role of telomeres in stress-induced premature senescence and design of anti-ageing strategies

243 Pages·2004·3.32 MB·English

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FUNDP
Faculté des Sciences
Unité de Recherche en Biologie Cellulaire Animale
Rue de Bruxelles, 61
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Belgique
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Modelling human ageing: role of telomeres in stress-induced premature
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senescence and design of anti-ageing strategies
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Rôle des télomères dans la sénescence sinduite prématurément par les stress et

design de stratégies anti-vieillissement
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rJoão Pedro de Magalhães
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Promoteur : Dr. O. Toussaint Dissertation présentée
pour l’obtention du diplôme de
docteur en sciences
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FUNDP
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Modelling human ageing: role of telomeres in stress-induced premature
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senescence and design of anti-ageing strategies

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João Pedro de Magalhães
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Adviser: Olivier Toussaint
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Modelling human ageing: role of telomeres in stress-induced premature
senescence and design of anti-ageing strategies
João Pedro de Magalhães, FUNDP
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Summary n
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Due to the duration of human ageing, researchers must rely on modelse such as animals and cells.
Replicative senescence and stress-induced premature senescence (ScIPS) are two cellular models
sharing many features. Although telomeres play a major role nin replicative senescence, their
involvement in SIPS is unclear. e
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In this work, we first wanted to investigate how accurates models of ageing are. We published a
new model of the evolution of human ageing, which eoffers a refined view of the evolution of
ageing in humans and suggests that human models nshould be favoured. Though studying other
mammals, reptiles, and birds may also be useful, wee conclude that lower life forms such as yeast
and invertebrates are not representative of the husman ageing process.

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Secondly, we wanted to elucidate the impoartance of telomeres in SIPS and study gene expression
and regulatory networks. Using a telom erase-immortalized cell line, we found no evidence that
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damage specific to the telomeres is at the origin of SIPS. In our published model, neither the
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TGF-(cid:533)1 pathway nor telomeres appear to play a crucial role in SIPS. We suggest that widespread
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damage to the DNA causes SIPSs and propose a rearrangement of gene expression networks as a
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result of stress. Moreover, we advise caution in using telomerase in anti-ageing therapies since
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telomerase expression may alter the normal cellular functions and promote tumorogenesis.
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Lastly, we published estrategies to integrate the modern computational approaches to research
ageing. Although wne find it unlikely that a full understanding of ageing may be achieved within
a near future, wei argue that understanding the structure and finding key regulatory genes of the
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human ageing process is possible.
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To Bernard Strehler, who passed away on May 13th, 2001,
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hoping I will one day justify his friendship and trust.
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Acknowledgments
First and foremost, I thank my adviser Olivier Toussaint. In his modesty, Olivier told me he was just doing
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his job. If so then it was a job marvellously done and I can only hope to work again with someone as
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professional as him. For me, it was a privilege and a fortune to profit from his dedicantion and knowledge.
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Je tiens à remercier les Professeurs José Remacle et Martine Raes dee m’avoir accueilli dans leur
laboratoire. Je remercie aussi mon ami Jean-Michel Fustin, pour rendre plucs facile mon adaptation au labo.
Mon travail à URBC a été plus facile grâce l’assistance de François, Valénrie, Andrée, et Anne. Mon travail à
GRAS a été plus agréable grâce à Olivier, bien sûr, mais aussi à Peatrick, Florence, Thierry, Christophe,
Jean-Jean, Friedel, et Véro. Il faut aussi remercier les gens de EAcT : Françoise, Vincent, Laurence, Muriel,
Nathalie, et Anna. Merci a tous ! s
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Merci aux membres du jury, les Professeurs Mouly, Rennard et Devos, et les Docteurs Van Huffel et
Toussaint d’avoir lu ce manuscrit et participé à son améelioration.
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Dans les Facultés-Universitaires Notre-Dame d e la Paix, il faut remercier Fabian Bastin pour son
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assistance dans Linux et un grand merci au Praofesseur Absil : maximas tibi gratias ago! Merci aussi à tout
le monde dans le centre sportif universitaire, premièrement à l’entraîneur Christian Maigret.
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Ces quatre ans en Belgique sont passés très vite car j’ai trouvé des amis qui m’ont aidé. Je commence par
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remercier Bernard Germain pour sosn infinie patience et dédication. Il faut aussi mentionner l’hospitalité de
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Madame et Monsieur Houart qui m’ont fait sentir chez moi, l’amabilité de Madame Mine et l’assistance de
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Patrick Vanackere et Denis Willems. Merci aussi aux restaurants Le Sand, Sorrento et Fac-Food, sans
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lesquels j’aurait passé faim.

