, Hannan RD, Pearson RB (2011) AKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: implications for tar- geting mTOR during malignancy. Oncogene 31:1949–1962 95. Inoki K, Ouyang H, Zhu T, Lindvall C, Wang Y, Zhang X, Yang Q, Bennett C, Harada Y, Stankunas K, Wang CY, He X, MacDougald OA, You M, Williams BO, Guan KL (2006) TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 126: 955–968 96. Castilho RM, Squarize CH, Chodosh LA, Williams BO, Gutkind JS (2009) mTOR mediates Wnt-induced epidermal stem cell exhaustion and aging. Cell Stem Cell 5:279–289 97. Chen C, Liu Y, Liu Y, Zheng P (2009) mTOR regulation and therapeutic rejuvenation of aging hematopoietic stem cells. Sci Signal 2:ra75 98. Vellai T, Takacs-Vellai K, Zhang Y, Kovacs AL, Orosz L, Muller F (2003) Genetics: influence of TOR kinase on lifespan in C. elegans. Nature 426:620 99. Kaeberlein M, Powers RW 3rd, Steffen KK, Westman EA, Hu D, Dang N, Kerr EO, Kirkland KT, Fields S, Kennedy BK (2005) Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science 310:1193–1196 100. Harrison DE, Strong R, Sharp ZD, Nelson JF, Astle CM, Flurkey K, Nadon NL, Wilkinson JE, Frenkel K, Carter CS, Pahor M, Javors MA, Fernandez E, Miller RA (2009) Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460:392–395 101. Kapahi P, Chen D, Rogers AN, Katewa SD, Li PW, Thomas EL, Kockel L (2010) With TOR, less is more: a key role for the conserved
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of our history, but they are Bank holidays. The closing of our banks is the one signal that for twenty-four hours we are free. The multifarious aspects of the theme are most bewildering. As Sir Henry Taylor said, “So manifold are the bearings of money upon the lives and character of mankind, that an insight which should search out the life of a man in his pecuniary relations would penetrate into almost every cranny of his nature. For if we take account of all the virtues with which money is mixed up, honesty, justice, charity, frugality, forethought, self-sacrifice, and of their correlative vices, it is a knowledge that goes near to cover the length and breadth of humanity.” It is certainly true that the amazingly extensive nature of the subject might lead one away into perfectly relevant discussions of almost every field of human activity; and nothing renders argument so unsatisfactory and inconclusive as to have unlimited scope. But in these pages the issue must be narrowed down and the question confined so far as possible to a very brief examination of one particular aspect of the subject, which will be created by formulating a deliberate contention and pursuing it by argument into some of the main channels of this perplexing problem. Even so it is likely that deep water will be reached, but, after all, a suggestion need not be driven to its utmost limits in all directions in order to establish its significance. In choosing a direct point of attack against this generally accepted belief we shall treat the matter more or less from a practical point of view. Without getting involved in abstract philosophic propositions, without entering too far into the sphere of economics and politics, without preaching high morality, though the words and teachings of preachers must be quoted, an endeavour will be made, by working out a definite line of reasoning, to submit as a whole some of the simpler and perhaps more personal considerations which have no doubt already occurred to many who have given the subject thought and reflection. No maxims will be laid down as to how money should be made, spent, saved, lent,
borrowed, invested, given or bequeathed, for the object is to strike at the root principle and shatter the ideal which underlies all those transactions, which colours men’s characters, influences their desires and aspirations, creates artificial class contrasts, and contributes largely to the general social confusion and chaos. Briefly, then, our contention is: That no individual is capable of possessing, spending, or administering more than a certain definite amount of money, which can be roughly described as a full competence, without producing positively harmful effects on himself as well as on those affected by his actions. In other words, the “rich man” is an impossibility in any decently organised economic State, and the accumulation of capital in individual hands is detrimental to the public good. That is what is meant by the saying from which the title of this volume is taken. It may appear at first sight to be an extreme view, because we have got so much accustomed to believing that a great deal of good can be done with money, and a great deal of happiness derived from it, that to be confronted with an uncompromising negation on such a time-honoured tradition may seem almost absurd. The argument is purposely intended to be completely comprehensive, and a case will be presented without exaggeration which will cover as much of the ground as possible, dealing with typical rather than exceptional instances by way of illustration. We find in human nature three characteristic impulses which serve as the mainspring and motive power in the gaining and spending of money: the passion for acquisition, the instinct for absolute property, and the desire to excel. No one would suggest that the passion for acquisition can be destroyed: it is neither possible nor desirable, but it can be prevented from running wild, and it can be controlled, though it does not seem to have occurred to many people that such control is expedient. The instinct for absolute property is very much overestimated, and this arises from the fact that we are accustomed to a system which hardly allows any satisfactory intermediate stage between property and positive need.
The craving for complete possession on any considerable scale only enters into the minds of those who covet their neighbours’ possessions. What a man wants and has every right to expect is security in the enjoyment of his necessaries and comforts, but this is precisely what in the vast majority of cases he does not get; and his want remaining unsatisfied is converted into a craving for absolute property. The desire to excel, which can undoubtedly be one of the finest human qualities, is in itself vitiated by the measure of money, which sets up an utterly false standard of excellence and converts pure ambition into a desire for material pre-eminence. However far we may travel, the problem will be continually resolving itself into some variation of the question as to how these impulses had best be regulated, and to what extent they have broken out beyond their legitimate bounds. But although the causes of the faith in money may be reduced to moral and psychological terms, there are economic as well as moral results, and it is not the metaphysical origins, but the practical results which must be looked into. At the outset we must acknowledge that our capacities of all kinds are strictly limited, whether moral, intellectual, or physical. An occasional saint, an occasional genius, or an occasional giant stretches the limit beyond its normal point, but the limit still remains. And yet we are foolish enough to believe that in regard to the possession, expenditure, and administering of riches there is no sum of money, however large, which we are not competent to deal with, and we are convinced that it is quite easy and unquestionably within the capacity of almost anyone to spend with benefit to himself and to others sums of money greatly in excess of what can cover in the widest sense his personal requirements. Whereas not only is it not easy, but as inquiry will show, it is purely and positively impossible, as impossible as it is to acquire vast knowledge with a limited brain capacity, or to endure more than a certain amount of physical strain with a limited muscular capacity. We are inclined also to think that men who have money and men who make money are ipso facto easily capable of spending the money properly, though we generally
make the mental reservation that if we had it ourselves we should spend it a great deal better. But the inheritance or accumulation of money does not imply by any means a special ability for spending it wisely. To put it plainly, such men have not, nor have we, nor has anyone this ability except in a very limited degree, far more limited than is generally supposed or ever admitted. The case against riches has been argued again and again on religious and moral grounds for over two thousand years, from Confucius to Tolstoy. But we are less impressed by the truth of it now than ever we were; and we still hear it stated by high authorities that it is a benefit to the community to contain men of great wealth. The whole delusion arises from the indestructible confidence in what money can do. And yet all of us see clearly enough by the roughest and most general observation that happiness does not increase with riches, that money indeed has very little to do with happiness, though it has a good deal to do with misery. But many of us are inclined to believe that our own individual case is rather different, and that more money added to our ample competence, and a consequent further enjoyment of material possessions, must undoubtedly make us happier. And when we have got the more and the desired result is not attained, we never pause in hesitation to consider whether perhaps the more has interfered with rather than augmented our happiness, but we are persuaded that the reason we find ourselves still discontented is simply that the more is not enough. Enough never comes to those who have encouraged the longing for more. Nothing short of actual experience can help to eradicate this belief, but there are few who would care to embark on an experiment in the direction of less. And yet it could quite well be demonstrated that a reduction of income, provided always that the loss does not reduce the income below a competence can lead to an increase in happiness—happiness being, of course, distinguished from pleasure. It may require a very rare philosophic resignation and an equally rare breadth of view to refuse to be deluded into regarding the possession of money as an absolute essential. Moreover, there are a
great many qualifications to be taken into account arising from natural characteristics, habit, temperament, and tastes. But broadly speaking, if a man has the courage to regard a reduction of income not as a loss but a gain, if he can use the opportunity to kill the instinctive but disturbing craving for more which unfortunately seems engrained in us all, in fact, if he can eradicate the germ of the disease, the limitation of his desire to satisfy transient and what are really artificial needs will certainly increase his power of enjoyment and his happiness. On the other hand, if he treats the lowering of his means as a calamity, which is the usual case, lamenting his fate, railing against fortune and encouraging the longing for gain—an attitude of mind which is only the outcome of his unlimited faith in the power of money—the result, naturally enough, will be despair. But it might be shown as well that a type of man does exist, exceptional no doubt, who, being capable of spending without hurt to himself or to others more money than he has actually got, can enrich his life in the broadest sense by an increase of fortune, and may therefore become the happier for it. He is a man who is indifferent to the enjoyment of material possessions and probably would be regarded in the eyes of the world as the last man who was competent to use money properly. But even he would be entirely overwhelmed by anything like a large increase of fortune, and would be as incapable as any one else of disposing of it without inflicting injury. “Could not riches be used well?” asks Jean Marie in Stevenson’s Treasure of Franchard. “In theory, yes,” replied the doctor. “But it is found in experience that no one does so. All the world imagine they will be exceptional when they grow wealthy; but possession is debasing, new desires spring up, and the silly taste for ostentation eats out the heart of pleasure.” Money is, after all, responsibility and nothing else. We are all of us capable of undertaking a certain amount. Some of us are capable of undertaking a good deal. No one is capable of undertaking more
than a relatively limited amount. But the trouble is that most of us think ourselves capable of undertaking far more than we properly can. Autocrats are ceasing to exist not so much because certain monarchs proved themselves dangerously incapable, but because the world has learned that no conceivable human being has the capacity to rule a country single-handed. We do not yet admit this incapacity with regard to the autocrats over capital, although it is equally true, and when we do so we shall find considerable difficulty in dethroning them. Another important inference to be deduced from the argument here set forth is that the surplus money which no individual does or can spend beneficially remains in his hands in stagnant unproductivity, is deflected from other remunerative channels, and is therefore the chief cause of the existence of some of the gravest economic ills which we have to face in our social life. Money cannot rest, it is an active instrument for producing good or for producing evil. Its presence in one quarter may not produce visible evil, but its consequent absence in another quarter will produce very visible and very positive evil. The word consequent must be emphasised because wealth is like water—to pump it up artificially on one side is to lower it automatically on the other. Money in its character of potential wealth seems also to have this peculiar characteristic. It has no positive value in itself. The greater part of its value is given to it by its possessor, and in proportion as it accumulates in the hands of an individual its value is rapidly depreciated. An electric current of a certain power will perform certain specified functions. Decrease the power and it ceases to produce the required effect. Increase the power tenfold or a hundredfold and you will be no nearer achieving the desired result. That is to say, in addition to the change in value effected by the change in individual ownership, there is actual deterioration, produced by accumulation, whoever the individual may be who is responsible for that accumulation.
As with individuals, so with the State. National wealth, which in the highest sense of the word means the enrichment of the lives of the people, depends not on how large a number of incomes there are of over ten thousand a year, but on how small a number there are of under two hundred a year. The real riches of a nation are not to be measured by vast calculations of commercial statistics, but by the absence of destitution and the high level of healthy life which the people enjoy. But we must accept the situation as it is. The rich have got their riches, and the problem to be considered here is not how to deprive them of their riches, but how to prevent all men, rich and poor alike, from confiding blindly in money, as they do at present, and from striving towards a false ideal which spoils their highest endeavours, blunts their moral susceptibilities, poisons their happiness, and produces a state of social disorder which is highly prejudicial to the common good. A just appreciation of the essential fact that money can only be made out of people’s labour and the wear and tear of their lives would in itself do much to prevent the growth of the spirit which leads to these alarming contrasts in riches and poverty. But men’s ideals and their moral outlook can only be altered in the long run by repeatedly exposing the actual fallacies in the views they now hold and constantly emphasising the disastrous results of the actions for which this waste of money is responsible.
A Chapter III Definition of the limit—Those whose means are above the limit—Income translated into terms of subsistence—The case of the rich man—His establishments—His servants— His luxuries—Extravagance—Vanity—Sport—Racing— Yachting—Condemnation of excess. MORE precise definition must be given of the limit of income referred to in the last chapter as “a definite amount of money which might be roughly described as a full competence.” Every man requires, though he by no means always gets a certain income to satisfy his own needs and those of his family. In addition to this he can profitably spend more so as to add to his general utility by conveniences and comforts, he can satisfy his artistic proclivities, his desire for further knowledge, his taste for sport or amusement, all to his own and the general benefit without hurt or hindrance to anyone. But after allowing the broadest scope for the satisfaction of these legitimate wants there is a definite point beyond which he cannot safely go. That is to say, if he acquires, or if by inheritance he finds himself burdened with money beyond this limit it will inevitably react detrimentally on himself and on others. And this for two reasons: firstly because he is, as a normal human being, incapable of dealing with so great a charge, and secondly because the money, while in his possession, is being drawn away from other channels where there is special need for it. So long as money encourages healthy effort a man may be sure the limit has not been reached, the moment money tends to relax
effort the limit has been passed. It must be described as healthy effort, as, of course, money-making may increase the undesirable efforts of the speculator, the gambler, and the thief. But who is to decide what is healthy effort? The man himself. No one else can. And he knows to a nicety. Every man or woman has a different standard, and the level of the limit varies in each individual case according to ideals, capacity, and temperament. But it will not depend at all on what is one of the strongest and often the most excusable inducements for spending money, namely, environment, or the conventions of the particular stratum of society to which the man belongs. The limit for one will not be the limit for another, and a man can only become aware that this limit exists at all by observing very closely what actually is the effect that his money is having on his life and character, instead of blindly accepting his already excessive income or every increase of his fortune as a natural and unquestionable blessing. The main brunt of the attack must clearly fall on those whose incomes are above the limit. They are in numbers a small minority, but the amount they possess is incredibly large. The present income of 1,250,000 people, assessed to income, reaches the vast sum of £850,000,000 a year. Taking the whole population of these islands, it is roughly estimated that there are 1½ millions who can be classed as rich, 3½ millions comfortably off, 38 millions as poor, of whom some 12 to 13 millions are in constant need. The existence of the 1½ millions is one of the chief causes of the condition of the 38 millions. In other words, excess above the limit causes want below the limit. The 3½ millions “comfortably off” are most of them occupied in trying to become identified with the select 1½ millions. If we could estimate the amounts in income which these classes represent the figures would be even more startling. The world has certainly never seen larger fortunes than exist to-day, nor has it seen more extensive and more inexcusable poverty. The average rate of luxurious living in the small minority is higher than it has ever been, and the dangerous and degrading effect of want on individuals and on the general community has never been so widespread or so
intense. “The rich,” to use a simple term, are nearly all actuated by the same motive. They accept what they have and what they make as their own, to be spent on themselves, according to their own caprice, or on others, if they are so inclined, casting an occasional sop to some charity or philanthropic scheme as a salve to their consciences. There are, it must be acknowledged, a few, a very few who regard their riches as a trust and endeavour to the best of their ability to divert the greater part of it back into remunerative channels without exceeding a reasonable sum for their own personal wants. But as a class they insist that efforts to alter our social system are fruitless, disturbing and doomed to failure, the division of the world into rich and poor being a Providential decree, and if the rich can get service from the poor without their grumbling, that is the most desirable arrangement that can be conceived. To this a reply may be given in the words of Professor Marshall: “Now at least we are setting ourselves seriously to inquire whether it is necessary that there should be any so-called ‘lower class’ at all: that is whether there need be large numbers of people doomed from their birth to hard work in order to provide for others the requisites of a refined and cultured life, while they themselves are prevented by their poverty and toil from having any share in that life.” The case would not be quite so bad as it is if it were only “the requisites of a refined and cultured life” that they were made to provide. But this point must be considered later. In order to appreciate fully the responsibility which the possession of riches entails, let us translate an income into terms of actual sustenance for human beings. By this means it is possible to arrive at a more or less positive measure. There is so much that is relative in most human requirements that they cannot serve as a standard or as a reliable quantity to be used in calculating any equation. But the requirements of a human being can be measured
in terms of actual sustenance, because they can be estimated with something approaching precision. Take a man with £20,000 a year, and say we deduct even as much as £3000 for himself and his family. With his remaining £17,000 he has the power of furnishing 170 people with £100 a year apiece. It is not for a moment suggested that he should do any such thing, as he would be quite unable to select 170 worthy people, and even if he could make the choice the 170 people, on the reception of this private dole, would soon become unworthy. This calculation is only taken to serve as a measure of his power. What might be the income or, more correctly speaking, the means of existence of 170 lives, is vested in one man, who is under the impression—and no one attempts to dispute it—that he is capable of disposing of this sum in a way that is generally beneficial. Now let us state the case fairly from the point of view of the rich man, taking a reasonable and more or less representative type. He may have £10,000, £50,000, or £100,000 a year—that only alters his activities in scope, not in quality. Let us say he has two or three country houses and a house in London. His “position” requires him to keep up a certain establishment, and this means the employment of some forty or fifty servants, grooms, gardeners, chauffeurs, etc., who, he readily tells you, will be thrown out of employment should any of his money be taken from him. If we take the case of a landlord, he will also have tenants, bailiffs, farm labourers, and gamekeepers dependent on him. He keeps the home farm and lets out the other farms on his estate to tenant farmers. Part of his land is built over and brings him in substantial returns in the shape of rent. His villages are in good order and the cottages kept in proper repair. Some thousands of acres or so he keeps for shooting. He may have a deer forest on one of his estates, and perhaps also a grouse moor or a river. Whether he keeps a racing stable, a pack of hounds, or a yacht depends on his particular fancy. He will acknowledge that he spends a certain amount on luxuries, but that “is good for trade,” as great numbers of people have to be employed in the manufacture of these luxuries. He is kind to his poor relations, whom he
entertains and helps; and his subscription list to hospitals, charities, and philanthropic works is a large one. He enjoys himself in an unostentatious but suitably expensive way, and his various responsibilities allow him to lead a life consisting of occasional rushes of activity and prolonged intervals of leisure. He most probably finds he can spare a certain amount of money for speculation, with a view to adding more to the sum total of his income. He looks forward to handing down to his children sufficient means to make each of them independent, and meanwhile has his boys educated in the large public schools, where they can associate with boys who are similarly situated. What possible harm can there be in all this? So far from being parasitic, he counts himself as a beneficent agent in the general industrial activity, at the same time appearing as a credit to his society and a notably refined product of the class of which he is a member. Above all, he is popular, and gains ostensibly the respect and regard of his friends, his neighbours, and his dependents. A favourable case has purposely been made for him, because if we accuse him of self-indulgence and greed, and describe him as a gambler, spending his substance on objects which are generally admitted to be pernicious and unworthy, the case could not be defended at all. The fundamental theory which makes this man’s position untenable has already been explained—namely, that after he has satisfied his legitimate requirements all the surplus money he keeps is being held back from serving urgent needs; and, moreover, the method in which he spends the surplus is directly or indirectly harmful to himself and others. We call the money his as if by some miracle he had made it. Often enough he has not helped even by the smallest exertion to create it. The wealth has been and is being daily and hourly produced by the exertion of numberless people who are either employed by him or employed in furthering enterprises in which he has invested his money. It will be said that his share as the wise
dispenser of capital, without which labour and enterprise could not be set in motion, is an all-important part of the general process of business. But he invests not to promote enterprise, but to get high dividends; and an elaborate system has been set up in order to tempt him to put his money into concerns that are by no means always sound or of the smallest public utility. Capital would exist and flow far more freely without the large capitalist. He acts as a dam to the stream; a certain amount escapes back into the main channel, but much more is checked and diverted into stagnant and putrefying pools of his own creation. The free flow of blood is life-giving; the clotting or coagulation of blood produces disease. Let us take the various points raised by his case seriatim. Many acutely controversial problems are opened, and it will be difficult to detach the particular actions of the rich man without generalising, to some extent, on the problems themselves. It is no argument against our main contention to say that people with costly tastes have, while gratifying them, been able to exercise powers of a high order, for, obviously, it is in spite of their shortcomings in this respect that they have succeeded, and not because of them. If some men with means have done valuable public service and performed admirable work in many different spheres of life, this they have done as men naturally gifted with high accomplishments, not as rich men. Here we are only concerned with their works and deeds in their latter capacity. It does not affect our argument whether our typical example has been brought up to regard this way of living as natural and necessary for a man of what is called his “position” (that is to say, the purely artificial place which a rich man is able to take up in the community solely on account of his riches), or whether he has made the money for himself and is simply aping the habits and customs of those who already possess it. The distinction between the vieux riches and the nouveaux riches is one they can fight out between themselves. The former scoffs at the latter while all the time he is setting him, and consciously setting him, the example he is to follow. It is not the gaining, but the spending of the money that must occupy our attention here.
