PDFlib PLOP: PDF Linearization, Optimization, Protection Page inserted by evaluation version www.pdflib.com – sales@pdflib.com Replicative Aging, Telomeres, and Oxidative Stress GABRIELE SARETZKI AND THOMAS VON ZGLINICKI Department of Gerontology, University of Newcastle, Newcastle upon Tyne NE6 4BE, United KIngdom ABSTRACT: Aging is a very complex phenomenon, both in vivo and in vitro. Free radicals and oxidative stress have been suggested for a long time to be involved in or even to be causal for the aging process. Telomeres are special structures at the end of chromosomes. They shorten during each round of replication and this has been characterized as a mitotic counting mechanism. Our experiments show that the rate of telomere shortening in vitro is modulated by oxidative stress as well as by differences in antioxidative defence capacity between cell strains. In vivo we found a strong correlation between short telomeres in blood lymphocytes and the incidence of vascular dementia. These data suggest that parameters that characterise replicative senescence in vitro offer potential for understanding of, and intervention into, the aging process in vivo. Keywords: senescence; telomeres; oxidative stress; dementia TELOMERES AND REPLICATIVE SENESCENCE In vitro aging of human fibroblasts is an established model for cellular aging, first described by Leonard Hayflick.1 Explanted mammalian cells perform in vitro a limited number of cell divisions and arrest then (at the Hayflick limit) in a state known as replicative senescence. Such cells are irreversibly blocked, mostly in the G1 phase of cell cycle, and are no longer sensitive to growth factor stimulation. Thus, replicative senescence was defined as a permanent and irreversible loss of replicative potential of cells. The Hayflick limit is usually a constant number under standardized culture conditions. However, the external change of oxidative stress levels can modulate the replicative life span of a given cell culture.2,3 Telomeres are repetitive structures of the sequence (TTAGGG)n at the ends of mammalian chromosomes. It has been shown that the average length of telomere repeats in human somatic cells decreases by 20–200 base pairs with each cell division.4,5 One reason for this shortening is the so-called “end replication problem”: during the replication of the lagging strand, the RNA primer for the most distal Okazaki fragment cannot be replaced by DNA. Accordingly, the newly synthesized lagging strand is shorter by at least the length of the primer (less than 12 nt), resulting Address for correspondence: Dr. Gabriele Saretzki, University of Newcastle, Department of Gerontology, Wolfson Research Centre, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE6 4BE, UK.Voice: +44-0191 256 3384; fax: +44-0191 2195074. gabriele.saretzki@ncl.ac.uk Ann. N.Y. Acad. Sci. 959: 24–29 (2002). © 2002 New York Academy of Sciences. 24 SARETZKI & ZGLINICKI: REPLICATIVE AGING 25 in an overhang of the parental G-rich strand at one end of the chromosome. Experimentally, G-rich overhangs 100–500 nt long have been found. However, whether these overhangs exist on one (as predicted by the end replication problem), or on both chromosome ends, is not clear,6,7 and neither is the contribution of a hypothetical 5′ exonuclease to telomere shortening. On the basis of regular shortening, telomeres have been connected with replicative aging in vitro and in vivo and were characterized as a “mitotic clock.” 8,9 Direct evidence linking telomere shortening causally to replicative senescence was given by overexpression of the telomere-elongating enzyme, telomerase, in mortal cells. In this case, telomeres are elongated and the transfected cells are immortalized without any evident karyotypic changes or perturbations of cell cycle checkpoints.10,11 DO EXTERNAL FACTORS CHANGE THE REPLICATIVE LIFE SPAN? The answer is yes: this is accomplished by changing the rate of telomere shortening. The end replication problem suggests a constant shortening of telomeres. However, we could show for the first time that telomere shortening is stress dependent. 2 There is a minimal shortening of less than 20 bp per cell division12 in cells with high antioxidative capacity, and telomere shortening rates are higher in cells with lower antioxidative defence.5 Cultivating cells under enhanced oxidative stress like mild hyperoxia (40% normobaric oxygen) shortens the telomeres prematurely and shortens the replicative life span accordingly.2 Most parameters of those prematurely aged fibroblasts are identical to normal fibroblast aging (e.g., morphology, lipofuscin accumulation, and specific changes in gene expression).2,13 Radical scavengers like phenyl-butyl-nitrone are able to lower the telomere shortening rate.14 We uncovered the mechanism of the enhanced telomere shortening rate as damage of free oxygen radicals to the telomeric DNA. More specifically, an accumulation of single-stranded regions takes place in the telomeres, which is caused by lower repair of those lesions in the telomeric compartment