ROS Damage Research Articles

NFκB1 is a suppressor of neutrophil-driven hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) develops on the background of chronic hepatitis. Leukocytes found within the HCC microenvironment are implicated as regulators of tumour growth. We show that diethylnitrosamine (DEN)-induced murine HCC is attenuated by antibody-mediated depletion of hepatic neutrophils, the latter stimulating hepatocellular ROS and telomere DNA damage. We additionally report a previously unappreciated tumour suppressor function for hepatocellular nfkb1 operating via p50:p50 dimers and the co-repressor HDAC1. These anti-inflammatory proteins combine to transcriptionally repress hepatic expression of a S100A8/9, CXCL1 and CXCL2 neutrophil chemokine network. Loss of nfkb1 promotes ageing-associated chronic liver disease (CLD), characterized by steatosis, neutrophillia, fibrosis, hepatocyte telomere damage and HCC. Nfkb1S340A/S340Amice carrying a mutation designed to selectively disrupt p50:p50:HDAC1 complexes are more susceptible to HCC; by contrast, mice lacking S100A9 express reduced neutrophil chemokines and are protected from HCC. Inhibiting neutrophil accumulation in CLD or targeting their tumour-promoting activities may offer therapeutic opportunities in HCC.

Enhanced fatty acid oxidation in adipocytes and macrophages reduces lipid-induced triglyceride accumulation and inflammation.

Lipid overload in obesity and type 2 diabetes is associated with adipocyte dysfunction, inflammation, macrophage infiltration, and decreased fatty acid oxidation (FAO). Here, we report that the expression of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme in mitochondrial FAO, is higher in human adipose tissue macrophages than in adipocytes and that it is differentially expressed in visceral vs. subcutaneous adipose tissue in both an obese and a type 2 diabetes cohort. These observations led us to further investigate the potential role of CPT1A in adipocytes and macrophages. We expressed CPT1AM, a permanently active mutant form of CPT1A, in 3T3-L1 CARΔ1 adipocytes and RAW 264.7 macrophages through adenoviral infection. Enhanced FAO in palmitate-incubated adipocytes and macrophages reduced triglyceride content and inflammation, improved insulin sensitivity in adipocytes, and reduced endoplasmic reticulum stress and ROS damage in macrophages. We conclude that increasing FAO in adipocytes and macrophages improves palmitate-induced derangements. This indicates that enhancing FAO in metabolically relevant cells such as adipocytes and macrophages may be a promising strategy for the treatment of chronic inflammatory pathologies such as obesity and type 2 diabetes.

Subcellular localization of six thioredoxins and their antioxidant activity and contributions to biological control potential in Beauveria bassiana

Thioredoxins (Trx) can detoxify sulfide or act as electron donors in the reduction of disulfide and dithiol to protect yeast cells from ROS damage but remain poorly explored in filamentous fungi. Here we show more Trx homologs in Beauveria bassiana than in many other fungi and examine their functions. This filamentous entomopathogen has six Trx homologs, including four (Txr1–4) evidently localized in cytoplasm, one (Trx5) in nuclear membrane and another (Trx6) in mitochondria. Deletion of each trx had no effect on radial growth on rich or minimal medium but resulted in remarkable transcriptional up-regulation of other partners for compensation. Compared with wild-type, only Δtrx2 was significantly more sensitive to menadione whereas none of six Δtrx mutants was responsive to other oxidants including H2O2. Intriguingly, Δtrx2 showed uniquely a significant increase in total Trx activity in normal cultures but a remarkable decrease in total SOD activity in the cultures grown normally or co-cultivated with menadione. The ratio of reduced/oxidized glutathione accumulated in hyphal cells stressed with menadione decreased to only 0.4 in Δtrx2 from ∼1.0 observed in wild-type and other mutants. The six Δtrx mutants displayed one or more phenotypic changes associated with the fungal biocontrol potential, including conidiation, and germination, thermotolerance, UV-B resistance and virulence of their conidia. All the changes were restored by trx complementation. Taken together, the greater Trx diversity evolutionarily gained by B. bassiana could help it to maintain cellular redox homeostasis and infect insect hosts in diverse habitats.