Our friend’s houses are only a detail in the upkeep of his position. They may be historic castles, sham “ancestral halls,” modern “palatial country residences,” or “fashionable mansions” in town. Does it ever strike the owner as, let us say, a curious arrangement that he should have several houses of fifty to a hundred rooms apiece while some millions of his fellow-men do not own one room? Does he know that in England and Wales alone 507,763 people occupy one-room tenements, 48.4% of which are classified as overcrowded, while 12,458,150 are occupying tenements of two, three, or four rooms? 4 In any case, he would indignantly refuse to admit that there was any remote connection between these two sets of circumstances. When we come to the staff necessary for the maintenance of these large establishments we touch a problem of employment which must be examined more closely. It is not sufficient to state baldly that these people are employed, and that if the opening were not available for them they would be unemployed. The immediate result of their being discharged would no doubt in some cases be unemployment. That is just the mischief of uneconomic employment. If a large number were simultaneously dismissed there might be temporary unemployment on a large scale, as it would amount to dislocation, like the extinction of some dying industry. But the eventual readjustment would subsequently be by that much the stronger and better adapted to the real requirements of the community. To employ a man in useless and unremunerative work can be regarded in some aspects as worse than not employing him at all. It is not intended, however—and, indeed, it would be impossible—here to enter into a discussion on the whole problem of unemployment, but there is undoubtedly a very great economic waste that largely contributes to the gravity of the problem, arising from the fact that a large number of people are being forced to devote their labour and energy to work which is, so to speak, final and sterile. It is precisely the same with regard to the production of expensive luxuries. The employment of a large retinue is only another form of the possession and enjoyment of articles of
excessive luxury. The employers and possessors have all disagreeable burdens and every sordid worry lifted from them, their smallest and their most extreme desires for pleasure met, their special appetites satisfied, their peculiar vanities titillated, and their artificial position safeguarded and maintained, without their giving more than a passing thought to the mass of people required to carry on this work. Plenty of examples might be quoted in contemporary as well as past history to show that after generations of the enjoyment of “the vile joys of tainting luxury” men deteriorate, both physically and mentally. As for the particular line of life which domestic service offers under modern circumstances, it is not too much to say that it is, as a rule, very demoralising, more especially for the men. And its demoralising tendency increases in proportion to the size of the establishment. The single general servant lives a life of hard work but genuine service on four to eight shillings a week, often living in friendly relations with master or mistress, and really lifting from them the burden of necessary domestic duties which they with limited incomes and professional work of their own cannot possibly find time to perform; and this remains true in other small households. In the large house the faithful old family servant, who is more of a friend than a servitor, is rare in these days of ostentation. The butler, on wages of fifty to sixty shillings a week, which together with board and lodging represents from £250 to £300 a year, has a life of leisure, ease, and excessive comfort, seldom having to exert himself even up to his limited capacities. Male house-servants are often chosen for their looks; their work is very light physically, they are overfed, and being under-educated, can hardly be blamed for becoming demoralised. These able-bodied men, whose muscles, if not their minds, might be devoted to some really serviceable purpose, are still increasing in numbers. Over 25,000 more male servants have got employment in the last ten years, the total number now being 227,995. Even deducting the single indoor servant, the single coachman or gardener, this means a large increase of ornamental male attendants. Female servants are
becoming more difficult to secure in the higher grades, because the class of women from which they are drawn value their liberty and are not so ready to sacrifice it for food and comforts. In fact, they are showing signs of impatience of control, and of preferring the risky though exhilarating struggle of independence. But still large retinues of men and women exist solely employed in keeping up huge houses to satisfy the vanity and minister to the comfort of a comparatively few rich people. No work of a more hopelessly barren, profitless, and, indeed, degrading character could be found for them. A system of tips deprives their smallest acts of what might be an obliging and disinterested intention. Arrangements are organised with tradesmen to defraud the employers in what is thought a perfectly legitimate way; the actual waste of food is appalling, and by extras, gratuities, perquisites, commissions, and pickings a considerable amount is added to the wages of the upper servants. In these large establishments immorality exists more as a rule than as an exception, but it can be kept secret, for these communities of private servants—like everything else connected with the lives of the rich—cannot be made the subject of investigation. If assistance to those who need it is the object of domestic service, it is striking to note that on the money basis, generally speaking, the wrong people are served. Who in the community most require and should specially have the help of servants? The old and infirm, the weak and ill, the very young and the hard-worked. Service under such conditions raises itself to the level of one of the highest occupations that can be imagined. But this is not our system. A man or woman may be ill, old or over-worked, without being able to get the assistance of a single soul. Another man or woman may be young and healthy and have at his or her command a retinue of thirty servants or more, solely because they have money and servants are forced, by economic pressure, to devote their lives to the menial task of furbishing up the endless and complicated appanage of wealth. Now let us turn to the inanimate luxuries, taking into account only indisputable luxuries—that is to say, articles of high price which
have no special artistic value, to which much labour has been devoted and which are not produced to serve any legitimately useful purpose. Luxury has been well defined as “that which creates imaginary needs, exaggerates real wants, diverts them from their true end, establishes a habit of prodigality in Society, and offers through the senses a satisfaction of self-love which puffs up but does not nourish the heart and which presents to others the picture of happiness they can never attain.” Bond Street catalogues abound with any quantity of examples. Furs at one thousand guineas, fifty-guinea dressing-bags, twenty- guinea hats, thousand-guinea tiaras, fruit and vegetables out of season, cigars at three shillings apiece, ruinously expensive wines, and fantastic foods of all descriptions. There is no need to exaggerate, for all those articles can be bought for much higher prices than those quoted. A great amount of skilled labour of a high order goes to the production of these luxuries, and a great amount of labour of the lowest and most cruelly sweated description is also enlisted for their production, and incredible as it may seem, it is on the ground that they give employment that these luxuries are defended. It was calculated in 1884 5 that, even giving a liberal extension of meaning to the term “necessaries” and “comforts” of life, over six millions of manual labourers, who with their families constitute thirteen millions of the population, were engaged in producing what, in contradistinction to the above, must be classified as luxuries. A prominent statesman, 6 expressing the views of his class, said a few years ago: “The more human wants are stimulated and multiplied, the more widespread will be the inducement to hire. Therefore all outcries and prejudices against the progress of wealth and what is called luxury are nothing but outcries of prejudice against the very sources and fountains of all employment.” On such an argument as this the defence of luxuries generally rests. The essence of the fallacy lies in the fact, which cannot be repeated too often, that labour spent on such articles is
unremunerative and unproductive, because its ultimate result is only to gratify various forms of vanity and greed. To exemplify by a concrete instance what is unremunerative and what is remunerative, let us take a hundred-guinea ball-gown and a pair of boots. It is not possible to estimate the number of people employed in producing the ball-gown. There is the silk, satin, or whatever the principal material may be; there are the trimmings of chiffon, hand- embroidery, lace, braid, beads, sequins, ribbons, etc., etc.; some hundreds of pairs of hands, including factory-workers, dressmakers, sempstresses, etc., will have touched some part of the gown before it is delivered to the wearer. To what end are all these specialised departments of labour concentrated? The gown is worn a few times in the one season; the wearer has the satisfaction of feeling as well dressed as A. to F., and far better dressed than F. to Z. In fact, the net result of all this expenditure of energy is the generating of a rather foolish pride, the encouragement of conceit on the one side and envy on the other, and the hardening of a nature into ways of worldliness and vanity. As for the boots. Again, many more hands than can be calculated have helped to produce them, but they are directly and immediately serviceable to the purchaser, to whose activity the wearing of boots is an essential, and in general they minister to the efficiency of human machines. But if balls are not wrong, ball-gowns must be worn. It is a question of degree; and here again we get to the theory of the limit which in this conjunction can be expressed thus: In relation to human needs, in relation to human powers of enjoyment, in relation to the beneficial effects of pleasure, even in relation to the dictates of fashion, there is a distinct limit not to be expressed in figures up to which expenditure (in this particular case on dress) is legitimate and relatively productive, beyond which it becomes progressively unremunerative and harmful. A hundred guineas, by any conceivable method of calculation, greatly exceeds this limit.
To assert that the purchase of luxuries is good for trade is quite as ridiculous as to say that a man can benefit the building and furnishing trade by burning down his house once a year. We do not want to create more artificial wants before we have satisfied the crying human needs which already exist. There is no loophole through which a reasonable defence of the senseless expenditure, which goes on in an increasing measure, can be made. Luxurious living has never been quite so blatant and unashamed as it is to-day, and the effete epicureanism and decadent effeminacy it produces stand out in rather sharp contrast to more hopeful signs of progress and moral and intellectual refinement and vigour which, happily, are visible around us. A lady writing in a review in the early ’seventies describes life in the country house, with its futile routine of heavy meals, sport, card- playing, and vacuous inanities which take the place of conversation, all very much as it is to-day. The writer speaks with dismay of gowns costing sixty guineas and of £1000 a year spent on clothes. But these figures are almost negligible compared with the sums spent nowadays. It is only through occasional actions in the courts that the outside public get an idea of what is actually spent, and it is surprising that there are not more disclosures, considering the mountainous debts that are piled up in West End shops. But the shopkeepers are very reluctant to lose a really leading customer, and they know how to meet the inconvenience of not being promptly paid. A typical case may be given of an article of clothing, the cost price of which was nine guineas, being sold for £28 7s. There may be delay in payment, but there appears to be compensation in the profit. When one hears of the woman who spent last year £36 5s. on a hat, or another who gave £1250 for a sable cape, it is not the isolated action of criminal folly that chiefly strikes one, but it is that the hat and the cape act as indicators of the sort of price such women are in the habit of paying for their clothes, a large supply of which are in the market ready to meet this artificial demand. Moreover, the habit of extravagance, especially as regards female
clothing, is catching and runs through all classes once the example is set. It is a common enough and very depressing sight to see absurdly elaborate clothes, which are cheaper imitations of the latest fashions, worn by women of the lower middle-class, whose deplorable want of education is shown by their inability even to pronounce their mother tongue. They watch the rich, and gather from what they see that fine feathers make fine birds, and it is not on them that the blame should rest. Vanity exists and insists on being satisfied. It is no good blinking the fact. Luxuries, in one form or another, will continue to be produced. But there is no reason why we should not stem the current lest it swell to danger point. There are many well-known historical examples of the enervating and degenerating effect of luxury on national life, and the modern tendency towards an increased production of these indulgences should be combated not only as a moral weakness, but as an integral factor in the general economic problem. When one considers what real comfort of living, with all the necessary intellectual and artistic equipage, opportunities for amusement, and domestic convenience, can be secured to-day at a comparatively moderate sum, it makes the wild and profligate extravagance the more inexcusable and the more futile. Anyhow, let us abandon once and for all the foolish and ignorant attitude of regarding this display as a desirable form of industrial stimulus which should be fostered and encouraged. Preaching and writing against it has never been of the smallest avail, but it has been necessary to deal with it here as a very important, if not predominating, element in the analysis of the rich man’s conceptions of his duties. In addition to luxuries of establishment, clothes, and food there is a complicated ritual of sport which in this country reaches an almost incredible pitch. It has been estimated that forty-five millions are permanently invested in the apparatus of sport, and an income of over forty millions spent annually upon it. We need not discuss all the intricacies of the numerous branches of sport, observing where
its effect is healthy and where harmful. No one will contend that the most expensive forms of it are by any manner of means the best. But the most obvious harm to be noted in this connection is the amount of land which is taken away from agriculture for sporting purposes. Landlords often keep up their shooting at a great loss, amounting to something like five to ten pounds per bird shot, all for the sake of having the shooting and asking friends down for a few days in the year to enjoy it. It is gravely regarded as an essential part of the education of a young man in this particular world to learn how to shoot. No question, even with respect to his education or possible professional career, is treated with more seriousness than the moment he first handles a gun, and family advice is sought as to how and when encouragement can be given to the development of this essential qualification which, coupled with a knowledge of bridge, will make him a desirable visitor in any country house. At card-playing, which occupies a vast amount of time in the lives of the rich, sums amounting to hundreds are often lost or gained by one person in one evening. But of the various sinks which help to drain away their money, horse-racing almost holds the first place. There are no statistics to show how many people have been ruined by it, or how many have been lured into a life of gambling by their success in the betting ring. But its popularity is certainly on the increase, as we can see by looking at the number of horses that have run under the rules of racing in the last thirty years. In 1878 there were 2097; in 1908 this figure had risen to 3706. The number of larger race meetings advertised in advance have more than doubled since 1881 (78 in 1881, 164 in 1909). Some sort of estimate of the money spent on it, apart from betting, can be gathered from the amounts won. In 1908 the winning owners secured between them nearly a quarter of a million pounds, the sums won by the first thirty-six amounting to £246,001 15s., the largest total secured by one owner being £26,246. 7 The populace are invited to join in this pursuit, though, of course, they must be railed off to prevent too close contact with
those who come in coaches and motor-cars. The crowd is vaguely supposed to be having a good time, and any attack on horse-racing is met by hackneyed arguments about “keeping up the national sport” or “improving the breed of horses,” and perhaps, again, the objection of unemployment for jockeys and bookies might be dragged in. It does not appear, however, to be a good method of improving the human breed. In observing the crowd on a race-course, whether it be the well-dressed portion or the ill-dressed, the betters or the bookies, neither a deep knowledge of humanity nor a very close power of observation into physiognomy is required to note the prevalence of a remarkably low type. But a still more vivid impression of what the pleasures of racing mean can be gained by going out on the road in the evening towards the scene of a large race meeting when the people are returning. Brakes and carts in endless procession will pass you loaded with men shouting in the excitement of semi-drunkenness, or with heaps of humanity sodden and silent in complete intoxication. Outside every public-house on the roadside traps await those who are squandering their gains on further refreshment or soothing the despair of losses in the temporary oblivion of drink. The localities where there is an annual race week suffer considerably, the inhabitants become infected by the gambling and betting mania, and during the actual days of the races the place is infested by the lowest dregs of the riff-raff who journey about from one race meeting to another. This so-called sport produces the lowest possible type; it degrades many who take part in it with sinister rapidity, it encourages fraud and deception, it is a canker of rottenness in public life, and it receives the highest sanction and patronage. Many people are present at a race meeting without being conscious that it is attended by any evil consequences. They go to meet their friends, perhaps putting an occasional sovereign on a horse to give them some interest in the racing. To them the crowd is a natural part of the proceedings, the heavy bets of the ring an amusement. To have been there is something to boast of, and
conveys the idea that they have associated with smart people. Thoughtless, as in so many of their other pursuits, they accept the whole proceeding as a recognised sport and they inquire no further. The philosophy of these people is the prevailing philosophy: “Do not examine below the surface, or you are bound to find something disagreeable. Take things as they come; skim the cream off the top; avoid that which is unpleasant or difficult to explain; and above all things, do what others do.” Yachting, which also runs away with a great deal of money, comes under a very different category. It is a health-giving and often strenuous occupation, and the seamen employed are, anyhow, deriving incidentally some positive benefit from the life they lead. Nevertheless, out of the 4655 private yachts registered in the current year (an increase of over 3500 in the last forty years), 8 only a very small proportion are actually navigated by owners who have any knowledge or love of seamanship. The great majority are floating houses of luxury (viz. a 700-ton steam yacht, for which £25 a day is paid for coal when in use), or racing yachts, mere toys used to minister to the fanciful pleasures of the rich. But in expressing the strongest disapproval of these excessive luxuries, it is not for a moment suggested that people should rush into the opposite extreme—live in discomfort and adopt the craze for “the simple life,” which is only an inverted form of vanity and ostentation. There are many of the lesser luxuries which give great pleasure and sufficient honest gratification to justify their existence. There may even be some reluctance in condemning extravagance, because the nature of the extravagant man is far preferable to the economical and cautious disposition which sometimes sinks into niggardly meanness. Moreover, any attempt at excessive restrictions and unnecessarily harsh discipline in the upbringing of children invariably leads to a violent reaction in the direction of profligacy and extravagance. Let human nature be allowed free play in all directions; but it is not taking up the attitude of an ascetic or of a prig to condemn
unhesitatingly unnatural excesses, reckless licence, the extremes of self-indulgence and greed, the exercise of which by some few involves the neglect, misery, and ruin of so many others. Thieves when they steal use violence and are pronounced enemies of society. These few people, by a silent conspiracy in which we all seem to acquiesce, are also stealing and are equally enemies of society.
W Chapter IV The rich man’s charities—His generosity—His hospitality—His land—The Feudal System—His responsibilities—The agricultural problem. E must now turn from what the rich man spends on himself and consider what good and what harm he does by his subscriptions and donations to philanthropic and charitable objects. In so far as he himself is concerned these gifts do not involve any element of personal sacrifice; the moral benefit which is by way of falling on a giver is therefore nil. The exertion of writing a cheque or banker’s order and the satisfaction of imposing a tax on himself complete the transaction on his side. Occasionally the sight of his name published at the head of a list with a large figure next it gives him a further agreeable sensation, and he can become famous as a household word of generous philanthropy without the very smallest personal inconvenience. But as an instance of pure charity—that is, loving sacrifice—the poor woman who gives a penny from her meagre store is on an entirely different plane. The picture presents itself to the present writer of a woman at the doorway of a wretched tenement, with her child in her arms, giving to a passing vagrant who was suffering from hunger and fatigue a penny from the few coins she had in her purse. The expression of her face as she handed him the money was the most sublime illumination of pure charity—no subscription list in the newspapers, no public recognition, and the sacrifice, not of luxuries, but of something that she and her baby needed. That something went with her penny, and
in return she received something else for which there is no price, no name, and no description. From such an experience as this the rich are for ever cut off. “Probably the most generous people in the world,” says J. D. Rockefeller, perhaps realising that charity is something he can never reach, “are the very poor, who assume each other’s burdens in the crises which come so often to the hard pressed.” The rich man’s so-called charity therefore must be to a large extent mechanical and conventional. He gives because others with the same means give, and the charity touts know how a list headed by Lord A. with a substantial sum will produce equally or perhaps even more substantial sums from Lord B., Sir. C. D., Alderman E., and Mr. F. The extraction of money from the rich is a business in itself, requiring considerable skill, and the rich are fleeced far more than they realise. In practical America they take the trouble to teach people professionally how to write what they call “letters of appeal.” When we hear of subscriptions to charities being stopped it may serve to remind us that it is most inexpedient that institutions such as hospitals should be at the mercy of the casual caprice of rich men. Nothing could eventually be more desirable than that every one of them should cut off their charitable contributions. It might entail a severe temporary shock to the funds of charitable institutions, as over seven millions a year is being spent in London alone on charities, but at the same time many ill-managed and misdirected endeavours would disappear, and the State would come to realise all the sooner its responsibilities in respect to the maintenance of really necessary institutions for the relief of suffering and the nursing of the sick, in the same way as it is beginning to recognise its duties towards poverty, old age, and unemployment. There are other enterprises which the State should undertake that are often delayed in their institution owing to the plea that the private munificence of rich men can be depended upon. It is certainly better that the funds should be expended thus than in sheer self-indulgence, but it is evident that the money would be far better spent and the object on which it is spent better served if the
source were not controlled by the whims and fancies of a single individual. In regard to the more private and personal aspects of the generosity attributed to riches: “Surely,” a critic will say, “if the rich man is benevolent and kindly disposed he can in a hundred thoughtful ways help his poorer friends by presents, by attention and timely help, by opening the doors of his houses, lending his conveyances, and showing many other attentions which his money allows him to do, thereby becoming justly popular and a source of great good.” The admiration, and just admiration, for open-handed generosity and the justifiable dislike of anything approaching miserliness in others cause an entirely erroneous impression that large gifts of money must unquestionably be praiseworthy and commendable. But this is not the question at issue. These are two moral qualities, the one admirable, the other objectionable. The generous disposition can show itself in many other ways besides money gifts, and the real man behind the rich man, though he may be one and the same individual, often comes forward with simple acts of thoughtful kindness because the finer qualities of human nature cannot be stifled even by money. But in so far as the rich man indulges his generosity in thoughtlessly giving away money broadcast, it amounts to a form of self-indulgence, and he is distinctly to blame for not estimating more precisely the effect of his actions. No doubt the harm of unwise and foolish actions is palliated by the purity and excellence of the motive. In so far as these people intend to show kindness they are amply justified in what they do. But let us consider for a moment what the effect of their benevolence is. In the first place they are made to occupy an entirely false position as dispensers of charity. Often, too, the desire to patronise and gain the power that patronage gives blights the spirit of genuine and unadulterated kindness, and further, the recipients are placed in the extremely uncomfortable and embarrassing situation of receiving benefits, presents, and comforts which they know they are not and probably never will be in a position to return. To force anyone to be
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  • 4. ME T H O D S I N MO L E C U L A R BI O L O G Y ™ Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651
  • 6. Cell Senescence Methods and Protocols Edited by Lorenzo Galluzzi, IlioVitale, Oliver Kepp, and Guido Kroemer InstitutGustaveRoussy(IGR),INSERMU848, Villejuif,France
  • 7. ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-62703-238-4 ISBN 978-1-62703-239-1 (eBook) DOI 10.1007/978-1-62703-239-1 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2012950333 © Springer Science+Business Media, LLC 2013 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. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. 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. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Editors Lorenzo Galluzzi Institut Gustave Roussy Université Paris Descartes Sorbonne Paris Cité Villejuif, France Oliver Kepp Institut Gustave Roussy INSERM U848 Villejuif, France Ilio Vitale Institut Gustave Roussy INSERM U848 Villejuif, France Guido Kroemer Institut Gustave Roussy INSERM U848 Villejuif, France
  • 8. v v Preface In Vitro and In Vivo Cell Senescence Cell senescence, i.e., the process whereby cells permanently lose the possibility to prolifer- ate without undergoing cell death, can be observed in vitro as well as in vivo, and occurs in a plethora of distinct model organisms. In both cases, cell senescence can be physiological, constituting a safeguard mechanism against cells that have accumulated potentially danger- ous genetic alterations, or can be triggered by exogenous perturbations, such as the admin- istration of DNA-damaging agents at low doses. This book provides a detailed description of the most common techniques for the investigation of cell senescence, in model organ- isms encompassing bacteria (Escherichia coli), fungi (Saccharomyces cerevisiae and Podospora anserina), worms (Caenorhabditis elegans), flies (Drosophila melanogaster), zebrafish (Danio rerio), and mammalian cells. The techniques presented in this book not only cover the study of all the biochemical and functional manifestations of senescence at the cellular level but also include protocols for population analysis and high-throughput approaches in suit- able model organisms, as described by worldwide renowned experts of the field. Chapter Organization The book is composed of three types of chapters. Four review chapters open the book to provide a solid theoretical background on cell senescence, its morphological and biochemi- cal manifestations and its pathophysiological relevance. Twenty-three protocol chapters follow, detailing the methods to investigate the morphological and biochemical features of senescence at the cellular level, in cultured mammalian cells. Finally, seven protocol chap- ters provide techniques for the study of cell senescence in lower model organisms, including methods for population studies. Each of these 30 protocols starts with an Abstract and includes four major sections: Introduction, Materials, Methods, and Notes. The “Abstract” presents an overview of the technique(s) detailed in the chapter. The “Introduction” pro- vides a short theoretical view of the procedure and of its applications. “Materials” recapitu- late the buffers, reagents, solutions, disposables, and equipments necessary to carry out the protocol(s). “Methods” describe step-by-step how the technique(s) must be carried out. Finally, the “Notes” section, which is the hallmark of Methods in Molecular Biology series, indicates not only the sources of problems and how to identify and overcome them, but also safety information, alternative procedures, and hints for the correct interpretation of experimental results.