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Heel, heel erg bedanknt, special voor Steff Vanpoucke en verder iedereen van 1-2-3 Comedy Club die mijn
dromen hebben waiargemaakt. May the laughter be with you.
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I must acknowledge the members of the Extropy Institute and its mailing-list for keeping me up-to-date with
the latest discoveries and always debating the most forefront topics. Particular thanks to Robert Bradbury
for our wonderful e-mail exchanges. Further thanks to Anders Sandberg, Damien Broderick, and Curt
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Adams for their opinions and criticisms. Big thanks to Frank Prengel and the organizers of TransVision
2001 for allowing me the chance to present my ideas and to Bruce Klein for his efforts at the Immortality
Institute. The biggest acknowledgments of them all go to the many visitors of senescence.info for their
feedback and encouragements.
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Huge thanks to Mikhail Soloviev for his kindness while I visited the beautiful St. fPetersburg as well as
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Vladimir Anisimov for helping me attend the 2nd European Congress on Biogerontology. Thanks to the
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many—and there were many because I can be really annoying—researchers w.orldwide who found the time
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and patience for sharing their ideas with me. Of these I highlight Judith Campisi, Leonid Gavrilov (or is it
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Gavilov? or Gravilov?), Thomas von Zglinicki, Thomas Kirkwood, Bruce Howard, Suresh Rattan, Simon
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Klebanov, Caleb Finch, Yasuhiro Furuichi, George Martin, Jerry Shay, Richard Miller, Gary Stormo, Steve
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Coles, Gordon Lithgow, David Kipling, Richard Farragher, Matt Kaeberlein, and Warren Ewens. This thesis
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would certainly be poorer without any of them. Special thanks to Justin Congdon, Marc Girondot, Kevin
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Wright, and James Christiansen for sharing their expertise regarding reptiles and amphibians with me.
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Further thanks to Jason Stajich, Ewan Birney, Van Savage, Jamie Gillooly, Andy Wuensche, and Ullrich
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Sigwanz for all their technical help. Big thanks to Aubrey de Grey for his creativity and the many profitable
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discussions we’ve had as well as Steven Austad for his intuition, Richard Cutler for his vision, and George
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Church for his knowledge. Thank you all!

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Gostava de agradecer aos meus pais por todo o apoio e atenção—vulgo, dinheiro—durante estes quatro

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anos. Tenho de agradecer principalmente à minha mãe por me ter ajudado sempre que precisei, não
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hesitando em deixar a bela cidade do Porto para se deslocar à feia e decadente Bélgica. Obrigado
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também à gaja que me raptou durante este último ano pelo seu apoio constante, incentivos e, acima de
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tudo, amizade. Acho que se chamra Joana qualquer-coisa. Vou ver . . . (cid:45) Obrigado também à minha tia Né
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e à minha prima Joana por me terem visitado na Bélgica e ajudado com o Francês. Obrigado a todos!
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Muito obrigado a Nuno Roby, Suely Costa e Rita Ramos pelo interesse demonstrado no meu trabalho.
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Uma palavra de apreço também aos Professores Alexandre Quintanilha e Pedro Moradas-Ferreira por
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tentarem que se faça algum trabalho na biologia do envelhecimento em Portugal. Finalmente, obrigado ao
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Professor Xaviern Malcata por me ter disponibilizado o seu laboratório na Escola Superior de Biotecnologia
e obrigado tamobém ao Professor António Rangel por todas as sugestões e conversas que tivemos.
Finalmente, tenho de agradecer à Fundação para a Ciência e a Tecnologia por terem financiado o meu
trabalho durante quatro anos assim como as minhas várias participações em congressos internacionais.
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Table of Contents
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Summary ii
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Abbreviations ix
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Introduction 1
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Chapter 1: Human ageing 2
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1.1. Definition and history 2
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1.2. The ageing phenotype 2
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1.3. Theories of ageing 3

1.3.1. Energy consumption hypothetsis 4
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1.3.2. Free radical theory 6

