- Email: [email protected]
Mechanisms and role of oxidative damage to telomeres
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Abstracts / Toxicology 226 (2006) 12–77
cytokine-suppressive anti-inflammatory drug that targets p38 activity (Davis et al., 2005). SB203580 treatment reverts the aged morphology of young WS fibroblasts to that seen in young normal fibroblasts. In addition, SB203580 increases the life span and growth rate of WS fibroblasts to within the normal range (Davis et al., 2005). These data suggest that the abbreviated replicative life span of WS cells is due to a stress-induced, p38mediated growth arrest that is independent of telomere erosion. With some p38 inhibitors already in clinical trials, our data suggest a potential route to drug intervention in a premature aging syndrome. References Baird, D.M., Davis, T., Rowson, J., Jones, C.J., Kipling, D., 2004. Hum. Mol. Genet. 13, 1515–1524. Davis, T., Baird, D.M., Haughton, M.F., Jones, C.J., Kipling, D., 2005. J. Gerontol. A Biol. Sci. Med. Sci. 60, 1386–1393. Kipling, D., Davis, T., Ostler, E.L., Faragher, R.G., 2004. Science 305, 1426–1431.
doi:10.1016/j.tox.2006.05.024 Checkpoint proteins determine organismal stress resistance and lifespan in C. elegans Anders Olsen, Maithili C. Vantipalli, Glenda A. Walker, Gordon J. Lithgow The Buck Institute, 8001 Redwood Blvd., Novato, CA 94945, USA E-mail address: [email protected] (G.J. Lithgow) There are considerable mechanistic links between organismal stress resistance and aging (Lithgow and Walker, 2002). We have shown previously that long-lived mutants of the nematode C. elegans are resistant to thermal stress and over-accumulate small heat shock proteins (shsps) which alone can extend lifespan (Lithgow et al., 1995; Walker et al., 2001; Walker and Lithgow, 2003). A large number of genes determine normal aging rate in the nematode and mutations in these genes tend to be highly pleiotropic including effects on organismal stress resistance and stress gene expression. We undertook genetic screen for mutations that conferred increased resistance to multiple stresses and were surprised when we tagged a gene shown in other organisms to affect checkpoint functions. Checkpoints are evolutionarily conserved signal transduction pathways that arrest cell division in response to DNA damage or stalled replication forks. We demonstrate that checkpoint proteins also determine
organismal stress resistance and lifespan. Inactivation of the checkpoint proteins CID-1, CHK-1 or CDC-25 in Caenorhabditis elegans results in up-regulation of stress response genes, increased stress resistance and lifespan extension. As adult nematode somatic tissues are post-mitotic, these results indicate a novel role for checkpoint functions in non-dividing cells that influences aging. To further explore the genetic influences on checkpoint function and lifespan, we undertook a whole genome screen (16,000 genes) by RNA interference to find genes that contributed to growth arrest by hydroxyurea which causes stalled replication forks. We present the complete list of genes that lead to HU resistance and show that some confer general stress resistance and lifespan extension. Our results have implications for how we think about the role of checkpoint pathways and other genome stability factors in post-mitotic cells. References Lithgow, G.J., Walker, G.A., 2002. Mech. Ageing Dev. 123, 765– 771. Lithgow, G.J., White, T.M., Melov, S., Johnson, T.E., 1995. Proc. Natl. Acad. Sci. U.S.A. 92, 7540–7544. Walker, G.A., White, T.M., McColl, G., Jenkins, N.L., Babich, S., Candido, E.P., Johnson, T.E., Lithgow, G.J., 2001. J. Gerontol. A Biol. Sci. Med. Sci. 56, B281–B287. Walker, G.A., Lithgow, G.J., 2003. Aging Cell 2, 131–139.