Mechanisms of hyperhomocysteinemia induced skeletal muscle myopathy after ischemia in the CBS-/+ mouse model.

Although hyperhomocysteinemia (HHcy) elicits lower than normal body weights and skeletal muscle weakness, the mechanisms remain unclear. Despite the fact that HHcy-mediated enhancement in ROS and consequent damage to regulators of different cellular processes is relatively well established in other organs, the nature of such events is unknown in skeletal muscles. Previously, we reported that HHcy attenuation of PGC-1α and HIF-1α levels enhanced the likelihood of muscle atrophy and declined function after ischemia. In the current study, we examined muscle levels of homocysteine (Hcy) metabolizing enzymes, anti-oxidant capacity and focused on protein modifications that might compromise PGC-1α function during ischemic angiogenesis. Although skeletal muscles express the key enzyme (MTHFR) that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE) make skeletal muscles more susceptible to the HHcy-induced myopathy. Our study indicates that elevated Hcy levels in the CBS−/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia. Furthermore, in the presence of NO donor SNP, either homocysteine (Hcy) or its cyclized version, Hcy thiolactone, not only increased PGC-1α specific protein nitrotyrosylation but also reduced its association with PPARγ in C2C12 cells. Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.

DNA damage and inhibition of akt pathway in mcf-7 cells and ehrlich tumor in mice treated with 1,4-naphthoquinones in combination with ascorbate.

The aim of this study was to enhance the understanding of the antitumor mechanism of 1,4-naphthoquinones and ascorbate. Juglone, phenylaminonaphthoquinone-7, and 9 (Q7/Q9) were evaluated for effects on CT-DNA and DNA of cancer cells. Evaluations in MCF-7 cells are DNA damage, ROS levels, viability, and proliferation. Proteins from MCF-7 lysates were immunoblotted for verifying PARP integrity, γH2AX, and pAkt. Antitumor activity was measured in Ehrlich ascites carcinoma-bearing mice. The same markers of molecular toxicity were assessed in vivo. The naphthoquinones intercalate into CT-DNA and caused oxidative cleavage, which is increased in the presence of ascorbate. Treatments caused DNA damage and reduced viability and proliferation of MCF-7 cells. Effects were potentiated by ascorbate. No PARP cleavage was observed. Naphthoquinones, combined with ascorbate, caused phosphorylation of H2AX and inhibited pAkt. ROS were enhanced in MCF-7 cells, particularly by the juglone and Q7 plus ascorbate. Ehrlich carcinoma was inhibited by juglone, Q7, or Q9, but the potentiating effect of ascorbate was reproduced in vivo only in the cases of juglone and Q7, which caused up to 60% inhibition of tumor and the largest extension of survival. Juglone and Q7 plus ascorbate caused enhanced ROS and DNA damage and inhibited pAkt also in Ehrlich carcinoma cells.

Oxidative Stress, Skin Aging and Antioxidant Therapy / Oksidacioni Stres, Starenje Kože I Antioksidaciona Terapija

Summary Skin aging is a dynamic process that occurs due to chronological and photo-aging caused by the influence of external factors, especially ultraviolet-UV radiation. Cumulative oxidative stress, formation of free radicals and their adverse effects on biological systems in the skin cells is a common mechanism of both skin aging processes. The formed reactive oxygen species-ROS can lead to the oxidation of cell`s building molecules. Lipid peroxidation, membrane protein damage and DNA mutation may lead to many structural, functional and aesthetic changes in the skin and can also initiate development of many diseases. One of the skin defense mechanisms is antioxidant defense where enzymes and other antioxidant substances react directly with ROS, preventing them from reaching their biological target. Antioxidant substances have the ability to bind free radicals, caused by oxidative stress, and may have significance in prevention and/or therapy of various skin diseases, as well as in slowing the skin aging process. Topical application of antioxidants, such as vitamin C and E, coenzyme Q10 and polyphenolic compounds may provide the strengthening of the skin endogenous protection system and protection from harmful effects of ROS and oxidative damage to the skin. This article outlines the impact of oxidative stress on the chronological and photo- aging, its mechanism of action, the changes that occur on/in the skin during the aging process. Substances with antioxidant characteristics, which can be used in the prevention of chronological and/or treatment of photo-aging of the skin are shown.

