Bone Aging in DNA Repair Deficient Trichothiodystrophy Mice Botveroudering in trichothiodystrophy muizen met een defect in DNA schadeherstel ISBN: 978-90-8559-915-9 Omslag: Ruud Koppenol en Karin Diderich Lay-out en drukwerk: Optima Grafische Communicatie Dit proefschrift kwam tot stand binnen de vakgroep Celbiologie en Genetica van de faculteit der Geneeskunde en Gezondheidswetenschappen van de Erasmus Universiteit Rotterdam. De vakgroep maakt deel uit van het Medisch Genetisch centrum Zuid-West Nederland. Het onderzoek is financieel ondersteund door de Nederlandse Organisatie voor Wetenschap- pelijk Onderzoek via het Research Institute for Diseases of the Elderly, the National Institutes of Health, the National Institute of Environmental Health Sciences, de Stichting Koningin Wilhelmina Fonds voor de Nederlandse Kankerbestrijding en de Europese Commissie. Bijdragen in de drukkosten zijn verkregen van de Erasmus Universiteit Rotterdam, de Neder- landse Paget patiënten vereniging en DNage BV, Leiden. Bone Aging in DNA Repair Deficient Trichothiodystrophy Mice Botveroudering in trichothiodystrophy muizen met een defect in DNA schadeherstel Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof.dr. H.G. Schmidt en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op woensdag 6 januari 2010 om 13.30 uur door Karin Elin Maria Diderich geboren te Rotterdam Promotiecommissie Promotoren: Prof.dr. J.H.J. Hoeijmakers Prof.dr. G.T.J. van der Horst Prof.dr. J.P.T.M. van Leeuwen Overige leden: Prof.dr. H. van Steeg Prof.dr.ir. A.P.N. Themmen Prof.dr.ir. H. Weinans “Als mijn proefschrift klaar is, zal er met geen woord gewag worden gemaakt van ontvelde schouders, geschaafde knieën, de beukende hoofdpijn, de muggen en de vleesetende vliegen.’’ Uit ‘’Nooit meer slapen’’ van Willem Frederik Hermans Voor mijn moeder In memoriam Ragnhild Hellestam Prof.dr. J. Sanders-Woudstra Contents Chapter 1 Introduction 9 1 Premature aging in DNA repair deficient disorders 11 2 Trichothiodystrophy and bone aging 24 3 Bone metabolism 25 4 Scope of this thesis 37 Chapter 2 Osteoporosis and decline in stem cells in prematurely aging DNA 51 repair deficient trichothiodystrophy mice Chapter 3 Age-related skeletal dynamics in DNA repair deficient male 75 trichothiodystrophy mice Chapter 4 Increased bone mass in vertebrae of DNA repair deficient 93 trichothiodystrophy mice strongly resembling patients Chapter 5 Endocrine parameters in relation to accelerated bone loss in 107 prematurely aging DNA repair deficient trichothiodystrophy mice Chapter 6 Concluding remarks and future perspectives 133 Summary/ Samenvatting 141 Appendix I Accelerated aging pathology in ad libitum fed XpdTTD mice is 147 accompanied by features suggestive of caloric restriction Appendix II Dysregulation of the peroxisome proliferator-activated receptor 169 target genes by XPD mutations Appendix III Impaired Genome Maintenance Suppresses the GH/IGF1 Axis in 195 Cockayne Syndrome Mice Curriculum vitae 231 Dankwoord 237 Colour figures 241 Chapter 1 Chapter 1 Chapter 1 Introduction Introduction 1 Premature aging in DNA repair deficient disorders 1.1.1 DNA damage Our genome is continuously damaged by environmental, endogenous agents as well as by the instrinsic instability of DNA. For example, UV light gives rise to helix-distorting cyclobu- tane pyrimidine dimers (CPDs) and pyrimidine-(6,4)-pyrimidone adducts (6-4PPs). Ionizing radiation can cause both single and double strand breaks in DNA and numerous types of oxi- dative lesions. Chemotherapeutics, that are used in cancer therapy, and other environmental chemical agents, which are present in e.g. polluted air and tobacco smoke, induce a plethora of DNA lesions, including intra- and inter-strand cross-links and mono-adducts. In addition, endogenous agents cause a wide variety of DNA lesions. Metabolic processes within our cells lead to reactive oxygen species (ROS), which react with proteins, lipids and DNA. Although ROS participate in beneficial physiological processes as growth factor signal transduction (1), these by-products of metabolism also underlie a broad spectrum of oxidative DNA lesions, including 8-oxo-2’-deoxyguanosine (8-oxodG), thymine glycols, cylcopurines, as well as single and double strand breaks (2). Finally, lesions in the DNA can also form without a direct damaging agent. E.g. spontaneous hydrolysis or modifications of nucleotides occurs in cells, which leaves non-informative a-basic sites or altered, miscoding nucleotides (3). 