Five dysfunctional telomeres predict onset of senescence in human cells

Replicative senescence is accompanied by a telomere-specific DNA damage response (DDR). We found that DDR+ telomeres occur spontaneously in early-passage normal human cells and increase in number with increasing cumulative cell divisions. DDR+ telomeres at replicative senescence retain TRF2 and RAP1 proteins, are not associated with end-to-end fusions and mostly result from strand-independent, postreplicative dysfunction. On the basis of the calculated number of DDR+ telomeres in G1-phase cells just before senescence and after bypassing senescence by inactivation of wild-type p53 function, we conclude that the accrual of five telomeres in G1 that are DDR+ but nonfusogenic is associated with p53-dependent senescence.     

化感胁迫诱导植物细胞损伤研究进展

化感胁迫(allelochemical stress)是指一种植物通过淋溶、挥发、根系分泌和残株腐解等途径释放化学物质,对另一种植物(包括微生物)产生直接或间接的伤害作用。有害化感物质对受体植物具有显著的细胞毒性,影响根边缘细胞的形成过程和活性,改变细胞壁和细胞膜的特性,破坏细胞内部结构,干扰细胞有丝分裂过程和基因表达模式;此外,化感胁迫往往伴随着氧化胁迫,受体植物细胞活性氧(ROS)水平升高,膜脂过氧化程度加剧,抗氧化系统被破坏,ROS影响与凋亡相关的信号调控过程,引起细胞大量死亡。因此,化感胁迫诱导的氧化胁迫可能是引起细胞凋亡的原因之一。阐明化感胁迫介导的氧化损伤和细胞损伤的相互关系以及根边缘细胞对化感胁迫的响应机制,是今后研究化感作用机制的一个方向。     

Prolonged swimming promotes cellular oxidative stress and p66Shc phosphorylation,but does not induce oxidative stress in mitochondria in the rat heart

Abstract

Exercise-induced changes in p66Shc-dependent signaling pathway are still not fully understood. The p66Shc protein is one of the key players in cell signaling, particularly in response to oxidative stress. Therefore, the aim of this study was to investigate the effect of prolonged swimming on the phosphorylation of p66Shc as well as the induction of mitochondrial and cellular oxidative stress in rat hearts.

Male Wistar rats were divided into a sedentary control group and an exercise group. The exercised rats swam for 3 hours and were burdened with an additional 3% of their body weight. After the cessation of exercise, their hearts were removed immediately for experiments.

The exercise protocol caused increased levels of the following oxidative stress parameters in cardiac cells: DNA damage, protein carbonyls, and lipid dienes. There was also increased phosphorylation of p66Shc without any alterations in Akt and extracellular signal-regulated kinases. Changes in the ferritin L levels and the L to H subunit ratio were also observed in the exercised hearts compared with the control hearts. Despite increased phosphorylation of p66Shc, no significant increase was observed in either mitochondrial H₂O₂ release or mitochondrial oxidative stress markers. Regardless of the changes in phosphorylation of p66Shc, the antioxidant enzyme activities (superoxide dismutase and catalase) and anti-apoptotic (Bcl2), and pro-apoptotic (Bax) protein levels were not affected by prolonged swimming. Further studies are required to investigate whether p66Shc phosphorylation is beneficial or detrimental to cardiac cells after exercise cessation.     

Crosstalk between Phosphoinositide 3-kinase/Akt signaling pathway with DNA damage response and oxidative stress in cancer

The phosphatidylinositol 3-kinases (PI3K)/Akt signaling pathway is one of the well-characterized and most important signaling pathways activated in response to DNA damage. This review discusses the most recent discoveries on the involvement of PI3K/Akt signaling pathway in cancer development, as well as stimulation of some important signaling networks involved in the maintenance of cellular homeostasis upon DNA damage, with an exploration of how PI3K/Akt signaling pathway contributes to the regulation of modulators and effectors underlying DNA damage response, the intricate, protein-based signal transduction network, which decides between cell cycle arrest, DNA repair, and apoptosis, the elimination of irreparably damaged cells to maintain homeostasis. The review continues by looking at the interplay between cell cycle checkpoints, checking the repair of damage inflicted to the DNA before entering DNA replication to facilitate DNA synthesis, and PI3K/Akt signaling pathway. We then investigate the challenges the cells overcome to ameliorate damages induced by oxidative activities, for example, the recruitment of many pathways and factors to maintain integrity and hemostasis. Finally, the review provides a discussion of how cells use the PI3K/Akt signaling pathway to regulate the balance between these networks.     