Effect of CO2, nutrients and light on coastal plankton. IV. Physiological responses

We studied the physiological response of phytoplankton to the interacting effects of 3 factors affected by global climate change: CO2, nutrient loading and irradiance. Treatments had a high and low level for each factor: CO2 was bubbled at 1000 ppm by volume versus present atmo spheric values; high nutrient treatments had a combination of inorganic and organic nutri- ents; and light treatments were obtained by covering the tanks with a single or double layer of screen. We measured esterase activity, oxidative stress (ROS), cell death, DNA damage, photosyn- thetic efficiency and 14 C assimilation as particulate or dissolved organic material (POC and DOC respectively). Conditions simulating future global change scenarios showed similar chlorophyll- normalized primary productivity as present conditions. The main effect driving phytoplankton physiology was the downregulation of the photosynthetic apparatus by elevated CO2, which decreased esterase activity, ROS, cell death and DNA damage. Nutrient concentration and light acted as additional modulators, upregulating or contributing to downregulation. The percentage of DO 14 C extracellular release (PER) was low (0 to 27%), significantly lower under ultraviolet radi- ation (UVR) than under photosynthetically active radiation (PAR), and acted mainly to re- equilibrate the internal balance when cells grown under UVR were exposed to PAR. PER was almost 3 times lower under high CO2, confirming a higher resource use efficiency of phytoplank- ton under future CO2 concentrations.

25-Hydroxyvitamin D3-deficiency enhances oxidative stress and corticosteroid resistance in severe asthma exacerbation.

Oxidative stress plays a significant role in exacerbation of asthma. The role of vitamin D in oxidative stress and asthma exacerbation remains unclear. We aimed to determine the relationship between vitamin D status and oxidative stress in asthma exacerbation. Severe asthma exacerbation patients with 25-hydroxyvitamin D3-deficiency (V-D deficiency) or 25-hydroxyvitamin D-sufficiency (V-D sufficiency) were enrolled. Severe asthma exacerbation with V-D-deficiency showed lower forced expiratory volume in one second (FEV1) compared to that with V-D-sufficiency. V-D-deficiency intensified ROS release and DNA damage and increased TNF-α, OGG1 and NFκB expression and NFκB phosphorylation in severe asthma exacerbation. Supplemental vitamin D3 significantly increased the rates of FEV1 change and decreased ROS and DNA damage in V-D-deficiency. Vitamin D3 inhibited LPS-induced ROS and DNA damage and were associated with a decline in TNF-α and NFκB in epithelial cells. H2O2 reduces nuclear translocation of glucocorticoid receptors in airway epithelial cell lines. V-D pretreatment enhanced the dexamethasone-induced nuclear translocation of glucocorticoid receptors in airway epithelial cell lines and monocytes from 25-hydroxyvitamin D3-deficiency asthma patients. These findings indicate that V-D deficiency aggravates oxidative stress and DNA damage, suggesting a possible mechanism for corticosteroid resistance in severe asthma exacerbation.

New cross talk between ROS, ABA and auxin controlling seed maturation and germination unraveled in APX6 deficient Arabidopsis seeds

Successful execution of germination program greatly depends on the seeds’ oxidative homeostasis. We recently identified new roles for the H2O2-reducing enzyme ascorbate peroxidase 6 (APX6) in germination control and seeds’ stress tolerance. APX6 replaces APX1 as the dominant APX in dry seeds, and its loss-of-function results in reduced germination due to over accumulation of ROS and oxidative damage. Metabolic analyses in dry apx6 seeds, revealed altered homeostasis of primary metabolites including accumulation of TCA cycle metabolites, ABA and auxin, supporting a novel role for APX6 in regulating cellular metabolism. Increased sensitivity of apx6 mutants to ABA or IAA in germination assays indicated impaired perception of these signals. Relative suppression of ABI3 and ABI5 expression, and induction of ABI4, suggested the activation of a signaling route inhibiting germination in apx6 seeds that is independent of ABI3. Here we provide additional evidence linking ABI4 with ABA- and auxin-controlled inhibition of germination and suggest a hypothetical model for the role of APX6 in the regulation of the crosstalk between these hormones and ROS.