  • 9. vi Brief Content of the Chapters Chapter 1 provides an overview on cell senescence and its dynamic links with autophagy, an important cytoprotective mechanism. Chapters 2 and 3 discuss the regulation of cell senes- cence by critical signaling molecules such as the mammalian target of rapamycin (mTOR) and p53. Chapter 4 summarizes the morphological and biochemical markers that have been associated with cell senescence. In Chapters 5–23, protocols for the investigation of senes- cence-associated alterations in cultured cells are provided, including the following: mor- phological features (Chapter 5), cell cycle blockage (Chapter 6), cell cycle-arresting proteins (Chapter 7), senescence-associated β-galactosidase (Chapters 8 and 9), senescence-associ- ated secretory phenotype and chemokyne signaling (Chapters 10 and 11), senescence- associated heterochromatin foci (Chapter 12), DNA damage (Chapter 13), telomerase activity and telomere length (Chapters 14 and 15), alterations of the nuclear envelope (Chapter 16), multiple markers of oxidative stress (Chapters 17–20), BRAF, sirtuin, and p66SHC signaling during senescence (Chapters 21–23). In Chapters 24–27, protocols for the study of cell senescence in global terms are detailed, including a method for the study of metabolomic alterations (Chapter 24), a technique to apply genome-wide RNAi approaches to cell senescence research (Chapter 25), and multiparametric strategies (Chapters 26 and 27). Finally, in Chapters 28–34, protocols applicable to lower model organisms are described, encompassing techniques to assess senescence in Escherichia coli (Chapter 28), Podospora anserina (Chapter 29), Saccharomyces cerevisiae (Chapter 30), Caenorhabditis elegans (Chapters 31 and 32), Drosophila melanogaster (Chapter 33), and Danio rerio (Chapter 34). Potential Audience of This Book In the first instance, this book will be of interest not only for undergraduate and graduate students but also for more experienced scientists who are approaching the study of cell senescence. In addition, the audience of this book encompasses: Libraries of universities and public biological/biomedical research institutions. ● Scientists interested in molecular and cell biology, biochemistry, pharmacology, genet- ● ics, systems biology, medicine, public health, and in life sciences in general. Specialists and experts in model organisms including bacteria, fungi, worms, flies, and ● mammals. Medical oncologists and scientists working in oncology. ● Pharmaceutical companies and developers of new drugs. ● Villejuif, France Lorenzo Galluzzi Ilio Vitale Oliver Kepp Guido Kroemer Preface
  • 10. vii Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 Cell Senescence as Both a Dynamic and a Static Phenotype. . . . . . . . . . . . . . . . . . 1 Andrew R.J. Young, Masako Narita, and Masashi Narita 2 Senescence Regulation by mTOR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Vjekoslav Dulic 3 Senescence Regulation by the p53 Protein Family. . . . . . . . . . . . . . . . . . . . . . . . . 37 Yingjuan Qian and Xinbin Chen 4 Markers of Cellular Senescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Amancio Carnero 5 Biomarkers of Cell Senescence Assessed by Imaging Cytometry . . . . . . . . . . . . . . 83 Hong Zhao and Zbigniew Darzynkiewicz 6 Cytofluorometric Assessment of Cell Cycle Progression . . . . . . . . . . . . . . . . . . . . 93 Ilio Vitale, Mohamed Jemaà, Lorenzo Galluzzi, Didier Metivier, Maria Castedo, and Guido Kroemer 7 Quantification of Cell Cycle-Arresting Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Oliver Kepp, Isabelle Martins, Laurie Menger, Mickaël Michaud, Sandy Adjemian, Abdul Qader Sukkurwala, Lorenzo Galluzzi, and Guido Kroemer 8 Colorimetric Detection of Senescence-Associated b Galactosidase . . . . . . . . . . . . . 143 Koji Itahana, Yoko Itahana, and Goberdhan P. Dimri 9 Chemiluminescent Detection of Senescence-Associated b Galactosidase . . . . . . . . 157 Vinicius Bassaneze, Ayumi Aurea Miyakawa, and José Eduardo Krieger 10 Detection of the Senescence-Associated Secretory Phenotype (SASP) . . . . . . . . . . 165 Francis Rodier 11 Unbiased Characterization of the Senescence-Associated Secretome Using SILAC-Based Quantitative Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Juan Carlos Acosta, Ambrosius P. Snijders, and Jesús Gil 12 Detection of Senescence-Associated Heterochromatin Foci (SAHF) . . . . . . . . . . . 185 Katherine M. Aird and Rugang Zhang 13 Monitoring DNA Damage During Cell Senescence . . . . . . . . . . . . . . . . . . . . . . . 197 Glyn Nelson and Thomas von Zglinicki 14 Assessment and Quantification of Telomerase Enzyme Activity . . . . . . . . . . . . . . . 215 Michelle F. Maritz, Laura A. Richards, and Karen L. MacKenzie 15 Methods for the Assessment of Telomere Status . . . . . . . . . . . . . . . . . . . . . . . . . . 233 Asako J. Nakamura
  • 11. viii Contents 16 Detection of Nuclear Envelope Alterations in Senescence . . . . . . . . . . . . . . . . . . . 243 Clea Bárcena, Fernando G. Osorio, and José Maria Pérez Freije 17 Measuring Reactive Oxygen Species in Senescent Cells . . . . . . . . . . . . . . . . . . . . . 253 João F. Passos, Satomi Miwa, and Thomas von Zglinicki 18 Quantification of Protein Carbonylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Nancy B. Wehr and Rodney L. Levine 19 Assays for the Measurement of Lipid Peroxidation . . . . . . . . . . . . . . . . . . . . . . . . 283 Ana Cipak Gasparovic, Morana Jaganjac, Branka Mihaljevic, Suzana Borovic Sunjic, and Neven Zarkovic 20 Raman Spectroscopy for the Detection of AGEs/ALEs . . . . . . . . . . . . . . . . . . . . 297 J. Renwick Beattie, John J. McGarvey, and Alan W. Stitt 21 Monitoring Oncogenic B-RAF-Induced Senescence in Melanocytes . . . . . . . . . . . 313 Sieu L. Tran and Helen Rizos 22 Methods to Investigate the Role of SIRT1 in Endothelial Senescence . . . . . . . . . . 327 Bo Bai and Yu Wang 23 Monitoring Nutrient Signaling Through the Longevity Protein p66SHC1 . . . . . . . . 341 Sofia Chiatamone Ranieri and Giovambattista Pani 24 Profiling the Metabolic Signature of Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . 355 Florian M. Geier, Silke Fuchs, Gabriel Valbuena, Armand M. Leroi, and Jacob G. Bundy 25 Genome-Wide RNAi Screening to Identify Regulators of Oncogene-Induced Cellular Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 Narendra Wajapeyee, Sara K. Deibler, and Michael R. Green 26 An Integrated Approach for Monitoring Cell Senescence . . . . . . . . . . . . . . . . . . . 383 Tatiana V. Pospelova, Zhanna V. Chitikova, and Valery A. Pospelov 27 Robust Multiparametric Assessment of Cellular Senescence. . . . . . . . . . . . . . . . . . 409 Clara Correia-Melo, Diana Jurk, and João F. Passos 28 Assessing Chronological Aging in Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421 Stavros Gonidakis and Valter D. Longo 29 Assessing Organismal Aging in the Filamentous Fungus Podospora anserina . . . . . 439 Heinz D. Osiewacz, Andrea Hamann, and Sandra Zintel 30 Assessing Chronological Aging in Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . 463 Jia Hu, Min Wei, Mario G. Mirisola, and Valter D. Longo 31 Assessing Aging and Senescent Decline in Caenorhabditis elegans: Cohort Survival Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473 Eirini Lionaki and Nektarios Tavernarakis 32 High-Throughput and Longitudinal Analysis of Aging and Senescent Decline in Caenorhabditis elegans . . . . . . . . . . . . . . . . . . . . . . . . . . 485 Eirini Lionaki and Nektarios Tavernarakis 33 Assessing Senescence in Drosophila Using Video Tracking . . . . . . . . . . . . . . . . . . 501 Reza Ardekani, Simon Tavaré, and John Tower 34 Assessing Vascular Senescence in Zebrafish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517 Sandra Donnini, Antonio Giachetti, and Marina Ziche Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
  • 12. ix Contributors JUAN CARLOS ACOSTA • Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College, London, UK SANDY ADJEMIAN • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France KATHERINE M. AIRD • Women’s Cancer Program, Epigenetics and Progenitor Cells Keystone Program, Fox Chase Cancer Center, Philadelphia, PA, USA REZA ARDEKANI • Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA BO BAI • Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China CLEA BÁRCENA • Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain VINICIUS BASSANEZE • Laboratory of Genetics and Molecular Cardiology/LIM 13, Heart Institute (InCor), University of Sao Paulo Medical School, Sao Paulo, Brazil J. RENWICK BEATTIE • Centre for Vision and Vascular Science, School of Medicine and Dentistry, Queen’s University, Belfast, UK JACOB G. BUNDY • Biomolecular Medicine, Department of Surgery and Cancer, Imperial College, London, UK AMANCIO CARNERO • Instituto de Biomedicina, Hospital Universitario Virgen del Rocio, Sevilla, Spain MARIA CASTEDO • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France XINBIN CHEN • Comparative Oncology Laboratory, University of California, Davis, CA, USA ZHANNA V. CHITIKOVA • Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia; St. Petersburg State University, St. Petersburg, Russia CLARA CORREIA-MELO • Ageing Research Laboratories, Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK ZBIGNIEW DARZYNKIEWICZ • Department of Pathology, Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA SARA K. DEIBLER • Howard Hughes Medical Institute, Chevy Chase, MD, USA; Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA GOBERDHAN P. DIMRI • Department of Biochemistry and Molecular Biology, The George Washington University Medical Center, Washington, DC, USA SANDRA DONNINI • Department of Biotechnology, University of Siena, Siena, Italy VJEKOSLAV DULIC • Institut de Génétique Moléculaire, Montpellier, France; CNRS, UMR5535, Montpellier, France; Université Montpellier 1, Montpellier, France JOSÉ MARIA PÉREZ FREIJE • Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain SILKE FUCHS • Cell and Molecular Biology, Department of Life Sciences, Imperial College, London, UK
  • 13. x Contributors LORENZO GALLUZZI • Institut Gustave Roussy, Villejuif, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France ANA CIPAK GASPAROVIC • Rudjer Boskovic Institute, Zagreb, Croatia FLORIAN M. GEIER • Biomolecular Medicine, Department of Surgery and Cancer, Imperial College, London, UK ANTONIO GIACHETTI • Department of Biotechnology, University of Siena, Siena, Italy JESÚS GIL • Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College, London, UK STAVROS GONIDAKIS • Department of Biological Sciences, Ethel Percy Andrus Gerontology Center, University of Southern California, Los Angeles, CA, USA MICHAEL R. GREEN • Howard Hughes Medical Institute, Chevy Chase, MD, USA; Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA ANDREA HAMANN • Faculty of Biosciences, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany; Frankfurt Cluster of Excellence “Macromolecular Complexes”, Frankfurt, Germany JIA HU • Ethel Percy Andrus Gerontology Center, Davis School of Gerontology, University of Southern California, Los Angeles, CA, USA KOJI ITAHANA • Duke-NUS Graduate Medical School Singapore, Singapore, Singapore YOKO ITAHANA • Duke-NUS Graduate Medical School Singapore, Singapore, Singapore MORANA JAGANJAC • Rudjer Boskovic Institute, Zagreb, Croatia MOHAMED JEMÀA • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France DIANA JURK • Ageing Research Laboratories, Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK OLIVER KEPP • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France JOSÉ EDUARDO KRIEGER • Laboratory of Genetics and Molecular Cardiology/LIM 13, Heart Institute (InCor), University of Sao Paulo Medical School, Sao Paulo, Brazil GUIDO KROEMER • Centre de Recherche des Cordeliers, Paris, France; INSERM, U848, Villejuif, France; Metabolomics Platform, Institut Gustave Roussy, Villejuif, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France ARMAND M. LEROI • Ecology and Evolution, Department of Life Sciences, Imperial College, London, UK RODNEY L. LEVINE • Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA EIRINI LIONAKI • Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Greece VALTER D. LONGO • Davis School of Gerontology, Ethel Percy Andrus Gerontology Center, University of Southern California, Los Angeles, CA, USA CARLOS LÓPEZ-OTÍN • Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain ISABELLE MARTINS • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France KAREN L. MACKENZIE • Cancer Cell Development Group, Children’s Cancer Institute Australia, Randwick, Australia; Lowy Cancer Research Centre, University of New South Wales, Randwick, Australia
  • 14. xi MICHELLE F. MARITZ • Cancer Cell Development Group, Children’s Cancer Institute Australia, Randwick, Australia; Lowy Cancer Research Centre, University of New South Wales, Randwick, Australia JOHN J. MCGARVEY • School of Chemistry and Chemical Engineering, Queen’s University, Belfast, UK LAURIE MENGER • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France DIDIER METIVIER • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France MICKAËL MICHAUD • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France BRANKA MIHALJEVIC • Rudjer Boskovic Institute, Zagreb, Croatia MARIO G. MIRISOLA • Department of Medical and Forensic Biopathology e Biotechnology, University of Palermo, Palermo, Italy SATOMI MIWA • Ageing Research Laboratories, Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK AYUMI AUREA MIYAKAWA • Laboratory of Genetics and Molecular Cardiology/LIM 13, Heart Institute (InCor), University of Sao Paulo Medical School, Sao Paulo, Brazil ASAKO J. NAKAMURA • Department of Anatomy and Cell Biology, Osaka Medical College, Takatsuki, Japan MASAKO NARITA • Cancer Research UK, Cambridge Research Institute, Cambridge, UK MASASHI NARITA • Cancer Research UK, Cambridge Research Institute, Cambridge, UK GLYN NELSON • Ageing Research Laboratories, Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK HEINZ D. OSIEWACZ • Faculty of Biosciences, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany; Frankfurt Cluster of Excellence “Macromolecular Complexes”, Frankfurt, Germany FERNANDO G. OSORIO • Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain GIOVAMBATTISTA PANI • Institute of General Pathology, Catholic University Medical School, Rome, Italy JOÃO F. PASSOS • Ageing Research Laboratories, Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK VALERY A. POSPELOV • Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia; St. Petersburg State University, St. Petersburg, Russia TATIANA V. POSPELOVA • Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia; St. Petersburg State University, St. Petersburg, Russia YINGJUAN QIAN • Comparative Oncology Laboratory, University of California, Davis, CA, USA SOFIA CHIATAMONE RANIERI • Clinical Chemistry, Laboratory and Endocrinology Unit, Departement of Laboratory Medicine, Azienda Ospedaliera ASMN, Istituto di Ricovero e Cura a Carattere Scientifico, Reggio Emilia, Italy LAURA A. RICHARDS • Cancer Cell Development Group, Children’s Cancer Institute Australia, Randwick, Australia; Lowy Cancer Research Centre, University of New South Wales, Randwick, Australia Contributors
  • 15. xii HELEN RIZOS • Melanoma Institute Australia, North Sydney, Australia University of Sydney, Sydney, Australia; Westmead Institute for Cancer Research, Westmead Millennium Institute for Medical Research, Westmead, Australia FRANCIS RODIER • Institut du cancer de Montréal, Centre de recherche du CHUM, Montréal, Canada AMBROSIUS P. SNIJDERS • Biomolecular Mass Spectrometry and Proteomics Laboratory, MRC Clinical Sciences Centre, Imperial College, London, UK ALAN W. STITT • Centre for Vision and Vascular Science, School of Medicine and Dentistry, Queen’s University, Belfast, UK ABDUL QADER SUKKURWALA • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France SUZANA BOROVIC SUNJIC • Rudjer Boskovic Institute, Zagreb, Croatia SIMON TAVARÉ • Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA; Department of Oncology, University of Cambridge, Cambridge, UK; Cancer Research UK, Cambridge Research Institute, Cambridge, UK NEKTARIOS TAVERNARAKIS • Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Greece JOHN TOWER • Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA SIEU L. TRAN • Melanoma Institute Australia, North Sydney, Australia; University of Sydney, Sydney, Australia; Westmead Institute for Cancer Research, Westmead Millennium Institute for Medical Research, Westmead, Australia GABRIEL VALBUENA • Biomolecular Medicine, Department of Surgery and Cancer, Imperial College, London, UK ILIO VITALE • INSERM, U848, Villejuif, France; Institut Gustave Roussy, Villejuif, France THOMAS VON ZGLINICKI • Ageing Research Laboratories, Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK NARENDRA WAJAPEYEE • Department of Pathology, Yale University School of Medicine, New Haven, CT, USA YU WANG • Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China NANCY B. WEHR • Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA MIN WEI • Davis School of Gerontology, Ethel Percy Andrus Gerontology Center, University of Southern California, Los Angeles, CA, USA ANDREW R.J. YOUNG • Cancer Research UK, Cambridge Research Institute, Cambridge, UK NEVEN ZARKOVIC • Rudjer Boskovic Institute, Zagreb, Croatia RUGANG ZHANG • Women’s Cancer Program, Epigenetics and Progenitor Cells Keystone Program, Fox Chase Cancer Center, Philadelphia, PA, USA HONG ZHAO • Department of Pathology, Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA MARINA ZICHE • Department of Biotechnology, University of Siena, Siena, Italy SANDRA ZINTEL • Faculty of Biosciences, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany; Frankfurt Cluster of Excellence “Macromolecular Complexes”, Frankfurt, Germany Contributors
  • 16. 1 Lorenzo Galluzzi et al. (eds.), Cell Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 965, DOI 10.1007/978-1-62703-239-1_1, © Springer Science+Business Media, LLC 2013 Chapter 1 Cell Senescence as Both a Dynamic and a Static Phenotype Andrew R.J. Young, Masako Narita, and Masashi Narita Abstract It has been 50 years since cellular senescence was first described in human diploid fibroblasts (HDFs), yet its mechanism as well as its physiological and clinical implications are still not fully appreciated. Recent progress suggests that cellular senescence is a collective phenotype, composed of complex networks of effector programs. The balance and quality within the effector network varies depending on the cell type, the nature of the stress as well as the context. Therefore, understanding each of these effectors in the con- text of the whole network will be necessary in order to fully understand senescence as a whole. Furthermore, searching for new effector programs of senescence will help to define this heterogeneous and complex phenotype according to cellular contexts. Key words: Autophagy, mTOR, Oncogene, SAHFs, SASP, Senescence, SMS, TASCC Cellular senescence is, fundamentally, a stress responsive cell cycle arrest. The senescence phenotype is extremely stable, and thus, senescence is often described as a state of “irreversible” or “permanent” cell cycle arrest, as opposed to the readily revers- ible quiescent state. The senescence phenotype was originally identified in genetically normal, primary, human diploid fibroblasts (HDFs), which “refused” to proliferate indefinitely in culture (1). At that time this behavior was attributed to the cul- ture medium lacking some essential nutrients (2), but it has since been formally attributed to the limits imposed by telomeres (3). When HDFs become senescent, in addition to their stable cell cycle exit, their morphology drastically changes. Typically the cells have enlarged cell and nuclear bodies, prominent nucleoli, and numerous cytoplasmic vacuoles. 1. Introduction
  • 17. 2 A.R.J. Young et al. While it is widely accepted that both the p53 and p16/Rb tumor suppressor pathways are involved in senescence, the precise mechanisms underlying the phenotype are still elusive. Cells respond to cytotoxic stimuli differently depending upon the cell type and the strength of the stress. HDFs are relatively resistant to apoptosis, and are highly prone to senescence. Compared to apop- tosis, senescence is a delayed and much longer process. The con- trast in these two stress responsive phenotypes may be due to the way the signals are conveyed. In apoptosis, a variety of triggers can converge to the executioner caspases through a common mecha- nism (4). In contrast, in senescence a number of effector mecha- nisms, each contributing to the process, have been proposed and it is likely that these effectors collectively define the phenotype. Thus, the senescence process can be viewed as a “matrix” composed of a series of signaling transductions, rather than the single “vector” of signals in apoptosis (Fig. 1). Such effector programs of senescence include senescence associated heterochromatic foci (SAHFs) and epigenetic gene regulation, the DNA damage response, senescence associated secretory phenotype (SASP)/Senescence-messaging secretome (SMS), and macroautophagy (5–16). Due to the highly heterogeneous nature of the senescence phenotype, it has not been possible to identify a universal marker for senescence that applies in all cases. However, the use of a combination of various markers often associated with effector programs of senescence has success- fully contributed to extending the concept of senescence outside the HDF system: a similar phenotype can be induced by various Fig. 1. The juxtaposition of senescence and apoptosis. The contrast between these two stress responsive phenotypes may be due to the way the signals are conveyed. In apop- tosis, a variety of triggers can converge to the executioner caspases through a common mechanism. In contrast, in senescence, a number of effector mechanisms, which can each contribute to the process, have been proposed and it is likely that these effectors collectively define the phenotype, and also that the eventual phenotype maybe different depending on the signals and effectors that induced it. In addition, senescence is typically a delayed and progressive process. Thus, as schematically illustrated here, the senes- cence process can be viewed as a “matrix” composed of a series of signal transductions, rather than the signal “vectors” of apoptosis.
  • 18. 3 1 Dynamic Interaction Between Autophagy and Senescence cellular stresses in different types of somatic cells, as well as different tissues in animals (17–20). Importantly, the effector mechanisms and markers used in these studies were mostly identified in experi- ments using HDFs, and thus, HDFs are still the best-characterized model system for this topic. Therefore, in this review, most descrip- tions are derived from this system unless specifically mentioned otherwise. We also use the term “senescence” to specifically describe the cellular phenotype, distinct from “aging” at the organism level. The complexity of senescence also implies a “time axis,” by which senescence can be understood as a delayed and progressive process. Therefore, it would be sensible to view senescence not only as a static endpoint, but also as a dynamic process of pheno- typic establishment (Fig. 1). This distinction becomes more rele- vant in acute types of senescence, such as oncogene-induced senescence (OIS). Here, we overview the static and dynamic aspects of senescence and discuss our current understanding of the com- plex interactions between the effector programs during senescence, with a particular focus on the regulation of gene expression. It was shown in 1997 that oncogenic Ras could induce premature senescence, in both HDFs and mouse embryonic fibroblasts (MEFs) (21). As the paradoxical name suggests, the Ras-induced senescence (RIS) phenotype is nonlinear. Due to the direct impact of the abnormal mitogenic stimuli, the cells initially proliferate more rapidly (22). This mitotic phenotype is then gradually replaced by the senescence phenotype. Interestingly, the in vivo counterpart of OIS is well represented by some pre-neoplastic or benign tumors, such as melanocytic nevi, lung adenomas, and pan- creatic intraductal neoplasias, and chemically induced skin papillo- mas (23–28) (see ref. 20). These tumors are likely to be a consequence of cell proliferation during this mitotic phase. Thus, OIS/RIS may be a model for the initial steps of tumorigenesis, which include the series of events from the first reaction of the cells to the mitotic stress, following activation of various effector pro- grams, and the maintenance of the phenotype. Importantly, each effector program is typically involved not only in senescence but also in its original, physiological, role outside of senescence. Thus, studying the OIS/RIS system should extend our understanding of each of these effector programs and provide insight into the more general concepts in dynamic alterations of phenotype, such as differentiation and transformation. 2. Oncogenic, Ras-Induced Senescence
  • 19. 4 A.R.J. Young et al. The strong stability of the phenotype, compared to the readily reversible G0 arrest of quiescence, is a critical aspect of senescence, implying its biological relevance in, for instance, tumor suppres- sion and therapy (29, 30), aging (31), and the efficiency of repro- gramming to pluripotent stem cells (32–37). Not surprisingly, one focus of the earlier studies on senescence was on the irreversibility of the cell cycle exit. For example, the Campisi group showed that the refractoriness of cells to mitogens is a hallmark of senescence, and that some growth-related genes are not responsive to growth factors (38, 39). The same group later examined the reversibility of senescence caused by telomere dysfunction using different types of HDFs, with varying levels of the CDK inhibitor p16. They found that the replicative senescence arrest could be reversed by p53 depletion in p16-low cells (such as BJ cells), whereas in p16-high cells (such as WI38 cells) senescence could not be so easily reversed (40). Thus, while p53 contributes to continuous cell cycle arrest signaling, the p16/Rb pathway provides a dominant barrier to the immortalization of human cells. A primary role for the p53 path- way has also been shown in OIS, where it is activated by a persis- tent DNA damage response (41). These data also reinforce the classical view that, in human cells, it is required to abrogate both the p53 and p16/Rb pathways in order to bypass senescence, and that most cancers do indeed have defects in both pathways. We and others have previously shown that HDFs exhibit dra- matic alterations in heterochromatin during senescence (SAHF) in a p16/Rb dependent manner, and have proposed that SAHF for- mation is involved in the stable silencing of at least some cell cycle genes (42, 43). Interestingly, senescent HDFs with high p16 (non- reversible by p53 depletion) show more prominent SAHFs com- pared to p16-low HDFs (reversible by p53 depletion), indicating a correlation between SAHF formation and the stability of senescence arrest (5). Furthermore, we have identified chromatin architectural proteins, HMGA1 and HMGA2, as SAHF components. SAHF dis- ruption by HMGA1 depletion made cellular senescence bypass easier, reinforcing the correlation between phenotype stability and high-order chromatin architecture (44). However, both hetero- chromatin structure and the related epigenetic marks can also be dynamic. Thus, it is conceivable that cells need to have additional mechanisms to achieve the ultimate irreversibility of senescence. The precise relationship between high-order SAHF structure and individual gene regulation is still unclear. To investigate this connection it would be necessary to effectively combine both imag- ing and biochemical data for SAHF markers, which to date has not been managed. It is tempting, however, to speculate that such a clear segregation between euchromatin and heterochromatin in 3. Transcriptional Regulation During Senescence
  • 20. 5 1 Dynamic Interaction Between Autophagy and Senescence the static condition might allow a rather fixed gene expression profile, giving up dynamic and complex gene expression regulation. In this sense, SAHFs may be important not only for gene repres- sion as originally proposed, but may also play a role in the stable maintenance of gene activation in the euchromatic regions. This interpretation would extend our understanding of SAHFs beyond senescence, and rather put the concept of SAHFs in the context of gene plasticity that is associated with distinct cellular phenotypes. Conceivably, senescent cells may exhibit a distinct mRNA expression profile compared to their growing or quiescent coun- terparts (45). A flurry of papers in the late 2000s defined the func- tionality of the plethora of factors secreted by senescent cells. This heterogenous collection of cytokines, chemokines and other fac- tors, termed the SASP or SMS has been shown to have autocrine and paracrine functions (10–13, 45, 46)—the autocrine function being to reinforce the senescence phenotype in the cell of origin and one potential paracrine function being to induce senescence in other cells sharing the same DNA damaging/tumorigenic microen- vironment. Another paracrine function may be the non-serendipi- tous promotion of the transformation of premalignant—but not of normal—cells within the microenvironment (47, 48). Amongtheseriesofpapersdescribingthefunctionalsignificance of SASP/SMS, the Peeper group notably identified SASP/SMS as a “secretome” specifically associated with OIS (10). Microarray experiments showed a set of transcripts highly upregulated in OIS cells, which were upregulated neither in cells that had bypassed OIS nor in quiescent cells (10). Thus, it is tempting to speculate that the “open” areas of chromatin generated by SAHF formation may permit and promote the expression of the secretome as a unit. Interestingly, SASP/SMS can also influence SAHF, in that deple- tion of IL-6 (a central component of SASP/SMS) in OIS corre- lated with reduced formation of SAHF as well as p15, a senescence-related CDK inhibitor (CDKI) (10). It has been shown that over-expression of p16, another CDKI, which is sufficient to induce senescence and SAHFs in HDFs, fails to trigger SASP/ SMS, indicating that SAHF formation per se cannot activate the secretome (49). However, in our time series microarray experi- ments in RIS HDFs, upregulation of SASP/SMS components starts during the transition phase of RIS, when SAHF formation is not yet fully established (15). Thus, the two processes may mutu- ally reinforce each other during the course of OIS establishment: SASP/SMS facilitating SAHF formation (possibly in part through p15 upregulation), while high-order chromatin reorganization might reinforce the stable expression of SASP/SMS components in the late stages of RIS. Thus, it is useful to think of OIS as a whole process, not just a static phenotype, and, along those lines, we recently identified another effector mechanism that affects SASP/SMS during the
  • 21. 6 A.R.J. Young et al. RIS-transition, namely macroautophagy (referred to hereafter as autophagy). As well as transcriptional effects, SASP/SMS is also facilitated at the posttranscription level. To study the process and the dynamic features of the senescence phenotype, 4OHT-inducible RIS is very useful. In the 4OHT RIS system in HDFs, it typically takes around 7 days to establish the senescence phenotype (Fig. 2) (15). In this system, much of the dynamic transcriptional alteration occurs during the RIS transition (15). However, if rapid protein turnover were to happen at the same time it would make achieving gene expression changes far more efficient, and perhaps allow emergent cell remodeling. Consistent with this idea, autophagy is highly active during the RIS transition (15). Autophagy is, essentially, the “trash collector” of the cell, or rather the “recycling collection truck.” Eukaryotic cells have two main known mechanisms for protein degradation: the proteasome and autophagy. The proteasome takes only one protein at a time and targets are signaled for selection, whereas autophagy can col- lect cellular components en masse (although, degrees of specificity are emerging for autophagy too (50)). In autophagy, vesicles form within the cytoplasm and can be seen to have enclosed cytoplasmic content. That cytoplasmic content is then trafficked along micro- tubules to the lysosome. Lysosomes contain the degradative enzymes of the cell: lipases, proteases, DNase, etc. (51). Autophagosomes then fuse with lysosomes, the product of which is termed an autolysosome, and pass their content inside for degra- dation and recycling. That content is then actively released from the lysosome (52), and thus, presumably a gradient of metabolites emanates from the lysosome. 4. Autophagy and SASP/SMS in Senescence Fig. 2. A schematic view of oncogenic Ras-induced senescence (RIS) in human diploid fibroblasts (HDFs). RIS can be acutely induced by adding 4OHT to HDFs stably expressing ER:Ras, mediated by retrovirus-mediated gene transfer. The whole process is typically completed in ~7 days.
