Stochastic variation in telomere shortening rate causes heterogeneity of human fibroblast replicative life span

The replicative life span of human fibroblasts is heterogeneous, with a fraction of cells senescing at every population doubling. To find out whether this heterogeneity is due to premature senescence, i.e. driven by a nontelomeric mechanism, fibroblasts with a senescent phenotype were isolated from growing cultures and clones by flow cytometry. These senescent cells had shorter telomeres than their cycling counterparts at all population doubling levels and both in mass cultures and in individual subclones, indicating heterogeneity in the rate of telomere shortening. Ectopic expression of telomerase stabilized telomere length in the majority of cells and rescued them from early senescence, suggesting a causal role of telomere shortening. Under standard cell culture conditions, there was a minor fraction of cells that showed a senescent phenotype and short telomeres despite active telomerase. This fraction increased under chronic mild oxidative stress, which is known to accelerate telomere shortening. It is possible that even high telomerase activity cannot fully compensate for telomere shortening in all cells. The data show that heterogeneity of the human fibroblast replicative life span can be caused by significant stochastic cell-to-cell variation in telomere shortening.     

Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress

The effect of supplementation with substances having antioxidant properties on the adaptive responses of human skin fibroblasts to UV-induced oxidative stress was studied in vitro. UVR was found to induce a substantial oxidative stress in fibroblasts, resulting in an increased release of superoxide anions and an increase in lipid peroxidation (shown by an elevated malonaldehyde content). Sub-lethal doses of UVR were also found to induce adaptive responses in the fibroblast antioxidant defences, with a transient rise in catalase and superoxide dismutase activities followed by a slower, large increase in cellular glutathione content. Supplementation of the fibroblasts with the antioxidants, Trolox (a water soluble analogue of alpha-tocopherol), ascorbic acid or beta-carotene, had differential effects on these responses. Trolox supplementation reduced the UVR-induced cellular oxidative stress and adaptive response in a predictable concentration-dependent manner. This was in contrast to ascorbic acid which increased superoxide release from fibroblasts. At low doses, ascorbate supplements also reduced the magnitude of the adaptive increases in catalase and superoxide dismutase activities and increase in glutathione content. Beta-carotene had a similar effect to ascorbic acid, reducing the extent of the adaptations to UVR at lower doses while simultaneously increasing superoxide release and malonaldehyde content. These in vitro data indicate that only the vitamin E analogue suppressed UVR-induced oxidative stress in a predictable manner and suggest that common dietary antioxidants may not be equally effective in reducing the potential deleterious effects of UVR-induced oxidative stress in skin.     

Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress

Abstract

The effect of supplementation with substances having antioxidant properties on the adaptive responses of human skin fibroblasts to UV-induced oxidative stress was studied in vitro. UVR was found to induce a substantial oxidative stress in fibroblasts, resulting in an increased release of superoxide anions and an increase in lipid peroxidation (shown by an elevated malonaldehyde content). Sub-lethal doses of UVR were also found to induce adaptive responses in the fibroblast antioxidant defences, with a transient rise in catalase and superoxide dismutase activities followed by a slower, large increase in cellular glutathione content. Supplementation of the fibroblasts with the antioxidants, Trolox (a water soluble analogue of α-tocopherol), ascorbic acid or β-carotene, had differential effects on these responses. Trolox supplementation reduced the UVR-induced cellular oxidative stress and adaptive response in a predictable concentration-dependant manner. This was in contrast to ascorbic acid which increased superoxide release from fibroblasts. At low doses, ascorbate supplements also reduced the magnitude of the adaptive increases in catalase and superoxide dismutase activities and increase in glutathione content. β-Carotene had a similar effect to ascorbic acid, reducing the extent of the adaptations to UVR at lower doses while simultaneously increasing superoxide release and malonaldehyde content. These in vitro data indicate that only the vitamin E analogue suppressed UVR-induced oxidative stress in a predictable manner and suggest that common dietary antioxidants may not be equally effective in reducing the potential deleterious effects of UVR-induced oxidative stress in skin.     