Hepatotoxicity in mice of a novel anti-parasite drug candidate hydroxymethylnitrofurazone: a comparison with Benznidazole.

BackgroundTreatment of Chagas disease, caused by Trypanosoma cruzi, relies on nifurtimox and benznidazole (BZL), which present side effects in adult patients, and natural resistance in some parasite strains. Hydroxymethylnitrofurazone (NFOH) is a new drug candidate with demonstrated trypanocidal activity; however, its safety is not known.MethodsHepG2 cells dose response to NFOH and BZL (5–100 µM) was assessed by measurement of ROS, DNA damage and survival. Swiss mice were treated with NFOH or BZL for short-term (ST, 21 d) or long-term (LT, 60 d) periods. Sera levels of cellular injury markers, liver inflammatory and oxidative stress, and fibrotic remodeling were monitored.ResultsHepG2 cells exhibited mild stress, evidenced by increased ROS and DNA damage, in response to NFOH, while BZL at 100 µM concentration induced >33% cell death in 24 h. In mice, NFOH ST treatment resulted in mild-to-no increase in the liver injury biomarkers (GOT, GPT), and liver levels of inflammatory (myeloperoxidase, TNF-α), oxidative (lipid peroxides) and nitrosative (3-nitrotyrosine) stress. These stress responses in NFOH LT treated mice were normalized to control levels. BZL-treated mice exhibited a >5-fold increase in GOT, GPT and TNF-α (LT) and a 20–40% increase in liver levels of MPO activity (ST and LT) in comparison with NFOH-treated mice. The liver inflammatory infiltrate was noted in the order of BZL>vehicle≥NFOH and BZL>NFOH≥vehicle, respectively, after ST and LT treatments. Liver fibrotic remodeling, identified after ST treatment, was in the order of BZL>vehicle>NFOH; lipid deposits, indicative of mitochondrial dysfunction and in the order of NFOH>vehicle>BZL were evidenced after LT treatment.ConclusionsNFOH induces mild ST hepatotoxicity that is normalized during LT treatment in mice. Our results suggest that additional studies to determine the efficacy and toxicity of NFOH are warranted.

Abstract 215: The Redox Activation of DUSP4 by N-Acetyl Cysteine Protects Endothelial Cells and the Myocardium against Oxidative Damage

Background: Redox imbalance is the primary cause for endothelial dysfunction (ED), obstructed blood flow, and subsequent heart attack and failure. Under oxidant stress, many critical proteins regulating endothelial function undergo oxidative modifications that lead to ED. Cellular levels of glutathione (GSH), the primary reducing source, can significantly regulate cell function via reversible protein thiol modification. N-Acetyl cysteine (NAC), a precursor for GSH biosynthesis, is beneficial for many vascular diseases; however, the detailed mechanism of these benefits is still not clear. Methods: We employed EPR spin-trapping, HPLC, fluorescent microscopy, immunoblotting, and qPCR of both in vitro and ex vivo experiments using either cultured cells or the Langendorff heart preparation. Results: NAC treatment increases NO generation from endothelial cells, as well as the enzyme and cofactor responsible for its production, ie eNOS and BH4. Interestingly, NAC treatment also increased the expression of DUSP4, an inducible nuclear dual-specificity phosphatase implicated in cardiovascular function. We hereby establish that DUSP4 redox modulates two important kinases (p38 and ERK1/2) of MAPK signaling pathways and provides protection against Cd2+-induced ROS damage as well as hypoxia-reoxygenation insult to endothelial cells. Furthermore, a four week oral NAC pre-treatment promotes DUSP4 both protein and mRNA expression in the rat myocardium and renders the heart less susceptible to ex vivo ischemia-reperfusion injury. Protein expression profiles in the myocardium closely mimic those observed with cultured endothelial cells. The infarct size of NAC-treated hearts is significantly reduced. The myocardial rate pressure product is much improved above vehicle treated rats. Conclusion: NAC serves as a direct antioxidant and as a regulator of transcription and translation of DUSP4, modulating the activity of its downstream effectors: ERK1/2 and p38, and thus protects against ROS-induced damage both in vitro and ex vivo. As such, NAC-derived drugs can provide a novel therapy to oxidant-induced diseases via the specific up-regulation of protective proteins such as DUSP4 and eNOS.