1.3.3. DNA damage theory n 9
1.4. Evolutionary theory of ageinog 11
1.5. Models of human ageing i 14
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Chapter 2: Replicative senescence and stress 16
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2.1. Hayflick’s limit e 16
2.1.1. Biomarkersv of RS 17
2.2. The theory of st ress 18
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2.3. Stress-induced premature senescence 19
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2.3.1. Biomarkers of SIPS induced by oxidative stress 21
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2.4. Relationl between ageing, RS, and SIPS 21
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Chapter 3: Cell cycle regulation by the telomeres 24
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3.1. Telomere shortening and RS 24
3.2. How telomere dysfunction induces senescence 25
3.2.1. Uncapped telomeres recognized as DNA damage 27
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3.3. Ageing, cancer, and the telomeres 31
Chapter 4: Mechanisms of SIPS 34
4.1. From DNA damage to SIPS 34
4.2. The telomeres 36
Chapter 5: Computational methods o 39
5.1. From genes to ageing f 39
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5.1.1. Comparative genomics 40
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5.1.2. Transcriptional regulation . 41
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5.1.3. Gene expression studies 44
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5.2. Systems biology 45
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Aim of the Work 47
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Results 49
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Chapter 6: Human ageing: evolution and research models 50
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6.1. Article 1: The evolution of mammalian aging (Experimental Gerontology,
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volume 37, pages 769-775, 2002) 50

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Chapter 7: Role of the telomeres in SIPS 59
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7.1. Article 2: Stress-induced premature senescence in BJ and hTERT-BJ1

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human foreskin fibroblasts (FEBS Letters, volume 523, pages 157-162,
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2002) 59
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Chapter 8: Gene expression in SIPS 67
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8.1. Article 3: All roadsr lead to Rome: How gene expression networks
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reorganize in premature senescence of human skin fibroblasts expressing or
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not telomerase (submitted for publication) 67

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Discussion 90
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Chapter 9: Mnodels of human ageing 91
9.1. Lessoons from the evolution of ageing 91
9.2. Animal models 93
9.3. Human models 95
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9.3.1. Centenarians 95
9.3.2. Werner’s syndrome 96
9.4. Cellular models 97
9.4.1. Senescence, RS, and SIPS 98
9.4.2. Comparative biology o 99
9.5. Computer models f 100
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9.6. Developing anti-ageing therapies 102
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9.6.1. Telomerase alters the normal cellular functions . 105
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Chapter 10: DNA damage, ageing, and SIPS 107
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10.1. Critical telomere shortening is not necessary for SIPS 107
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10.2. DNA damage induces cellular senescence through complementary
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pathways 109
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10.3. RS versus SIPS 112
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10.4. DNA metabolism and ageing 113
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Chapter 11: How bioinformatics can help reverse engineer human aging 116
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11.1. Introduction 116
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11.2. Data-mining methods 117
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11.2.1. Comparative genomics of ageing 117

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11.2.2.Transcriptional regulation of ageing 119
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11.2.3. DNA microarrays 120

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11.3. Modelling human ageing 124
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11.3.1.System structure and identification 124
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11.3.2.System-level perturbations in model organisms 125
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11.3.3. Reconstructingr the genetic network of human aging 127
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11.4. Conclusion: is it possible to reverse engineer human aging? 128
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General Conclusion 130
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Appendix: How the genome regulates aging: development of a comparative 133
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genomics methnod to study human aging and a test of theories of aging (submitted
for publicatioon)
Bibliography 150
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Abbreviations
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ARCT Ageing Research Computational Tools
ATF-2 activating transcription factor-2 n
ATM ataxia telangiectasia mutated
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ATP adenosine tri-phosphate
bp base pairs c
CDK cyclin-dependent kinase
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CDKI cyclin-dependent kinase inhibitor
cDNA complementary DNA e
CPDs cumulative population doublings
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CR caloric restriction
Cy3 cyanine 3 e
Cy5 cyanine 5
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DNA desoxyribonucleic acid

g gram(s)
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G1 gap 1 (cell cycle)
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G2 gap 2 (cell cycle)
GHR growth hormone recepto r
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dUTP deoxy-uridine-tri-phosphate
H2O2 hydrogen peroxide o
HDFs human diploid fibroblasts
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hrs hours
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hTERT catalytic subunit of human telomerase
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IGF-1 insulin-like growth factor I
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IMR initial mortality rate
kbp kilobase pvairs
kcal kilocalories

MAPK mitogen-activated protein kinase
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ml milliliter(s)
MRDT morntality rate doubling time
mRNA messenger RNA
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MSRA lmethionine sulfoxide reductase A
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mtDNA mitochondrial DNA
O ooxygen
2
PBA sodium 4-phenylbutyrate
PD population doubling
pRb retinoblastoma protein
ROS reactive oxygen species
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