Fanconi anaemia proteins: major roles in cell protection against oxidative damage

Fanconi anaemia (FA) is a cancer-prone genetic disorder that is characterised by cytogenetic instability and redox abnormalities. Although rare subtypes of FA (B, D1 and D2) have been implicated in DNA repair through links with BRCA1 and BRCA2, such a role has yet to be demonstrated for gene products of the common subtypes. Instead, these products have been strongly implicated in xenobiotic metabolism and redox homeostasis through interactions of FANCC with cytochrome P-450 reductase and with glutathione S-transferase, and of FANCG with cytochrome P-450 2E1, as well as redox-dependent signalling through an interaction between FANCA and Akt kinase. We hypothesise that FA proteins act directly (via FANCC and FANCG) and indirectly (via FANCA, BRCA2 and FANCD2) with the machinery of cellular defence to modulate oxidative stress. The latter interactions may co-ordinate the link between the response to DNA damage and oxidative stress parameters (3, 6-12).     

Defective mitochondrial peroxiredoxin-3 results in sensitivity to oxidative stress in Fanconi anemia

Cells from patients with Fanconi anemia (FA), an inherited disorder that includes bone marrow failure and cancer predisposition, have increased sensitivity to oxidative stress through an unknown mechanism. We demonstrate that the FA group G (FANCG) protein is found in mitochondria. Wild-type but not G546R mutant FANCG physically interacts with the mitochondrial peroxidase peroxiredoxin-3 (PRDX3). PRDX3 is deregulated in FA cells, including cleavage by a calpainlike cysteine protease and mislocalization. FA-G cells demonstrate distorted mitochondrial structures, and mitochondrial extracts have a sevenfold decrease in thioredoxin-dependent peroxidase activity. Transient overexpression of PRDX3 suppresses the sensitivity of FA-G cells to H2O2, and decreased PRDX3 expression increases sensitivity to mitomycin C. Cells from the FA-A and -C subtypes also have PRDX3 cleavage and decreased peroxidase activity. This study demonstrates a role for the FA proteins in mitochondria witsh sensitivity to oxidative stress resulting from diminished peroxidase activity. These defects may lead to apoptosis and the accumulation of oxidative DNA damage in bone marrow precursors.     

From clinical description,to in vitro and animal studies,and backward to patients: Oxidative stress and mitochondrial dysfunction in Fanconi anemia

Fanconi anemia (FA) is a rare genetic disease associated with deficiencies in DNA repair pathways. A body of literature points to a pro-oxidant state in FA patients, along with evidence for oxidative stress (OS) in the FA phenotype reported by in vitro, molecular, and animal studies. A highlight arises from the detection of mitochondrial dysfunction (MDF) in FA cell lines of complementation groups A, C, D2, and G. As yet lacking, in vivo studies should focus on FA-associated MDF, which may help in the understanding of the mitochondrial basis of OS detected in cells and body fluids from FA patients. Beyond the in vitro and animal databases, the available analytical devices may prompt the direct observation of metabolic and mitochondrial alterations in FA patients. These studies should evaluate a set of MDF-related endpoints, to be related to OS endpoints. The working hypothesis is raised that, parallel to OS, nitrosative stress might be another, so far unexplored, hallmark of the FA phenotype. The expected results may shed light on the FA pathogenesis and might provide grounds for pilot chemoprevention trials using mitochondrial nutrients.     

Drought acclimation confers oxidative stress tolerance by inducing co-ordinated antioxidant defense at cellular and subcellular level in leaves of wheat seedlings

Wheat ( Triticum aestivum L.) seedlings of a drought-resistant cv. C306 were subjected to severe water deficit directly or through stress cycles of increasing intensity with intermittent recovery periods (drought acclimation). The antioxidant defense in terms of redox metabolites and enzymes in leaf cells, chloroplasts, and mitochondria was examined in relation to ROS-induced membrane damage. Drought-acclimated seedlings modulated growth by maintaining favorable turgor potential and RWC and were able to limit H₂O₂ accumulation and membrane damage as compared with non-acclimated plants during severe water stress conditions. This was due to systematic upregulation of H₂O₂-metabolizing enzymes especially ascorbate peroxidase (APX, EC 1.11.1.11) and by maintaining ascorbate–glutathione redox pool in acclimated plants. By contrast, failure in the induction of APX and ascorbate–glutathione cycle enzymes makes the chloroplast susceptible to oxidative stress in non-acclimated plants. Non-acclimated plants protected the leaf mitochondria from oxidative stress by upregulating superoxide dismutase (SOD, EC 1.15.1.1), APX, and glutathione reductase (GR, EC 1.6.4.2) activities. Rewatering led to rapid enhancement in all the antioxidant defense components in non-acclimated plants, which suggested that the excess levels of H₂O₂ during severe water stress conditions might have inhibited or downregulated the antioxidant enzymes. Hence, drought acclimation conferred enhanced oxidative stress tolerance by well-co-ordinated induction of antioxidant defense both at the chloroplast and at the mitochondrial level.     