Mössbauer, EPR, and modeling study of iron trafficking and regulation in Δccc1 and CCC1-up Saccharomyces cerevisiae.

Strains lacking and overexpressing the vacuolar iron (Fe) importer CCC1 were characterized using Mössbauer and EPR spectroscopies. Vacuolar Fe import is impeded in Δccc1 cells and enhanced in CCC1-up cells, causing vacuolar Fe in these strains to decline and accumulate, respectively, relative to WT cells. Cytosolic Fe levels should behave oppositely. The Fe content of Δccc1 cells grown under low-Fe conditions was similar to that in WT cells. Most Fe was mitochondrial with some nonheme high spin (NHHS) FeII present. Δccc1 cells grown with increasing Fe concentration in the medium contained less total Fe, less vacuolar HS FeIII, and more NHHS FeII than in comparable WT cells. As the Fe concentration in the growth medium increased, the concentration of HS FeIII in Δccc1 cells increased to just 60% of WT levels, while NHHS FeII increased to twice WT levels, suggesting that the NHHS FeII was cytosolic. Δccc1 cells suffered more oxidative damage than WT cells, suggesting that the accumulated NHHS FeII promoted Fenton chemistry. The Fe concentration in CCC1-up cells was higher than in WT cells; the extra Fe was present as NHHS FeII and FeIII and as FeIII oxyhydroxide nanoparticles. These cells contained less mitochondrial Fe and exhibited less ROS damage than Δccc1 cells. CCC1-up cells were adenine-deficient on minimal medium; supplementing with adenine caused a decline of NHHS FeII suggesting that some of the NHHS FeII that accumulated in these cells was associated with adenine deficiency rather than the overexpression of CCC1. A mathematical model was developed that simulated changes in Fe distributions. Simulations suggested that only a modest proportion of the observed NHHS FeII in both strains was the cytosolic form of Fe that is sensed by the Fe import regulatory system. The remainder is probably generated by the reduction of the vacuolar NHHS FeIII species.

Effect of advancing paternal age on semen parameters and seminal oxidative stress markers in infertile men

BackgroundHigh rate of subfertility and adverse pregnancy related out-comes associated with childbearing are seen after age 40.In contrast to oogenesis, spermatogenesis continues inelderly men. Adult male germ cells pass through signifi-cantly more mitotic replications than the germ cells inadult female. In men, there is an age associated increase inthe incidence of breaks in sperm DNA, decrease in apopto-sis, and a higher frequency of point mutations. Advancedpaternal age is associated with an increased time to preg-nancy and decreased pregnancy rates. After adjusting forfemale age, conception during a 12-month period was > 30percent less likely for men over 40 years of age as com-pared to men 45years compared to men 40 years (n = 105). We evaluated the conven-tional semen parameters (WHO, 2010) [5] and OS mar-kers: seminal ROS (chemiluminescence assay), totalantioxidant capacity (TAC) and sperm DNA damage(TUNEL assay) in all patients.ResultsThe mean age of the study subjects was 36.8 ± 6.7 years.No age-related differences were seen in conventionalsemen parameters (volume, concentration, motility, andmorphology) (Table 1). ROS and antioxidant levels werecomparable in the 3 groups. Significantly higher levels ofsperm DNA damage (19.94 ± 15.30%) was seen in infertilemen >40 years compared to men in younger age groups(P = 0.028 and P = 0.027, respectively).ConclusionsSperm DNA damage increases with advancing paternalage. Evaluation of sperm DNA damage will help diagnosethe underlying cause of poor fertility in some men andassist the clinician in offering correct treatment modality.