1.1.2 Consequences of DNA damage Lesions in DNA have immediate effects on cell function as well as long-term consequences. For instance, DNA lesions will interfere with transcription and replication (4,5). This causes dysfunctioning of cells and -depending on the damage load- will lead to cell cycle arrest or programmed cell death (apoptosis) (6). Apoptosis is a way of eliminating cells that are at risk of malignant transformation. In addition to apoptosis, cellular senescence, i.e. limited growth potential followed by growth arrest, can neutralize potentially malignant cells (7). De Waard and co-workers found that different cell types exhibit different responses to DNA damage (8). For instance, pluripotent embryonic stem cells show increased apoptosis after treatment with several types of DNA damaging agents compared to differentiated keratinocytes (8). Persistent DNA lesions that are misinterpreted by the replication machinery, result in the induction of mutations. These mutations, as well as other changes in the DNA that result from genome instability or miss-segregation (rearrangements, deletions, insertions, loss of heterozygosity and numerical aberrations), can on the long-term give rise to cancer or inborn diseases. Furthermore, cellular dysfunction or depletion of proliferative capacity of cells by senescence or apoptosis can contribute to aging (9,10). These processes lead to compromised tissue homeostasis, most likely through diminished self-renewal or altered tissue structure 11 Chapter 1 (11). For example, cells that are lost via apoptosis might be replaced by progenitors and in time this may exhaust the regenerative capacity of a tissue. 1.2.1 Repair mechanisms To counteract the deleterious effects of DNA damage, the cell is equipped with a wide variety of genome caretaking mechanisms (12,13) (Figure 1). On the one hand, a transient block in the cell cycle can provide an extended time window for repair to take place (14). Various DNA repair machineries exist to repair the different DNA lesions (reviewed by (12,13)). On the other hand, cells with persisting DNA damage may bypass DNA damage during replication at the risk of mutation induction. This process is called trans-lesion synthesis and involves several polymerases that are more or less error-prone (15-17). Nucleotide excision repair (NER), which is discussed in more detail below, functions by a ‘cut and patch’-like mechanism, in which damage recognition, opening of the DNA helix around the lesion, damage excision, and gap-filling are the successive steps (reviewed by (12,18,19)). This repair mechanism can remove numerous types of helix-distorting and bulky lesions. Damaging agent Oxygen radicals Ionizing radiation Antitumor agents UV-light Replication errors Alkylating agents (MMC, cis-Pt) Polycyclic aromatic Spontaneous hydrocarbons T U T T G C T A G* G G T Uracil Double-strand Interstrand 6-4 PP A-G mismatch Abasic site break crosslink CPD T-C mismatch 8-oxodG Bulky adduct Insertion Single strand break deletion BER DSB repair Crosslink NER Mismatch (HR, NHEJ) repair repair Repair process Figure 1: DNA lesions and repair mechanisms. At the top of the figure, examples of common DNA damaging agents are depicted. As indicated by the arrows, many DNA damage inducing agents produce a spectrum of different (classes of) lesions. Oppositely, different DNA damaging agents can cause similar DNA lesions. The middle part of the figure shows the DNA helix, with several DNA lesions, as depicted under the figure. The lower part of the figure shows the various repair pathways that cells use to remove these lesions (adapted from de Boer et al., Carcinogenesis 2000). 12 Figure 1 Introduction Base excision repair (BER) is another ‘cut and patch’-like mechanism that involves the con- certed interplay of different specialized proteins. A battery of glycosylases with overlapping lesion specificity recognize and remove the lesions from the DNA. These different glycosyl- ases allow BER to remove a wide variety of (non helix-distorting) nucleotide modifications (reviewed by (20,21)). As BER removes base adducts from ROS, methylation, deamination, and hydroxylation, it is considered as the main guardian against DNA lesions caused by cel- lular metabolism. Mismatch repair is a ‘cut and patch’-like mechanism that removes single base-base mis- matches and small insertion/deletion loops caused by erroneous base incorporation, and slippage of DNA polymerases during replication or recombination (reviewed by (22)). As such it prevents the accumulation of these mutagenic lesions and is important in the prevention of cancer. This is illustrated by the fact that mutations in mismatch repair genes cause Lynch syndrome (previously called hereditary non-polyposis colorectal cancer or HNPCC) (23). Double strand breaks (DSBs) are deleterious lesions that arise from ionizing radiation, free radicals or chemicals, or are formed during replication of