Inorganic arsenic compounds cause oxidative damage to DNA and protein by inducing ROS and RNS generation in human keratinocytes

Arsenic is a naturally occurring element that is present in food, soil, and water. Inorganic arsenic can accumulate in human skin and is associated with increased risk of skin cancer. Oxidative stress due to arsenic exposure is proposed as one potential mode of carcinogenic action. The purpose of this study is to investigate the specific reactive oxygen and nitrogen species that are responsible for the arsenic-induced oxidative damage to DNA and protein. Our results demonstrated that exposure of human keratinocytes to trivalent arsenite caused the generation of 8-hydroxyl-2′-deoxyguanine (8-OHdG) and 3-nitrotyrosine (3-NT) in a concentration- and time-dependent manner. Pentavalent arsenate had similar effects, but to a significantly less extent. The observed oxidative damage can be suppressed by pre-treating cells with specific antioxidants. Furthermore, we found that pre-treating cells with Nω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide synthase (NOS), or with 5,10,15,20-tetrakis (N-methyl-4′-pyridyl) porphinato iron (III) chloride (FeTMPyP), a decomposition catalyst of peroxynitrite, suppressed the generation of both 8-OHdG and 3-NT, which indicated that peroxynitrite, a product of the reaction of nitric oxide and superoxide, played an important role in arsenic-induced oxidative damage to both DNA and protein. These findings highlight the involvement of peroxynitrite in the molecular mechanism underlying arsenic-induced human skin carcinogenesis.     

Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage

Our study first proposed that curcumin could protect human endothelial cells from the damage caused by oxidative stress via autophagy. Furthermore, our results revealed that curcumin causes some novel cellular mechanisms that promote autophagy as a protective effect. Pretreatment with curcumin remarkably improves the survival of human umbilical vein endothelial cells (HUVECs) from H₂O₂-induced viability loss, which specifically evokes an autophagic response. Exposed to H₂O₂, curcumin-treated HUVECs upregulate the level of microtubule-associated protein 1 light chain 3-II (LC3-II), the number of autophagosomes, and the degradation of p62. We show that this compound promotes BECN1 expression and inhibits the phosphatidylinositol 3-kinase (PtdIns3K)-AKT-mechanistic target of rapamycin (MTOR) signaling pathway. Curcumin can also reverse FOXO1 (a mediator of autophagy) nuclear localization along with causing an elevated level of cytoplasmic acetylation of FOXO1 and the interaction of acetylated FOXO1 and ATG7, under the circumstance of oxidative stress. Additionally, knockdown of FOXO1 by shRNA inhibits not only the protective effects that curcumin induced, but the autophagic process, from the quantity of LC3-II to the expression of RAB7. These results suggest that curcumin induces autophagy, indicating that curcumin has the potential for use as an autophagic-related antioxidant for prevention and treatment of oxidative stress. These data uncover a brand new protective mechanism involving FOXO1 as having a critical role in regulating autophagy in HUVECs, and suggest a novel role for curcumin in inducing a beneficial form of autophagy in HUVECs, which may be a potential multitargeted therapeutic avenue for the treatment of oxidative stress-related cardiovascular diseases.     

An investigation of the effects of MitoQ on human peripheral mononuclear cells

Abstract

MitoQ is a ubiquinone derivative targeted to mitochondria which is known to have both antioxidant and anti-apoptotic properties within mammalian cells. Previous research has suggested that the age-related increase in oxidative DNA damage in T lymphocytes might contribute to their functional decline with age. This paper describes the impact of mitoQ on unchallenged or oxidatively challenged ex vivo human peripheral blood mononuclear cells from healthy 25–30 or 55–60 year old volunteers. When cells were challenged with hydrogen peroxide (H₂O₂), following mitoQ treatment (0.1–1.0 μM), the ratio of reduced to oxidized forms of glutathione increased, the levels of oxidative DNA damage decreased and there was an increase in the mitochondrial membrane potential. Low levels of mitoQ (0.1 or 0.25 μM) had no impact on endogenous DNA damage, whilst higher levels (0.5 and 1.0 μM) of mitoQ significantly reduced endogenous levels of DNA damage. The results of this investigation suggest that mitoQ may have anti-immunosenescent potential.     