Protein restriction in lactation confers nephroprotective effects in the male rat and is associated with increased antioxidant expression

Telomere shortening has been implicated in the aging process and various age-associated disorders, including renal disease. Moreover, oxidative stress has been identified as an initiator of accelerated telomere shortening. We have shown previously that maternal protein restriction during lactation leads to reduced renal telomere shortening, reduced albuminuria, and increased longevity in rats. Here we address the hypothesis that maternal protein restriction during lactation is nephroprotective and associated with increased expression of antioxidative enzymes and decreased age-dependent renal telomere shortening. Newborn rats were suckled by a dam fed either a control (20% protein) or low-protein (8% protein) diet. All animals were weaned onto standard chow. Offspring that had been suckled by protein-restricted mothers had reduced albuminuria, N-acetyl-glucosaminidase, and urinary aldosterone excretion. These animals also did not show significant age-dependent renal telomere shortening and hence had significantly longer telomeres at 12 mo of age. This lack of renal telomere shortening was associated with increased levels of the antioxidant enzymes manganese superoxide dismutase, glutathione peroxidase, and glutathione reductase. These findings suggest that beneficial effects of slow growth during lactation are associated with increased antioxidant capacity and prevention of age-dependent telomere shortening in the kidney.     

Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts

Telomere shortening triggers replicative senescence in human fibroblasts. The inability of DNA polymerases to replicate a linear DNA molecule completely (the end replication problem) is one cause of telomere shortening. Other possible causes are the formation of single-stranded overhangs at the end of telomeres and the preferential vulnerability of telomeres to oxidative stress. To elucidate the relative importance of these possibilities, amount and distribution of telomeric single-strand breaks, length of the G-rich overhang, and telomere shortening rate in human MRC-5 fibroblasts were measured. Treatment of nonproliferating cells with hydrogen peroxide increases the sensitivity to S1 nuclease in telomeres preferentially and accelerates their shortening by a corresponding amount as soon as the cells proliferate. A reduction of the activity of intracellular peroxides using the spin trap alpha-phenyl-t-butyl-nitrone reduces the telomere shortening rate and increases the replicative life span. The length of the telomeric single-stranded overhang is independent of DNA damaging stresses, but single-strand breaks accumulate randomly all along the telomere after alkylation. The telomere shortening rate and the rate of replicative aging can be either accelerated or decelerated by a modification of the amount of oxidative stress. Quantitatively, stress-mediated telomere damage contributes most to telomere shortening under standard conditions.     

Telomere shortening in human fibroblasts is not dependent on the size of the telomeric-3'-overhang

Telomeres shorten in human somatic cells with each round of DNA replication, and this shortening is thought to ultimately trigger replicative senescence. Telomere shortening is caused partly by the inability of semiconservative DNA replication to copy a linear strand of DNA to its very end. Post-replicative processing of telomeric ends, producing single-stranded G-rich 3' overhangs, has also been suggested to contribute to telomere shortening. This suggestion implies that a positive correlation should exist between the length of 3' overhangs and the rate of telomere shortening. We confirmed shortening of overhangs as human lung (MRC5) and foreskin (BJ) fibroblasts approach senescence by measuring overhang length using in-gel hybridization. However, a large study of fibroblast strains from 21 donors maintained under conditions which lead to two orders of magnitude of variation in telomere shortening rate failed to show any correlation between telomere overhang length and shortening rate, suggesting that overhang length is neither a cause nor a correlate of telomere shortening.     

BJ fibroblasts display high antioxidant capacity and slow telomere shortening independent of hTERT transfection

Human foreskin BJ fibroblasts are well protected against oxidative stress as shown by their low intracellular peroxide content, low levels of protein carbonyls, and low steady-state lipofuscin content as compared to other primary human fibroblasts. This correlates with a long replicative life span of the parental cells of about 90 population doublings and a telomere-shortening rate of only 15-20 bp/PD. This value might define the upper limit of a telomere-shortening rate that can still be explained by the end replication problem alone. In BJ clones immortalized by transfection with hTERT, the catalytic subunit of telomerase, the same telomere-shortening rate as in parental cells is observed over a long time despite strong telomerase activity. Hyperoxia, which induces oxidative stress and accelerates telomere shortening in a variety of human fibroblast strains, does not do so in BJ cells. It is possible that the high antioxidative capacity of BJ cells, by minimizing the accumulation of genomic damage, is instrumental in the successful immortalization of these cells by telomerase.     