  • 22. 7 1 Dynamic Interaction Between Autophagy and Senescence The emerging dogma is thus that autophagy represents the “guardian” of the proteome (53), which could be broadened out to the macromolecule-ome. As macromolecules become damaged, for instance through oxidation, they become misfunctional. Hence constant degradation and resynthesis of macromolecules is required, and is especially required in dire times of cellular stress, such as oxidative or metabolic stress. In addition to that role, however, we have shown that autophagy can have quite unforeseen conse- quences for the cell, impacting on cell fate, in that it can facilitate the establishment of senescence both cell autonomously and non- cell-autonomously through its role in the production of SASP/ SMS in RIS, and beyond. As mentioned in the introduction, the characteristics of senescent cells are that they typically have enlarged cell and nuclear bodies, prominent nucleoli, as well as numerous cytoplasmic vacuoles. Part of this vacuolation may come from enlargement of the ER (54) but a good part comes from the accumulation of autolysosomes (15). Upon the induction of Ras, the HDFs at first proliferate more often, or in other words they go through a “proliferative burst,” and then gradually proliferation declines as they approach the “per- manent” proliferative arrested state of senescence. It is during the transition phase (from this proliferative phase to the senescent, proliferative arrest) that autophagy becomes active. Inhibition of autophagy causes the downregulation of SASP/SMS, such as IL6 and IL8, at the posttranscriptional level. Presumably autophagy plays a role in delivering the “raw material,” the proteins for deg- radation, for their reuse in the synthesis of these SASP/SMS fac- tors, thus supporting the translation of the upregulated SASP/ SMS transcripts. A similar positive role for autophagy in senescence associated secretory protein synthesis has recently been observed in senescent HDFs induced by oxidative stress (55). The authors showed that H2 O2 induced senescence with activated autophagy, and that the inhibition of autophagy not only delayed senescence but also reduced the secretory phenotype. Furthermore, autophagy seems to affect SASP/SMS outside fibroblasts. Sasaki et al. (56) showed that autophagy can mediate cellular senescence in primary biliary cirrhosis (PBC). PBC is a liver-specific autoimmune disease that eventually leads to the extensive loss of small bile ducts. The authors had previously demonstrated cellular senescence in the damaged small bile ducts and suggested that it may be involved in the patho- genesis of progressive bile duct loss in PBC (57). They went on to look for autophagy in their bank of biopsied or surgically resected human liver specimens. Immunohistochemistry staining for autophagy and lysosomal markers showed large vesicles in PBC, chronic viral hepatitis, nonalcoholic steatohepatitis and extrahe- patic biliary obstruction. By far the highest level of these vesicles 4.1. Autophagy in RIS
  • 23. 8 A.R.J. Young et al. was seen in PBC. The autophagy positive small bile ducts also stained for markers of senescence. Moving into cultured biliary epithelial cells (BECs), Sasaki et al. showed that if autophagy was pharmacologically inhibited then the extent of senescence was significantly decreased, as was the BEC SASP/SMS. These studies support the idea that active protein turnover can promote the en masse production of secretory proteins during senes- cence. However, it is also possible that autophagy modulates the senescence phenotype through its ability to maintain the quality of macromolecules. Gamerdinger et al. identified activated autophagy during replicative senescence in HDFs as well as in the neurons of aged rodent brains (14, 58). The authors showed that BAG3, a co- chaperone, together with other chaperons, Hsp70 and HspB8, pro- moted the targeting of misfolded and aggregation prone proteins to autophagosomes. Thus, damaged proteins accumulated during senescence or aging can be eliminated by autophagy. Of note, rep- licative senescence is also associated with activation of a secretome (49), although how autophagy affects SASP/SMS in the context of replicative senescence remains to be tested. Therefore, autophagy would seem to have, at least, two distinct levels of influence on senescence; through its specific role in SASP/SMS and also through its general activity in the quality control of macromolecules. How can we reconcile these roles, or does this suggest another contro- versy, as in the association between autophagy and cell death: do cells become senescent due to autophagy or despite autophagy? The potentially mixed roles for autophagy in senescence might reflect the complex effector networks of senescence mentioned above (Fig. 1). The collective outcome of the combinatorial consequences of those effector programs would shift within the range of the senes- cence phenotype depending on the situation. Therefore, the domi- nant effect of autophagy might be different between RIS (a highly dynamic process) and replicative senescence (a slow and adaptive process). Consistent with this idea, we have recently identified an additional mechanism occurring during RIS by which autophagy can support SASP/SMS, which involves a TOR-autophagy spatial coupling compartment (TASCC) (15). TASCC—more defined mechanistic insight into the relationship between autophagy and senescence. Autophagy and protein synthesis are typically regulated in opposite directions. For example, when cells are starved, autophagy is activated while protein synthesis is suppressed, allowing the degradation products (such as amino acids) to be used as an alternative energy source. This reciprocal regulation of anabolic and catabolic processes is achieved through target of rapamycin (TOR), the master regulator of protein synthe- sis (59). TOR coordinately senses cellular nutrient and energy sta- tus as well as growth factor signals. TOR then integrates those signals and “decides” whether sufficient metabolites and energy are available to synthesize protein (60). TOR is a protein kinase
  • 24. 9 1 Dynamic Interaction Between Autophagy and Senescence and, if conditions are right, it in turn phosphorylates its substrates, such as eIF4E binding protein (4EBP) and S6 kinase (S6K), and facilitates protein synthesis (59). However, it is possible that the simultaneous activation of pro- tein degradation and synthesis may allow cells to “regenerate” themselves very efficiently, and this seems to be occurring during RIS, where both autophagy and protein synthesis are active (15). In addition, it has also been shown that Rapamycin, which inhibits TOR, can divert senescence into quiescence; thus, mTOR is required for senescence maintenance (61). One potential explana- tion for the apparently paradoxical simultaneous activation of pro- tein degradation and protein synthesis may be the spatial separation of these processes within the cell. By immunostaining, we identified a unique cellular compartment, the TASCC, in which both mam- malian TOR (mTOR) and autolysosomes (the end stage of autophagy) are enriched in the vicinity of the rER-Golgi apparatus (GA). Aside from growth factor signaling, mTOR can also be acti- vated by amino acids (the ultimate end product of autophagic pro- tein degradation). The inhibitory point where mTOR acts on autophagy is the initial step of autophagosome formation; thus, it is possible that the physical coupling of these two opposing pro- cesses causes them to reinforce each other. Indeed, we have shown that amino acids derived from autolysosomes are required for mTOR localization to the TASCC, and that early stage autophago- somes are only detected outside of the TASCC. Our data is highly consistent with emerging evidence from the Sabatini group, indi- cating that mTOR is recruited to, and activated at, the lysosomal surface, where Rheb resides, and that this process is dependent on Rag GTPases, which also localize to lysosomes (62, 63). Furthermore, the TASCC is consistently surrounded by the trans-side of the GA. The rER-GA is the place where both secre- tory proteins as well as lysosomal and other membrane compo- nents are synthesized and processed. Thus, the encompassing of the TASCC by the rER-GA raises an interesting possibility: that the TASCC, together with the associated rER-GA, may form a protein “factory” for both the SASP/SMS to be secreted, and lys- osomal and other membrane proteins (which would, in turn, rein- force TASCC formation itself). This could be tested using the technique of fluorescent noncanonical amino acid tagging (FUNCAT) to specifically visualize newly synthesized proteins in situ (FUNCAT was developed by the Schuman laboratory (64– 66), and is now licensed to Invitrogen). This system is essentially a more modern alternative to radioactive, e.g., 35 S, methionine met- abolic labeling. Pulse–chase experiments with the “tagged” amino acid revealed that as early as 3 min the nascent proteins are enriched around the GA, and subsequently move to the GA, and about 2 h later are detected in the TASCC. In addition, by immunolabelling, IL6 and IL8 were specifically detected in the TASCC as well as the
  • 25. 10 A.R.J. Young et al. surrounding GA, suggesting that the same compartment facilitates the synthesis of proteins for both the degradation machinery and for SASP/SMS. Consistently, inhibition of Rag GTPase activity during RIS blocked mTOR enrichment to the TASCC and reduced the synthesis of SASP/SMS components (15). Although direct evidence for the regulation of protein synthesis within the TASCC by mTOR remains to be shown, the TASCC demonstrates addi- tional complexity in terms of its functional associations between SASP/SMS, senescence and autophagy. Importantly, we also found that the TASCC was in close asso- ciation with the GA in other cellular conditions, such as mac- rophage-like differentiation in culture, during which cells acutely produce abundant IL8 (15). In addition, TASCC-like compart- ments were detected specifically in the glomerular podocytes of mouse kidneys (15). Podocytes are highly enriched for constitutive autophagy as well as prominent GA, and produce abundant secre- tory proteins to support the constant turnover of the glomerular basement membrane (67, 68). The TASCC may thus be associated more generally with the acute, abundant production of secretory proteins, rather than senescence per se. Consistent with this inter- pretation, replicative senescent HDFs do not show a prominent TASCC (Masako Narita & Masashi Narita, unpublished data), sug- gesting that the rate or amount of synthesis of SASP/SMS compo- nents in this adaptive condition may not require such self-reinforcing machinery. Although the exact molecular basis for the necessity of the TASCC in facilitating the production of secretory proteins in some cases but not in others remains to be elucidated, the TASCC might provide a mechanism to modulate the downstream effects of activated autophagy. Taken together, the studies outlined in this review reinforce the higher-order network of the different effector programs of senes- cence and its complexity, in which the same effector might even produce different outcomes through the different aspects— dynamic or static—of senescence. Furthermore when it is consid- ered, as mentioned in Subheading 3, that SASP can affect SAHF, then the new role for autophagy in the regulation of SASP/SMS and its spatial association with mTOR implies a functional coop- eration between nuclear (transcription) and cytoplasmic pheno- types (protein turnover) toward the expression of a subset of genes. While we only focus on the limited aspects of gene regulation in RIS in this review, there are other mechanisms that are also involved in the phenotype and we expect, in this model system, additional unexpected interactions between them, as well as new effector mechanisms to be identified in the future. 5. Conclusions
  • 26. 11 1 Dynamic Interaction Between Autophagy and Senescence Acknowledgments Our work is supported by Cancer Research UK. References 1. Hayflick L (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25: 585–621 2. Shay JW, Wright WE (2000) Hayflick, his limit, and cellular ageing. Nat Rev Mol Cell Biol 1:72–76 3. Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345:458–460 4. Strasser A, Cory S, Adams JM (2011) Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. EMBO J 30:3667–3683 5. Narita M, Nũnez S, Heard E, Narita M, Lin AW, Hearn SA, Spector DL, Hannon GJ, Lowe SW (2003) Rb-mediated heterochromatin for- mation and silencing of E2F target genes dur- ing cellular senescence. Cell 113:703–716 6. Zhang R, Poustovoitov MV, Ye X, Santos HA, ChenW,DaganzoSM,ErzbergerJP,Serebriiskii IG, Canutescu AA, Dunbrack RL, Pehrson JR, Berger JM, Kaufman PD, Adams PD (2005) Formation of MacroH2A-containing senes- cence-associated heterochromatin foci and senescence driven by ASF1a and HIRA. Dev Cell 8:19–30 7. Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, Luise C, Schurra C, Garre M, Giovanni Nuciforo P, Bensimon A, Maestro R, Giuseppe Pelicci P, D’adda Di Fagagna F (2006) Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 444:638–642 8. Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, Vassiliou L-VF, Kolettas E, Niforou K, Zoumpourlis VC, Takaoka M, Nakagawa H, Tort F, Fugger K, Johansson F, Sehested M, Andersen CL, Dyrskjot L, Ørntoft T, Lukas J, Kittas C, Helleday T, Halazonetis TD, Bartek J, Gorgoulis VG (2006) Oncogene- induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 444:633–637 9. Mallette FA, Gaumont-Leclerc M-F, Ferbeyre G (2007) The DNA damage signaling pathway is a critical mediator of oncogene-induced senescence. Genes Dev 21:43–48 10. Kuilman T, Michaloglou C, Vredeveld LCW, Douma S, van Doorn R, Desmet CJ, Aarden LA, Mooi WJ, Peeper DS (2008) Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell 133:1019–1031 11. Acosta JC, O’Loghlen A, Banito A, Guijarro MV, Augert A, Raguz S, Fumagalli M, Da Costa M, Brown C, Popov N, Takatsu Y, Melamed J, D’adda Di Fagagna F, Bernard D, Hernando E, Gil J (2008) Chemokine signal- ing via the CXCR2 receptor reinforces senes- cence. Cell 133:1006–1018 12. Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR (2008) Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell 132:363–374 13. Coppé J-P, Patil CK, Rodier F, Sun Y, Muñoz DP, Goldstein J, Nelson PS, Desprez P-Y, Campisi J (2008) Senescence-associated secre- tory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6:2853–2868 14. Gamerdinger M, Hajieva P, Kaya AM, Wolfrum U, Hartl FU, Behl C (2009) Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3. EMBO J 28:889–901 15. Young ARJ, Narita M, Ferreira M, Kirschner K, Sadaie M, Darot JFJ, Tavaré S, Arakawa S, Shimizu S, Watt FM, Narita M (2009) Autophagy mediates the mitotic senescence transition. Genes Dev 23:798–803 16. Narita M, Young ARJ, Arakawa S, Samarajiwa SA, Nakashima T, Yoshida S, Hong S, Berry LS, Reichelt S, Ferreira M, Tavaré S, Inoki K, Shimizu S, Narita M (2011) Spatial coupling of mTOR and autophagy augments secretory phenotypes. Science 332(6032):966–970 17. Romanov SR, Kozakiewicz BK, Holst CR, Stampfer MR, Haupt LM, Tlsty TD (2001) Normal human mammary epithelial cells spon- taneously escape senescence and acquire genomic changes. Nature 409:633–637 18. Lin AW, Lowe SW (2001) Oncogenic ras acti- vates the ARF-p53 pathway to suppress epithe- lial cell transformation. Proc Natl Acad Sci U S A 98:5025–5030 19. Shay JW, Roninson IB (2004) Hallmarks of senescence in carcinogenesis and cancer ther- apy. Oncogene 23:2919–2933 20. Narita M, Lowe SW (2005) Senescence comes of age. Nat Med 11:920–922
  • 27. 12 A.R.J. Young et al. 21. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW (1997) Oncogenic ras provokes pre- mature cell senescence associated with accumu- lation of p53 and p16INK4a. Cell 88:593–602 22. Lin AW, Barradas M, Stone JC, Van Aelst L, Serrano M, Lowe SW (1998) Premature senes- cence involving p53 and p16 is activated in response to constitutive MEK/MAPK mito- genic signaling. Genes Dev 12:3008–3019 23. Lazzerini Denchi E, Attwooll C, Pasini D, Helin K (2005) Deregulated E2F activity induces hyperplasia and senescence-like fea- tures in the mouse pituitary gland. Mol Cell Biol 25:2660–2672 24. Braig M, Lee S, Loddenkemper C, Rudolph C, Peters AH, Schlegelberger B, Stein H, Dörken B, Jenuwein T, Schmitt CA (2005) Oncogene- induced senescence as an initial barrier in lym- phoma development. Nature 436:660–665 25. Chen Z, Trotman LC, Shaffer D, Lin H-K, Dotan ZA, Niki M, Koutcher JA, Scher HI, Ludwig T, Gerald W, Cordon-Cardo C, Paolo Pandolfi P (2005) Crucial role of p53-depen- dent cellular senescence in suppression of Pten- deficient tumorigenesis. Nature 436:725–730 26. Collado M, Gil J, Efeyan A, Guerra C, Schuhmacher AJ, Barradas M, Benguría A, Zaballos A, Flores JM, Barbacid M, Beach D, Serrano M (2005) Tumour biology: senescence in premalignant tumours. Nature 436:642 27. Michaloglou C, Vredeveld LCW, Soengas MS, Denoyelle C, Kuilman T, Van Der Horst CMAM, Majoor DM, Shay JW, Mooi WJ, Peeper DS (2005) BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436:720–724 28. DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K, Mangal D, Yu KH, Yeo CJ, Calhoun ES, Scrimieri F, Winter JM, Hruban RH, Iacobuzio-Donahue C, Kern SE, Blair IA, Tuveson DA (2011) Oncogene- induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 475: 106–109 29. Schmitt CA, Fridman JS, Yang M, Lee S, Baranov E, Hoffman RM, Lowe SW (2002) A senescence program controlled by p53 and p16INK4a contributes to the outcome of can- cer therapy. Cell 109:335–346 30. te Poele RH, Okorokov AL, Jardine L, Cummings J, Joel SP (2002) DNA damage is able to induce senescence in tumor cells in vitro and in vivo. Cancer Res 62:1876–1883 31. Sharpless NE, Depinho RA (2007) How stem cells age and why this makes us grow old. Nat Rev Mol Cell Biol 8:703–713 32. Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Nakagawa M, Okita K, Yamanaka S (2009) Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460:1132–1135 33. Kawamura T, Suzuki J, Wang YV, Menendez S, Morera LB, Raya A, Wahl GM, Belmonte JCI (2009) Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460:1140–1144 34. Marión RM, Strati K, Li H, Murga M, Blanco R, Ortega S, Fernandez-Capetillo O, Serrano M, Blasco MA (2009) A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 460: 1149–1153 35. Li H, Collado M, Villasante A, Strati K, Ortega S, Cañamero M, Blasco MA, Serrano M (2009) The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 460:1136–1139 36. Krizhanovsky V, Lowe SW (2009) Stem cells: the promises and perils of p53. Nature 460:1085–1086 37. Banito A, Rashid ST, Acosta JC, Li S, Pereira CF, Geti I, Pinho S, Silva JC, Azuara V, Walsh M, Vallier L, Gil J (2009) Senescence impairs successful reprogramming to pluripotent stem cells. Genes Dev 23:2134–2139 38. Seshadri T, Campisi J (1989) Growth-factor- inducible gene expression in senescent human fibroblasts. Exp Gerontol 24:515–522 39. Seshadri T, Campisi J (1990) Repression of c-fos transcription and an altered genetic pro- gram in senescent human fibroblasts. Science 247:205–209 40. Beauséjour CM, Krtolica A, Galimi F, Narita M, Lowe SW, Yaswen P, Campisi J (2003) Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J 22: 4212–4222 41. D’adda Di Fagagna F (2008) Living on a break: cellular senescence as a DNA-damage response. Nat Rev Cancer 8:512–522 42. Narita M (2007) Cellular senescence and chro- matin organisation. Br J Cancer 96:686–691 43. Adams PD (2007) Remodeling of chromatin structure in senescent cells and its potential impact on tumor suppression and aging. Gene 397:84–93 44. Narita M, Narita M, Krizhanovsky V, Nuñez S, Chicas A, Hearn SA, Myers MP, Lowe SW (2006) A novel role for high-mobility group a proteins in cellular senescence and heterochro- matin formation. Cell 126:503–514 45. Shelton DN, Chang E, Whittier PS, Choi D, Funk WD (1999) Microarray analysis of repli- cative senescence. Curr Biol 9:939–945 46. Young ARJ, Narita M (2009) SASP reflects senescence. EMBO Rep 10:228–230
  • 28. 13 1 Dynamic Interaction Between Autophagy and Senescence 47. Krtolica A, Parrinello S, Lockett S, Desprez PY, Campisi J (2001) Senescent fibroblasts pro- mote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci U S A 98:12072–12077 48. Campisi J, D’adda Di Fagagna F (2007) Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 8:729–740 49. Rodier F, Coppé J-P, Patil CK, Hoeijmakers WAM, Muñoz DP, Raza SR, Freund A, Campeau E, Davalos AR, Campisi J (2009) Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion. Nature 11:973–979 50. Kraft C, Peter M, Hofmann K (2010) Selective autophagy: ubiquitin-mediated recognition and beyond. Nature 12:836–841 51. Holtzman E (1989) Lysosomes. Springer, New York, p 439 52. Mancini GM, Havelaar AC, Verheijen FW (2000) Lysosomal transport disorders. J Inherit Metab Dis 23:278–292 53. Ryan KM (2011) p53 and autophagy in cancer: guardian of the genome meets guardian of the proteome. Eur J Cancer 47:44–50 54. Denoyelle C, Abou-Rjaily G, Bezrookove V, Verhaegen M, Johnson TM, Fullen DR, Pointer JN, Gruber SB, Su LD, Nikiforov MA, Kaufman RJ, Bastian BC, Soengas MS (2006) Anti- oncogenic role of the endoplasmic reticulum differentially activated by mutations in the MAPK pathway. Nat Cell Biol 8:1053–1063 55. Luo Y, Zou P, Zou J, Wang J, Zhou D, Liu L (2011) Autophagy regulates ROS-induced cel- lular senescence via p21 in a p38 MAPKα dependent manner. Exp Gerontol 46:860–867 56. Sasaki M, Miyakoshi M, Sato Y, Nakanuma Y (2010) Autophagy mediates the process of cel- lular senescence characterizing bile duct dam- ages in primary biliary cirrhosis. Lab Invest 90:835–843 57. Sasaki M, Ikeda H, Haga H, Manabe T, Nakanuma Y (2005) Frequent cellular senes- cence in small bile ducts in primary biliary cir- rhosis: a possible role in bile duct loss. J Pathol 205:451–459 58. Gamerdinger M, Kaya AM, Wolfrum U, Clement AM, Behl C (2011) BAG3 mediates chaperone-based aggresome-targeting and selective autophagy of misfolded proteins. EMBO Rep 12:149–156 59. Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism. Cell 124:471–484 60. Sengupta S, Peterson TR, Sabatini DM (2010) Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell 40:310–322 61. Korotchkina LG, Leontieva OV, Bukreeva EI, Demidenko ZN, Gudkov AV, Blagosklonny MV (2010) The choice between p53-induced senescence and quiescence is determined in part by the mTOR pathway. Aging (Albany NY) 2:344–352 62. Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, Sabatini DM (2008) The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320:1496–1501 63. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM (2010) Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141:290–303 64. Dieterich DC, Link AJ, Graumann J, Tirrell DA, Schuman EM (2006) Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc Natl Acad Sci U S A 103:9482–9487 65. Dieterich DC, Lee JJ, Link AJ, Graumann J, Tirrell DA, Schuman EM (2007) Labeling, detection and identification of newly synthe- sized proteomes with bioorthogonal non- canonical amino-acid tagging. Nat Protoc 2: 532–540 66. Dieterich DC, Hodas JJL, Gouzer G, Shadrin IY, Ngo JT, Triller A, Tirrell DA, Schuman EM (2010) In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat Neurosci 13:897–905 67. Hartleben B, Gödel M, Meyer-Schwesinger C, Liu S, Ulrich T, Köbler S, Wiech T, Grahammer F, Arnold SJ, Lindenmeyer MT, Cohen CD, Pavenstädt H, Kerjaschki D, Mizushima N, Shaw AS, Walz G, Huber TB (2010) Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. J Clin Invest 120:1084–1096 68. Pavenstädt H, Kriz W, Kretzler M (2003) Cell biology of the glomerular podocyte. Physiol Rev 83:253–307
  • 29. 15 Lorenzo Galluzzi et al. (eds.), Cell Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 965, DOI 10.1007/978-1-62703-239-1_2, © Springer Science+Business Media, LLC 2013 Chapter 2 Senescence Regulation by mTOR Vjekoslav Dulic Abstract The senescence program is activated in response to diverse stress stimuli potentially compromising genetic stability and leads to an irreversible cell cycle arrest. The mTOR pathway plays a crucial role in the regula- tion of cell metabolism and cellular growth. The goal of this chapter is to present evidence linking these two processes, which have one common regulator—the tumor suppressor p53. While the role of mTOR in senescence is still controversial, recent papers have shed new light onto this issue. This review, far from being exhaustive given the complexity of the field, will hopefully stimulate further research in this domain, whose relevance for ageing is becoming increasingly documented. Key words: AKT, CDK, CKI, Cell cycle exit, D-type cyclins, mTOR, pRB, Quiescence, Senescence Abbreviations ARF Alternative reading frame ATM Ataxia telangiectasia mutated ATR ATM and Rad3-related kinase Bmi1 B lymphoma Mo-MLV insertion region 1 Cdc25 Cell division cycle 25 (CDK-activating phosphatase) CDK Cyclin-dependent kinase Chk1/2 Checkpoint kinase 1/2 CKI CDK inhibitor ERK1/2 Extracellular regulated kinase (also called MAPK) FOXO Forkhead box protein O FKBP12 Peptidyl-prolyl cis/trans isomerase that forms a complex with rapamycin HES1 Hairy enhancer of Split1 IGF Insulin-like growth factors IRS Insulin receptor substrate INK4A Inhibitor of Cdk4 A MAPK Mitogen-activated protein kinase mTOR Mechanistic (mammalian) target of rapamycin TORC1/2 TOR complex 1 and 2 PcG Polycomb group
  • 30. 16 V. Dulic PDK1 3-Phosphoinositide-dependent kinase 1 PI3K Phosphoinositide 3-kinase PIKK Phosphoinositide 3-kinase (PI3K)-related protein serine/threonine kinase family PML Promyelocytic leukemia protein PTEN Phosphatase and tensin homologue pRb Retinoblastoma protein RHEB Ras homologue enriched in brain SAHF Senescence-associated heterochromatin foci SASP Senescence-associated secretory phenotype SGK Serum- and glucocorticoid-regulated kinase TSC1/2 Tuberous sclerosis 1,2 GTPase TGFb Transforming growth factor b Cellular senescence was originally described by Leonard Hayflick, who showed that, after a finite number of population doublings (mitotic clock), primary human cells irreversibly cease to prolifer- ate in vitro (1). This so-called Hayflick limit was later shown to be provoked, at least in part, by telomere erosion (2), the gradual loss of DNA at the ends of chromosomes, a consequence of the “replication problem” predicted by Olovnikov in the early 1970— hence “replicative senescence” (3). It has been therefore proposed that telomere erosion could activate a DNA damage response (DDR) and cause G1 cell cycle arrest similar to the one elicited by ionizing radiation (4). Moreover, following an excellent intuition, Hayflick associated cellular senescence with both cancer and ageing— with cancer, because cells must acquire certain characteristics of tumor cells to escape senescence, and with ageing, because accu- mulation of senescent cells could contribute to the global deterio- ration of an organism. We now know that these original hypotheses were fundamentally right, and since then, senescence, from a “tis- sue culture artifact,” became an ever-expanding field of intense research largely due to its tumor suppression potential and contri- bution to age-related pathologies. In addition to telomere dys- function, senescence can be induced by a large variety of stress stimuli, including strong mitogenic signals generated by hyper- activation of certain oncogenes (Ras, Raf, Myc), irreparable DNA damage, or oxidative stress. This invariably inhibits cyclin- dependent kinases (CDK), key cell division regulators, leading to the cell cycle arrest that requires activities of two major tumor suppressors, p53 and pRb (Fig. 1). As a number of excellent recent reviews extensively covered this topic (5–7), we shall here briefly outline the aspects of cellular senescence and the pathways implicated in its implementation that are relevant for this chapter. 1. Introduction
  • 31. 17 2 mTOR, Metabolism and Cell Senescence Senescence is a viable and metabolically active state, which is char- acterized by a virtually permanent (irreversible) cessation of cell division. It is associated with dramatic changes in cell morphology (large flat cells), metabolism, gene expression and secretion patterns (senescence-associated secretory phenotype or SASP), originally described by Campisi and colleagues (8). Senescence has recently been shown to play a crucial role in age-related pathologies associ- ated with accumulation of senescent cells (9, 10). Both in vitro and in vivo, senescent cells can be detected by virtue of β-galactosidase activity (β-gal) (11), a lysosomal enzyme, which probably does not play a direct role in senescence (12), but serves as a reliable biomarker 2. How is Permanent Cell Cycle Arrest Achieved? Fig. 1. Molecular pathways leading to senescence-associated irreversible cell cycle arrest. Pro-senescence stimuli activate DNA damage response pathway (DDR) leading to perma- nent inactivation of cyclin-dependent kinases (CDK) that control DNA replication and mito- sis (K1/2). In addition, together with Cdk4/6 (K4/6), they keep inactive tumor suppressor pRb (and related pocket proteins p107 and p130). Genotoxic stress and activated onco- genes induce expression of CDK inhibitors p21Waf1/Cip1 (p21) and p16Ink4A (p16) that block cell cycle progression and pRb inactivation. Active pRb blocks expression of genes controlling cell cycle (in part by sequestering E2F family of transcription factors) and contributes to chromatin reorganization in the form of SAHF or PML nuclear bodies.The pathways leading to induction of p16 are not entirely elucidated but, in the case of OIS (oncogene-induced senescence), it has been shown that demethylase JMJD3 blocks repression of Ink4A locus by PcG protein Bmi1.Arf (p19ARF ), another product of this locus, plays a major role in senes- cence in mice, while p16 is more important in humans. Figure adapted from ref. 119.