in comparison to other repetitive nontranscribed regions in the genome, like minisatellite structures, and to the bulk of the genome.15 The single-strand breaks in the telomeric regions result in telomere shortening after DNA replication.16 The mechanism is probably the transient stalling of the replication fork.17 Telomeres are not simple linear stretches of DNA but are highly organized structures. The G-rich telomeric strand forms an overhang, which is more than 100 nt long. There is still controversy as to whether the length of these overhangs relates to telomere shortening rates in different cell lines.14,18 When we treated fibroblasts with different concentrations of hydrogen peroxide and other agents that damage telomeric DNA preferentially, we could not find a difference in the overhang length.14 CAN THE G-RICH OVERHANG BE CONNECTED WITH SIGNALING PATHWAYS INITIATING SENESCENCE? Not only the overhang, but also different telomere binding proteins, like TRF1 and TRF2, are responsible for a characteristic loop structure of telomeres19 and are involved in telomere length control.20 In this way the single strand sequence of the 26 ANNALS NEW YORK ACADEMY OF SCIENCES overhang is protected and probably not accessible to signal-transducing proteins and enzymes. Recently the existence of the overhang binding protein pot1 has been described,21 but its function is not yet known. There are at least three events that unfold the telomeric loop structure. The first one is the normal DNA replication. A second process takes place in cells with active telomerase during elongation of the G-rich overhang of a telomere. These two events are both regulated and coordinated. By contrast, unfolding of the loop structure due to critical telomere shortening or telomere-specific DNA damage is unscheduled and might activate a cell cycle checkpoint signal. We modeled this unscheduled exposure of the single-stranded overhang by treating cells with short oligonucleotides of telomeric G- and C-rich sequences and compared them with unrelated sequences like scrambled or minisatellite sequences. Those oligonucleotides (12mers) are fast internalized into the cell nucleus. Only the G-rich oligonucleotides cause a proliferation arrest, which is p53 dependent.22 This arrest is long lasting in human fibroblasts but is of shorter duration in the telomerase-positive human glioblastoma cell line U87. U87 cells that overcome the arrest have activated telomerase above normal levels and elongated telomeres. The same effect was detected after arrests by hydrogen peroxide treatment or chronic hyperoxia. p53 seemed necessary for both the transient proliferation arrest and the telomere elongation by telomerase in cells overcoming the arrest, because both effects were abrogated in cells expressing a dominant negative p53 mutation.22 The simplest interpretation of these findings is that treatment with G-rich oligonucletides mimics the unscheduled unfolding of the telomeric loop and that single-stranded G-rich overhangs activate p53 either directly or indirectly. The telomeric loop structure might also be the cause for the decreased repair of telomeres, because it might make telomeres less accessible for repair enzymes than other parts of the genome. In this way the unrepaired single-strand breaks accumulated in the telomeres contribute to a telomere shortening, which is additional to the one caused by the end replication problem. Therefore, oxidative damage and free radicals can modulate the telomere length plus their structural and functional integrity. Additional evidence for the importance of a stabilized telomeric structure comes from telomerase inhibition experiments. Telomerase not only elongates telomeres but stabilizes the ends of chromosomes via a “capping” function as well.23 Inhibition of capping, for instance, by prevention of the synthesis of the catalytic subunit of telomerase by a ribozyme approach24 appears to destabilize the telomere structure in tumor cells. The surprising result of telomerase inhibition via viral transduction of an anti-hTERT ribozyme was the fast induction of apoptosis in the treated tumor cells, irrespective of their telomere length or p53 status.25 We hypothesize that the withdrawal of telomerase in this model activates p53-independent apoptotic pathways. TELOMERE SHORTENING AND ANTIOXIDATIVE DEFENCE An age-dependent telomere shortening has been demonstrated in different selfrenewing human tissues like peripheral blood monocytes, fibroblasts, endothelial cells, and others.9,26 The variation of telomere length between different individuals seems to be mainly genetically determined.27 SARETZKI & ZGLINICKI: REPLICATIVE AGING 27 We measured telomere shortening rates over at least 20 population doublings in different fibroblast strains. From all examined strains, BJ foreskin fibroblasts had the longest replicative life span (80–100 PD) and maintained their telomeres best both under normoxic and hyperoxic conditions. By contrast, MRC-5 and WI38 displayed a high telomere shortening rate and shorter replicative life span under both conditions.12 Measuring both telomere-shortening rate and antioxidative capacity (using DCF fluorescence as an indicator of intracellular peroxide levels), we found a significant inverse correlation between telomere-shortening rate and antioxidative capacity in more than 20 human fibroblast strains. 