The cellular control enzyme polyADP ribosyl transferase is eliminated in cultured Fanconi anemia fibroblasts at confluency

Fanconi anemia (FA) is a hereditary disease of unknown pathogenic mechanisms, although mutations in seven different genes can be causative. Six of these genes have been cloned and sequenced. Only slight homology to the DNA of any other known gene has been found with the exception of FANCG which is identical to XRCC9. The function of these genes, including XRCC9, is presently unknown. Since pADP ribosyl transferase (pADPRT) plays a role in apoptosis, and apoptosis is affected in FA cells, we studied the correlation between pADPRT and FA cells. We reinvestigated the previously reported lack of pADPRT activity in fibroblasts from patients with Fanconi anemia. Here we describe the role of the lower redox potential of FA cells and demonstrate that this is an efficient strategy in the prevention of cell death due to the lack of energy under oxidative stress. This strategy is advantageous for the cells under the nonreplicative condition of confluency in which the risk of mutation is low and the prevention of apoptosis permits cell survival. pADPRT is not diminished to the same extent in all complementation groups of FA. It is prominent in FANCA, FANCG and FANCF cells, indicating that these genes control pADPRT diminution. Our experiments suggest that the pADPRT level is linked with the oxidoreduction reactions seen in FA.     

New insights into redox response modulation in Fanconi's anemia cells by hydrogen peroxide and glutathione depletors

Fanconi's anemia (FA) patients face severe pathological consequences. Bone marrow failure, the major cause of death in FA, accounting for as much as 80-90% of FA mortality, appears to be significantly linked to excessive apoptosis of hematopoietic cells induced by oxidative stress. However, 20-25% of FA patients develop malignancies of myeloid origin. A survival strategy for bone marrow and hematopoietic cells under selective pressure evidently exists. This study reports that lymphoblastoid cell lines derived from two FA patients displayed significant resistance to oxidative stress induced by treatments with H(2) O(2) and various glutathione (GSH) inhibitors that induce production of reactive oxygen species, GSH depletion and mitochondrial membrane depolarization. Among the various GSH inhibitors employed, FA cells appear particularly resistant to menadione (5 μm) and ethacrynic acid (ETA, 50 μm), two drugs that specifically target mitochondria. Even after pre-treatment with buthionine sulfoximine, a GSH synthesis inhibitor that induces enhanced induction of reactive oxygen species, FA cells maintain significant resistance to these drugs. These data suggest that the resistance to oxidative stress and the altered mitochondrial and metabolic functionality found in the FA mutant cells used in this study may indicate the survival strategy that is adopted in FA cells undergoing transformation. The study of redox and mitochondria regulation in FA may be of assistance in diagnosis of the disease and in the care of patients.     

Fanconi anemia FANCG protein in mitigating radiation- and enzyme-induced DNA double-strand breaks by homologous recombination in vertebrate cells

The rare hereditary disorder Fanconi anemia (FA) is characterized by progressive bone marrow failure, congenital skeletal abnormality, elevated susceptibility to cancer, and cellular hypersensitivity to DNA cross-linking chemicals and sometimes other DNA-damaging agents. Molecular cloning identified six causative genes (FANCA, -C, -D2, -E, -F, and -G) encoding a multiprotein complex whose precise biochemical function remains elusive. Recent studies implicate this complex in DNA damage responses that are linked to the breast cancer susceptibility proteins BRCA1 and BRCA2. Mutations in BRCA2, which participates in homologous recombination (HR), are the underlying cause in some FA patients. To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40. FANCG-deficient DT40 cells resemble mammalian fancg mutants in that they are sensitive to killing by cisplatin and mitomycin C (MMC) and exhibit increased MMC and radiation-induced chromosome breakage. We find that the repair of I-SceI-induced chromosomal double-strand breaks (DSBs) by HR is decreased approximately 9-fold in fancg cells compared with the parental and FANCG-complemented cells. In addition, the efficiency of gene targeting is mildly decreased in FANCG-deficient cells, but depends on the specific locus. We conclude that FANCG is required for efficient HR-mediated repair of at least some types of DSBs.     