  • 32. 18 V. Dulic (see Chapter 4 and Fig. 2). In addition, senescent cells often exhibit dramatic rearrangement of chromatin structure, in the forms of PML (promyelocytic leukemia protein) nuclear bodies (13, 14) and senescence-associated heterochromatin foci (SAHF) (15). These heterochromatin structures, whose formation requires active pRb, are thought to play an important role in conferring irreversibility of the cell cycle arrest by repressing the genes controlling cell prolif- eration (Figs. 1 and 2). However, SAHFs are not observed in all cell types and in all forms of senescence (15), and recent work associates SAHF formation with oncogene induced senescence and repression of DNA damage signaling (16). The pro-senescence stimuli all converge on one major pathway to permanently arrest the cell cycle (Fig. 1). The senescence pro- gram is initiated by inactivation of CDK (17), the key cell cycle regulators. CDKs play two distinct roles. First, they control the onset and progression of DNA replication and mitosis, thus enabling and orchestrating the cell cycle. Second, CDKs, particu- larly Cdk4/6 associated with D-type cyclins, phosphorylate, thus keeping inactive, the retinoblastoma (pRb) tumor suppressor and related “pocket-proteins” (p107, p130). This is essential for cell cycle progression, since active (hypo-phosphorylated) pocket pro- teins drive cell cycle exit by sequestering E2F family transcription factors and by repressing the genes required for cell division (18). Fig. 2. Senescence versus quiescence. Quiescence (or G0) is reversible and hypotrophic cell cycle exit that occurs in G1 prior to restriction (R) point. It is mediated preferentially by p27-dependent CDK inactivation and cyclin D1 downregulation. Some results suggest that genes expressed in quiescent cells can block senescence (HES1). By contrast, senescence is irreversible and hypertrophic cell cycle exit that takes place either in G1 or G2, depending on the activated checkpoint. Cell cycle arrest is mediated by p16/p21-dependent CDK inactivation (Fig. 1) and senescent cells contain high levels of G1 cyclins (cyclin D and cyclin E) associated with inactive CDK.While the cytoplasm could be frequently stained for β-galacto- sidase (see Chapters 4, 8 and 9), senescent nuclei are often large and sometimes binuclear (due to endoreplication or defective cytokinesis) and could contain heterochromatin in the forms of SAHF (see Chapter 12) or PML bodies. Both events require active pRb family pocket proteins but in senescence only pRb is essential (ref. 19). GF growth factors.
  • 33. 19 2 mTOR, Metabolism and Cell Senescence Despite often-overlapping functions between different members of the pocket protein family, pRb was shown to play a unique and nonredundant role in senescence by repressing E2F target genes involved in DNA replication (19) (Fig. 1). Recent data suggest that this process requires a recruitment of promoter-bound pRb and E2Fs to PML nuclear bodies (14). More recently, p130/E2F4 complex was also implicated in senescence by repressing, via PML, T-box protein 2 (TBX2) frequently overexpressed in cancer (20). The presence of diverse genotoxic stresses that induce senescence activates a DDR network, controlled by ATM/ATR kinases, which blocks CDK activation via CDC25 family phosphatases, thereby temporarily halting cell cycle progression (Fig. 1). While not required for this transient arrest, p53 and its transcriptional target p21Waf1/Cip1/Sdi1 (p21), a CDK inhibitor (CKI), are essential parts of irreversible cell cycle arrest by the senescence program (21, 22). Permanent CDK inactivation by p21 stably blocks DNA synthesis and mitotic entry and, via activation of pRb, drives exit from the cell cycle (19, 23) (Fig. 1). Another CKI, p16Ink4A (p16), which targets specifically pRb kinases Cdk4/6, also plays an important role in senescence and, along with β-gal, it is often used as a bio- marker both in vitro and in vivo (9, 24). However, unlike p21, p16 does not seem to be involved in the cell cycle arrest associated with replicative senescence (21) and its late induction, which occurs after p21-mediated cell cycle arrest, might promote a stabilization of senescent state (25, 26). Nevertheless, p16 is a key regulator of oncogene-induced senescence (OIS) (27) and recent work in a mouse model strongly implicated this inhibitor in acquisition of age-related pathologies (9). Although p16 is widely considered as an essential part of the senescence program, the pathways regulat- ing its induction in response to senescence-promoting stimuli are still not entirely elucidated. It has been suggested that, in the case of OIS, activation of histone demethylase JMJD3 suppresses the repression of the INK4A-ARF locus by Polycomb group proteins (PcG), Bmi1; (5). Another protein encoded by this locus is tumor suppressor p19ARF (ARF) that plays a prominent role in senescence in mice but not in humans (27). ARF stabilizes p53 by inhibiting MDM2 and, via its effector p21 blocks cell cycle. Thus, ultimate targets of both products of the INK4A-ARF locus are CDKs that inactivate pRb, and their dysregulation invariably compromises genome integrity (Fig. 1). What distinguishes senescence from another non-proliferating state, quiescence, also referred to as G0 or G0/G1 phase (28)? In contrast to senescence, quiescence is a reversible cell cycle arrest, 3. Quiescence Versus Senescence
  • 34. 20 V. Dulic which is induced by the absence of mitogens or growth factors, nutrient starvation, or increasing cell density (confluence) (Fig. 2). Typical examples of quiescent cells are lymphocytes, whose activation is part of the immune response, adult stem cells, or dermal fibroblasts, which actively participate in wound healing (29). In general, quiescence is characterized by low metabolism and protein synthesis, lack of cellular growth, and, the absence of global het- erochromatin structures such as PML bodies (30) or SAHF (15). The latter feature is probably responsible for the reversible nature of quiescence. However, much against the prevailing view, recent results showed that fibroblasts rendered quiescent by contact inhi- bition exhibit comparable metabolic activity to actively proliferat- ing cells. In addition to promote recycling of damaged macromolecules via autophagy, high metabolic activity might serve for biosynthesis and secretion of extracellular matrix proteins (31). Importantly, while downregulating expression of genes involved in cell division, quiescent cells upregulate genes, such as HES1, that inhibit senescence, differentiation and apoptosis (32, 33). Unlike senescence, which can occur either in the G1 or G2 phase of the cell cycle (34), depending on when the damage is detected and the efficiency of the checkpoints (23, 35), quiescence essentially takes place in G1, prior to the restriction (R) checkpoint (36). A teleo- logical explanation for this observation could be given by the uni- directionality of the cell cycle—G0 state always precedes DNA replication (S phase). Therefore, if cells became quiescent in the G2 phase, cell cycle entry upon stimulation would result in genome reduplication giving rise to undesirable tetraploidy. Like senescence, quiescence is characterized by CDK inactivation, the absence of S-phase- and Mitosis-promoting cyclins and the pres- ence of hypo-phosphorylated pocket proteins. However, unlike senescence, where CDKs are inhibited by p21 or/and p16, the major CKI involved in quiescent arrest is p27Kip1 (p27) (37–39). Unlike p21, p27 induction is independent of the p53 pathway (39) and its levels/activity is primarily regulated by translation, phos- phorylation, and Skp2-mediated degradation (40). Although p53 and p21 were also implicated in the cell cycle arrest following growth factor removal (41, 42), p53 integrity does not seem to be essential for quiescence (43) (Fig. 2). However, a redundancy of p27 with p21 might explain why contact inhibition is not impaired in p27−/− cells (44). Interestingly, the expression of D-type cyclins, regulatory subunits of Cdk4/6 kinases and key signal inte- grators, strikingly differs between quiescence and senescence (Fig. 2). Downregulation of cyclin Ds is a hallmark of quiescence and their rapid mitogen-dependent induction is invariably associated with cell cycle entry (42, 45, 46). In sharp contrast, D cyclins are stabi- lized (cyclin D1) and even overexpressed (cyclin D2) in senescent cells (17, 35, 43, 47). In conjunction with the large-cell phenotype that is observed in senescent cells, these results suggest that the
  • 35. 21 2 mTOR, Metabolism and Cell Senescence mitogenic pathways involved in cell growth are active in senescent cells (see below) and might even have a positive role in senescence regulation. Indeed, several recent publications have connected the “hypertrophic” phenotype of senescent cells with activity of mTOR, a master cell growth regulator (48), although this topic is still controversial (49). Originally identified in budding yeast mutants conferring resis- tance to a potent antifungal metabolite (50), the target of rapamy- cin (TOR) is at the core of a vast signaling pathway regulating cell growth and metabolism in virtually all eukaryotes. The complexity of this pathway, which couples energy and nutrient abundance to the execution of cellular growth and division, relies on the fact that TOR simultaneously senses energy (ATP state), nutrients, stress, as well as growth factors (51, 52). In addition to its growth-related functions, the TOR pathway is also wired to the regulators of cell cycle machinery such as G1 cyclins (D-type and E-type) control- ling G1/S-phase progression (see below and Fig. 3). It is therefore not surprising that TOR is essential for many developmental and physiological processes (53) while deregulation in its signaling has been implicated in a wide variety of diseases (52). mTOR (“m” stands for mammalian or, more recently, mechanistic), which belongs to the phosphoinositide 3-kinase (PI3K)-related protein serine/threonine kinase family (PIKK), forms two distinct multi- protein complexes, having distinctive physical structures and functions: mTOR complex 1 (mTORC1), which is sensitive to rapamycin owing to association with the peptidyl-prolyl cis/trans isomerase FKBP12 (50), and mTORC2, which is not sensitive to rapamycin in most cases. Their assembly, substrate specificity, and regulation are defined by specific accessory proteins: Raptor (regu- latory associated protein of mTOR) being the most prominent for mTORC1, and Rictor (rapamycin-insensitive companion of mTOR), which is specific for mTORC2. Since the regulatory inputs and the cellular actions of mTOR are far too complex for the scope of this chapter (and are reviewed in ref. 53, 54), I outline only some “essentials” focusing mainly on mTORC1, the better characterized of the two. mTORC1, which is activated by nutrients, growth factors, and cellular energy status, regulates temporal aspects of cellular growth including protein synthesis, ribosome biogenesis, lipid synthesis, nutrient import, and autophagy. Best-known substrates of mTORC1 are p70 ribosomal protein S6 kinases (S6K1 and S6K2) and the eIF4E binding proteins (4E-BP1 and 4E-BP2) by which it controls protein synthesis. The positive and negative control of 4. The mTOR Pathway Essentials
  • 36. 22 V. Dulic mTORC1 in response to intracellular and extracellular stimuli is mediated by the phosphorylation and subsequent inhibition of Tuberous sclerosis 1 (TSC1, hamartin)/TSC2 (tuberin) complex (Fig. 3). TSC2 subunit, a hub for a variety of cues impinging on mTORC1, serves as GTPase activating protein (GAP), which itself inhibits the small GTPase Rheb, a direct mTORC1 activator (51, 53, 55). TSC2 activity is regulated by several kinases: negatively, by Akt and ERK1/2 (activate mTORC1) and positively, by AMPK and GSK3β (inhibit mTORC1). Importantly, the Wnt pathway also regulates TSC1/TSC2, via inhibition of GSK3β (53) (see Fig. 4). mTORC2 is thought to regulate mainly cytoskeleton orga- nization and cell survival by phosphorylating of AGC kinase family members, including Akt, SGK1, and PKC (52, 53) (Fig. 3). Although little is known about upstream regulators of mTORC2 (56), recent reports showing that its activation requires association with ribosomes suggest that it is active only in growing cells (57). Fig. 3. Interactions between mTOR signaling, p53 and cell cycle regulators.While mTORC1 controls cell growth/division and metabolism,TORC2 mainly controls cell shape and activates Akt.TSC1/TSC2 complex plays a pivotal role as a hub for both positive and negative cues that control mTORC1 activity via signaling kinases (see ref. 53). mTOR controls cell division by regulating synthesis of cyclin D and activity/localization of CDK inhibitor p27. While in the nucleus, p27 inhibits CDKs (cD- K4/K6 and cE-K2) thereby blocking cell cycle progression (like in quiescence). Growth factors stimulate mTOR and ERK1/2 that, via Akt, RSK and SGK, mediate p27 phosphorylation leading to its cytoplasmic sequestration. Cytoplasmic p27 pro- motes proliferation by stabilizing cyclin D-Cdk4/6 assembly and cell motility by inhibiting RhoA/ROCK (see ref. 71). In response to DNA damage or other stresses, p53 negatively regulates mTORC1 by activating AMPK and PTEN and inducing TSC2. Constitutive activation of mTOR (asterisk) could induce p53 translation (see ref. 86). Figure adapted from ref. 119. GF growth factors (insulin, IGF1); Sesn, sestrin.
  • 37. 23 2 mTOR, Metabolism and Cell Senescence As eluded above, mTOR pathway is also implicated in cell division, in part by controlling the synthesis of D-type cyclins (58, 59), major regulators of G1-S phase progression, whose induction in response to growth factors is regulated by the Ras-ERK1/2 path- way (60, 61) (Fig. 3). Recent data show that mTORC1 controls cell proliferation, but not growth, via its targets 4E-BPs, by stimu- lating the translation of proteins involved in cell cycle progression, including cyclin D3 (62). In addition, in the fission yeast model, TOR was also shown to control mitotic entry in response to nutrients. In this system, both rapamycin (by inhibiting TOR) and poor nitrogen sources stimulate the stress MAPK pathway (Spc1/Sty1), which, by activating Polo kinase and Cdc2 (Cdk1 ortholog, the main mitotic regulator) accelerated mitosis with reduced cell size as consequence (63). While no equivalent path- way was yet reported in other models, these observations are con- sistent with earlier results that linked mTOR signaling with cell-size control (64). Nevertheless, despite general downregulation of 5. mTOR and Cell Cycle Fig. 4. Possible roles of p53 and mTOR in senescence. Genotoxic stress (via p53–p21) and activated oncogenes (via p16) induce senescence by inactivating CDK and activating pRb (see Fig. 1). Classically, p53 and activated oncogenes induce autophagy by inhibiting mTOR pathway (grey box).Autophagy is implicated in senescence by stimulating specific secretory phenotype (SASP) that also promotes senescence. However, recent data suggest that mTOR might positively regulate senescence (grey dotted lines) as in the case of persistent Wnt1 signaling or PTEN knockdown. Pro-senescence mTOR activity could be also activated via oncogenic stress—by inducing autophagy, or via low p53 levels (grey dotted cross)—by promoting cell growth and D-type cyclin synthesis. Hypertrophy of senescent cells could be explained by deregulated cell signaling (not responding to extracellular cues) resulting in permanent mTOR activation. This is in agreement with results showing that in senescent cells cell growth and cell division are uncoupled events. For more details regarding mTOR regu- lation, see Fig. 3 and ref. 53. Figure adapted from ref. 119. GF growth factors.
  • 38. 24 V. Dulic protein translation in mitosis (65), several recent papers suggest that mTORC1 might also control protein synthesis in mitosis by regulating expression of human Cdk1, which phosphorylates eukaryotic elongation factor 2 (eEF2); (66). Moreover, it seems that, at G2/M transition, Cdk1 might also regulate mTORC1 activity by phosphorylating Raptor (67, 68). The mTOR pathway also positively controls cell cycle by regulating the activity and subcellular localization of the CKI p27, negative regulator of G1/S progression (Fig. 3). In the presence of growth factors, Akt (activated by mTORC2), RSK (activated by ERK1/2), and SKG (activated by mTORC1, mTORC2 and Pdk1) phosphorylate p27, resulting in its cyto- plasmic retention (69–71). This not only prevents p27 to exert its nuclear role as CDK inhibitor, by inactivating CDKs control- ling DNA replication (as cyclin E-Cdk2), but also promote cell proliferation by stabilizing assembly of cyclin D-CDK4/6 com- plexes and increasing cell motility, via inhibition of RhoA signal- ing (71) (Fig. 3). In addition, mTORC2 regulates cell proliferation via AKT-mediated inhibition of forkhead box pro- tein O (FOXO) transcription factors playing a key role in pro- moting apoptosis (72). Importantly, the FOXO family members were also shown to block cell cycle by inducing p27 (73) or repressing D-type cyclins (74). In the light of increasing evidence that altered metabolism is not only a common feature of many cancer cells but that it can also greatly contribute to malignant transformation (75), the discov- ery that p53 also regulates metabolic pathways and interacts with mTOR/AKT pathways is not that surprising (reviewed in refs. 76, 77) (Fig. 3 and 4). Indeed, it has been shown that, in response to diverse stress signals, activation of p53 directly or indirectly inhibits mTOR activity, thus regulating its downstream targets, including those involved in the activation of autophagy, a recently described tumor suppression mechanism that is involved in senes- cence (54, 78) (see also Chapter 3). The role of autophagy in senescence is discussed in more detail in the chapter by M. Narita (see Chapter 1). In addition to its housekeeping role in the main- tenance of energy homeostasis, autophagy, which is negatively regulated by mTOR, is also induced by a variety of stress stimuli, such as nutrient depletion (when mTOR is inhibited), playing a vital role in preserving cellular viability through the degradation of cellular proteins and organelles (53, 79). 6. TOR, p53, and Genotoxic Stress
  • 39. 25 2 mTOR, Metabolism and Cell Senescence The mechanism by which p53 negatively regulates mTOR involves, in part, the activation of AMPK and requires its target TSC1/TSC2 complex (Fig. 3), both of which respond to energy deprivation in cells (53, 80). In response to genotoxic stress caused by DNA damage, p53 inhibits mTORC1 pathway, by phosphory- lating Sestrin 1 and 2 that activate AMPK (81), as well as the IGF-1/Akt pathway, by inducing transcription of PTEN (82) (Fig. 3). In addition, p53 can repress mTORC1 by inducing directly TSC2 (83) or AMPK, which suppresses translation by acti- vating TSC2 (80) or by directly inhibiting mTORC1 (54, 84). However, it has been reported that AMPK might also control the p53 activity, like in the case of glucose starvation, which induces the transient AMPK–mediated phosphorylation and activation of p53 leading to reversible cell cycle arrest (85, 86). There are sev- eral examples showing that conversely, mTOR can also control p53. Upon glucose removal, mTOR negatively regulates p53 by activating PP2A (87). However, some results suggest that deregu- lated (constitutive) mTOR activity might, in response to nutrient or genomic stress, induce p53 translation, which in conjunction with activation by AMPK, induces apoptosis (88). This implies that, at least in certain biological contexts, mTOR downregula- tion/inactivation is required to prevent cell death caused by stress stimuli. Thus, p53 and mTOR signaling networks can cross-talk and coordinately regulate cell growth, proliferation and death. The mTOR pathway has been shown to have opposing roles on cellular senescence. On the one hand, by inhibiting autophagy (78, 86, 89), mTOR is considered as a negative regulator of senescence (Fig. 4), which is in agreement with its widely accepted negative role in organismal ageing (90). Moreover, as mentioned above, mTOR is inhibited by p53, a bona fide pro-senescence positive reg- ulator (Figs. 3 and 4). On the other hand, the hypertrophic pheno- type of senescent cells (Fig. 2) and increased production of secretory proteins (SASP; Fig. 4), which requires high metabolic activity, are in apparent contradiction with cellular shrinkage that is usually associated with autophagy and mTOR inhibition (as in quiescence). Indeed, in the case of oncogene or DNA damage-induced senes- cence, the onset of both senescence and autophagy correlates with inhibition of mTORC1 and mTORC2 activity (91). This might suggest a negative feedback between mTOR and senescence. These findings are also in agreement with earlier observations that acti- vation of p53 by genotoxic stress inhibits mTOR pathway by acti- vating both AMPK and PTEN (Fig. 3) (81, 86). Likewise, persistent 7. mTOR’ s Role in Senescence: Repressor or Inducer?