Relief of oxidative stress by ascorbic acid delays cellular senescence of normal human and Werner syndrome fibroblast cells

Primary human cells have a definite life span and enter into cellular senescence before ceasing cell growth. Oxidative stress produced by aerobic metabolism has been shown to accelerate cellular senescence. Here, we demonstrated that ascorbic acid, used as an antioxygenic reagent, delayed cellular senescence in a continuous culture of normal human embryonic cells, human adult skin fibroblast cells, and Werner syndrome (WS) cells. The results using human embryonic cells showed that treatment with ascorbic acid phospholic ester magnesium salt (APM) decreased the level of oxidative stress, and extended the replicative life span. The effect of APM to extend the replicative life span was also shown in normal human adult cells and WS cells. To understand the mechanism of extension of cellular life span, we determined the telomere lengths of human embryonic cells, both with and without APM treatment, and demonstrated that APM treatment reduced the rate of telomere shortening. The present results indicate that constitutive oxidative stress plays a role in determining the replicative life span and that suppression of oxidative stress by an antioxidative agent, APM, extends the replicative life span by reducing the rate of telomere shortening.     

Glycosaminoglycans reduce oxidative damage induced by copper (Cu+2), iron (Fe+2) and hydrogen peroxide (H2O2) in human fibroblast cultures

Acid glycosaminoglycans (GAGs) antioxidant activity was assessed in a fibroblast culture system by evaluating reduction of oxidative system-induced damage. Three different methods to induce oxidative stress in human skin fibroblast cultures were used. In the first protocol cells were treated with CuSO4 plus ascorbate. In the second experiment fibroblasts were exposed to FeSO4 plus ascorbate. In the third system H2O2 was utilised. The exposition of fibroblasts to each one of the three oxidant systems caused inhibition of cell growth and cell death, increase of lipid peroxidation evaluated by the analysis of malondialdehyde (MDA), decrease of reduced glutathione (GSH) and superoxide dismutase (SOD) levels, and rise of lactate dehydrogenase activity (LDH). The treatment with commercial GAGs at different doses showed beneficial effects in all oxidative models. Hyaluronic acid (HA) and chondroitin-4-sulphate (C4S) exhibited the highest protection. However, the cells exposed to CuSO4 plus ascorbate and FeSO4 plus ascorbate were better protected by GAGs compared to those exposed to H2O2. These outcomes confirm the antioxidant properties of GAGs and further support the hypothesis that these molecules may function as metal chelators.     

Developmental differences in the immortalization of lung fibroblasts by telomerase

The role of ambient (21%) and physiological oxygen (2-5%) in the immortalization of fetal vs. adult human lung fibroblasts was examined. Growth in low oxygen and antioxidants extended the lifespan of both fetal and adult strains. As the ectopic expression of telomerase could immortalize adult lung fibroblasts cultured in ambient oxygen, the lifespan-shortening effects of 21% oxygen must have been largely limited to telomeres. By contrast, fetal lung fibroblasts could not be immortalized in ambient oxygen in spite of telomere elongation by telomerase, suggesting more widespread oxidative damage. The long-term culture requirements for the immortalization of WI-38 fetal lung fibroblasts included supplementation with N-(tert) butyl hydroxylamine, dexamethasone, zinc and vitamin B12, in addition to growth in physiological oxygen. The mechanisms regulating telomere shortening remain controversial. The present results suggest that both end-replication and oxidative damage events contribute to telomere shortening in lung fibroblasts in vitro. These observations emphasize the need for better analytical techniques to distinguish whether the correlation of short telomeres with disease and mortality in humans reflects the consequences of increased proliferation, telomere shortening as a result of oxidative damage or some combination of these processes.     

Cryptococcus neoformans mitochondrial superoxide dismutase: an essential link between antioxidant function and high-temperature growth

Manganese superoxide dismutase is an essential component of the mitochondrial antioxidant defense system of most eukaryotes. In the present study, we used a reverse-genetics approach to assess the contribution of the Cryptococcus neoformans manganese superoxide dismutase (Sod2) for antioxidant defense. Strains with mutations in the SOD2 gene exhibited increased susceptibility to oxidative stress as well as poor growth at elevated temperatures compared to isogenic wild-type strains. The sod2Delta mutants were also avirulent in a murine model of inhaled cryptococcosis. Reconstitution of a sod2Delta mutant restored Sod2 activity, eliminated the oxidative stress and temperature-sensitive (ts) phenotypes, and complemented the virulence phenotype. Characterization of the ts phenotype revealed a dependency between Sod2 antioxidant activity and the ability of C. neoformans cells to adapt to growth at elevated temperatures. The ts phenotype could be suppressed by the addition of either ascorbic acid (10 mM) or Mn salen (200 muM) at 30 degrees C, but not at 37 degrees C. Furthermore, sod2Delta mutant cells that were incubated for 24 h at 37 degrees C under anaerobic, but not aerobic, conditions were viable when shifted to the permissive conditions of 25 degrees C in the presence of air. These data suggest that the C. neoformans Sod2 is a major component of the antioxidant defense system in this human fungal pathogen and that adaptation to growth at elevated temperatures is also dependent on Sod2 activity.     