The p66Shc Adaptor Protein Controls Oxidative Stress Response in Early Bovine Embryos

The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2–4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2–4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos.

Phosphine-induced physiological and biochemical responses in rice seedlings

Paddy fields have been demonstrated to be one of the major resources of atmospheric phosphine and may have both positive and negative effects on rice plants. To elucidate the physiological and biochemical responses of rice plants to phosphine, rice seedlings (30d old) were selected as a model plant and were treated with different concentrations of phosphine (0, 1.4, 4.2, and 7.0mgm−3). Antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and lipid peroxidation measured via malondialdehyde (MDA) were determined as indicators of the physiological and biochemical responses of the rice seedlings to phosphine exposure. Increasing concentrations of phosphine treatment enhanced the activity of SOD, POD, and CAT. In addition, the MDA content increased with increasing concentrations of phosphine. These results suggested that antioxidant enzymes played important roles in protecting rice seedlings from ROS damage. Moreover, rice seedlings were able to cope with the oxidative stress induced by low concentrations of phosphine via an increase in antioxidant enzymatic activities. However, oxidative stress may not fully be prevented when the plants were exposed to higher concentrations of phosphine.

Accumulation of Microsatellite Instability in Superoxide Dismutase-1 Knockout Mice: A Possible Predictor of Germ Cell Development or Tumorigenesis

The superoxide dismutase 1 gene (Sod1) is one of the cell protective mechanisms to eliminate reactive oxygen species damage in order to protect cell components and avoid germinal failure or tumorigenesis. Mouse primordial germinal cells are a population of cells originating from the proximal margin of the epiblast in close proximity to the extra embryonic ectoderm, and from which some cells differentiate into male or female germinal cells. During cell migration across the genital ridge, primordial germinal cells display an extensive proliferation rate and they are exposed to ROS damage. In absence of cell antioxidant mechanisms, primordial germinal cells have increased risk to accumulate specific mutations by free radical damage. We hypothesize that during cell migration, Sod1 knockout primordial germinal cells accumulate microsatellite instability and this could be a signal for primordial germinal cells pool selection, premature ovarian failure or tumor transformation. We dissected and isolated primordial germinal cellsfrom Sod1+/+ (wild type), Sod1+/- (heterozygous), and Sod1-/- (homozygous) mouse strain (B6; 129S7-Sod1tm1Leb/J), from the genital ridge at 10. 5 and 18 days post coitum to determine and quantify microsatellite instability in 10 single tandem repeat markers by single cell PCR methods. Our results demonstrate that primordial germinal cells from Sod1knockout mice accumulated microsatellite instability in five markers (4-4-19844, D13mit16, D13mit78, D19mit68, and DXmit249) located near important genes involved in cell proliferation and differentiation. Taken together, this study shows that microsatellite instability is directly related to Sod1 gene disruption that could induce abnormal migration characteristics seen in primordial germinal cells such as germinal premature failure or tumor invasion and metastasis. In conclusion, microsatellite instability could be a signal for primordial germinal cells pool selection, or tumorigenesis.

Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin

Reactive oxygen species (ROS)-induced DNA damage is repaired by the base excision repair pathway. However, the effect of chromatin structure on BER protein recruitment to DNA damage sites in living cells is poorly understood. To address this problem, we developed a method to specifically produce ROS-induced DNA damage by fusing KillerRed (KR), a light-stimulated ROS-inducer, to a tet-repressor (tetR-KR) or a transcription activator (TA-KR). TetR-KR or TA-KR, bound to a TRE cassette (∼90 kb) integrated at a defined genomic locus in U2OS cells, was used to induce ROS damage in hetero- or euchromatin, respectively. We found that DNA glycosylases were efficiently recruited to DNA damage in heterochromatin, as well as in euchromatin. PARP1 was recruited to DNA damage within condensed chromatin more efficiently than in active chromatin. In contrast, recruitment of FEN1 was highly enriched at sites of DNA damage within active chromatin in a PCNA- and transcription activation-dependent manner. These results indicate that oxidative DNA damage is differentially processed within hetero or euchromatin.