Fanconi anemia protein, FANCG, is a phosphoprotein and is upregulated with FANCA after TNF-alpha treatment

Fanconi anemia (FA) is a genetic syndrome characterized by bone marrow failure, birth defects, and a predisposition to malignancy. At this time, six FA genes have been identified, and several gene products have been found to interact in a protein complex. FA cells appear to overexpress the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). We therefore examined the effects of TNF-alpha on the regulation of FA complementation group proteins, FANCG and FANCA. We found that treatment with TNF-alpha induced FANCG protein expression. FANCA was induced concurrently with FANCG, and the FANCA/FANCG complex was increased in the nucleus following TNF-alpha treatment. Inactivation of inhibitory kappa B kinase-2 modulated the expression of FANCG. We also found that both nuclear and cytoplasmic FANCG fractions were phosphorylated. These results show that FANCG is a phosphoprotein and suggest that the cellular accumulation of FA proteins is subject to regulation by TNF-alpha signaling.     

Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase

Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.     

SkQ1 Controls <Emphasis Type="Italic">CASP3</Emphasis> Gene Expression and Caspase-3-Like Activity in the Brain of Rats under Oxidative Stress

Here, we studied the effect of the mitochondria-targeted antioxidant SkQ1 (plastoquinone cationic derivative) on the CASP3 gene expression and caspase-3 activity in rat cerebral cortex and brain mitochondria under normal conditions and in oxidative stress induced by hyperbaric oxygenation (HBO). Under physiological conditions, SkQ1 administration (50 nmol/kg, 5 days) did not affect the CASP3 gene expression and caspase-3-like activity in the cortical cells, as well as caspase-3-like activity in brain mitochondria, but caused a moderate decrease in the content of primary products of lipid peroxidation (LPO) and an increase in the reduced glutathione (GSH) level. HBO-induced oxidative stress (0.5 MPa, 90 min) was accompanied by significant upregulation of CASP3 mRNA and caspase-3-like activity in the cerebral cortex, activation of the mitochondrial enzyme with simultaneous decrease in the GSH content, increase in the glutathione reductase activity, and stimulation of LPO. Administration of SkQ1 before the HBO session maintained the basal levels of the CASP3 gene expression and enzyme activity in the cerebral cortex cells and led to the normalization of caspase-3-like activity and redox parameters in brain mitochondria. We hypothesize that SkQ1 protects brain cells from the HBO-induced oxidative stress due to its antioxidant activity and stimulation of antiapoptotic mechanisms.     

Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae

Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H(2) O(2) ) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress.     

Chronic melatonin treatment prevents age-dependent cardiac mitochondrial dysfunction in senescence-accelerated mice

Heart mitochondria from female senescence-accelerated (SAMP8) and senescence-resistant (SAMR1) mice of 5 or 10 months of age, were studied. Mitochondrial oxidative stress was determined by measuring the levels of lipid peroxidation, glutathione and glutathione disulfide and glutathione peroxidase and reductase activities. Mitochondrial function was assessed by measuring the activity of the respiratory chain complexes and ATP content. The results show that the age-dependent mitochondrial oxidative damage in the heart of SAMP8 mice was accompanied by a reduction in the electron transport chain complex activities and in ATP levels. Chronic melatonin administration between 1 and 10 months of age normalized the redox and the bioenergetic status of the mitochondria and increased ATP levels. The results support the presence of significant mitochondrial oxidative stress in SAM mice at 10 months of age, and they suggest a beneficial effect of chronic pharmacological intervention with melatonin, which reduces the deteriorative and functional oxidative changes in cardiac mitochondria with age.     

Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2

Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG-XRCC3 and FANCG-BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein-protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.     