  • 40. 26 V. Dulic AMPK activation leads to senescence in mouse embryo fibroblasts (MEF) in the presence of functional p53 (85), which is consistent with earlier observation that AMP levels strongly increase in senes- cent cells (92). However, it is also possible that mTOR activation via different stimuli could be involved in the induction of senescence. This pro- senescence role of mTOR was first uncovered by manipulation of its upstream regulators or downstream targets. Pandolfi and col- leagues found that overexpression of an mTOR target eIF-4E induced senescence both in vitro and in vivo (93). More recently, activation of the PI3K/Akt and mTOR pathways by PTEN knock- down has been shown to induce senescence by promoting accu- mulation of p53 and p21 (94). Similarly, in an elegant in vivo skin model, Gutkind and colleagues showed that continuous mTOR activation induced by persistent Wnt1 signaling (via inhibiting GSK3; (95)) promoted epithelial stem cell exhaustion by provok- ing senescence (96). Importantly, senescence and hair loss were abolished by the presence of the mTOR inhibitor rapamycin sug- gesting that mTOR stimulation is a direct consequence of Wnt1 expression (96) (Fig. 4). These results connect persistent mitogen stimulation and mTOR activation with senescence. Moreover, they are in agreement with accumulated evidence connecting accumulation of senescent cells with ageing (7, 9). Indeed, increased mTOR activity was also observed in hematopoietic stem cells from old mice (97), whereas mTOR inhibition by rapamycin could extend lifespan both in invertebrates (98, 99) and in mice (97, 100, 101). In the context of OIS, mTOR promotes lysosome biogenesis, which is required for autophagy and senescence-specific secretory phenotype, in a newly identified cytoplasmic compartment (termed TOR-autophagy spatial coupling compartment or TASCC) (102). While these results clearly indicate that mTOR has pro-senescence functions (Fig. 4), they are surprising in light of earlier report, con- necting the induction of senescence by oncogenic ras with mTOR downregulation (91). However, in this previous report it was clearly shown that increased phosphorylation of both mTORC1 and mTORC2 substrates (S6K and FoxO3a, respectively) coin- cided more with the onset of cycle arrest (and senescence) than with ras-induced cell cycle entry, as proposed by the authors. Indeed, transient activation of mTOR was also observed in cells in which senescence was triggered by the genotoxic agent etoposide. Therefore, while the results showing that appearance of late senes- cence and autophagy markers coincided with downregulation of mTOR activity, overall this work supports a positive role of mTOR pathway in the induction of autophagy at the onset of senescence. Although mainly focused on autophagy-related roles of mTOR, the above results are consistent with the model proposed by Blagosklonny and coworkers, whose recent experiments suggest
  • 41. 27 2 mTOR, Metabolism and Cell Senescence that cellular growth and persistent mTOR activity might be implicated in the onset of the senescence program. Consistent with their finding that serum stimulation is required for both the senes- cent phenotype (large flat cells) and irreversibility of the cell cycle arrest induced by overexpression of the CDK inhibitor p21 or DNA damage, they observed that a key mTOR target, S6K, is strongly phosphorylated in senescent cells (103). Moreover, in agreement with our earlier observation (17), senescent cells also accumulated another mTOR target, cyclin D1, but the biological significance of its presence is not clear (see below). Interestingly, while rapamycin, like serum starvation, prevented S6K phosphory- lation and diminished the senescent marker β-gal staining, it failed to completely abolish large flat cell morphology and to inhibit cyclin D1 induction. While these results imply that mTORC1 is not essential for these events, it would be interesting to test whether they are controlled by mTORC2, which is less sensitive to rapamy- cin and which is implicated in both cell shape and, via its target Akt, in cyclin D1 stability (Figs. 3 and 4). In addition, rapamycin prevents the onset of senescence if added simultaneously with senescence inducers (p21 induction, genotoxic agents), but fails to revert the already established senescence (104). Taken together, the above results suggest that (transient?) mTOR activity might positively control senescence but it is still unclear how DDR network controls activity of this pathway. How one can reconcile the data suggesting a positive role of mTOR in senescence with those showing that p53 inhibits the mTOR pathway (76, 77)? In the work of Narita’s team the possible inter- actions between p53 and the mTOR pathway were not examined, because in OIS cell cycle arrest is mainly mediated by the CDK inhibitor p16, which is not regulated by p53 (91) (Fig. 1). In the quest to decipher the respective roles of mTOR and p53 in the senescence program, Blagosklonny and coworkers took advantage of nutlin-3A, a specific small-molecule MDM2 antagonist, which strongly induces p53 expression thereby triggering its transcrip- tional activity in a non-genotoxic manner (105). Surprisingly, while previous work has shown that nutlin-3A induces senescence in mouse fibroblasts (106), in human fibrosarcoma cells HT1080 and WI-38 human fibroblasts, nutlin-3A induces reversible cell cycle exit resembling quiescence (107). By exploiting a HT1080- derived cell line in which p21 is expressed from an inducible promoter, Demidenko et al. showed that nutlin-3A or p53 overex- pression could “convert” p21-induced senescence into a quies- cence-like state (108). Nutlin-3A was, however, much less efficient 8. Connections Between mTOR and p53 in Senescence— Lessons from Nutlin-3A
  • 42. 28 V. Dulic in preventing senescence in the presence of DNA damage by H2 O2 , which also stimulates the phosphorylation of mTOR (108). In addition, both nutlin-3A and rapamycin suppress senescence with- out interfering with cell cycle arrest, which led the authors to sug- gest that high p53 levels preclude the onset of senescence by inhibitingmTORandinducingquiescence(108,109).Accordingly, knockdown of TSC2, a negative mTORC1 regulator (Fig. 3), par- tially compromises nutlin-3-induced quiescence, resulting in accu- mulation of senescent cells (110). In agreement with the positive role of mTOR in senescence, in cell lines in which nutlin-3A did induce senescence (termed “senescence-prone”), mTOR was not inhibited, whereas the quiescence was “restored” in the presence of rapamycin (110). Unfortunately, these studies did not provide insight into molecular mechanisms explaining differences between “senescence-prone” and “quiescence-prone” cells. In agreement with the above experiments, when exposed to etoposide, serum- deprived or rapamycin-treated fibroblasts (or epithelial cells) failed to become senescent despite p21 induction, as drug removal, con- comitant with serum addition, enabled proliferation (111). In con- trast, serum addition in the presence of etoposide induced senescence, presumably by activating mTOR. These results sup- port the hypothesis that quiescence (or mTOR inhibition) com- promises senescence. However, it is not clear why the checkpoints were not activated once cells exposed to etoposide reentered the cell cycle (assuming that the DNA damage had not been repaired) and how the cells got rid of high p21 levels. This work, however, did not address the role of the CKI p27 in p53-induced quiescence. This point is relevant for understand- ing the mechanism that induces quiescence, since mTOR inhibi- tion by rapamycin or p53 overexpression might also block SGK/ Akt-mediated p27 phosphorylation and cytoplasmic localization leading to its nuclear accumulation and activation (70, 71) (Fig. 3). One could imagine that, in the presence of rapamycin or absence of serum, p27-dependent CDK inactivation together with cyclin D1 down-regulation could also contribute to quiescence. Reactivation of mTOR (by serum addition) would revert this pro- cess, enabling cell cycle entry and progression, probably by degrad- ing p27 and p21 and inducing G1 cyclins (D- and E-type). Overall, the results of Blagosklonny’s team are consistent with the idea that mTOR positively regulates senescence, which might be because p53 levels are not sufficient to inhibit mTOR-dependent cell growth. According to their hypothesis, the “true role” of p53 (when highly expressed) would be to induce quiescence by inhibit- ing mTOR, which in turn would block the onset of senescence. Consequently, senescence occurs in situations where “the condi- tions for quiescence are not met” (low p53 levels) and where p53 “fails” to suppress the mTOR pathway (108, 112). This hypothesis is consistent with the data showing that quiescence prevents
  • 43. 29 2 mTOR, Metabolism and Cell Senescence p21-induced senescence or inappropriate differentiation due to expression of the transcriptional repressor HES1, playing a key role in the reversibility of this non-proliferative state (33) (Fig. 2). There are, however, several caveats regarding the use of nutlin- 3A as a tool to study the role of p53 and mTOR in senescence. For example, strong p53 induction by nutlin-3A is not usually observed in either quiescence or senescence and it is not clear which biologi- cal event produces equivalent amounts of p53. As a matter of fact, primary fibroblasts expressing the HPV16-E6 (hereafter E6) onco- gene that degrades p53 (113) can become quiescent in the absence of serum or confluence, whereas strong p21 induction in senescent cells occurs in the absence of significant increases in p53 protein levels (43). Therefore, the hypothesis that high p53 levels induce quiescence while low p53 levels lead to senescence (108, 109) should be verified in other experimental models. For example, our recent results showed that irreversible cell cycle arrest by genotoxic drugs in non-transformed human cells is independent on the degree of p53 activation (or levels) or even p21 levels but rather on the efficiency of p21 to inhibit different CDKs (Lossaint, 2011). Finally, as mentioned above, this model is in apparent contradic- tion with the results showing that in other cell lines nutlin-3A does not induce quiescence but instead elicits endoreduplication, giving rise to tetraploid cells (114), senescence—due to persistent p21 expression (106, 115), or even apoptosis (116). One explanation could be that nutlin-3A might have other targets than p53 (pRb?) or that its effects might be dependent on the experimental proto- col and cell type. The above-mentioned work mostly addressed the role of mTOR in premature (induced) senescence but less is known regarding its implication in replicative senescence. Several earlier observations, however, are consistent with the notion that the mTOR pathway might be active in senescent cells and that it is not controlled by p53 or affected by its status. In fact, in human fibroblasts aged in vitro a suppression of the p53/p21 pathway compromises cell cycle arrest, senescence-specific cyclin D2 induction (see below), as well as formation of SAHF, but it does not prevent large flat cell morphology or cyclin D1 accumulation (43). This implies that these two sets of key events associated with senescence, i.e. irre- versible cell cycle arrest and cell growth, might be uncoupled, which can also explain the senescent phenotype (Fig. 4). In addi- tion, some data suggest that mitogenic pathways in senescent cells might be also deregulated. Firstly, in sharp contrast to early passage fibroblasts, serum withdrawal in senescent fibroblasts does not 9. mTOR and Senescence: Constitutive Activation or Deregulation?
  • 44. 30 V. Dulic affect cyclin D1 mRNA or cyclin E1 expression or protein synthesis. The CDKs associated with these cyclins are, however, inhibited by p21, explaining the absence of DNA replication (17, 26) (Fig. 1). Interestingly, in agreement with other publications (35, 117), late senescence is associated with strong accumulation of cyclin D2, whose levels are not affected by serum withdrawal. The presence of G1 cyclins, and especially of cyclin D2, in senescent cells is intrigu- ing and it is not clear whether they might play a role in senescence or they merely reflect deregulated mTOR activity. One possibility is that cyclin D1 is part of the DNA repair machinery, as suggested recently (118). Secondly, late-passage p53-deficient fibroblasts (E6) failed to become quiescent upon serum withdrawal, which is documented by both the absence of cyclin D1 or cyclin A down- regulation and p27 induction (or activation). Consequently, CDKs controlling DNA replication were highly active even in the absence of serum. However, these cells failed to proliferate due to deleteri- ous effects of DNA damage resulting in aberrant mitoses, endorep- lication, or cell death (43). It is therefore possible that, concomitant with increased population doubling, gradual deregulation of the mTOR pathway contributes to the senescent phenotype. In the absence of the p53/pRb safeguard system, this deregulation pro- vides a fertile ground for tumorigenesis. In conclusion, the role of the mTOR pathway in senescence is still controversial, partly due to the various models and experimen- tal designs employed in different studies. Apart from the studies manipulating upstream mTOR regulators (PTEN, Wnt1) that reveal pro-senescence mTOR-functions, most of the work has focused on the autophagy-related aspects of senescence, which appeared to exclude mTOR as an important regulator of senes- cence. Few researchers, however, considered a hypertrophic phe- notype of senescent cells and elevated levels of D-type cyclins as an evidence of mTOR activity. Future work will show whether or not this activity is actually required for the onset of senescence, as some investigators proposed, or it is merely the result of senescence- associated deregulation of the mTOR pathway. If the former is the case, it will be interesting to learn whether, and if so, how p53 and/or pRb networks contribute to its induction. Acknowledgments I am grateful to Drs Anne Brunet (Stanford University, Palo Alto, USA) and Jacques Piette (CRBM, Montpellier, France) for their insightful comments.
  • 45. 31 2 mTOR, Metabolism and Cell Senescence References 1. Hayflick L (1965) The limited in vitro life- time of human diploid cell strains. Exp Cell Res 37:614–636 2. Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345:458–460 3. Olovnikov AM (1973) A theory of margino- tomy. The incomplete copying of template margin in enzymic synthesis of polynucle- otides and biological significance of the phe- nomenon. J Theor Biol 41:181–190 4. Stein GH, Dulic V (1995) Origins of G1 arrest in senescent human fibroblasts. Bioessays 17:537–543 5. Kuilman T, Michaloglou C, Mooi WJ, Peeper DS (2010) The essence of senescence. Genes Dev 24:2463–2479 6. Adams PD (2009) Healing and hurting: molecular mechanisms, functions, and pathol- ogies of cellular senescence. Mol Cell 36: 2–14 7. Rodier F, Campisi J (2011) Four faces of cel- lular senescence. J Cell Biol 192:547–556 8. Rodier F, Coppe JP, Patil CK, Hoeijmakers WA, Munoz DP, Raza SR, Freund A, Campeau E, Davalos AR, Campisi J (2009) Persistent DNA damage signalling triggers senescence- associated inflammatory cytokine secretion. Nat Cell Biol 11:973–979 9. Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B, Kirkland JL, van Deursen JM (2011) Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479:232–236 10. Kang TW, Yevsa T, Woller N, Hoenicke L, Wuestefeld T, Dauch D, Hohmeyer A, Gereke M, Rudalska R, Potapova A, Iken M, Vucur M, Weiss S, Heikenwalder M, Khan S, Gil J, Bruder D, Manns M, Schirmacher P, Tacke F, Ott M, Luedde T, Longerich T, Kubicka S, Zender L (2011) Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 479:547–551 11. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O et al (1995) A bio- marker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92:9363–9367 12. Lee BY, Han JA, Im JS, Morrone A, Johung K, Goodwin EC, Kleijer WJ, DiMaio D, Hwang ES (2006) Senescence-associated beta- galactosidase is lysosomal beta-galactosidase. Aging Cell 5:187–195 13. Bernardi R, Pandolfi PP (2007) Structure, dynamics and functions of promyelocytic leu- kaemia nuclear bodies. Nat Rev Mol Cell Biol 8:1006–1016 14. Vernier M, Bourdeau V, Gaumont-Leclerc MF, Moiseeva O, Begin V, Saad F, Mes-Masson AM, Ferbeyre G (2011) Regulation of E2Fs and senescence by PML nuclear bodies. Genes Dev 25:41–50 15. Narita M, Nunez S, Heard E, Narita M, Lin AW, Hearn SA, Spector DL, Hannon GJ, Lowe SW (2003) Rb-mediated heterochromatin forma- tion and silencing of E2F target genes during cellular senescence. Cell 113:703–716 16. Di Micco R, Sulli G, Dobreva M, Liontos M, Botrugno OA, Gargiulo G, Dal Zuffo R, Matti V, d’Ario G, Montani E, Mercurio C, Hahn WC, Gorgoulis V, Minucci S, d’Adda di Fagagna F (2011) Interplay between onco- gene-induced DNA damage response and heterochromatin in senescence and cancer. Nat Cell Biol 13:292–302 17. Dulic V, Drullinger LF, Lees E, Reed SI, Stein GH (1993) Altered regulation of G1 cyclins in senescent human diploid fibroblasts: accumulation of inactive cyclin E-Cdk2 and cyclin D1-Cdk2 complexes. Proc Natl Acad Sci U S A 90:11034–11038 18. Burkhart DL, Sage J (2008) Cellular mecha- nisms of tumour suppression by the retino- blastoma gene. Nat Rev Cancer 8:671–682 19. Chicas A, Wang X, Zhang C, McCurrach M, Zhao Z, Mert O, Dickins RA, Narita M, Zhang M, Lowe SW (2010) Dissecting the unique role of the retinoblastoma tumor sup- pressor during cellular senescence. Cancer Cell 17:376–387 20. Martin N, Benhamed M, Nacerddine K, Demarque MD, van Lohuizen M, Dejean A, Bischof O (2012) Physical and functional interaction between PML and TBX2 in the establishment of cellular senescence. EMBO J 31:95–109 21. Herbig U, Jobling WA, Chen BP, Chen DJ, Sedivy JM (2004) Telomere shortening trig- gers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a). Mol Cell 14:501–513 22. Vousden KH, Prives C (2009) Blinded by the light: the growing complexity of p53. Cell 137:413–431 23. Baus F, Gire V, Fisher D, Piette J, Dulic V (2003) Permanent cell cycle exit in G2 phase after DNA damage in normal human fibroblasts. EMBO J 22:3992–4002
  • 46. 32 V. Dulic 24. Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L, Sharpless NE (2004) Ink4a/Arf expression is a biomarker of aging. J Clin Invest 114:1299–1307 25. Alcorta DA, Xiong Y, Phelps D, Hannon G, Beach D, Barrett JC (1996) Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc Natl Acad Sci U S A 93:13742–13747 26. Stein GH, Drullinger LF, Soulard A, Dulic V (1999) Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mecha- nisms of senescence and differentiation in human fibroblasts. Mol Cell Biol 19: 2109–2117 27. Gil J, Peters G (2006) Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol 7:667–677 28. Coller HA (2011) Cell biology. The essence of quiescence. Science 334:1074–1075 29. Coller HA (2007) What’s taking so long? S-phase entry from quiescence versus prolif- eration. Nat Rev Mol Cell Biol 8:667–670 30. Ferbeyre G, de Stanchina E, Querido E, Baptiste N, Prives C, Lowe SW (2000) PML is induced by oncogenic ras and promotes premature senescence. Genes Dev 14: 2015–2027 31. Lemons JM, Feng XJ, Bennett BD, Legesse- Miller A, Johnson EL, Raitman I, Pollina EA, Rabitz HA, Rabinowitz JD, Coller HA (2010) Quiescent fibroblasts exhibit high metabolic activity. PLoS Biol 8:e1000514 32. Coller HA, Sang L, Roberts JM (2006) A new description of cellular quiescence. PLoS Biol 4:e83 33. Sang L, Coller HA, Roberts JM (2008) Control of the reversibility of cellular quies- cence by the transcriptional repressor HES1. Science 321:1095–1100 34. Blagosklonny MV (2006) Cell senescence: hypertrophic arrest beyond the restriction point. J Cell Physiol 209:592–597 35. Shelton DN, Chang E, Whittier PS, Choi D, Funk WD (1999) Microarray analysis of repli- cative senescence. Curr Biol 9:939–945 36. Pardee AB (1989) G1 events and regulation of cell proliferation. Science 246:603–608 37. Polyak K, Kato JY, Solomon MJ, Sherr CJ, Massague J, Roberts JM, Koff A (1994) p27Kip1, a cyclin-Cdk inhibitor, links trans- forming growth factor-beta and contact inhi- bition to cell cycle arrest. Genes Dev 8:9–22 38. Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM (1996) A syndrome of multior- gan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 85:733–744 39. Sherr CJ, Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13:1501–1512 40. Chu IM, Hengst L, Slingerland JM (2008) The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to antican- cer therapy. Nat Rev Cancer 8:253–267 41. Itahana K, Dimri GP, Hara E, Itahana Y, Zou Y, Desprez PY, Campisi J (2002) A role for p53 in maintaining and establishing the qui- escence growth arrest in human cells. J Biol Chem 277:18206–18214 42. Pajalunga D, Mazzola A, Salzano AM, Biferi MG, De Luca G, Crescenzi M (2007) Critical requirement for cell cycle inhibitors in sus- taining nonproliferative states. J Cell Biol 176:807–818 43. Dulic V, Beney GE, Frebourg G, Drullinger LF, Stein GH (2000) Uncoupling between phe- notypic senescence and cell cycle arrest in aging p21-deficient fibroblasts. Mol Cell Biol 20:6741–6754 44. Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, Horii I, Loh DY (1996) Mice lacking p27(Kip1) display increased body size, multiple organ hyperpla- sia, retinal dysplasia, and pituitary tumors. Cell 85:707–720 45. Matsushime H, Roussel MF, Ashmun RA, Sherr CJ (1991) Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell 65:701–713 46. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G (1993) Cyclin D1 is a nuclear pro- tein required for cell cycle progression in G1. Genes Dev 7:812–821 47. Atadja P, Wong H, Veillete C, Riabowol K (1995) Overexpression of cyclin D1 blocks proliferation of normal diploid fibroblasts. Exp Cell Res 217:205–216 48. Pani G (2011) From growing to secreting: new roles for mTOR in aging cells. Cell Cycle 10:2450–2453 49. Blagosklonny MV (2011) Cell cycle arrest is not senescence. Aging (Albany NY) 3:94–101 50. Heitman J, Movva NR, Hall MN (1991) Targets for cell cycle arrest by the immuno- suppressant rapamycin in yeast. Science 253:905–909 51. Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism. Cell 124:471–484
  • 47. 33 2 mTOR, Metabolism and Cell Senescence 52. Dazert E, Hall MN (2011) mTOR signaling in disease. Curr Opin Cell Biol 23:744–755 53. Zoncu R, Efeyan A, Sabatini DM (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 12:21–35 54. Sengupta S, Peterson TR, Sabatini DM (2010) Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell 40:310–322 55. Conn CS, Qian SB (2011) mTOR signaling in protein homeostasis: less is more? Cell Cycle 10:1940–1947 56. Oh WJ, Jacinto E (2011) mTOR complex 2 signaling and functions. Cell Cycle 10: 2305–2316 57. Zinzalla V, Stracka D, Oppliger W, Hall MN (2011) Activation of mTORC2 by association with the ribosome. Cell 144:757–768 58. Averous J, Fonseca BD, Proud CG (2008) Regulation of cyclin D1 expression by mTORC1 signaling requires eukaryotic initi- ation factor 4E-binding protein 1. Oncogene 27:1106–1113 59. Balcazar N, Sathyamurthy A, Elghazi L, Gould A, Weiss A, Shiojima I, Walsh K, Bernal-Mizrachi E (2009) mTORC1 activa- tion regulates beta-cell mass and proliferation by modulation of cyclin D2 synthesis and sta- bility. J Biol Chem 284:7832–7842 60. Lavoie JN, L’Allemain G, Brunet A, Muller R, Pouyssegur J (1996) Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK path- way. J Biol Chem 271:20608–20616 61. Hitomi M, Stacey DW (1999) Cyclin D1 pro- duction in cycling cells depends on ras in a cell- cycle- specific manner. Curr Biol 9:1075–1084 62. Dowling RJ, Topisirovic I, Alain T, Bidinosti M, Fonseca BD, Petroulakis E, Wang X, Larsson O, Selvaraj A, Liu Y, Kozma SC, Thomas G, Sonenberg N (2010) mTORC1- mediated cell proliferation, but not cell growth, controlled by the 4E-BPs. Science 328:1172–1176 63. Petersen J, Nurse P (2007) TOR signalling regulates mitotic commitment through the stress MAP kinase pathway and the Polo and Cdc2 kinases. Nat Cell Biol 9:1263–1272 64. Fingar DC, Salama S, Tsou C, Harlow E, Blenis J (2002) Mammalian cell size is con- trolled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev 16: 1472–1487 65. Pyronnet S, Dostie J, Sonenberg N (2001) Suppression of cap-dependent translation in mitosis. Genes Dev 15:2083–2093 66. Smith EM, Proud CG (2008) cdc2-cyclin B regulates eEF2 kinase activity in a cell cycle- and amino acid-dependent manner. EMBO J 27:1005–1016 67. Ramirez-Valle F, Badura ML, Braunstein S, Narasimhan M, Schneider RJ (2010) Mitotic raptor promotes mTORC1 activity, G(2)/M cell cycle progression, and internal ribosome entry site-mediated mRNA translation. Mol Cell Biol 30:3151–3164 68. Gwinn DM, Asara JM, Shaw RJ (2010) Raptor is phosphorylated by cdc2 during mitosis. PLoS One 5:e9197 69. Hong F, Larrea MD, Doughty C, Kwiatkowski DJ, Squillace R, Slingerland JM (2008) mTOR-raptor binds and activates SGK1 to regulate p27 phosphorylation. Mol Cell 30: 701–711 70. Toker A (2008) mTOR and Akt signaling in cancer: SGK cycles in. Mol Cell 31:6–8 71. Wander SA, Zhao D, Slingerland JM (2011) p27: a barometer of signaling deregulation and potential predictor of response to tar- geted therapies. Clin Cancer Res 17:12–18 72. Guertin DA, Stevens DM, Thoreen CC, Burds AA, Kalaany NY, Moffat J, Brown M, Fitzgerald KJ, Sabatini DM (2006) Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. Dev Cell 11: 859–871 73. Medema RH, Kops GJ, Bos JL, Burgering BM (2000) AFX-like Forkhead transcription fac- tors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404:782–787 74. Schmidt M, Fernandez de Mattos S, van der Horst A, Klompmaker R, Kops GJ, Lam EW, Burgering BM, Medema RH (2002) Cell cycle inhibition by FoxO forkhead transcrip- tion factors involves downregulation of cyclin D. Mol Cell Biol 22:7842–7852 75. Kroemer G, Pouyssegur J (2008) Tumor cell metabolism: cancer’s Achilles’ heel. Cancer Cell 13:472–482 76. Vousden KH, Ryan KM (2009) p53 and metabolism. Nat Rev Cancer 9:691–700 77. Feng Z (2010) p53 regulation of the IGF-1/ AKT/mTOR pathways and the endosomal compartment. Cold Spring Harb Perspect Biol 2:a001057 78. Young AR, Narita M (2010) Connecting autophagy to senescence in pathophysiology. Curr Opin Cell Biol 22:234–240 79. Kroemer G, Marino G, Levine B (2010) Autophagy and the integrated stress response. Mol Cell 40:280–293
  • 48. 34 V. Dulic 80. Inoki K, Zhu T, Guan KL (2003) TSC2 mediates cellular energy response to control cell growth and survival. Cell 115:577–590 81. Budanov AV, Karin M (2008) p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling. Cell 134: 451–460 82. Feng Z, Hu W, de Stanchina E, Teresky AK, Jin S, Lowe S, Levine AJ (2007) The regula- tion of AMPK beta1, TSC2, and PTEN expression by p53: stress, cell and tissue specificity, and the role of these gene products in modulating the IGF-1-AKT-mTOR path- ways. Cancer Res 67:3043–3053 83. Feng Z, Zhang H, Levine AJ, Jin S (2005) The coordinate regulation of the p53 and mTOR pathways in cells. Proc Natl Acad Sci U S A 102:8204–8209 84. Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk BE, Shaw RJ (2008) AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 30:214–226 85. Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, Birnbaum MJ, Thompson CB (2005) AMP-activated protein kinase induces a p53- dependent metabolic checkpoint. Mol Cell 18:283–293 86. Feng Z, Levine AJ (2010) The regulation of energy metabolism and the IGF-1/mTOR pathways by the p53 protein. Trends Cell Biol 20:427–434 87. Kong M, Fox CJ, Mu J, Solt L, Xu A, Cinalli RM, Birnbaum MJ, Lindsten T, Thompson CB (2004) The PP2A-associated protein alpha4 is an essential inhibitor of apoptosis. Science 306:695–698 88. Lee CH, Inoki K, Karbowniczek M, Petroulakis E, Sonenberg N, Henske EP, Guan KL (2007) Constitutive mTOR activa- tion in TSC mutants sensitizes cells to energy starvation and genomic damage via p53. EMBO J 26:4812–4823 89. Kim J, Kundu M, Viollet B, Guan KL (2011) AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 13:132–141 90. Selman C, Tullet JM, Wieser D, Irvine E, Lingard SJ, Choudhury AI, Claret M, Al-Qassab H, Carmignac D, Ramadani F, Woods A, Robinson IC, Schuster E, Batterham RL, Kozma SC, Thomas G, Carling D, Okkenhaug K, Thornton JM, Partridge L, Gems D, Withers DJ (2009) Ribosomal pro- tein S6 kinase 1 signaling regulates mamma- lian life span. Science 326:140–144 91. Young AR, Narita M, Ferreira M, Kirschner K, Sadaie M, Darot JF, Tavare S, Arakawa S, Shimizu S, Watt FM, Narita M (2009) Autophagy mediates the mitotic senescence transition. Genes Dev 23:798–803 92. Zwerschke W, Mazurek S, Stockl P, Hutter E, Eigenbrodt E, Jansen-Durr P (2003) Metabolic analysis of senescent human fibroblasts reveals a role for AMP in cellular senescence. Biochem J 376:403–411 93. Ruggero D, Montanaro L, Ma L, Xu W, Londei P, Cordon-Cardo C, Pandolfi PP (2004) The translation factor eIF-4E pro- motes tumor formation and cooperates with c-Myc in lymphomagenesis. Nat Med 10: 484–486 94. Astle MV, Hannan KM, Ng PY, Lee RS, George AJ, Hsu AK, Haupt Y, Hannan RD, Pearson RB (2011) AKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: implications for tar- geting mTOR during malignancy. Oncogene 31:1949–1962 95. Inoki K, Ouyang H, Zhu T, Lindvall C, Wang Y, Zhang X, Yang Q, Bennett C, Harada Y, Stankunas K, Wang CY, He X, MacDougald OA, You M, Williams BO, Guan KL (2006) TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 126: 955–968 96. Castilho RM, Squarize CH, Chodosh LA, Williams BO, Gutkind JS (2009) mTOR mediates Wnt-induced epidermal stem cell exhaustion and aging. Cell Stem Cell 5:279–289 97. Chen C, Liu Y, Liu Y, Zheng P (2009) mTOR regulation and therapeutic rejuvenation of aging hematopoietic stem cells. Sci Signal 2:ra75 98. Vellai T, Takacs-Vellai K, Zhang Y, Kovacs AL, Orosz L, Muller F (2003) Genetics: influence of TOR kinase on lifespan in C. elegans. Nature 426:620 99. Kaeberlein M, Powers RW 3rd, Steffen KK, Westman EA, Hu D, Dang N, Kerr EO, Kirkland KT, Fields S, Kennedy BK (2005) Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science 310:1193–1196 100. Harrison DE, Strong R, Sharp ZD, Nelson JF, Astle CM, Flurkey K, Nadon NL, Wilkinson JE, Frenkel K, Carter CS, Pahor M, Javors MA, Fernandez E, Miller RA (2009) Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460:392–395 101. Kapahi P, Chen D, Rogers AN, Katewa SD, Li PW, Thomas EL, Kockel L (2010) With TOR, less is more: a key role for the conserved
