Accumulation of oxidative DNA damage, 8-oxo-2′-deoxyguanosine, and change of repair systems during in vitro cellular aging of cultured human skin fibroblasts

Kaneko, Takao; Tahara, Shoichi; Taguchi, Takahiko; Kondo, Hiroshi

doi:10.1016/s0921-8777(01)00100-8

Cited by 52 publications

(34 citation statements)

References 49 publications

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“…To do so, we manipulated the levels of the most frequently occurring oxidized DNA lesion, 8-oxoguanine (8-oxoG), which is found at elevated levels not only in senescent cells (21)(22)(23) but also in the frontal brain cortex (24) of elderly individuals. Deoxyguanosines (dG) may suffer oxidation at two stages-either when present in the soluble nucleotide pool or after incorporation into DNA.…”

mentioning

confidence: 99%

Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence

Rai

Önder

Young

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

154

163

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Reactive oxygen species (ROS) appear to play a role in limiting both cellular and organismic lifespan. However, because of their pleiotropic effects, it has been difficult to ascribe a specific role to ROS in initiating the process of cellular senescence. We have studied the effects of oxidative DNA damage on cell proliferation, believing that such damage is of central importance to triggering senescence. To do so, we devised a strategy to decouple levels of 8-oxoguanine, a major oxidative DNA lesion, from ROS levels. Suppression of MTH1 expression, which hydrolyzes 8-oxo-dGTP, was accompanied by increased total cellular 8-oxoguanine levels and caused early-passage primary and telomerase-immortalized human skin fibroblasts to rapidly undergo senescence, doing so without altering cellular ROS levels. This senescent phenotype recapitulated several salient features of replicative senescence, notably the presence of senescence-associated beta-galactosidase (SA beta-gal) activity, apparently irreparable genomic DNA breaks, and elevation of p21 Cip1 , p53, and p16 INK4A tumor suppressor protein levels. Culturing cells under low oxygen tension (3%) largely prevented the shMTH1-dependent senescent phenotype. These results indicate that the nucleotide pool is a critical target of intracellular ROS and that oxidized nucleotides, unless continuously eliminated, can rapidly induce cell senescence through signaling pathways very similar to those activated during replicative senescence.8-oxoguanine ͉ DNA damage ͉ p53 ͉ reactive oxygen species (ROS) W hen propagated in culture, normal somatic cells achieve a limited number of divisions before undergoing the loss of proliferative capacity termed replicative senescence (1). Several studies have suggested that cell senescence plays a role in organismic aging (2, 3) and that activation of senescence programs in cancer cells block tumor progression (4, 5). Consequently, elucidating the biochemical mechanisms of cellular senescence is critical for understanding the physiologic basis of aging and the mechanisms of tumorigenesis.Several lines of evidence indicate that cumulative damage to cellular constituents sustained during culture in vitro eventually triggers senescence (6, 7). Such damage can be inflicted by reactive oxygen species (ROS), which are byproducts of incomplete mitochondrial electron transfer (8). Despite the actions of detoxifying enzymes, such as superoxide dismutases (SOD1 and SOD2) and catalase, and low molecular weight antioxidants, increasing oxidative stress due to age-related mitochondrial dysfunction may eventually exceed the capacity of cellular antioxidant defenses (9). Indeed, both ROS levels and oxidative damage levels are found to be higher in late-passage cells relative to early-passage cells (10). Additionally, increased oxidative stress in the form of hyperoxia (11), treatment with hydrogen peroxide (12), shRNA-mediated knockdown of SOD1 (13), or oncogenic Ras overexpression (14, 15), causes cells to enter senescence prematurely. Conversely, culturing...

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mentioning

confidence: 99%

Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence

Rai

Önder

Young

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

154

163

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“…MDSCs can be maintained in culture through 200 population doublings with no evidence of neoplastic transformation, chromosome abnormalities, or decrease in regenerative capacity (Deasy et al, 2005). There is no evidence that MDSCs obtain increased antioxidant capacity with increasing passage number; indeed, just the opposite has been reported in multiple groups that have demonstrated that low passage cell populations have an increased resistance to stress, antioxidant capacity, and ability to withstand adverse environmental conditions compared with higher passaged cell populations (Luce and Cristofalo, 1992;Yuan et al, 1996;Kaneko et al, 2001;Gurjala et al, 2005;Dowling et al, 2006).Inflammation clearly is one of the most important hurdles to overcome in order to increase the efficacy of cellular therapies. A major source of the destructive power of inflammation is the direct and indirect generation of ROS and free radicals after the inflammatory cytokine response (Dhalla et al, 1999).…”