2 Fibroblasts with low antioxidative defence capacity shorten their telomeres faster and vice versa. These data are in good concordance with those showing an important role of the antioxidative enzymes gluthathione peroxidase and Cu/Zn-superoxide dismutase for the telomere shortening rate in human fibroblasts.28 These data confirm that telomere length is mainly determined by the relation between oxidative stress and antioxidative defence capacity. Thus, the age-corrected telomere length is a cumulative measure of the history of oxidative damage a cell line has experienced over its life span. While this has been shown for in vitro experiments so far, it might hold true for the in vivo situation as well. A correlation between oxidative stress and enhanced telomere shortening rate has been shown earlier for inherited respiratory chain disorders29 and for patients with Down syndrome.30 If antioxidative defence determines telomere length in vivo as well, one would expect a good correlation between telomere lengths in different tissues from the same donor, and such correlation should not deteriorate with age. In fact, telomere lengths in blood lymphocytes and fibroblasts from the same donor were significantly correlated irrespective of different replicative histories and the presence of telomerase in lymphocytes. Similar correlations were found between telomere lengths in four different tissues in cattle (Serra et al., in prep.). There was no evidence for a deterioration of the correlations with age. Recently, these results were independently confirmed.31 Taken together, our results suggest that telomere length in one (proliferating) tissue should be a cumulative indicator of the relative amount of oxidative stress even in other tissues, and of the ability of the individual to cope with such stress. In other words, the relative telomere length might indicate the probability of a successful defence against an oxidative insult. TELOMERE LENGTH AND DEMENTIA The involvement of telomeres has been implicated in several conditions: progeria, Werner syndrome,32 and hyper- and hypoproliferative diseases.33,34,26 We sought to examine whether degenerative neurological conditions might be related to telomeres and oxidative stress. To test the idea that telomere length is not only important for the replicative life span of a cell population in vitro but might represent a valuable diagnostic or even prognostic factor for an in vivo situation, we analyzed telomeres of lymphocytes from 186 individuals between 18–98 years of age. Among them were 16 patients with Alzheimer’s dementia; 97 patients displayed severe vascular symptoms like stroke, myocardial infarction, severe peripheral arterial occlusions, coronary heart diseases, and other vascular risk factors. Out of these 97 patients, 56 were cognitively unimpaired, while a possible or probable vascular de- 28 ANNALS NEW YORK ACADEMY OF SCIENCES mentia was diagnosed in 41 cases. The estimation of telomere length revealed that none of the patients with vascular dementia had telomeres longer than the age-dependent average of the healthy study participants. The average telomere length in the vascular dementia group was about 400 bp shorter than in the control group.2 This difference was highly significant (P < 0.001), while telomere lengths in stroke or infarct patients without cognitive decline and in Alzheimer’s patients were not significantly different from controls. In our patients, there was no correlation between dementia and presence of the ApoEe4 allele or gluthation-S-transferase polymorphisms. So the conclusion is that the correlation between short telomeres in blood lymphocytes and vascular dementia is much more robust than that of established genetic risk markers for dementia. Telomere length might actually be a prognostic factor for vascular dementia. Oxidative stress and insufficient antioxidative defence is most probably an important pathogenetic factor for vascular as well as so-called mixed dementias.35 The interesting question now is whether our in vitro data, which define telomere length as a marker for antioxidative defence capacity, can be confirmed in vivo. Experiments are in progress to test this suggestion. REFERENCES 1. HAYFLICK, L. 1965. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37: 614–636. 2. VON ZGLINICKI, T., et al. 1995. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp. Cell Res. 220: 186–193. 3. TOUSSAINT, O., et al. 2000. Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes. Exp. Gerontol. 35:927–945. 4. HARLEY, C.B., et al. 1990. Telomeres shorten during ageing of human fibroblasts. Nature 345: 458–460. 5. VON ZGLINICKI, T., et al. 2000. 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