  • 49. Discovering Diverse Content Through Random Scribd Documents
  • 50. of our history, but they are Bank holidays. The closing of our banks is the one signal that for twenty-four hours we are free. The multifarious aspects of the theme are most bewildering. As Sir Henry Taylor said, “So manifold are the bearings of money upon the lives and character of mankind, that an insight which should search out the life of a man in his pecuniary relations would penetrate into almost every cranny of his nature. For if we take account of all the virtues with which money is mixed up, honesty, justice, charity, frugality, forethought, self-sacrifice, and of their correlative vices, it is a knowledge that goes near to cover the length and breadth of humanity.” It is certainly true that the amazingly extensive nature of the subject might lead one away into perfectly relevant discussions of almost every field of human activity; and nothing renders argument so unsatisfactory and inconclusive as to have unlimited scope. But in these pages the issue must be narrowed down and the question confined so far as possible to a very brief examination of one particular aspect of the subject, which will be created by formulating a deliberate contention and pursuing it by argument into some of the main channels of this perplexing problem. Even so it is likely that deep water will be reached, but, after all, a suggestion need not be driven to its utmost limits in all directions in order to establish its significance. In choosing a direct point of attack against this generally accepted belief we shall treat the matter more or less from a practical point of view. Without getting involved in abstract philosophic propositions, without entering too far into the sphere of economics and politics, without preaching high morality, though the words and teachings of preachers must be quoted, an endeavour will be made, by working out a definite line of reasoning, to submit as a whole some of the simpler and perhaps more personal considerations which have no doubt already occurred to many who have given the subject thought and reflection. No maxims will be laid down as to how money should be made, spent, saved, lent,
  • 51. borrowed, invested, given or bequeathed, for the object is to strike at the root principle and shatter the ideal which underlies all those transactions, which colours men’s characters, influences their desires and aspirations, creates artificial class contrasts, and contributes largely to the general social confusion and chaos. Briefly, then, our contention is: That no individual is capable of possessing, spending, or administering more than a certain definite amount of money, which can be roughly described as a full competence, without producing positively harmful effects on himself as well as on those affected by his actions. In other words, the “rich man” is an impossibility in any decently organised economic State, and the accumulation of capital in individual hands is detrimental to the public good. That is what is meant by the saying from which the title of this volume is taken. It may appear at first sight to be an extreme view, because we have got so much accustomed to believing that a great deal of good can be done with money, and a great deal of happiness derived from it, that to be confronted with an uncompromising negation on such a time-honoured tradition may seem almost absurd. The argument is purposely intended to be completely comprehensive, and a case will be presented without exaggeration which will cover as much of the ground as possible, dealing with typical rather than exceptional instances by way of illustration. We find in human nature three characteristic impulses which serve as the mainspring and motive power in the gaining and spending of money: the passion for acquisition, the instinct for absolute property, and the desire to excel. No one would suggest that the passion for acquisition can be destroyed: it is neither possible nor desirable, but it can be prevented from running wild, and it can be controlled, though it does not seem to have occurred to many people that such control is expedient. The instinct for absolute property is very much overestimated, and this arises from the fact that we are accustomed to a system which hardly allows any satisfactory intermediate stage between property and positive need.
  • 52. The craving for complete possession on any considerable scale only enters into the minds of those who covet their neighbours’ possessions. What a man wants and has every right to expect is security in the enjoyment of his necessaries and comforts, but this is precisely what in the vast majority of cases he does not get; and his want remaining unsatisfied is converted into a craving for absolute property. The desire to excel, which can undoubtedly be one of the finest human qualities, is in itself vitiated by the measure of money, which sets up an utterly false standard of excellence and converts pure ambition into a desire for material pre-eminence. However far we may travel, the problem will be continually resolving itself into some variation of the question as to how these impulses had best be regulated, and to what extent they have broken out beyond their legitimate bounds. But although the causes of the faith in money may be reduced to moral and psychological terms, there are economic as well as moral results, and it is not the metaphysical origins, but the practical results which must be looked into. At the outset we must acknowledge that our capacities of all kinds are strictly limited, whether moral, intellectual, or physical. An occasional saint, an occasional genius, or an occasional giant stretches the limit beyond its normal point, but the limit still remains. And yet we are foolish enough to believe that in regard to the possession, expenditure, and administering of riches there is no sum of money, however large, which we are not competent to deal with, and we are convinced that it is quite easy and unquestionably within the capacity of almost anyone to spend with benefit to himself and to others sums of money greatly in excess of what can cover in the widest sense his personal requirements. Whereas not only is it not easy, but as inquiry will show, it is purely and positively impossible, as impossible as it is to acquire vast knowledge with a limited brain capacity, or to endure more than a certain amount of physical strain with a limited muscular capacity. We are inclined also to think that men who have money and men who make money are ipso facto easily capable of spending the money properly, though we generally
  • 53. make the mental reservation that if we had it ourselves we should spend it a great deal better. But the inheritance or accumulation of money does not imply by any means a special ability for spending it wisely. To put it plainly, such men have not, nor have we, nor has anyone this ability except in a very limited degree, far more limited than is generally supposed or ever admitted. The case against riches has been argued again and again on religious and moral grounds for over two thousand years, from Confucius to Tolstoy. But we are less impressed by the truth of it now than ever we were; and we still hear it stated by high authorities that it is a benefit to the community to contain men of great wealth. The whole delusion arises from the indestructible confidence in what money can do. And yet all of us see clearly enough by the roughest and most general observation that happiness does not increase with riches, that money indeed has very little to do with happiness, though it has a good deal to do with misery. But many of us are inclined to believe that our own individual case is rather different, and that more money added to our ample competence, and a consequent further enjoyment of material possessions, must undoubtedly make us happier. And when we have got the more and the desired result is not attained, we never pause in hesitation to consider whether perhaps the more has interfered with rather than augmented our happiness, but we are persuaded that the reason we find ourselves still discontented is simply that the more is not enough. Enough never comes to those who have encouraged the longing for more. Nothing short of actual experience can help to eradicate this belief, but there are few who would care to embark on an experiment in the direction of less. And yet it could quite well be demonstrated that a reduction of income, provided always that the loss does not reduce the income below a competence can lead to an increase in happiness—happiness being, of course, distinguished from pleasure. It may require a very rare philosophic resignation and an equally rare breadth of view to refuse to be deluded into regarding the possession of money as an absolute essential. Moreover, there are a
  • 54. great many qualifications to be taken into account arising from natural characteristics, habit, temperament, and tastes. But broadly speaking, if a man has the courage to regard a reduction of income not as a loss but a gain, if he can use the opportunity to kill the instinctive but disturbing craving for more which unfortunately seems engrained in us all, in fact, if he can eradicate the germ of the disease, the limitation of his desire to satisfy transient and what are really artificial needs will certainly increase his power of enjoyment and his happiness. On the other hand, if he treats the lowering of his means as a calamity, which is the usual case, lamenting his fate, railing against fortune and encouraging the longing for gain—an attitude of mind which is only the outcome of his unlimited faith in the power of money—the result, naturally enough, will be despair. But it might be shown as well that a type of man does exist, exceptional no doubt, who, being capable of spending without hurt to himself or to others more money than he has actually got, can enrich his life in the broadest sense by an increase of fortune, and may therefore become the happier for it. He is a man who is indifferent to the enjoyment of material possessions and probably would be regarded in the eyes of the world as the last man who was competent to use money properly. But even he would be entirely overwhelmed by anything like a large increase of fortune, and would be as incapable as any one else of disposing of it without inflicting injury. “Could not riches be used well?” asks Jean Marie in Stevenson’s Treasure of Franchard. “In theory, yes,” replied the doctor. “But it is found in experience that no one does so. All the world imagine they will be exceptional when they grow wealthy; but possession is debasing, new desires spring up, and the silly taste for ostentation eats out the heart of pleasure.” Money is, after all, responsibility and nothing else. We are all of us capable of undertaking a certain amount. Some of us are capable of undertaking a good deal. No one is capable of undertaking more
  • 55. than a relatively limited amount. But the trouble is that most of us think ourselves capable of undertaking far more than we properly can. Autocrats are ceasing to exist not so much because certain monarchs proved themselves dangerously incapable, but because the world has learned that no conceivable human being has the capacity to rule a country single-handed. We do not yet admit this incapacity with regard to the autocrats over capital, although it is equally true, and when we do so we shall find considerable difficulty in dethroning them. Another important inference to be deduced from the argument here set forth is that the surplus money which no individual does or can spend beneficially remains in his hands in stagnant unproductivity, is deflected from other remunerative channels, and is therefore the chief cause of the existence of some of the gravest economic ills which we have to face in our social life. Money cannot rest, it is an active instrument for producing good or for producing evil. Its presence in one quarter may not produce visible evil, but its consequent absence in another quarter will produce very visible and very positive evil. The word consequent must be emphasised because wealth is like water—to pump it up artificially on one side is to lower it automatically on the other. Money in its character of potential wealth seems also to have this peculiar characteristic. It has no positive value in itself. The greater part of its value is given to it by its possessor, and in proportion as it accumulates in the hands of an individual its value is rapidly depreciated. An electric current of a certain power will perform certain specified functions. Decrease the power and it ceases to produce the required effect. Increase the power tenfold or a hundredfold and you will be no nearer achieving the desired result. That is to say, in addition to the change in value effected by the change in individual ownership, there is actual deterioration, produced by accumulation, whoever the individual may be who is responsible for that accumulation.
  • 56. As with individuals, so with the State. National wealth, which in the highest sense of the word means the enrichment of the lives of the people, depends not on how large a number of incomes there are of over ten thousand a year, but on how small a number there are of under two hundred a year. The real riches of a nation are not to be measured by vast calculations of commercial statistics, but by the absence of destitution and the high level of healthy life which the people enjoy. But we must accept the situation as it is. The rich have got their riches, and the problem to be considered here is not how to deprive them of their riches, but how to prevent all men, rich and poor alike, from confiding blindly in money, as they do at present, and from striving towards a false ideal which spoils their highest endeavours, blunts their moral susceptibilities, poisons their happiness, and produces a state of social disorder which is highly prejudicial to the common good. A just appreciation of the essential fact that money can only be made out of people’s labour and the wear and tear of their lives would in itself do much to prevent the growth of the spirit which leads to these alarming contrasts in riches and poverty. But men’s ideals and their moral outlook can only be altered in the long run by repeatedly exposing the actual fallacies in the views they now hold and constantly emphasising the disastrous results of the actions for which this waste of money is responsible.
  • 57. A Chapter III Definition of the limit—Those whose means are above the limit—Income translated into terms of subsistence—The case of the rich man—His establishments—His servants— His luxuries—Extravagance—Vanity—Sport—Racing— Yachting—Condemnation of excess. MORE precise definition must be given of the limit of income referred to in the last chapter as “a definite amount of money which might be roughly described as a full competence.” Every man requires, though he by no means always gets a certain income to satisfy his own needs and those of his family. In addition to this he can profitably spend more so as to add to his general utility by conveniences and comforts, he can satisfy his artistic proclivities, his desire for further knowledge, his taste for sport or amusement, all to his own and the general benefit without hurt or hindrance to anyone. But after allowing the broadest scope for the satisfaction of these legitimate wants there is a definite point beyond which he cannot safely go. That is to say, if he acquires, or if by inheritance he finds himself burdened with money beyond this limit it will inevitably react detrimentally on himself and on others. And this for two reasons: firstly because he is, as a normal human being, incapable of dealing with so great a charge, and secondly because the money, while in his possession, is being drawn away from other channels where there is special need for it. So long as money encourages healthy effort a man may be sure the limit has not been reached, the moment money tends to relax
  • 58. effort the limit has been passed. It must be described as healthy effort, as, of course, money-making may increase the undesirable efforts of the speculator, the gambler, and the thief. But who is to decide what is healthy effort? The man himself. No one else can. And he knows to a nicety. Every man or woman has a different standard, and the level of the limit varies in each individual case according to ideals, capacity, and temperament. But it will not depend at all on what is one of the strongest and often the most excusable inducements for spending money, namely, environment, or the conventions of the particular stratum of society to which the man belongs. The limit for one will not be the limit for another, and a man can only become aware that this limit exists at all by observing very closely what actually is the effect that his money is having on his life and character, instead of blindly accepting his already excessive income or every increase of his fortune as a natural and unquestionable blessing. The main brunt of the attack must clearly fall on those whose incomes are above the limit. They are in numbers a small minority, but the amount they possess is incredibly large. The present income of 1,250,000 people, assessed to income, reaches the vast sum of £850,000,000 a year. Taking the whole population of these islands, it is roughly estimated that there are 1½ millions who can be classed as rich, 3½ millions comfortably off, 38 millions as poor, of whom some 12 to 13 millions are in constant need. The existence of the 1½ millions is one of the chief causes of the condition of the 38 millions. In other words, excess above the limit causes want below the limit. The 3½ millions “comfortably off” are most of them occupied in trying to become identified with the select 1½ millions. If we could estimate the amounts in income which these classes represent the figures would be even more startling. The world has certainly never seen larger fortunes than exist to-day, nor has it seen more extensive and more inexcusable poverty. The average rate of luxurious living in the small minority is higher than it has ever been, and the dangerous and degrading effect of want on individuals and on the general community has never been so widespread or so
  • 59. intense. “The rich,” to use a simple term, are nearly all actuated by the same motive. They accept what they have and what they make as their own, to be spent on themselves, according to their own caprice, or on others, if they are so inclined, casting an occasional sop to some charity or philanthropic scheme as a salve to their consciences. There are, it must be acknowledged, a few, a very few who regard their riches as a trust and endeavour to the best of their ability to divert the greater part of it back into remunerative channels without exceeding a reasonable sum for their own personal wants. But as a class they insist that efforts to alter our social system are fruitless, disturbing and doomed to failure, the division of the world into rich and poor being a Providential decree, and if the rich can get service from the poor without their grumbling, that is the most desirable arrangement that can be conceived. To this a reply may be given in the words of Professor Marshall: “Now at least we are setting ourselves seriously to inquire whether it is necessary that there should be any so-called ‘lower class’ at all: that is whether there need be large numbers of people doomed from their birth to hard work in order to provide for others the requisites of a refined and cultured life, while they themselves are prevented by their poverty and toil from having any share in that life.” The case would not be quite so bad as it is if it were only “the requisites of a refined and cultured life” that they were made to provide. But this point must be considered later. In order to appreciate fully the responsibility which the possession of riches entails, let us translate an income into terms of actual sustenance for human beings. By this means it is possible to arrive at a more or less positive measure. There is so much that is relative in most human requirements that they cannot serve as a standard or as a reliable quantity to be used in calculating any equation. But the requirements of a human being can be measured
  • 60. in terms of actual sustenance, because they can be estimated with something approaching precision. Take a man with £20,000 a year, and say we deduct even as much as £3000 for himself and his family. With his remaining £17,000 he has the power of furnishing 170 people with £100 a year apiece. It is not for a moment suggested that he should do any such thing, as he would be quite unable to select 170 worthy people, and even if he could make the choice the 170 people, on the reception of this private dole, would soon become unworthy. This calculation is only taken to serve as a measure of his power. What might be the income or, more correctly speaking, the means of existence of 170 lives, is vested in one man, who is under the impression—and no one attempts to dispute it—that he is capable of disposing of this sum in a way that is generally beneficial. Now let us state the case fairly from the point of view of the rich man, taking a reasonable and more or less representative type. He may have £10,000, £50,000, or £100,000 a year—that only alters his activities in scope, not in quality. Let us say he has two or three country houses and a house in London. His “position” requires him to keep up a certain establishment, and this means the employment of some forty or fifty servants, grooms, gardeners, chauffeurs, etc., who, he readily tells you, will be thrown out of employment should any of his money be taken from him. If we take the case of a landlord, he will also have tenants, bailiffs, farm labourers, and gamekeepers dependent on him. He keeps the home farm and lets out the other farms on his estate to tenant farmers. Part of his land is built over and brings him in substantial returns in the shape of rent. His villages are in good order and the cottages kept in proper repair. Some thousands of acres or so he keeps for shooting. He may have a deer forest on one of his estates, and perhaps also a grouse moor or a river. Whether he keeps a racing stable, a pack of hounds, or a yacht depends on his particular fancy. He will acknowledge that he spends a certain amount on luxuries, but that “is good for trade,” as great numbers of people have to be employed in the manufacture of these luxuries. He is kind to his poor relations, whom he
  • 61. entertains and helps; and his subscription list to hospitals, charities, and philanthropic works is a large one. He enjoys himself in an unostentatious but suitably expensive way, and his various responsibilities allow him to lead a life consisting of occasional rushes of activity and prolonged intervals of leisure. He most probably finds he can spare a certain amount of money for speculation, with a view to adding more to the sum total of his income. He looks forward to handing down to his children sufficient means to make each of them independent, and meanwhile has his boys educated in the large public schools, where they can associate with boys who are similarly situated. What possible harm can there be in all this? So far from being parasitic, he counts himself as a beneficent agent in the general industrial activity, at the same time appearing as a credit to his society and a notably refined product of the class of which he is a member. Above all, he is popular, and gains ostensibly the respect and regard of his friends, his neighbours, and his dependents. A favourable case has purposely been made for him, because if we accuse him of self-indulgence and greed, and describe him as a gambler, spending his substance on objects which are generally admitted to be pernicious and unworthy, the case could not be defended at all. The fundamental theory which makes this man’s position untenable has already been explained—namely, that after he has satisfied his legitimate requirements all the surplus money he keeps is being held back from serving urgent needs; and, moreover, the method in which he spends the surplus is directly or indirectly harmful to himself and others. We call the money his as if by some miracle he had made it. Often enough he has not helped even by the smallest exertion to create it. The wealth has been and is being daily and hourly produced by the exertion of numberless people who are either employed by him or employed in furthering enterprises in which he has invested his money. It will be said that his share as the wise
  • 62. dispenser of capital, without which labour and enterprise could not be set in motion, is an all-important part of the general process of business. But he invests not to promote enterprise, but to get high dividends; and an elaborate system has been set up in order to tempt him to put his money into concerns that are by no means always sound or of the smallest public utility. Capital would exist and flow far more freely without the large capitalist. He acts as a dam to the stream; a certain amount escapes back into the main channel, but much more is checked and diverted into stagnant and putrefying pools of his own creation. The free flow of blood is life-giving; the clotting or coagulation of blood produces disease. Let us take the various points raised by his case seriatim. Many acutely controversial problems are opened, and it will be difficult to detach the particular actions of the rich man without generalising, to some extent, on the problems themselves. It is no argument against our main contention to say that people with costly tastes have, while gratifying them, been able to exercise powers of a high order, for, obviously, it is in spite of their shortcomings in this respect that they have succeeded, and not because of them. If some men with means have done valuable public service and performed admirable work in many different spheres of life, this they have done as men naturally gifted with high accomplishments, not as rich men. Here we are only concerned with their works and deeds in their latter capacity. It does not affect our argument whether our typical example has been brought up to regard this way of living as natural and necessary for a man of what is called his “position” (that is to say, the purely artificial place which a rich man is able to take up in the community solely on account of his riches), or whether he has made the money for himself and is simply aping the habits and customs of those who already possess it. The distinction between the vieux riches and the nouveaux riches is one they can fight out between themselves. The former scoffs at the latter while all the time he is setting him, and consciously setting him, the example he is to follow. It is not the gaining, but the spending of the money that must occupy our attention here.