High doses of sodium tungstate can promote mitochondrial dysfunction and oxidative stress in isolated mitochondria

Tungstate (W) is recognized as an agent of environmental pollution and a substitute to depleted uranium. According to some preliminary studies, tungstate toxicity is related to the formation of reactive oxygen species (ROS) under abnormal pathological conditions. The kidneys and liver are the main tungstate accumulation sites and important targets of tungstate toxicity. Since the mitochondrion is the main ROS production site, we evaluated the mechanistic toxicity of tungstate in isolated mitochondria for the first time, following a two‐step ultracentrifugation method. Our findings demonstrated that tungstate‐induced mitochondrial dysfunction is related to the increased formation of ROS, lipid peroxidation, and potential membrane collapse, correlated with the amelioration of adenosine triphosphate and glutathione contents. The present study indicated that mitochondrial dysfunction was associated with disruptive effects on the mitochondrial respiratory chain and opening of mitochondrial permeability transition (MPT) pores, which is correlated with cytochrome c release. Our findings suggest that high concentrations of tungstate (2 mM)‐favored MPT pore opening in the inner membranes of liver and kidney mitochondria of rats. Besides, the results indicated higher tungstate susceptibility in the kidneys, compared with the liver.     

Simulated microgravity decreases DNA repair capacity and induces DNA damage in human lymphocytes

The effect of simulated microgravity on DNA damage and apoptosis is still controversial. The objective of this study was to test whether simulated microgravity conditions affect the expression of genes for DNA repair and apoptosis. To achieve this objective, human lymphocyte cells were grown in a NASA‐developed rotating wall vessel (RWV) bioreactor that simulates microgravity. The same cell line was grown in parallel under normal gravitational conditions in culture flasks. The effect of microgravity on the expression of genes was measured by quantitative real‐time PCR while DNA damage was examined by comet assay. The result of this study revealed that exposure to simulated microgravity condition decreases the expression of DNA repair genes. Mismatch repair (MMR) class of DNA repair pathway were more susceptible to microgravity condition‐induced gene expression changes than base excision repair (BER) and nucleotide excision repair (NER) class of DNA repair genes. Downregulation of genes involved in cell proliferation (CyclinD1 and PCNA) and apoptosis (Bax) was also observed. Microgravity‐induced changes in the expression of some of these genes were further verified at the protein level by Western blot analysis. The findings of this study suggest that microgravity may induce alterations in the expression of these DNA repair genes resulting in accumulation of DNA damage. Reduced expression of cell‐cycle genes suggests that microgravity may cause a reduction in cell growth. Downregulation of pro‐apoptotic genes further suggests that extended exposure to microgravity may result in a reduction in the cells' ability to undergo apoptosis. Any resistance to apoptosis seen in cells with damaged DNA may eventually lead to malignant transformation of those cells. J. Cell. Biochem. 107: 723–731, 2009. © 2009 Wiley‐Liss, Inc.     

Voluntary locomotor activity mitigates oxidative damage associated with isolation stress in the prairie vole (Microtus ochrogaster)

Organismal performance directly depends on an individual''s ability to cope with a wide array of physiological challenges. For social animals, social isolation is a stressor that has been shown to increase oxidative stress. Another physiological challenge, routine locomotor activity, has been found to decrease oxidative stress levels. Because we currently do not have a good understanding of how diverse physiological systems like stress and locomotion interact to affect oxidative balance, we studied this interaction in the prairie vole (Microtus ochrogaster). Voles were either pair housed or isolated and within the isolation group, voles either had access to a moving wheel or a stationary wheel. We found that chronic periodic isolation caused increased levels of oxidative stress. However, within the vole group that was able to run voluntarily, longer durations of locomotor activity were associated with less oxidative stress. Our work suggests that individuals who demonstrate increased locomotor activity may be better able to cope with the social stressor of isolation.     

Tempol protection of spinal cord mitochondria from peroxynitrite-induced oxidative damage

Peroxynitrite (PN)-mediated mitochondrial dysfunction has been implicated in the secondary injury process after traumatic spinal cord injury (SCI). This study investigated the detrimental effects of the PN donor SIN-1 (3-morpholinosydnonimine) on isolated healthy spinal cord mitochondria and the protective effects of tempol, a catalytic scavenger of PN-derived radicals. A 5 min exposure of the mitochondria to SIN-1 caused a dose-dependent decrease in the respiratory control ratio (RCR) that was accompanied by significant increases in complex I-driven states II and IV respiration rates and decreases in states III and V. These impairments occurred together with an increase in mitochondrial protein 3-nitrotyrosine (3-NT), but not in lipid peroxidation (LP)-related 4-hydroxynonenal (4-HNE). Tempol significantly antagonized the respiratory effects of SIN-1 in parallel with an attenuation of 3-NT levels. These results show that the exogenous PN donor, SIN-1, rapidly causes mitochondrial oxidative damage and complex I dysfunction identical to traumatic spinal cord mitochondrial impairment and that this is mainly due to tyrosine nitration. Consistent with that, the protection of mitochondrial respiratory function by tempol is associated with a decrease in 3-NT levels in mitochondrial proteins also similar to the previously reported antioxidant actions of tempol in traumatically-injured spinal cord mitochondria.     