doi:10.1016/j.tox.2006.05.025 Mechanisms and role of oxidative damage to telomeres Joao Passos, Torsten Thomas von Zglinicki
Richter, Gabriele
Saretzki,
Institute for Ageing and Health, University of Newcastle, Newcastle upon Tyne, NE4 6BE, UK E-mail address: [email protected] (T. von Zglinicki) Telomere shortening is the major determinant of the replicative lifespan of primary human cells, with short telomeres inducing a DNA damage response leading to a p53/p21/pRb-mediated permanent cell cycle arrest (d’Adda di Fagagna et al., 2003; von Zglinicki, 2002). The rate of telomere shortening is greatly modified by oxidative stress-induced single strand break accumulation because of a TRF2-dependent telomerespecific deficiency of single-strand break repair. Thus, we hypothesised that the regulation of intrinsic, predominantly mitochondrial production of reactive oxy-
Abstracts / Toxicology 226 (2006) 12–77
gen species (ROS) might be important for telomeredependent replicative senescence and its cell-to-cell variation. We show that mitochondrial superoxide production increases with replicative age in human fibroblasts despite an adaptive retrograde response including UCP-2-dependent mitochondrial uncoupling. Uncoupling extends replicative lifespan by reducing mitochondrial superoxide generation, slowing-down telomere shortening and delaying formation of telomeric DNA damage foci. This indicates mitochondrial ROS production as one of the causes of replicative senescence. By sorting early senescent (SES) cells from young proliferating fibroblast cultures we show that SES cells have higher ROS levels, dysfunctional mitochondria, shorter telomeres and telomeric DNA damage foci. Thus, we propose that mitochondrial redox regulation is a major determinant of telomere-dependent senescence at the single cell level that is responsible for cell-to-cell variation in replicative lifespan. References d’Adda di Fagagna, F., Reaper, P.M., Clay-Farrace, L., Fiegler, H., Carr, P., von Zglinicki, T., Saretzki, G., Carter, N.P., Jackson, S.P., 2003. Nature 426, 194–198. von Zglinicki, T., 2002. Trends Biochem. Sci. 27, 339–344.
doi:10.1016/j.tox.2006.05.026 Genome instability and accelerated aging: The relevance for normal aging and life span extension Jan H.J. Hoeijmakers 1 , George Garinis 1 , Ingrid van der Pluijm 1 , Jay Mitchell 1 , Jaan-Olle Andressoo 1 , Karin Diderich 1 , Astrid Lalai 1 , Harm de Waard 1 , Dolf Beems 2 , Harry van Steeg 2 , Laura Niedernhofer 1,3 , Bert van der Horst 1 1 MGC, CBG, Department of Cell Biology and Genetics,
Erasmus MC, 3000 DR Rotterdam, The Netherlands; 2 RIVM, Bilthoven, The Netherlands; 3 University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA 15213-1863, USA E-mail address: [email protected] (J.H.J. Hoeijmakers) One of the most versatile DNA damage repair systems is nucleotide excision repair (NER), which removes a wide class of helix-distorting lesions in a complex ‘cut and patch’ reaction. Most NER lesions are of exogenous origin (e.g. UV-induced DNA pyrimidine dimers), but importantly also some endogenously generated oxidative lesions are removed by this process.
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There are two sub-pathways: global genome NER operates genome-wide and is critical for preventing mutations whereas transcription-coupled repair (TCR) counteracts the cytotoxic effects of DNA injury by rescuing gene expression blocked by DNA damage. Inherited NER defects are associated with sun (UV) hypersensitive syndromes, including xeroderma pigmentosum (XP) which is characterized by a high predisposition to skin cancer, and the severe conditions Cockayne syndrome (CS) and trichothiodystrophy (TTD), which seem to be protected from cancer but instead display many neurodevelopmental abnormalities. Mutations in the multifunctional NER/TCR XPB and XPD helicases are associated with an extreme clinical heterogeneity, ranging from XP to XP combined with CS and TTD. Defects in the NER and crosslink repair endonuclease, ERCC1/XPF, cause XP or XP with multi-system dysfunction. Mouse models have provided important insights into the impact of the NER sub-pathways on human health and the complex genotype-phenotype relationship. XPDTTD mice, with a partial defect in both global and TCR have reduced cancer susceptibility, but exhibit wide spread premature ageing. XPDXP/CS mutant mice are highly predisposed to cancer, with an additional ageing phenotype. Complete repair deficiency in TTDxXPA mice aggravates many premature ageing symptoms, reducing life span to ∼3 weeks. Similar findings are made when TCR-deficient CS mouse mutants are crossed with GG-NER deficient XP mice. Mutations in the ERCC1 gene involved in NER as well as cross-link repair induce a distinct set of accelerated ageing features, with a rate of onset depending on the severity of the mutation. The striking correlation between severity of the repair defect and the clinical progeroid manifestations provides strong evidence for the DNA damage theory of ageing. We propose that endogenous oxidative lesions compromise transcription, inactivate genes, and trigger apoptosis/senescence inducing ageing. Very cytotoxic interstrand cross-links may also cause cell death (particularly in proliferating cells), as well as senescence and features of ageing. In contrast, lesions or defects in genetic stability mechanisms causing enhanced levels of DNA damage-induced mutagenesis correlate with increased carcinogenesis. Microarray analysis has revealed the involvement of the IGF1/GH somatotrophic axis that controls metabolism and antioxidant systems in the organismal response to intrinsic cytotoxic DNA damage. Interestingly, these pathways have previously been associated with life span extension in various species. Data concerning the parallels between short life span, natural aging and extended life
Abstracts / Toxicology 226 (2006) 12–77
cytokine-suppressive anti-inflammatory drug that targets p38 activity (Davis et al., 2005). SB203580 treatment reverts the aged morphology of young WS fibroblasts to that seen in young normal fibroblasts. In addition, SB203580 increases the life span and growth rate of WS fibroblasts to within the normal range (Davis et al., 2005). These data suggest that the abbreviated replicative life span of WS cells is due to a stress-induced, p38mediated growth arrest that is independent of telomere erosion. With some p38 inhibitors already in clinical trials, our data suggest a potential route to drug intervention in a premature aging syndrome. References Baird, D.M., Davis, T., Rowson, J., Jones, C.J., Kipling, D., 2004. Hum. Mol. Genet. 13, 1515–1524. Davis, T., Baird, D.M., Haughton, M.F., Jones, C.J., Kipling, D., 2005. J. Gerontol. A Biol. Sci. Med. Sci. 60, 1386–1393. Kipling, D., Davis, T., Ostler, E.L., Faragher, R.G., 2004. Science 305, 1426–1431.