Lack of protection of prior heat shock against UV-induced oxidative stress in human skin fibroblasts

The effect of prior hyperthermia on UV-induced oxidative stress was studied in human skin fibroblasts. UV radiation alone induced an increased release of superoxide anions and increased lipid peroxidation in skin fibroblasts accompanied by a rise in catalase and superoxide dismutase activities. Hyperthermia was found to induce a significant rise in the cell content of heat-shock proteins, HSP60 and HSP70, but this treatment prior to UV radiation did not influence any indicators of oxidative stress in the fibroblasts. In contrast, the combination of heat shock prior to UV-exposure reduced fibroblast cell viability compared with UV radiation-exposure alone.     

Lack of protection of prior heat shock against UV-induced oxidative stress in human skin fibroblasts

Abstract

The effect of prior hyperthermia on UV-induced oxidative stress was studied in human skin fibroblasts. UV radiation alone induced an increased release of superoxide anions and increased lipid peroxidation in skin fibroblasts accompanied by a rise in catalase and superoxide dismutase activities. Hyperthermia was found to induce a significant rise in the cell content of heat-shock proteins, HSP60 and HSP70, but this treatment prior to UV radiation did not influence any indicators of oxidative stress in the fibroblasts. In contrast, the combination of heat shock prior to UV-exposure reduced fibroblast cell viability compared with UV radiation-exposure alone.     

Changes in antioxidant gene expression and induction of oxidative stress in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) under Al stress

Aluminium toxicity has been recognized as a primary growth-limiting factor in acid soil, resulting in a decrease in plant growth and production. In this experiment we have studied the induction of oxidative stress and changes in antioxidant gene expression in pea (var. ALASKA) under aluminium (Al) stress. We have found that Al treatment affected the growth of pea plant and induced oxidative stress with a change in antioxidant gene expression profile. While the expression of glutathione-s-transferase (GST) and catalase (CAT) was more in root, cytosolic Ascorbate peroxidase (cAPX) expression increased in shoot under aluminium stress. Copper- Zinc Superoxide dismutase (Cu-Zn SOD) gene expression was higher after 24 h but decreased after 48 h along with elevated expression of manganese superoxide dismutase (MnSOD) and iron-superoxide dismutase (FeSOD) at higher aluminium contentrations after 24 and 48 h. Aluminium stress elevated hydrogen peroxide (H₂O₂) level and affected the growth. The proline content did not change significantly, whereas glutathione content increased with a decreased ascorbate content under Al stress. The present study indicates that aluminium treatment affected the antioxidant gene expression and induced oxidative stress in pea plant.     

Coenzyme Q10 depletion is comparatively less detrimental to human cultured skin fibroblasts than respiratory chain complex deficiencies

The oxidative stress possibly resulting from an inherited respiratory chain (RC) deficiency was investigated in a series of human cultured skin fibroblasts presenting either ubiquinone depletion or isolated defect of the various RC complexes. Taken as an index for superoxide overproduction, a significant induction of superoxide dismutase activity was observed in complex V-deficient fibroblasts harboring the NARP-mutation in the ATPase 6 gene. Superoxide dismutase induction was also noticed, albeit to a lesser extent, in complex II-deficient fibroblasts with a mutation in the nuclear gene encoding the flavoprotein subunit of the succinate dehydrogenase. No sign of oxidative stress could be found in ubiquinone-depleted fibroblasts. In all cases but complex IV-defect, increased oxidative stress was associated with increased cell death. In glucose-rich medium, apoptosis appeared as the main cell death process associated with all types of RC defect. However, similar to the great variations in oxidative stress associated with the various types of RC defect, we found that apoptotic features differed noticeably between defects. No indication of increased cell death was found in ubiquinone-depleted fibroblasts.     

Role of p14(ARF) in replicative and induced senescence of human fibroblasts

Following a proliferative phase of variable duration, most normal somatic cells enter a growth arrest state known as replicative senescence. In addition to telomere shortening, a variety of environmental insults and signaling imbalances can elicit phenotypes closely resembling senescence. We used p53(-/-) and p21(-/-) human fibroblast cell strains constructed by gene targeting to investigate the involvement of the Arf-Mdm2-p53-p21 pathway in natural as well as premature senescence states. We propose that in cell types that upregulate p21 during replicative exhaustion, such as normal human fibroblasts, p53, p21, and Rb act sequentially and constitute the major pathway for establishing growth arrest and that the telomere-initiated signal enters this pathway at the level of p53. Our results also revealed a number of significant differences between human and rodent fibroblasts in the regulation of senescence pathways.     