FANCD1/BRCA2 plays predominant role in the repair of DNA damage induced by ACNU or TMZ

Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG(-/-) and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ.     

Role of Ferulic Acid in the Amelioration of Ionizing Radiation Induced Inflammation: A Murine Model

Ionizing radiation is responsible for oxidative stress by generating reactive oxygen species (ROS), which alters the cellular redox potential. This change activates several redox sensitive enzymes which are crucial in activating signaling pathways at molecular level and can lead to oxidative stress induced inflammation. Therefore, the present study was intended to assess the anti-inflammatory role of ferulic acid (FA), a plant flavonoid, against radiation-induced oxidative stress with a novel mechanistic viewpoint. FA was administered (50 mg/kg body wt) to Swiss albino mice for five consecutive days prior to exposing them to a single dose of 10 Gy ⁶⁰Co γ-irradiation. The dose of FA was optimized from the survival experiment and 50 mg/kg body wt dose showed optimum effect. FA significantly ameliorated the radiation induced inflammatory response such as phosphorylation of IKKα/β and IκBα and consequent nuclear translocation of nuclear factor kappa B (NF-κB). FA also prevented the increase of cycloxygenase-2 (Cox-2) protein, inducible nitric oxide synthase-2 (iNOS-2) gene expression, lipid peroxidation in liver and the increase of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in serum. It was observed that exposure to radiation results in decreased activity of superoxide dismutase (SOD), catalase (CAT) and the pool of reduced glutathione (GSH) content. However, FA treatment prior to irradiation increased the activities of the same endogenous antioxidants. Thus, pretreatment with FA offers protection against gamma radiation induced inflammation.     

Oxidative stress-induced apoptosis in neurons correlates with mitochondrial DNA base excision repair pathway imbalance

Neurodegeneration can occur as a result of endogenous oxidative stress. Primary cerebellar granule cells were used in this study to determine if mitochondrial DNA (mtDNA) repair deficiencies correlate with oxidative stress-induced apoptosis in neuronal cells. Granule cells exhibited a significantly higher intracellular oxidative state compared with primary astrocytes as well as increases in reductants, such as glutathione, and redox sensitive signaling molecules, such as AP endonuclease/redox effector factor-1. Cerebellar granule cultures also exhibited an increased susceptibility to exogenous oxidative stress. Menadione (50 μM) produced twice as many lesions in granule cell mtDNA compared with astrocytes, and granule cell mtDNA repair was significantly less efficient. A decreased capacity to repair oxidative mtDNA damage correlates strongly with mitochondrial initiated apoptosis in these neuronal cultures. Interestingly, the mitochondrial activities of initiators for base excision repair (BER), the bifunctional glycosylase/AP lyases as well as AP endonuclease, were significantly higher in cerebellar granule cells compared with astrocytes. The increased mitochondrial AP endonuclease activity in combination with decreased polymerase γ activity may cause an imbalance in oxidative BER leading to an increased production and persistence of mtDNA damage in neurons when treated with menadione. This study provides evidence linking neuronal mtDNA repair capacity with oxidative stress-related neurodegeneration.     

Seizure-induced changes in mitochondrial redox status

The aim of this study was to determine seizure-induced oxidative stress by measuring hippocampal glutathione (GSH) and glutathione disulfide (GSSG) levels in tissue and mitochondria. Kainate-induced status epilepticus (SE) in rats resulted in a time-dependent decrease of GSH/GSSG ratios in both hippocampal tissue and mitochondria. However, changes in GSH/GSSG ratios were more dramatic in the mitochondrial fractions compared to hippocampal tissue. This was accompanied by a mild increase in glutathione peroxidase activity and a decrease in glutathione reductase activity in hippocampal tissue and mitochondria, respectively. Since coenzyme A (CoASH) and its disulfide with GSH (CoASSG) are primarily compartmentalized within mitochondria, their measurement in tissue was undertaken to overcome problems associated with GSH/GSSG measurement following subcellular fractionation. Hippocampal tissue CoASH/CoASSG ratios were decreased following kainate-induced SE, the time course and magnitude of change paralleling mitochondrial GSH/GSSG levels. Cysteine, a rate-limiting precursor of glutathione was decreased following kainate administration in both hippocampal tissue and mitochondrial fractions. Together these changes in altered redox status provide further evidence for seizure-induced mitochondrial oxidative stress.