doi:10.1016/j.tox.2006.05.024 Checkpoint proteins determine organismal stress resistance and lifespan in C. elegans Anders Olsen, Maithili C. Vantipalli, Glenda A. Walker, Gordon J. Lithgow The Buck Institute, 8001 Redwood Blvd., Novato, CA 94945, USA E-mail address: [email protected] (G.J. Lithgow) There are considerable mechanistic links between organismal stress resistance and aging (Lithgow and Walker, 2002). We have shown previously that long-lived mutants of the nematode C. elegans are resistant to thermal stress and over-accumulate small heat shock proteins (shsps) which alone can extend lifespan (Lithgow et al., 1995; Walker et al., 2001; Walker and Lithgow, 2003). A large number of genes determine normal aging rate in the nematode and mutations in these genes tend to be highly pleiotropic including effects on organismal stress resistance and stress gene expression. We undertook genetic screen for mutations that conferred increased resistance to multiple stresses and were surprised when we tagged a gene shown in other organisms to affect checkpoint functions. Checkpoints are evolutionarily conserved signal transduction pathways that arrest cell division in response to DNA damage or stalled replication forks. We demonstrate that checkpoint proteins also determine
organismal stress resistance and lifespan. Inactivation of the checkpoint proteins CID-1, CHK-1 or CDC-25 in Caenorhabditis elegans results in up-regulation of stress response genes, increased stress resistance and lifespan extension. As adult nematode somatic tissues are post-mitotic, these results indicate a novel role for checkpoint functions in non-dividing cells that influences aging. To further explore the genetic influences on checkpoint function and lifespan, we undertook a whole genome screen (16,000 genes) by RNA interference to find genes that contributed to growth arrest by hydroxyurea which causes stalled replication forks. We present the complete list of genes that lead to HU resistance and show that some confer general stress resistance and lifespan extension. Our results have implications for how we think about the role of checkpoint pathways and other genome stability factors in post-mitotic cells. References Lithgow, G.J., Walker, G.A., 2002. Mech. Ageing Dev. 123, 765– 771. Lithgow, G.J., White, T.M., Melov, S., Johnson, T.E., 1995. Proc. Natl. Acad. Sci. U.S.A. 92, 7540–7544. Walker, G.A., White, T.M., McColl, G., Jenkins, N.L., Babich, S., Candido, E.P., Johnson, T.E., Lithgow, G.J., 2001. J. Gerontol. A Biol. Sci. Med. Sci. 56, B281–B287. Walker, G.A., Lithgow, G.J., 2003. Aging Cell 2, 131–139.