  • 63. Our friend’s houses are only a detail in the upkeep of his position. They may be historic castles, sham “ancestral halls,” modern “palatial country residences,” or “fashionable mansions” in town. Does it ever strike the owner as, let us say, a curious arrangement that he should have several houses of fifty to a hundred rooms apiece while some millions of his fellow-men do not own one room? Does he know that in England and Wales alone 507,763 people occupy one-room tenements, 48.4% of which are classified as overcrowded, while 12,458,150 are occupying tenements of two, three, or four rooms? 4 In any case, he would indignantly refuse to admit that there was any remote connection between these two sets of circumstances. When we come to the staff necessary for the maintenance of these large establishments we touch a problem of employment which must be examined more closely. It is not sufficient to state baldly that these people are employed, and that if the opening were not available for them they would be unemployed. The immediate result of their being discharged would no doubt in some cases be unemployment. That is just the mischief of uneconomic employment. If a large number were simultaneously dismissed there might be temporary unemployment on a large scale, as it would amount to dislocation, like the extinction of some dying industry. But the eventual readjustment would subsequently be by that much the stronger and better adapted to the real requirements of the community. To employ a man in useless and unremunerative work can be regarded in some aspects as worse than not employing him at all. It is not intended, however—and, indeed, it would be impossible—here to enter into a discussion on the whole problem of unemployment, but there is undoubtedly a very great economic waste that largely contributes to the gravity of the problem, arising from the fact that a large number of people are being forced to devote their labour and energy to work which is, so to speak, final and sterile. It is precisely the same with regard to the production of expensive luxuries. The employment of a large retinue is only another form of the possession and enjoyment of articles of
  • 64. excessive luxury. The employers and possessors have all disagreeable burdens and every sordid worry lifted from them, their smallest and their most extreme desires for pleasure met, their special appetites satisfied, their peculiar vanities titillated, and their artificial position safeguarded and maintained, without their giving more than a passing thought to the mass of people required to carry on this work. Plenty of examples might be quoted in contemporary as well as past history to show that after generations of the enjoyment of “the vile joys of tainting luxury” men deteriorate, both physically and mentally. As for the particular line of life which domestic service offers under modern circumstances, it is not too much to say that it is, as a rule, very demoralising, more especially for the men. And its demoralising tendency increases in proportion to the size of the establishment. The single general servant lives a life of hard work but genuine service on four to eight shillings a week, often living in friendly relations with master or mistress, and really lifting from them the burden of necessary domestic duties which they with limited incomes and professional work of their own cannot possibly find time to perform; and this remains true in other small households. In the large house the faithful old family servant, who is more of a friend than a servitor, is rare in these days of ostentation. The butler, on wages of fifty to sixty shillings a week, which together with board and lodging represents from £250 to £300 a year, has a life of leisure, ease, and excessive comfort, seldom having to exert himself even up to his limited capacities. Male house-servants are often chosen for their looks; their work is very light physically, they are overfed, and being under-educated, can hardly be blamed for becoming demoralised. These able-bodied men, whose muscles, if not their minds, might be devoted to some really serviceable purpose, are still increasing in numbers. Over 25,000 more male servants have got employment in the last ten years, the total number now being 227,995. Even deducting the single indoor servant, the single coachman or gardener, this means a large increase of ornamental male attendants. Female servants are
  • 65. becoming more difficult to secure in the higher grades, because the class of women from which they are drawn value their liberty and are not so ready to sacrifice it for food and comforts. In fact, they are showing signs of impatience of control, and of preferring the risky though exhilarating struggle of independence. But still large retinues of men and women exist solely employed in keeping up huge houses to satisfy the vanity and minister to the comfort of a comparatively few rich people. No work of a more hopelessly barren, profitless, and, indeed, degrading character could be found for them. A system of tips deprives their smallest acts of what might be an obliging and disinterested intention. Arrangements are organised with tradesmen to defraud the employers in what is thought a perfectly legitimate way; the actual waste of food is appalling, and by extras, gratuities, perquisites, commissions, and pickings a considerable amount is added to the wages of the upper servants. In these large establishments immorality exists more as a rule than as an exception, but it can be kept secret, for these communities of private servants—like everything else connected with the lives of the rich—cannot be made the subject of investigation. If assistance to those who need it is the object of domestic service, it is striking to note that on the money basis, generally speaking, the wrong people are served. Who in the community most require and should specially have the help of servants? The old and infirm, the weak and ill, the very young and the hard-worked. Service under such conditions raises itself to the level of one of the highest occupations that can be imagined. But this is not our system. A man or woman may be ill, old or over-worked, without being able to get the assistance of a single soul. Another man or woman may be young and healthy and have at his or her command a retinue of thirty servants or more, solely because they have money and servants are forced, by economic pressure, to devote their lives to the menial task of furbishing up the endless and complicated appanage of wealth. Now let us turn to the inanimate luxuries, taking into account only indisputable luxuries—that is to say, articles of high price which
  • 66. have no special artistic value, to which much labour has been devoted and which are not produced to serve any legitimately useful purpose. Luxury has been well defined as “that which creates imaginary needs, exaggerates real wants, diverts them from their true end, establishes a habit of prodigality in Society, and offers through the senses a satisfaction of self-love which puffs up but does not nourish the heart and which presents to others the picture of happiness they can never attain.” Bond Street catalogues abound with any quantity of examples. Furs at one thousand guineas, fifty-guinea dressing-bags, twenty- guinea hats, thousand-guinea tiaras, fruit and vegetables out of season, cigars at three shillings apiece, ruinously expensive wines, and fantastic foods of all descriptions. There is no need to exaggerate, for all those articles can be bought for much higher prices than those quoted. A great amount of skilled labour of a high order goes to the production of these luxuries, and a great amount of labour of the lowest and most cruelly sweated description is also enlisted for their production, and incredible as it may seem, it is on the ground that they give employment that these luxuries are defended. It was calculated in 1884 5 that, even giving a liberal extension of meaning to the term “necessaries” and “comforts” of life, over six millions of manual labourers, who with their families constitute thirteen millions of the population, were engaged in producing what, in contradistinction to the above, must be classified as luxuries. A prominent statesman, 6 expressing the views of his class, said a few years ago: “The more human wants are stimulated and multiplied, the more widespread will be the inducement to hire. Therefore all outcries and prejudices against the progress of wealth and what is called luxury are nothing but outcries of prejudice against the very sources and fountains of all employment.” On such an argument as this the defence of luxuries generally rests. The essence of the fallacy lies in the fact, which cannot be repeated too often, that labour spent on such articles is
  • 67. unremunerative and unproductive, because its ultimate result is only to gratify various forms of vanity and greed. To exemplify by a concrete instance what is unremunerative and what is remunerative, let us take a hundred-guinea ball-gown and a pair of boots. It is not possible to estimate the number of people employed in producing the ball-gown. There is the silk, satin, or whatever the principal material may be; there are the trimmings of chiffon, hand- embroidery, lace, braid, beads, sequins, ribbons, etc., etc.; some hundreds of pairs of hands, including factory-workers, dressmakers, sempstresses, etc., will have touched some part of the gown before it is delivered to the wearer. To what end are all these specialised departments of labour concentrated? The gown is worn a few times in the one season; the wearer has the satisfaction of feeling as well dressed as A. to F., and far better dressed than F. to Z. In fact, the net result of all this expenditure of energy is the generating of a rather foolish pride, the encouragement of conceit on the one side and envy on the other, and the hardening of a nature into ways of worldliness and vanity. As for the boots. Again, many more hands than can be calculated have helped to produce them, but they are directly and immediately serviceable to the purchaser, to whose activity the wearing of boots is an essential, and in general they minister to the efficiency of human machines. But if balls are not wrong, ball-gowns must be worn. It is a question of degree; and here again we get to the theory of the limit which in this conjunction can be expressed thus: In relation to human needs, in relation to human powers of enjoyment, in relation to the beneficial effects of pleasure, even in relation to the dictates of fashion, there is a distinct limit not to be expressed in figures up to which expenditure (in this particular case on dress) is legitimate and relatively productive, beyond which it becomes progressively unremunerative and harmful. A hundred guineas, by any conceivable method of calculation, greatly exceeds this limit.
  • 68. To assert that the purchase of luxuries is good for trade is quite as ridiculous as to say that a man can benefit the building and furnishing trade by burning down his house once a year. We do not want to create more artificial wants before we have satisfied the crying human needs which already exist. There is no loophole through which a reasonable defence of the senseless expenditure, which goes on in an increasing measure, can be made. Luxurious living has never been quite so blatant and unashamed as it is to-day, and the effete epicureanism and decadent effeminacy it produces stand out in rather sharp contrast to more hopeful signs of progress and moral and intellectual refinement and vigour which, happily, are visible around us. A lady writing in a review in the early ’seventies describes life in the country house, with its futile routine of heavy meals, sport, card- playing, and vacuous inanities which take the place of conversation, all very much as it is to-day. The writer speaks with dismay of gowns costing sixty guineas and of £1000 a year spent on clothes. But these figures are almost negligible compared with the sums spent nowadays. It is only through occasional actions in the courts that the outside public get an idea of what is actually spent, and it is surprising that there are not more disclosures, considering the mountainous debts that are piled up in West End shops. But the shopkeepers are very reluctant to lose a really leading customer, and they know how to meet the inconvenience of not being promptly paid. A typical case may be given of an article of clothing, the cost price of which was nine guineas, being sold for £28 7s. There may be delay in payment, but there appears to be compensation in the profit. When one hears of the woman who spent last year £36 5s. on a hat, or another who gave £1250 for a sable cape, it is not the isolated action of criminal folly that chiefly strikes one, but it is that the hat and the cape act as indicators of the sort of price such women are in the habit of paying for their clothes, a large supply of which are in the market ready to meet this artificial demand. Moreover, the habit of extravagance, especially as regards female
  • 69. clothing, is catching and runs through all classes once the example is set. It is a common enough and very depressing sight to see absurdly elaborate clothes, which are cheaper imitations of the latest fashions, worn by women of the lower middle-class, whose deplorable want of education is shown by their inability even to pronounce their mother tongue. They watch the rich, and gather from what they see that fine feathers make fine birds, and it is not on them that the blame should rest. Vanity exists and insists on being satisfied. It is no good blinking the fact. Luxuries, in one form or another, will continue to be produced. But there is no reason why we should not stem the current lest it swell to danger point. There are many well-known historical examples of the enervating and degenerating effect of luxury on national life, and the modern tendency towards an increased production of these indulgences should be combated not only as a moral weakness, but as an integral factor in the general economic problem. When one considers what real comfort of living, with all the necessary intellectual and artistic equipage, opportunities for amusement, and domestic convenience, can be secured to-day at a comparatively moderate sum, it makes the wild and profligate extravagance the more inexcusable and the more futile. Anyhow, let us abandon once and for all the foolish and ignorant attitude of regarding this display as a desirable form of industrial stimulus which should be fostered and encouraged. Preaching and writing against it has never been of the smallest avail, but it has been necessary to deal with it here as a very important, if not predominating, element in the analysis of the rich man’s conceptions of his duties. In addition to luxuries of establishment, clothes, and food there is a complicated ritual of sport which in this country reaches an almost incredible pitch. It has been estimated that forty-five millions are permanently invested in the apparatus of sport, and an income of over forty millions spent annually upon it. We need not discuss all the intricacies of the numerous branches of sport, observing where
  • 70. its effect is healthy and where harmful. No one will contend that the most expensive forms of it are by any manner of means the best. But the most obvious harm to be noted in this connection is the amount of land which is taken away from agriculture for sporting purposes. Landlords often keep up their shooting at a great loss, amounting to something like five to ten pounds per bird shot, all for the sake of having the shooting and asking friends down for a few days in the year to enjoy it. It is gravely regarded as an essential part of the education of a young man in this particular world to learn how to shoot. No question, even with respect to his education or possible professional career, is treated with more seriousness than the moment he first handles a gun, and family advice is sought as to how and when encouragement can be given to the development of this essential qualification which, coupled with a knowledge of bridge, will make him a desirable visitor in any country house. At card-playing, which occupies a vast amount of time in the lives of the rich, sums amounting to hundreds are often lost or gained by one person in one evening. But of the various sinks which help to drain away their money, horse-racing almost holds the first place. There are no statistics to show how many people have been ruined by it, or how many have been lured into a life of gambling by their success in the betting ring. But its popularity is certainly on the increase, as we can see by looking at the number of horses that have run under the rules of racing in the last thirty years. In 1878 there were 2097; in 1908 this figure had risen to 3706. The number of larger race meetings advertised in advance have more than doubled since 1881 (78 in 1881, 164 in 1909). Some sort of estimate of the money spent on it, apart from betting, can be gathered from the amounts won. In 1908 the winning owners secured between them nearly a quarter of a million pounds, the sums won by the first thirty-six amounting to £246,001 15s., the largest total secured by one owner being £26,246. 7 The populace are invited to join in this pursuit, though, of course, they must be railed off to prevent too close contact with
  • 71. those who come in coaches and motor-cars. The crowd is vaguely supposed to be having a good time, and any attack on horse-racing is met by hackneyed arguments about “keeping up the national sport” or “improving the breed of horses,” and perhaps, again, the objection of unemployment for jockeys and bookies might be dragged in. It does not appear, however, to be a good method of improving the human breed. In observing the crowd on a race-course, whether it be the well-dressed portion or the ill-dressed, the betters or the bookies, neither a deep knowledge of humanity nor a very close power of observation into physiognomy is required to note the prevalence of a remarkably low type. But a still more vivid impression of what the pleasures of racing mean can be gained by going out on the road in the evening towards the scene of a large race meeting when the people are returning. Brakes and carts in endless procession will pass you loaded with men shouting in the excitement of semi-drunkenness, or with heaps of humanity sodden and silent in complete intoxication. Outside every public-house on the roadside traps await those who are squandering their gains on further refreshment or soothing the despair of losses in the temporary oblivion of drink. The localities where there is an annual race week suffer considerably, the inhabitants become infected by the gambling and betting mania, and during the actual days of the races the place is infested by the lowest dregs of the riff-raff who journey about from one race meeting to another. This so-called sport produces the lowest possible type; it degrades many who take part in it with sinister rapidity, it encourages fraud and deception, it is a canker of rottenness in public life, and it receives the highest sanction and patronage. Many people are present at a race meeting without being conscious that it is attended by any evil consequences. They go to meet their friends, perhaps putting an occasional sovereign on a horse to give them some interest in the racing. To them the crowd is a natural part of the proceedings, the heavy bets of the ring an amusement. To have been there is something to boast of, and
  • 72. conveys the idea that they have associated with smart people. Thoughtless, as in so many of their other pursuits, they accept the whole proceeding as a recognised sport and they inquire no further. The philosophy of these people is the prevailing philosophy: “Do not examine below the surface, or you are bound to find something disagreeable. Take things as they come; skim the cream off the top; avoid that which is unpleasant or difficult to explain; and above all things, do what others do.” Yachting, which also runs away with a great deal of money, comes under a very different category. It is a health-giving and often strenuous occupation, and the seamen employed are, anyhow, deriving incidentally some positive benefit from the life they lead. Nevertheless, out of the 4655 private yachts registered in the current year (an increase of over 3500 in the last forty years), 8 only a very small proportion are actually navigated by owners who have any knowledge or love of seamanship. The great majority are floating houses of luxury (viz. a 700-ton steam yacht, for which £25 a day is paid for coal when in use), or racing yachts, mere toys used to minister to the fanciful pleasures of the rich. But in expressing the strongest disapproval of these excessive luxuries, it is not for a moment suggested that people should rush into the opposite extreme—live in discomfort and adopt the craze for “the simple life,” which is only an inverted form of vanity and ostentation. There are many of the lesser luxuries which give great pleasure and sufficient honest gratification to justify their existence. There may even be some reluctance in condemning extravagance, because the nature of the extravagant man is far preferable to the economical and cautious disposition which sometimes sinks into niggardly meanness. Moreover, any attempt at excessive restrictions and unnecessarily harsh discipline in the upbringing of children invariably leads to a violent reaction in the direction of profligacy and extravagance. Let human nature be allowed free play in all directions; but it is not taking up the attitude of an ascetic or of a prig to condemn
  • 73. unhesitatingly unnatural excesses, reckless licence, the extremes of self-indulgence and greed, the exercise of which by some few involves the neglect, misery, and ruin of so many others. Thieves when they steal use violence and are pronounced enemies of society. These few people, by a silent conspiracy in which we all seem to acquiesce, are also stealing and are equally enemies of society.
  • 74. W Chapter IV The rich man’s charities—His generosity—His hospitality—His land—The Feudal System—His responsibilities—The agricultural problem. E must now turn from what the rich man spends on himself and consider what good and what harm he does by his subscriptions and donations to philanthropic and charitable objects. In so far as he himself is concerned these gifts do not involve any element of personal sacrifice; the moral benefit which is by way of falling on a giver is therefore nil. The exertion of writing a cheque or banker’s order and the satisfaction of imposing a tax on himself complete the transaction on his side. Occasionally the sight of his name published at the head of a list with a large figure next it gives him a further agreeable sensation, and he can become famous as a household word of generous philanthropy without the very smallest personal inconvenience. But as an instance of pure charity—that is, loving sacrifice—the poor woman who gives a penny from her meagre store is on an entirely different plane. The picture presents itself to the present writer of a woman at the doorway of a wretched tenement, with her child in her arms, giving to a passing vagrant who was suffering from hunger and fatigue a penny from the few coins she had in her purse. The expression of her face as she handed him the money was the most sublime illumination of pure charity—no subscription list in the newspapers, no public recognition, and the sacrifice, not of luxuries, but of something that she and her baby needed. That something went with her penny, and
  • 75. in return she received something else for which there is no price, no name, and no description. From such an experience as this the rich are for ever cut off. “Probably the most generous people in the world,” says J. D. Rockefeller, perhaps realising that charity is something he can never reach, “are the very poor, who assume each other’s burdens in the crises which come so often to the hard pressed.” The rich man’s so-called charity therefore must be to a large extent mechanical and conventional. He gives because others with the same means give, and the charity touts know how a list headed by Lord A. with a substantial sum will produce equally or perhaps even more substantial sums from Lord B., Sir. C. D., Alderman E., and Mr. F. The extraction of money from the rich is a business in itself, requiring considerable skill, and the rich are fleeced far more than they realise. In practical America they take the trouble to teach people professionally how to write what they call “letters of appeal.” When we hear of subscriptions to charities being stopped it may serve to remind us that it is most inexpedient that institutions such as hospitals should be at the mercy of the casual caprice of rich men. Nothing could eventually be more desirable than that every one of them should cut off their charitable contributions. It might entail a severe temporary shock to the funds of charitable institutions, as over seven millions a year is being spent in London alone on charities, but at the same time many ill-managed and misdirected endeavours would disappear, and the State would come to realise all the sooner its responsibilities in respect to the maintenance of really necessary institutions for the relief of suffering and the nursing of the sick, in the same way as it is beginning to recognise its duties towards poverty, old age, and unemployment. There are other enterprises which the State should undertake that are often delayed in their institution owing to the plea that the private munificence of rich men can be depended upon. It is certainly better that the funds should be expended thus than in sheer self-indulgence, but it is evident that the money would be far better spent and the object on which it is spent better served if the
  • 76. source were not controlled by the whims and fancies of a single individual. In regard to the more private and personal aspects of the generosity attributed to riches: “Surely,” a critic will say, “if the rich man is benevolent and kindly disposed he can in a hundred thoughtful ways help his poorer friends by presents, by attention and timely help, by opening the doors of his houses, lending his conveyances, and showing many other attentions which his money allows him to do, thereby becoming justly popular and a source of great good.” The admiration, and just admiration, for open-handed generosity and the justifiable dislike of anything approaching miserliness in others cause an entirely erroneous impression that large gifts of money must unquestionably be praiseworthy and commendable. But this is not the question at issue. These are two moral qualities, the one admirable, the other objectionable. The generous disposition can show itself in many other ways besides money gifts, and the real man behind the rich man, though he may be one and the same individual, often comes forward with simple acts of thoughtful kindness because the finer qualities of human nature cannot be stifled even by money. But in so far as the rich man indulges his generosity in thoughtlessly giving away money broadcast, it amounts to a form of self-indulgence, and he is distinctly to blame for not estimating more precisely the effect of his actions. No doubt the harm of unwise and foolish actions is palliated by the purity and excellence of the motive. In so far as these people intend to show kindness they are amply justified in what they do. But let us consider for a moment what the effect of their benevolence is. In the first place they are made to occupy an entirely false position as dispensers of charity. Often, too, the desire to patronise and gain the power that patronage gives blights the spirit of genuine and unadulterated kindness, and further, the recipients are placed in the extremely uncomfortable and embarrassing situation of receiving benefits, presents, and comforts which they know they are not and probably never will be in a position to return. To force anyone to be
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