Coenzyme Q 10 Depletion is Comparatively Less Detrimental to Human Cultured Skin Fibroblasts than Respiratory Chain Complex Deficiencies

The oxidative stress possibly resulting from an inherited respiratory chain (RC) deficiency was investigated in a series of human cultured skin fibroblasts presenting either ubiquinone depletion or isolated defect of the various RC complexes. Taken as an index for superoxide overproduction, a significant induction of superoxide dismutase activity was observed in complex V-deficient fibroblasts harboring the NARP-mutation in the ATPase 6 gene. Superoxide dismutase induction was also noticed, albeit to a lesser extent, in complex II-deficient fibroblasts with a mutation in the nuclear gene encoding the flavoprotein subunit of the succinate dehydrogenase. No sign of oxidative stress could be found in ubiquinone-depleted fibroblasts. In all cases but complex IV-defect, increased oxidative stress was associated with increased cell death. In glucose-rich medium, apoptosis appeared as the main cell death process associated with all types of RC defect. However, similar to the great variations in oxidative stress associated with the various types of RC defect, we found that apoptotic features differed noticeably between defects. No indication of increased cell death was found in ubiquinone-depleted fibroblasts.     

Oxidative Stress Induces Persistent Telomeric DNA Damage Responsible for Nuclear Morphology Change in Mammalian Cells

One main function of telomeres is to maintain chromosome and genome stability. The rate of telomere shortening can be accelerated significantly by chemical and physical environmental agents. Reactive oxygen species are a source of oxidative stress and can produce modified bases (mainly 8-oxoG) and single strand breaks anywhere in the genome. The high incidence of guanine residues in telomeric DNA sequences makes the telomere a preferred target for oxidative damage. Our aim in this work is to evaluate whether chromosome instability induced by oxidative stress is related specifically to telomeric damage. We treated human primary fibroblasts (MRC-5) in vitro with hydrogen peroxide (100 and 200 µM) for 1 hr and collected data at several time points. To evaluate the persistence of oxidative stress-induced DNA damage up to 24 hrs after treatment, we analysed telomeric and genomic oxidative damage by qPCR and a modified comet assay, respectively. The results demonstrate that the genomic damage is completely repaired, while the telomeric oxidative damage persists. The analysis of telomere length reveals a significant telomere shortening 48 hrs after treatment, leading us to hypothesise that residual telomere damage could be responsible for the telomere shortening observed. Considering the influence of telomere length modulation on genomic stability, we quantified abnormal nuclear morphologies (Nucleoplasmic Bridges, Nuclear Buds and Micronuclei) and observed an increase of chromosome instability in the same time frame as telomere shortening. At subsequent times (72 and 96 hrs), we observed a restoration of telomere length and a reduction of chromosome instability, leaving us to conjecture a correlation between telomere shortening/dysfunction and chromosome instability. We can conclude that oxidative base damage leads to abnormal nuclear morphologies and that telomere dysfunction is an important contributor to this effect.     

ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium

To further elucidate the impact of fermentative stress on Saccharomyces cerevisiae wine strains, we have here evaluated markers of oxidative stress, oxidative damage and antioxidant response in four oenological strains of S. cerevisiae, relating these to membrane integrity, ethanol production and cell viability during fermentation in high-sugar-containing medium. The cells were sampled at different fermentation stages and analysed by flow cytometry to evaluate membrane integrity and accumulation of reactive oxygen species (ROS). At the same time, catalase and superoxide dismutase activities, trehalose accumulation, and protein carbonylation and degradation were measured. The results indicate that the stress conditions occurring during hypoxic fermentation in high-sugar-containing medium result in the production of ROS and trigger an antioxidant response. This involves superoxide dismutase and trehalose for the protection of cell structures from oxidative damage, and protein catabolism for the removal of damaged proteins. Cell viability, membrane integrity and ethanol production depend on the extent of oxidative damage to cellular components. This is, in turn, related to the 'fitness' of each strain, which depends on the contribution of individual cells to ROS accumulation and scavenging. These findings highlight that the differences in individual cell resistances to ROS contribute to the persistence of wine strains during growth under unfavourable culture conditions, and they provide further insights into our understanding of yeast behaviour during industrial fermentation.