The crosstalk between trace elements with DNA damage response,repair, and oxidative stress in cancer

DNA damage response (DDR) is a regulatory system responsible for maintaining genome integrity and stability, which can sense and transduce DNA damage signals. The severity of damage appears to determine DDRs, which can include damage repair, cell-cycle arrest, and apoptosis. Furthermore, defective components in DNA damage and repair machinery are an underlying cause for the development and progression of various types of cancers. Increasing evidence indicates that there is an association between trace elements and DDR/repair mechanisms. In fact, trace elements seem to affect mediators of DDR. Besides, it has been revealed that oxidative stress (OS) and trace elements are associated with cancer development. In this review, we discuss the role of some critical trace elements in the risk of cancer. In addition, we provide a brief introduction on DDR and OS in cancer. Finally, we will further review the interactions between some important trace elements including selenium, zinc, chromium, cadmium, and arsenic, and DDR, and OS in cancer.     

Mammalian MutY homolog (MYH or MUTYH) protects cells from oxidative DNA damage

MutY DNA glycosylase homologs (MYH or MUTYH) reduce G:C to T:A mutations by removing misincorporated adenines or 2-hydroxyadenines paired with guanine or 8-oxo-7,8-dihydroguanine (8-oxo-G). Mutations in the human MYH (hMYH) gene are associated with the colorectal cancer predisposition syndrome MYH-associated polyposis. To examine the function of MYH in human cells, we regulated MYH gene expression by knockdown or overproduction. MYH knockdown human HeLa cells are more sensitive to the killing effects of H₂O₂ than the control cells. In addition, hMYH knockdown cells have altered cell morphology, display enhanced susceptibility to apoptosis, and have altered DNA signaling activation in response to oxidative stress. The cell cycle progression of hMYH knockdown cells is also different from that of the control cells following oxidative stress. Moreover, hMYH knockdown cells contain higher levels of 8-oxo-G lesions than the control cells following H₂O₂ treatment. Although MYH does not directly remove 8-oxo-G, MYH may generate favorable substrates for other repair enzymes. Overexpression of mouse Myh (mMyh) in human mismatch repair defective HCT15 cells makes the cells more resistant to killing and refractory to apoptosis by oxidative stress than the cells transfected with vector. In conclusion, MYH is a vital DNA repair enzyme that protects cells from oxidative DNA damage and is critical for a proper cellular response to DNA damage.     

Upregulation of miR-24 is associated with a decreased DNA damage response upon etoposide treatment in highly differentiated CD8(+) T cells sensitizing them to apoptotic cell death

The life-long homeostasis of memory CD8(+) T cells as well as persistent viral infections have been shown to facilitate the accumulation of highly differentiated CD8(+) CD28(-) T cells, a phenomenon that has been associated with an impaired immune function in humans. However, the molecular mechanisms regulating homeostasis of CD8(+) CD28(-) T cells have not yet been elucidated. In this study, we demonstrate that the miR-23～24～27 cluster is up-regulated during post-thymic CD8(+) T-cell differentiation in humans. The increased expression of miR-24 in CD8(+) CD28(-) T cells is associated with decreased expression of the histone variant H2AX, a protein that plays a key role in the DNA damage response (DDR). Following treatment with the classic chemotherapeutic agent etoposide, a topoisomerase II inhibitor, apoptosis was increased in CD8(+) CD28(-) when compared to CD8(+) CD28(+) T cells and correlated with an impaired DDR in this cell type. The reduced capacity of CD8(+) CD28(-) T cell to repair DNA was characterized by the automated fluorimetric analysis of DNA unwinding (FADU) assay as well as by decreased phosphorylation of H2AX at Ser139, of ATM at Ser1981, and of p53 at Ser15. Interleukin (IL)-15 could prevent etoposide-mediated apoptosis of CD8(+) CD28(-) T cells, suggesting a role for IL-15 in the survival and the age-dependent accumulation of CD8(+) CD28(-) T cells in humans.     