Abstract 185: Additional Benefit of Cyclosporine-Assisted Adipose-Derived Mesenchymal Stem Cell Therapy for Against Acute Kidney Ischemia-Reperfusion Injury

Objectives: This study tested the hypothesis that cyclsporine (CsA)-supported adipose-derived mesenchymal stem cell (ADMSC) therapy offered superior attenuation of acute ischemia-reperfusion (IR) kidney injury than either therapy alone. Methods: Adult SD-rats (n=40) were equally divided into group 1 (sham controls), group 2 (IR injury), group 3 [IR + CsA (20 mg/kg at 1 and 24 hr after procedure)], group 4 [ADMSC (1.2x106) at 1, 6 and 24 h after procedure], and group 5 (IR + CsA-ADMSC). Results: By 72 h after IR procedure, creatinine level and the ratio of urine protein to creatinine were highest in group 2 and lowest in group 1, significantly higher in groups 3 and 4 than in group 5, but they showed no differences between groups 3 and 4 (all p<0.0001). The inflammatory biomarkers at mRNA (MMP-9, RANTES, TNF-α), protein (TNF-α, NF-κB, ICAM-1, PDGF), and cellular (CD68+) levels of IR kidney showed an identical pattern to creatinine level in all groups (all p<0.0001). The protein expressions of oxidative stress (oxidized protein), ROS (NOX-1, NOX-2), apoptosis (Bax, caspase-3 and PARP) and DNA damage markers (γH2AX+, PNCA+ cells) exhibited a similar pattern to inflammatory mediators (all p<0.0001) amongst all groups (all p<0.0001). Anti-oxidant biomarkers at cellular (GPx, GR, HO-1) and protein (NQO-1, HO-1, eNOS) levels, and endothelial progenitor cell markers (CXCR4+, SDF-1α+) were lowest in groups 1 and 2, higher in groups 3 and 4 and highest in group 5 (all p<0.0001). Conclusions: Combination therapy using cyclosporine-plus-ADMSCs offers improved protection against acute IR kidney injury. Key words: cyclosporine, adipose-derived mesenchymal stem cell, acute kidney ischemia-reperfusion injury, combination therapy, inflammation, oxidative stress, apoptosis

Normal ABL1 Is a Tumor Suppressor and Therapeutic Target In BCR-ABL1–positive Leukemias