doi:10.1016/j.tox.2006.05.025 Mechanisms and role of oxidative damage to telomeres Joao Passos, Torsten Thomas von Zglinicki
Richter, Gabriele
Saretzki,
Institute for Ageing and Health, University of Newcastle, Newcastle upon Tyne, NE4 6BE, UK E-mail address: [email protected] (T. von Zglinicki) Telomere shortening is the major determinant of the replicative lifespan of primary human cells, with short telomeres inducing a DNA damage response leading to a p53/p21/pRb-mediated permanent cell cycle arrest (d’Adda di Fagagna et al., 2003; von Zglinicki, 2002). The rate of telomere shortening is greatly modified by oxidative stress-induced single strand break accumulation because of a TRF2-dependent telomerespecific deficiency of single-strand break repair. Thus, we hypothesised that the regulation of intrinsic, predominantly mitochondrial production of reactive oxy-
Abstracts / Toxicology 226 (2006) 12–77
gen species (ROS) might be important for telomeredependent replicative senescence and its cell-to-cell variation. We show that mitochondrial superoxide production increases with replicative age in human fibroblasts despite an adaptive retrograde response including UCP-2-dependent mitochondrial uncoupling. Uncoupling extends replicative lifespan by reducing mitochondrial superoxide generation, slowing-down telomere shortening and delaying formation of telomeric DNA damage foci. This indicates mitochondrial ROS production as one of the causes of replicative senescence. By sorting early senescent (SES) cells from young proliferating fibroblast cultures we show that SES cells have higher ROS levels, dysfunctional mitochondria, shorter telomeres and telomeric DNA damage foci. Thus, we propose that mitochondrial redox regulation is a major determinant of telomere-dependent senescence at the single cell level that is responsible for cell-to-cell variation in replicative lifespan. References d’Adda di Fagagna, F., Reaper, P.M., Clay-Farrace, L., Fiegler, H., Carr, P., von Zglinicki, T., Saretzki, G., Carter, N.P., Jackson, S.P., 2003. Nature 426, 194–198. von Zglinicki, T., 2002. Trends Biochem. Sci. 27, 339–344.
doi:10.1016/j.tox.2006.05.026 Genome instability and accelerated aging: The relevance for normal aging and life span extension Jan H.J. Hoeijmakers 1 , George Garinis 1 , Ingrid van der Pluijm 1 , Jay Mitchell 1 , Jaan-Olle Andressoo 1 , Karin Diderich 1 , Astrid Lalai 1 , Harm de Waard 1 , Dolf Beems 2 , Harry van Steeg 2 , Laura Niedernhofer 1,3 , Bert van der Horst 1 1 MGC, CBG, Department of Cell Biology and Genetics,
Erasmus MC, 3000 DR Rotterdam, The Netherlands; 2 RIVM, Bilthoven, The Netherlands; 3 University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA 15213-1863, USA E-mail address: [email protected] (J.H.J. Hoeijmakers) One of the most versatile DNA damage repair systems is nucleotide excision repair (NER), which removes a wide class of helix-distorting lesions in a complex ‘cut and patch’ reaction. Most NER lesions are of exogenous origin (e.g. UV-induced DNA pyrimidine dimers), but importantly also some endogenously generated oxidative lesions are removed by this process.
17
There are two sub-pathways: global genome NER operates genome-wide and is critical for preventing mutations whereas transcription-coupled repair (TCR) counteracts the cytotoxic effects of DNA injury by rescuing gene expression blocked by DNA damage. Inherited NER defects are associated with sun (UV) hypersensitive syndromes, including xeroderma pigmentosum (XP) which is characterized by a high predisposition to skin cancer, and the severe conditions Cockayne syndrome (CS) and trichothiodystrophy (TTD), which seem to be protected from cancer but instead display many neurodevelopmental abnormalities. Mutations in the multifunctional NER/TCR XPB and XPD helicases are associated with an extreme clinical heterogeneity, ranging from XP to XP combined with CS and TTD. Defects in the NER and crosslink repair endonuclease, ERCC1/XPF, cause XP or XP with multi-system dysfunction. Mouse models have provided important insights into the impact of the NER sub-pathways on human health and the complex genotype-phenotype relationship. XPDTTD mice, with a partial defect in both global and TCR have reduced cancer susceptibility, but exhibit wide spread premature ageing. XPDXP/CS mutant mice are highly predisposed to cancer, with an additional ageing phenotype. Complete repair deficiency in TTDxXPA mice aggravates many premature ageing symptoms, reducing life span to ∼3 weeks. Similar findings are made when TCR-deficient CS mouse mutants are crossed with GG-NER deficient XP mice. Mutations in the ERCC1 gene involved in NER as well as cross-link repair induce a distinct set of accelerated ageing features, with a rate of onset depending on the severity of the mutation. The striking correlation between severity of the repair defect and the clinical progeroid manifestations provides strong evidence for the DNA damage theory of ageing. We propose that endogenous oxidative lesions compromise transcription, inactivate genes, and trigger apoptosis/senescence inducing ageing. Very cytotoxic interstrand cross-links may also cause cell death (particularly in proliferating cells), as well as senescence and features of ageing. In contrast, lesions or defects in genetic stability mechanisms causing enhanced levels of DNA damage-induced mutagenesis correlate with increased carcinogenesis. Microarray analysis has revealed the involvement of the IGF1/GH somatotrophic axis that controls metabolism and antioxidant systems in the organismal response to intrinsic cytotoxic DNA damage. Interestingly, these pathways have previously been associated with life span extension in various species. Data concerning the parallels between short life span, natural aging and extended life