Arsenic induces oxidative DNA damage in mammalian cells

Although arsenic is a well-established human carcinogen, the underlying carcinogenic mechanism(s) is not known. Using the human-hamster hybrid (A_L) cell mutagenic assay that is sensitive in detecting mutagens that induce predominately multilocus deletions, we showed previously that arsenite is indeed a potent gene and chromosomal mutagen and that oxyradicals may be involved in the mutagenic process. In the present study, the effects of free radical scavenging enzymes on the cytotoxic and mutagenic potential of arsenic were examined using the A_L cells. Concurrent treatment of cells with either superoxide dismutase or catalase reduced both the cytotoxicity and mutagenicity of arsenite by an average of 2–3 fold, respectively. Using immunoperoxidase staining with a monoclonal antibody specific for 8-hydroxy-2-deoxyguanosine (8-OHdG), we demonstrated that arsenic induced oxidative DNA damage in A_L cells. This induction was significantly reduced in the presence of the antioxidant enzymes. Furthermore, reducing the intracellular levels of non-protein sulfhydryls (mainly glutathione) using buthionine S-R-Sulfoximine increased the total mutant yield by more than 3-fold as well as the proportion of mutants with multilocus deletions. Taken together, our data provide clear evidence that reactive oxygen species play an important causal role in the genotoxicity of arsenic in mammalian cells.     

SMG1 heterozygosity exacerbates haematopoietic cancer development in Atm null mice by increasing persistent DNA damage and oxidative stress

Suppressor of morphogenesis in genitalia 1 (SMG1) and ataxia telangiectasia mutated (ATM) are members of the PI3‐kinase like–kinase (PIKK) family of proteins. ATM is a well‐established tumour suppressor. Loss of one or both alleles of ATM results in an increased risk of cancer development, particularly haematopoietic cancer and breast cancer in both humans and mouse models. In mice, total loss of SMG1 is embryonic lethal and loss of a single allele results in an increased rate of cancer development, particularly haematopoietic cancers and lung cancer. In this study, we generated mice deficient in Atm and lacking one allele of Smg1, Atm^?/?Smg1^gt/+ mice. These mice developed cancers more rapidly than either of the parental genotypes, and all cancers were haematopoietic in origin. The combined loss of Smg1 and Atm resulted in a higher level of basal DNA damage and oxidative stress in tissues than loss of either gene alone. Furthermore, Atm^?/?Smg1^gt/+ mice displayed increased cytokine levels in haematopoietic tissues compared with wild‐type animals indicating the development of low‐level inflammation and a pro‐tumour microenvironment. Overall, our data demonstrated that combined loss of Atm expression and decreased Smg1 expression increases haematopoietic cancer development.     

Mitochondrial oxidative stress elicits chromosomal instability after exposure to isocyanates in human kidney epithelial cells

The role of oxidative stress is often attributed in environmental renal diseases. Isocyanates, a ubiquitous chemical group with diverse industrial applications, are known to undergo bio-transformation reactions upon accidental and occupational exposure. This study delineates the role of isocyanate-mediated mitochondrial oxidative stress in eliciting chromosomal instability in cultured human kidney epithelial cells. Cells treated with 0.005 µM concentration of methyl isocyanate displayed morphological transformation and stress-induced senescence. Along the time course, an increase in DCF fluorescence indicative of oxidative stress, depletion of superoxide dismutase (SOD) and glutathione reductase (GR) and consistent accumulation of 8-oxo-dG were noticed. Thus, endogenous oxidative stress resulted in aberrant expression of p53, p21, cyclin E and CDK2 proteins, suggestive of deregulated cell cycle, chromosomal aberrations, centromeric amplification, aneuploidy and genomic instability.     

Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres

Cells respond to DNA double-strand breaks (DSBs) and uncapped telomeres by recruiting checkpoint and repair factors to the site of lesions. Single-stranded DNA (ssDNA) is an important intermediate in the repair of DSBs and is produced also at uncapped telomeres. Here, we provide evidence that binding of the checkpoint protein Rad9, through its Tudor domain, to methylated histone H3-K79 inhibits resection at DSBs and uncapped telomeres. Loss of DOT1 or mutations in RAD9 influence a Rad50-dependent nuclease, leading to more rapid accumulation of ssDNA, and faster activation of the critical checkpoint kinase, Mec1. Moreover, deletion of RAD9 or DOT1 partially bypasses the requirement for CDK1 in DSB resection. Interestingly, Dot1 contributes to checkpoint activation in response to low levels of telomere uncapping but is not essential with high levels of uncapping. We suggest that both Rad9 and histone H3 methylation allow transmission of the damage signal to checkpoint kinases, and keep resection of damaged DNA under control influencing, both positively and negatively, checkpoint cascades and contributing to a tightly controlled response to DNA damage.