Abstract BCR-ABL1 results from t(9;22)(q34;q11) reciprocal translocation resulting in BCR-ABL1 kinase expression, initiating chronic myeloid leukemia in chronic phase (CML-CP). At the initial stages of CML-CP both oncogenic BCR-ABL1 kinase and normal ABL1 kinase are expressed, however, loss of ABL1 kinase expression in CML-CP can result from an interstitial deletion in the normal chromosome 9 [del(9q34)] which may be combined with the transcriptional silencing of the alternative ABL1 promoter within the translocation eventually leading to disease progression and drug resistance. We found that BCR-ABL1 Abl1-/- cells generated a CML-blast phase (BP)-like disease phenotype in SCID mice compared to CML-CP-like disease from BCR-ABL1 Abl1+/+ cells. To determine the mechanisms responsible for blastic transformation of BCR-ABL1 Abl1-/- cells, we examined the role of ABL1 in proliferation, differentiation, apoptosis, genomic instability, and stemness. The presence of ABL1 inhibited proliferation in BCR-ABL1 cells as BCR-ABL1 Abl1-/- cells had higher clonogenic activity and proliferative rate compared to their wild-type counterparts. ABL1 is essential for myeloid differentiation since BCR-ABL1 Abl1-/- cells showed an immature blast phenotype when stained with Wright-Giemsa and myeloid differentiation markers Gr-1 and CD11b. ABL1 promoted apoptosis in response to genotoxic stress as revealed by reduced clonogenicity and elevated expression of p53, phosphoserine-15 p53 and activated caspase 3 in BCR-ABL1 Abl1 +/+ compared to knock-out cells. Although the absence of ABL1 did not enhance ROS and oxidative DNA damage, it appears that an impaired DNA damage response may be responsible for higher chromosome numbers and an accumulation of high numbers of chromosomal aberrations in BCR-ABL1 Abl1-/- cells. We detected an expansion of Lin-c-Kit+Sca-1+ leukemia stem cells (LSCs) in BCR-ABL1 Abl1-/- cells compared to BCR-ABL1 Abl1+/+ or non-transformed counterparts; among the LSCs, there was a higher percentage of CD34-Flt3- long-term and CD34+Flt3-short-term stem cells. These results showed that ABL1 is involved in regulating the LSC compartment in BCR-ABL1 cells. DNA microarray analysis revealed changes in mRNA levels of several genes involved in proliferation, myeloid differentiation, apoptosis, DNA damage response and stemness in BCR-ABL1 Abl1-/- cells in comparison to BCR-ABL1 Abl1+/+ cells. Together, these results demonstrated a critical role of ABL1 in BCR-ABL1-induced leukemia, prolonging survival in mice by suppressing proliferation and expansion of LSC, inducing myeloid differentiation, apoptosis and DNA damage response in BCR-ABL1 cells. Thus, it appears that ABL1 acts as a tumor suppressor in BCR-ABL1 –positive CML cells. Moreover, we hypothesized that the enhancement of the tumor suppressor function of ABL1 may have a significant impact on CML treatment. A small molecule activator of ABL1 kinase, 5-(1,3-diaryl-1H-pyrazol-4-yl)hydantoin (DPH), had been reported to interact with the myristoyl-binding site of ABL1 and destabilize the bent conformation of the α-1 helix, thereby preventing the auto-inhibitory conformation. DPH partially restored ABL1 activity in imatinib-treated cells. DPH-mediated stimulation of ABL1 tumor suppressor activity enhanced the effect of imatinib and ponatinib against CML CD34+ cells, Philadelphia chromosome-positive B-ALL (Ph+B-ALL) cells and relapsed Ph+B-ALL cells harboring T315I mutation without affecting normal counterparts. In summary, ABL1 is a potential tumor suppressor in BCR-ABL1-induced leukemia and stimulation of its function may play a significant role in the development of novel therapeutic strategies for CML and Ph+ALL. Disclosures: No relevant conflicts of interest to declare.

Compost enhances plant resistance against the bacterial wilt pathogen Ralstonia solanacearum via up-regulation of ascorbate-glutathione redox cycle

The interactions between the pathogen Ralstonia solanacearum and potato Solanum tuberosum plants were studied to investigate the reactive oxygen species metabolic system and ascorbate (ASC)-glutathione (GSH) redox cycle in response to compost application. Single potato eyepieces were germinated and grown in pots containing sandy soil with or without compost at a rate of 7.5 g kg−1 soil. Non-compost- and compost-treated plants (CTP) were inoculated with R. solanacearum 25 days after planting and then analyzed after 10 days, unless otherwise stated. The present results revealed that pathogen infection caused a remarkable decrease in plant growth related parameters and productivity and an increase in disease incidence. However, under these conditions compost had substantially improved plant growth and decreased disease incidence and bacterial population. R. solanacearum resulted in significant enhancement in the activities of NADPH oxidase, lipoxygenase, the production rate of superoxide and hydroxyl radicals, levels of hydrogen peroxide, membrane lipid peroxidation, and protein oxidation indicating the induction of oxidative stress in potato roots. However, the pathogen-mediated enhancement in indices of oxidative stress was considerably decreased by compost application, which enhanced the activities of ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) in infected potato plants, implying a better ROS-scavenging activity. Data also indicated that there were general increases in ASC and GSH content in infected compost treated plants, but non-compost treated ones significantly had lower levels of such redox metabolites. In addition, significantly higher ratios of ASC/DHA (dehydroascorbate) and GSH/GSSG (glutathione disulphide) were generally found in CTP than in non-compost treated-ones. The obtained results suggest that compost provides effective protection against the Ralstonia bacterial pathogen via up-regulation of the capacity of the ASC-GSH cycle and modulation of the cellular redox status, thereby eliminating ROS damage and sustaining membrane stability.