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confidence: 99%

Antioxidant Levels Represent a Major Determinant in the Regenerative Capacity of Muscle Stem Cells

Urish

Vella

Okada³

et al. 2009

MBoC

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Stem cells are classically defined by their multipotent, long-term proliferation, and self-renewal capabilities. Here, we show that increased antioxidant capacity represents an additional functional characteristic of muscle-derived stem cells (MDSCs). Seeking to understand the superior regenerative capacity of MDSCs compared with myoblasts in cardiac and skeletal muscle transplantation, our group hypothesized that survival of the oxidative and inflammatory stress inherent to transplantation may play an important role. Evidence of increased enzymatic and nonenzymatic antioxidant capacity of MDSCs were observed in terms of higher levels of superoxide dismutase and glutathione, which appears to confer a differentiation and survival advantage. Further when glutathione levels of the MDSCs are lowered to that of myoblasts, the transplantation advantage of MDSCs over myoblasts is lost when transplanted into both skeletal and cardiac muscles. These findings elucidate an important cause for the superior regenerative capacity of MDSCs, and provide functional evidence for the emerging role of antioxidant capacity as a critical property for MDSC survival post-transplantation. INTRODUCTIONMyogenic cell transplantation has been proposed as a promising therapeutic approach in the treatment of skeletal and cardiac muscle injury. Early studies of myoblast transplantation into dystrophic skeletal muscle yielded limited regeneration of dystrophin-expressing muscle fibers (Beauchamp et al., 1994;Gussoni et al., 1997;Qu-Petersen et al., 2002). Similarly, given the limited regenerative ability of cardiac muscle, cell transplantation has been proposed as an alternative to heart transplantation (Assmus et al., 2006;Lunde et al., 2006;Schachinger et al., 2006).A major obstacle in both cardiac and skeletal myogenic therapies is the poor rate of engraftment of myogenic cells after transplantation (Taylor et al., 1998;Oshima et al., 2005). In skeletal muscle, numerous groups have observed a rapid inflammatory response that appears to contribute to rapid cell loss and limit therapeutic success (Beauchamp et al., 1994;Gussoni et al., 1997;Beauchamp et al., 1999). Multiple groups have postulated that the small number of injected cells that survive transplantation in both cardiac and skeletal muscle may represent a special subpopulation of stem-like cells (Qu et al., 1998;Beauchamp et al., 1999;Qu-Petersen et al., 2002).For these reasons, our group attempted to isolate this putative subpopulation of cells using a modified preplate technique. This procedure was initially developed to purify myoblasts from nonmyogenic cells, including fibroblasts, of whole tissue preparations based on the differential adhesion characteristics of the cells to a collagen coated flask (Rando and Blau, 1994). Our group modified this technique to isolate various populations of myogenic cells, including a population of early adhering preplate (EP) cells and a late adhering preplate (LP) cell population from which a subpopulation of long-term proliferating (LTP) cells,...

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“…3), and esculin also suppressed the increase in 8-oxodG content, but the change was not significant. Since it has been observed that esculin in culture medium is partly converted to esculetin during the 24 h-pretreatment, 16) esculetin formed through the hydrolysis of esculin during treatment may be producing the protective effect.…”

Section: Discussionmentioning

confidence: 99%

“…16) In brief, cells were trypsinized and centrifuged at 1000 rpm for 5 min. Cell pellets were washed with phosphate-buffered saline (PBS; pH 7.2), resuspended in an aqueous solution of proteinase K (25 mg/ml) and 1% sodium dodecyl sulfate (SDS)/1 mM EDTA (pH 8.0), and incubated at 37°C for 1 h under an argon atmosphere.…”

Section: Treatment Of Tig-7 Cells With Esculetin or Esculinmentioning

confidence: 99%

Suppression of Lipid Hydroperoxide-Induced Oxidative Damage to Cellular DNA by Esculetin

Kaneko

Tahara

Takabayashi

2003

Biological & Pharmaceutical Bulletin

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Accumulation of oxidative DNA damage, 8-oxo-2′-deoxyguanosine, and change of repair systems during in vitro cellular aging of cultured human skin fibroblasts

Cited by 52 publications

References 49 publications

Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence

Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence

Antioxidant Levels Represent a Major Determinant in the Regenerative Capacity of Muscle Stem Cells

Suppression of Lipid Hydroperoxide-Induced Oxidative Damage to Cellular DNA by Esculetin

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